Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides Patent Application (2025)

U.S. patent application number 14/365063 was filed with the patent office on 2014-11-27 for mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides. The applicant listed for this patent is Universiteit Gent. Invention is credited to Joeri Beauprez, Gaspard Lequeux, Jo Maertens.

Mutant microorganisms to synthesize colanic acid, mannosylatedand/or fucosylated oligosaccharides

Abstract

The present invention relates to mutated and/or transformedmicroorganisms for the synthesis of various compounds. Morespecifically, the present invention discloses microorganismsmutated in the genes encoding for the regulators ArcA and IclR. Thelatter mutations result in a significant upregulation of the genesthat are part of the colanic acid operon. Hence, saidmicroorganisms are useful for the synthesis of any compound beingpart of the colanic acid pathway such as GDP-fucose, GDP-mannoseand colanic acid, and/or, can be further used--starting formGDP-fucose as a precursor--to synthesize fucosylatedoligosaccharides or--starting from GDP-mannose as a precursor--tosynthesize mannosylated oligosaccharides. In addition, mutations inthe genes coding for the transcriptional regulators ArcA and IclRlead to an acid resistance phenotype in the exponential growthphase allowing the synthesis of pH sensitive molecules or organicacids.

Foreign Application Data

Claims

1. Use of a mutated and/or transformed microorganism comprising agenetic change leading to a modified expression of thetranscriptional regulators the aerobic respiration control proteinArcA and the isocitrate lyase regulator IclR, to upregulate atleast one of the genes of the colanic acid operon, wherein saidoperon comprises the genes cpsG, cpsB, gmd and fcl that code for aphosphomannomutase, a mannose-1-phosphate guanylyltransferase,GDP-mannose 4,6-dehydratase and GDP-fucose synthase,respectively.

2. Use according to claim 1, wherein said upregulation of at leastone of the genes of the colanic acid operon is preceded by theupregulation of the transcriptional regulator of said colanic acidoperon rcsA.

3. Use according to claim 1 or 2, wherein said microorganism is anEscherichia coli strain, wherein said E. coli strain isspecifically a K12 strain or, wherein said K12 strain is morespecifically E. coli MG1655.

4. Use according to claims 1-3, wherein said genetic change, isdisrupting the genes encoding for ArcA and IclR, is replacing theendogenous promoters of the genes encoding for ArcA and IclR byartificial promoters, or is replacing the endogenous ribosomebinding site by an artificial ribosome binding site.

5. Use according to claims 1-4 wherein said modified expression isa decreased expression, or wherein said decreased expressionspecifically is an abolished expression.

6. Use according to any of claims 1-5, wherein at least one of thegenes of the colanic acid operon are upregulated 6 to 8 times incomparison to the expression of the colanic acid operon in thecorresponding wild type microorganism.

7. A process for the synthesis of colanic acid and/or GDP-fucoseand/or fucosylated oligosaccharides comprising: geneticallychanging the transcriptional regulators, the aerobic respirationcontrol protein ArcA, and the isocitrate lyase regulator IclR, toupregulate at least one of the genes of the colanic acid operon,wherein said operon comprises the genes cpsG, cpsB, gmd andfcl.

8. A process according to claim 7, wherein the mutations accordingto claim 1 are applied in combination with at least one mutationthat enhances the production of fucosylated compounds.

9. A process according to claim 8, wherein said at least onemutation that enhance the production of fucosylated compounds isselected from the group consisting of: the deletion of the wcaJgene; knocking-out the colanic acid operon genes gmm, wcaA, wcaB,wcaC, wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzcand/or wcaM; knocking-out lacZ; introducing a sucrose phosporylaseor invertase; knocking out the genes pgi, pfkA and pfkB; knockingout the gene Ion; introducing a fucosyltransferase and/or a lactosepermease; or combinations thereof.

10. A process according to claim 7, for the synthesis ofGDP-mannose and/or for the synthesis of mannosylatedoligosaccharides.

11. A process according to claim 10 wherein the genes cpsG and cpsBof the colanic acid operon are upregulated and wherein: a) the genegmd of the colanic acid operon is deleted, and/or, b) wherein thegene gmm is deleted, and/or c) wherein the colanic acid operongenes fcl, gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI,wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM are knocked outand/or, d) wherein a gene encoding for a sucrose phosphorylase oran invertase is introduced, and/or, e) wherein the genes pgi, pfkAand pfkB are deleted, and/or, f) knocking out the gene Ion, and/org) wherein a gene encoding for a mannosyltransferase isintroduced.

12. A mutated and/or transformed organism in which the regulatorsArcA and IclR, in combination with the genes encoding for theenzymes phosphoglucose isomerase and phosphofructokinase, areknocked out or are rendered less functional.

13. A mutated and/or transformed organism according to claim 12wherein the enzyme phosphoglucose isomerase is encoded by the genepgi and wherein the enzyme phosphofructokinase is encoded by thegene(s) pfkA and/or pfkB.

14. A mutated and/or transformed organism according to claim 12 or13 wherein said organism is further transformed with a geneencoding for a sucrose phosphorylase or invertase.

15. A mutated and/or transformed organism according to any one ofclaims 12 to 14 wherein the activity of the gene encoding for alactose permease is increased.

16. A mutated and/or transformed organism according to any one ofclaims 12 to 15 wherein at least one of the following genes isknocked out or is rendered less functional: a gene encoding for abeta-galactosidase, a gene encoding for a glucose-1-phosphateadenylyltransferase, a gene encoding for a glucose-1-phosphatase, agene encoding for phosphogluconate dehydratase, a gene encoding for2-keto-3-deoxygluconate-6-phosphate aldolase, a gene encoding for aglucose-1-phosphate uridyltransferase, a gene encoding for anUDP-glucose-4-epimerase, a gene encoding for anUDP-glucose:galactose-1-phosphate uridyltransferase, a geneencoding for an UDPgalactopyranose mutase, a gene encoding for anUDP-galactose:(glucosyl)lipopolysaccharide-1,6-galactosyltransferase,a gene encoding for an UDP-galactosyltransferase, a gene encodingfor an UDP-glucosyltransferase, a gene encoding for anUDP-glucuronate transferase, a gene encoding for an UDP-glucoselipid carrier transferase, a gene encoding for a GDP-mannosehydrolase, a gene encoding for an UDP-sugar hydrolase, a geneencoding for a mannose-6-phosphate isomerase, a gene encoding foran UDP-N-acetylglucosamine enoylpyruvoyl transferase, a geneencoding for an UDP-Nacetylglucosamine acetyltransferase, a geneencoding for an UDP-Nacetylglucosamine-2-epimerase, a gene encodingfor an undecaprenyl-phosphate alfa-N-acetylglucosaminyltransferase, a gene encoding for aglucose-6-phosphate-1-dehydrogenase, and/or, a gene encoding for aL-glutamine:D-fructose-6-phosphate aminotransferase, a geneencoding for a mannose-6-phosphate isomerase, a gene encoding for asorbitol-6-phosphate dehydrogenase, a gene encoding for amannitol-1-phosphate 5-dehydrogenase, a gene encoding for aallulose-6-phosphate 3-epimerase, a gene encoding for an invertase,a gene incoding for a maltase, a gene encoding for a trehalase, agene encoding for a sugar transporting phosphotransferase, a geneencoding for a protease, or a gene encoding for a hexokinase.

17. Use of a mutated and/or transformed Escherichia coli straincomprising a genetic change leading to a modified expression of thetranscriptional regulators the aerobic respiration control proteinArcA and the isocitrate lyase regulator IclR, to upregulate atleast one of the following acid resistance related genes: ydeP,ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or slp.

18. Use according to claim 17 for the synthesis of acids, sialicacid, sialylated oligosaccharides or glucosamine.

19. A process for the synthesis of acids, sialic acid, sialylatedoligosaccharides or glucosamine comprising genetically changing thetranscriptional regulators the aerobic respiration control proteinArcA and the isocitrate lyase regulator IclR to upregulate at leastone of the following acid resistance related genes: ydeP, ydeO,hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or slp.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to mutated and/or transformedmicroorganisms for the synthesis of various compounds. Morespecifically, the present invention discloses microorganismsmutated in the genes encoding for the regulators ArcA and IclR. Thelatter mutations result in a significant upregulation of the genesthat are part of the colanic acid operon. Hence, saidmicroorganisms are useful for the synthesis of any compound beingpart of the colanic acid pathway such as GDP-fucose, GDP-mannoseand colanic acid, and/or, can be further used--starting fromGDP-fucose as a precursor--to synthesize fucosylatedoligosaccharides or--starting from GDP-mannose as a precursor--tosynthesize mannosylated oligosaccharides. In addition, mutations inthe genes coding for the transcriptional regulators ArcA and IclRlead to an acid resistance phenotype in the exponential growthphase allowing the synthesis of pH sensitive molecules and organicacids.

BACKGROUND OF THE INVENTION

[0002] The genes arcA encoding for the aerobic respiration controlprotein and iclR encoding the isocitrate lyase regulator are knownto regulate the central carbon metabolism. ArcA is a globaltranscriptional regulator that regulates a wide variety of genes,while IclR is a local transcriptional regulator that regulates theglyoxylate pathway. ArcA is known to regulate the central carbonmetabolism in response to oxygen deprivation and has no connectionwith IclR other than that it also regulates the glyoxylate pathway(24, 28, 29, 37, 38). In an earlier study the combined effect of.DELTA.iclR.DELTA.arcA mutant strains on the central carbonmetabolism has been observed. Increased fluxes were shown in thetricarboxylic acid (TCA) cycle and glyoxylate pathway and aninteresting and surprising phenotype appeared when both genes whereknocked out, namely the double mutant strain formed biomass with ayield that approached the maximal theoretical yield (4, 39).

[0003] Some compounds, such as GDP-fucose, are in high demand. Thelatter compound is indeed a precursor of fucosylatedoligosaccharides such as fucosyllactose, fucosyllactoNbiose andlewis X oligosaccharides, or, of fucosylated proteins. These sugarsare components of human mother milk in which they haveanti-inflammatory and prebiotic effects and/or have applications intherapeutics as nutraceutical, anti-inflammatory agent orprebiotic, in addition, fucosylated proteins find applications inthe pharmaceutics (5, 8, 27). However, an efficient method toproduce the latter high-value compounds is still needed.

[0004] In addition GDP-mannose is also an intermediate of thepathway towards GDP-fucose. Interrupting the pathway prematurelyleads to the accumulation of this compound, which is a precursor ofmannosylated oligosaccharides. These oligosaccharides find forexample applications in the treatment of gram-negative bacterialinfections, in addition, GDP-mannose is important for thehumanization of protein glycosylations, which is essential for theproduction of certain therapeutic proteins (18, 30). Mannosylatedoligosaccharides and mannosylated glycoconjugates are also used fordrug targeting, for instance mannosylated antivirals canspecifically target the liver and kidneys (7).

[0005] The present invention provides microorganisms which aregenetically changed in such a manner that they can efficientlyproduce the latter compounds.

[0006] Moreover, the synthesis of pH sensitive molecules, suchas--but not limited to--glucosamine, and organic acids, suchas--but not limited to--pyruvic acid, succinic acid, adipic, sialicacid, sialylated oligosaccharides . . . are preferably produced atlow pH, either to stabilize the product or for downstreamprocessing reasons (4, 12, 40). Therefore, strains that can grow atlow pH are beneficial for these production processes. E. coli is anorganism that can adapt easily to various conditions, for instanceit can easily adapt to the harsh pH conditions in the stomach,which is about pH 2 (14). Nonetheless, E. coli does not seem togrow at these conditions, but induces its acid resistancemechanisms in the stationary phase (40). During this phase the celldoes not multiply anymore and therefore hampers productivity. Up tonow, no solution was found to this problem. However, in the presentinvention, a genetically engineered microorganism is provided thatcan induce acid resistance mechanisms in the exponential growthphase, which is the phase that is mostly used for production oforganic acids and pH instable products.

BRIEF DESCRIPTION OF FIGURES

[0007] FIG. 1: Relative gene expression pattern of the wild type,the .DELTA.iclR and .DELTA.arcA mutant strain to the.DELTA.arcA.DELTA.iclR mutant strain of genes involved in colanicacid biosynthesis in batch fermentation conditions. The genesinvolved in colanic acid biosynthesis are presented in FIGS. 3 and4.

[0008] FIG. 2: Gene expression pattern of the colanic acid operonof the wild type, the .DELTA.iclR and .DELTA.arcA mutant strain inchemostat fermentation conditions relative to the.DELTA.arcA.DELTA.iclR mutant strain.

[0009] FIG. 3: The gene organisation of the colanic acid operon andan overview of the function of these genes:

TABLE-US-00001 Gene: Function: wza Component of capsularpolysaccharide export apparatus wzb Tyrosine phosphatase wzcTyrosine kinase wcaA Glycosyltransferase wcaB Acyltransferase wcaCGlycosyltransferase wcaD Colanic acid polymerase wcaEGlycosyltransferase wcaF Acyltransferase gmdGDP-mannose-4,6-dehydratase fcl GDP-fucose synthase gmm GDP-mannosehydrolase wcal Glycosyltransferase cpsB Mannose-1-phosphateguanylyltransferase cpsG Phosphomannomutase wcaJ UDP-glucose lipidcarrier transferase wzxC Putative transporter wcaKPyruvyltransferase wcaL Glycosyltransferase wcaM Predicted proteinin colanic acid biosynthesis

[0010] FIG. 4: The colanic acid biosynthesis pathway.

[0011] FIG. 5: Regulatory network of the colanic acid operon. Thisnetwork was constructed with Pathway tools v 13.0.

[0012] FIG. 6: Effect of the .DELTA.arcA.DELTA.iclR mutations onthe GDP-fucose biosynthesis route.

[0013] FIG. 7: Overview of the genetic modifications needed toenhance fucosyllactose and fucosylated oligosaccharides productionstarting from glucose as a substrate.

[0014] FIG. 8: Starting from sucrose, fucosylated sugar derivatessuch as fucosyllactose and more specifically 1,2-fucosyllactose areproduced. The strain is modified to force the cell to producefrucose-6-phosphate which is an intermediate in the synthesis ofGDP-fucose. Glucose or glucose-1-phosphate (if the starting enzymeis either a sucrase or a sucrose phosphorylase) is then fed to thecentral carbon metabolism via the pentose phosphate pathway.

[0015] FIG. 9: Overview of the genetic modifications needed toenhance fucosyllactose and fucosylated oligosaccharides productionstarting from glucose as a substrate in a split metabolism.

[0016] FIG. 10: Detail of the pentose phosphate pathway flux in astrain in which the genes coding for phosphoglucose isomerase andphosphofructokinase are knocked out.

[0017] FIG. 11: Starting from sucrose, mannosylated sugar derivatesare produced. The strain is modified to force the cell to producefrucose-6-phosphate which is an intermediate in the synthesis ofGDP-fucose. Glucose or glucose-1-phosphate (if the starting enzymeis either a sucrase or a sucrose phosphorylase) is then fed to thecentral carbon metabolism via the pentose phosphate pathway.

[0018] FIG. 12: Gene expression pattern acid resistance relatedgenes of the wild type, the .DELTA.iclR and .DELTA.arcA mutantstrain in batch culturing conditions relative to the.DELTA.arcA.DELTA.iclR mutant strain.

[0019] FIG. 13: LC MSMS analysis chromatograms of culture broth anda 2-fucosyllactose standard. A. LC MSMS analysis of the standard,B. LC MSMS analysis of a sample of the culture broth of a mutantstrain expressing a H. pylori fucosyltransferase, C. LC MSMSanalysis of a sample of the culture broth of a mutant strainexpressing a H. pylori fucosyltransferase.

[0020] FIG. 14: LC MSMS analysis mass spectrum from thechromatograms shown in FIG. 13 of culture broth and a2-fucosyllactose standard. A. Mass (m/z) of the standard, B. Mass(m/z) of the sample of the culturing broth of a mutant strainexpressing a H. pylori fucosyltransferase, C. Mass (m/z) of thesample of the culturing broth of a mutant strain expressing a H.pylori fucosyltransferase.

[0021] FIG. 15: The sequence of the artificial hybrid promoter asgiven by SEQ ID No 6 (the combination of the native and anartificial promoter) that was cloned in front of the colanic acidoperon.

DESCRIPTION OF INVENTION

[0022] The present invention provides microorganisms such asEnterobacteriaceae which are genetically changed in such a mannerthat they can efficiently produce compounds which are part of thecolanic acid pathway. A particular compound of interest isGDP-fucose which is used as a precursor to synthesize fucosylatedoligosaccharides. The latter have health-promoting effects asindicated above but there is no efficient production methodavailable to produce said compounds.

[0023] The present invention thus provides for the usage of amutated and/or transformed microorganism comprising a geneticchange leading to a modified expression and/or activity of thetranscriptional regulators the aerobic respiration control proteinArcA and the isocitrate lyase regulator IclR to upregulate at leastone of the genes of the colanic acid operon, wherein said operoncomprises the genes cpsG, cpsB, gmd and fcl that code for aphosphomannomutase, a mannose-1-phosphate guanylyltransferase,GDP-mannose 4,6-dehydratase and GDP-fucose synthase, respectively.The latter operon may also comprise the genes cpsG, cpsB, gmd, fcland wza. In addition the expression of the gene rcsA is increased.This gene is a transcriptional regulator of the colanic acidoperon. Enhanced expression of this gene increases transcription ofthe colanic acid operon (13, 36).

[0024] Hence the present invention relates to the usage of amutated and/or transformed microorganism comprising a geneticchange leading to a modified expression and/or activity of thetranscriptional regulator, the aerobic respiration control protein,ArcA and the isocitrate lyase regulator IclR to upregulate thetranscriptional regulator of the colanic acid operon, rcsA, whichin turn upregulates at least one of the genes of the colanic acidoperon.

[0025] Hence, the present invention relates to a mutated and/ortransformed microorganism such as--but not limited toEnterobacteriaceae such as an Escherichia coli (E. coli) straincomprising a genetic change leading to a modified expression of thetranscriptional regulators: the aerobic respiration control proteinArcA and the isocitrate lyase regulator IclR.

[0026] A mutated and/or transformed microorganism such as E. colias used here can be obtained by any method known to the personskilled in the art, including but not limited to UV mutagenesis andchemical mutagenesis. A preferred manner to obtain the lattermicroorganism is by disrupting (knocking-out) the genes (arcA andiclR) encoding for the proteins ArcA and IclR, or, by replacing theendogenous promoters of said genes by artificial promoters orreplacing the endogenous ribosome binding site by an artificialribosome binding site. The term `artificial promoters` relates toheterologous or non-natural or in silico designed promoters withknown expression strength, these promoters can be derived fromlibraries as described by Alper et al. (2005), Hammer et al.(2006), or De Mey et al. (2007) (3, 11, 15). The term heterologouspromoter refers to any promoter that does not naturally occur infront of the gene. The term `artificial promoter` may also refer topromoters with DNA sequences that are combinations of the native(autologous) promoter sequence with parts of different (autologousor heterologous) promoter sequences as for example shown further inthe examples. Sequences of such `artificial promoters` can be foundin databases such as for example partsregistry.org (6). The term`artificial ribosome binding site` relates to heterologous ornon-natural or in silico designed ribosome binding sites with knownor measurable translation rates, these libraries can be derivedfrom libraries or designed via algorithms as described by Salis etal (2009) (26). Hence, the present invention specifically relatesto a mutated and/or transformed microorganism as indicated abovewherein said genetic change is disrupting the genes encoding forArcA and IclR, or, reducing or eliminating the function of ArcA andIclR via mutations in the coding sequence of the genes coding forArcA and IclR, or, is replacing the endogenous promoters of thegenes encoding for ArcA and IclR by artificial promoters; or, isreplacing the endogenous ribosome binding site by an artificialribosome binding site. It is further clear that the mutant and/ortransformant according to the present invention may furthercomprise additional genetic changes in one or more other geneswithin its genome as is also described further. The termmicroorganism specifically relates to a bacterium, morespecifically a bacterium belonging to the family ofEnterobacteriaceae. The latter bacterium preferably relates to anystrain belonging to the species Escherichia coli such as but notlimited to Escherichia coli B, Escherichia coli C, Escherichia coliW, Escherichia coli K12, Escherichia coli Nissle. Morespecifically, the latter term relates to cultivated Escherichiacoli strains--designated as E. coli K12 strains--which arewell-adapted to the laboratory environment, and, unlike wild typestrains, have lost their ability to thrive in the intestine.Well-known examples of the E. coli K12 strains are K12 Wild type,W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111and AA200. Hence, the present invention specifically relates to amutated and/or transformed Escherichia coli strain as indicatedabove wherein said E. coli strain is a K12 strain. Morespecifically, the present invention relates to a mutated and/ortransformed Escherichia coli strain as indicated above wherein saidK12 strain is E. coli MG1655.

[0027] The terms `leading to a modified expression or activity`indicates that the above described mutations/transformationsaffects the transcription and/or translation and/orpost-translational modification of said genes (arcA and iclR) intothe transcriptional regulator proteins of the present invention(ArcA and IclR) in such a way that the latter transcription hassignificantly decreased or has even been completely abolishedcompared to a wild type strain, which has not been mutated ortransformed with regard to both particular genes of the presentinvention. Hence, the present invention relates to a mutated and/ortransformed microorganism such as an Escherichia coli strain asindicated above wherein said modified expression is a decreasedexpression, and, to a mutated and/or transformed microorganism suchas an Escherichia coli strain as indicated above wherein saiddecreased expression is an abolished expression.

[0028] The terms `upregulating at least one of the genes of thecolanic acid operon` indicates that the expression of at least 1,2, 3, 4, . . . , or all of the genes belonging to the colanic acidoperon are significantly (=P>0.05) upregulated when compared tothe expression of said genes within a corresponding wild typemicroorganism which is cultivated under the same conditions as themutated and/or transformed microorganism. The genes which belong tothe colanic acid operon are wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD,wcaE, wcaF, gmd, fcl, gmm, wcaI, cpsB, cpsG, wcaJ, wzxC, wcaK, wcaLand wcaM as indicated in FIG. 3 and/or as described in (35).Furthermore, the gene rcsA, coding for the transcriptionalregulator of the colanic acid operon is upregulated (13, 36). Morespecifically the terms `upregulating at least one of the genes ofthe colanic acid operon` or the transcriptional regulator of thecolanic acid operon indicates that at least one of the genes of thecolanic acid operon is 6 to 8 times upregulated in comparison tothe expression of the genes of the colanic acid operon in thecorresponding wild type microorganism. In addition the presentinvention relates to upregulating genes of the colanic acid operonas described above by replacing the native promoter by an`artificial promoter`. More specifically, the present inventionrelates to a combination of the sequence of the native promoterwith sequences of other artificial promoter sequences. Thecombination of the sequence of the native promoter with thesequence of other artificial promoter sequences is morespecifically the replacement of the sigma factor binding site ofthe native promoter with a stronger sigma factor binding site.Sigma factors, such as but not limited to sigma70, sigmaS, sigma24,. . . , are described (41), subunits of RNA polymerase thatdetermine the affinity for promoter sequences and the transcriptionrate of genes. The present invention provides microorganisms whichare genetically changed in such a manner that they can efficientlyproduce compounds which are part of the colanic acid pathway. Theterms `compounds which are part of the colanic acid pathway` referto all compounds as indicated on FIG. 4 starting from fructose-6-Pand resulting in extracellular colanic acid. More specifically thelatter terms refer to the compounds mannose-6-P, mannose-1-P,GDP-mannose, GDP-4-dehydro-6deoxy-mannose, GDP-fucose and colanicacid. Hence the present invention specifically relates to the usageas indicated for the synthesis of colanic acid and/or for thesynthesis of GDP-fucose. As GDP-fucose is a precursor forfucosylated oligosaccharides such as fucosyllactose,fucosyllactoNbiose and lewis X oligosaccharide or fucosylatedproteins, and as these sugars have therapeutical, nutraceutical,anti-inflammatory and prebiotic effects, the present inventionspecifically relates to the usage as described above for thesynthesis of fucosylated oligosaccharides. In other words, thepresent invention relates to a process for the synthesis of colanicacid and/or GDP-fucose and/or fucosylated oligosaccharidescomprising genetically changing the transcriptional regulators theaerobic respiration control protein ArcA and the isocitrate lyaseregulator IclR to upregulate at least one of the genes of thecolanic acid operon, wherein said operon comprises the genes cpsG,cpsB, gmd and fcl or genes cpsG, cpsB, gmd, fcl and wza. Morespecifically, the present invention relates to a process asdescribed wherein the mutations for ArcA and IclR are applied incombination with at least one mutation that enhances the productionof fucosylated compounds. In order to efficiently producefucosylated oligosaccharides (see FIGS. 1, 2 and 5-10), the abovedescribed mutations in arcA and iclR can be applied in combinationwith other mutations which further enhance the production offucosylated compounds. Some of these--non-limiting--other mutationsare: a) the deletion of wcaJ from the colanic operon, stopping theinitiation of the colanic acid biosynthesis and thus allowing theaccumulation of GDP-fucose; b) the introduction of afucosyltransferase to link fucose with different acceptor moleculessuch as lactose; c) for the accumulation of the precursor of theGDP-fucose biosynthetic pathway and additional to the deletion ofwcaJ, at least one of the following colanic acid operon genes thatdo not code for GDP-fucose biosynthesis is knocked out: gmm, wcaA,wcaB, wcaC, wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL, wzx, wza,wzb, wzc, and/or, wcaM; d) for the production of fucosyllactose,lacZ coding for .beta.-galactosidase, is knocked out to avoidlactose degradation; e) to accumulate the precursor fructose andfructose-6-phosphate, a sucrose phosphorylase or invertase isintroduced; f) because fructose-6-phosphate is easily degraded inthe glycolysis, the glycolysis has to be interrupted in order tosteer all fructose-6-phosphate in the direction of GDP-fucose andthe genes pgi, pfkA and pfkB (coding for glucose-6-phosphateisomerase and phosphofructokinase A and B) are thus knocked out; g)reducing protein degradation by knocking out a protease coded by agene such as Ion; h) By constitutively expressing a lactosepermease, subpopulations are avoided in the production processwhich are common for lactose induced gene expression systems (19).In other words, the present invention relates to a process asdescribed above for the synthesis of fucosylated oligosaccharideswherein said at least one mutation that enhance the production offucosylated compounds is: the deletion of the wcaJ gene, and/or,knocking-out the colanic acid operon genes gmm, wcaA, wcaB, wcaC,wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc,and/or, wcaM, and/or, knocking-out lacZ, and/or, introducing asucrose phosporylase or invertase, and/or, knocking out the genespgi, pfkA and pfkB, and/or, knocking out the gene Ion, and/orintroducing a fucosyltransferase, and/or a lactose permease. Theterm `introducing a fucosyltransferase` relates to upregulating orheterologous expression of fucosyltransferases which are within,but not limited to the enzymes in enzyme classes classesEC2.4.1.65, 2.4.1.68, 2.4.1.69, 2.4.1.152, 2.4.1.214, and/or2.4.1.221 and/or the glycosyltransferase families GT1, GT2, GT10GT11, GT23, GT37, GT65, GT68, and/or GT74 and/or originating frombut not limited to Helicobacter pylori, Campylobacter jejuni,Dictyostellium discoideum, Mus musculus, Homo sapiens, . . . andthese fucosyltransferases catalyse the formation of .alpha.(1,2),.alpha.(1,3), .alpha.(1,4), or .alpha.(1,6) bonds on other sugarssuch as but not limited to galactose, lactose, lactoNbiose,lactoNtetraose, lactosamine, lactoNtetraose, sialyllactoses,disialyllactoses, or fucosylated proteins, or fucosylated fattyacids., or fucosylated aglycons such as, but not limited to,antivirals, antibiotics, . . . .

[0029] The present invention provides for the usage of a mutatedand/or transformed microorganism comprising a genetic changeleading to a modified expression and/or activity of thetranscriptional regulators the aerobic respiration control proteinArcA and the isocitrate lyase regulator IclR to upregulate at leastone of the genes of the colanic acid operon, wherein said operoncomprises the genes cpsG and cpsB, coding for phosphomannomutaseand mannose-1-phosphate guanylyltransferase, which are needed forthe biosynthesis of GDP-mannose. As GDP-mannose is a precursor formannosyllated oligosaccharides and mannosylated glycoconjugates.These oligosaccharides and glycoconjugates find for exampleapplications in the treatment of gram-negative bacterialinfections, in addition, GDP-mannose is important for thehumanization of protein glycosylations, which is essential for theproduction of certain therapeutic proteins (18, 30). Mannosylatedoligosaccharides and mannosylated glycoconjugates are also used fordrug targeting, for instance mannosylated antivirals canspecifically target the liver and kidneys (7). In order toefficiently produce mannosylated oligosaccharides (see FIGS. 1, 2,5, 6 and 11), the above described mutations in arcA and iclR can beapplied in combination with other mutations which further enhancethe production of mannosylated compounds. Some ofthese--non-limiting--other mutations are: a) the gene gmd of thecolanic acid operon is deleted, and/or, b) wherein the gene gmmcoding for GDP-mannose hydrolase is deleted, and/or, c) wherein thecolanic acid operon genes that do not code for GDP-mannosebiosynthesis reactions, the genes gmm, wcaA, wcaB, wcaC, wcaD,wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL, fcl, gmd, wzx, wza, wzb and/or,wcaM, are deleted, and/or, d) wherein a gene encoding for a sucrosephosphorylase or an invertase is introduced, and/or, e) wherein thegenes pgi, pfkA and pfkB, coding for phosphoglucose isomerase,phosphofructokinase A and phosphofructokinase B respectively, aredeleted, and/or, f) knocking out the gene Ion encoding for aprotease, and/or f) wherein a gene encoding for amannosyltransferase is introduced. In other words, the presentinvention relates to a process as described above for the synthesisof colanic acid and/or GDP-fucose and/or fucosylatedoligosaccharides for the synthesis of GDP-mannose and/or for thesynthesis of mannosylated oligosaccharides. The present inventionfurther relates to said process wherein the genes cpsG and cpsB ofthe colanic acid operon are upregulated and wherein: a) the genegmd of the colanic acid operon is deleted, and/or, b) wherein thegene gmm is deleted, and/or c) wherein the colanic acid operongenes fcl, gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI,wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM are knocked outand/or, d) wherein a gene encoding for a sucrose phosphorylase oran invertase is introduced, and/or, e) wherein the genes pgi, pfkAand pfkB are deleted, and/or, f) knocking out the gene Ion, and/org) wherein a gene encoding for a mannosyltransferase is introduced.The term `introducing a mannosyltransferase` relates toupregulating or heterologous expression of mannosyltransferaseswhich are within, but not limited to the enzymes in enzyme classesEC 2.4.1.32, 2.4.1.B27, 2.4.1.B44, 2.4.1.48, 2.4.1.54, 2.4.1.57,2.4.1.83, 2.4.1.109, 2.4.1.110, 2.4.1.119, 2.4.1.130, 2.4.1.131,2.4.1.132, 2.4.1.142, 2.4.1.199, 2.4.1.217, 2.4.1.232, 2.4.1.246,2.4.1.251, 2.4.1.252, 2.4.1.257, 2.4.1.258, 2.4.1.259, 2.4.1.260,2.4.1.265, and/or 2.4.1.270 and/or the glycosyltransferase familiesGT1, GT2, GT4, GT15, GT22, GT32, GT33, GT39, GT50 and/or GT58and/or originating from but not limited to Helicobacter pylori,Campylobacter jejuni, Dictyostellium discoideum, Mus musculus, Homosapiens, . . . and these mannosyltransferases catalyse theformation of .alpha.(1,2), .alpha.(1,3), .alpha.(1,4), or.alpha.(1,6) bonds on other sugars such as but not limited togalactose, N-acetylglucosamine, Rhamnose, lactose, lactoNbiose,lactoNtetraose, lactosamine, lactoNtetraose, sialyllactoses,disialyllactoses, or mannosylated proteins, or mannosylated fattyacids, or mannosylated aglycons such as, but not limited to,antivirals, antibiotics, . . . .

[0030] The term `heterologous expression` relates to the expressionof genes that are not naturally present in the production host,genes which can be synthesized chemically or be picked up fromtheir natural host via PCR, genes which can be codon optimized forthe production host or in which point mutation can be added toenhance enzyme activity or expression. Expressing heterologousand/or native genes can either be done on the chromosome,artificial chromosomes or plasmids and transcription can becontrolled via inducible, constitutive, native or artificialpromoters and translation can be controlled via native orartificial ribosome binding sites.

[0031] Consequently, the present invention further relates tomutated and/or transformed organisms in which the regulators ArcAand IclR as describe above, in combination with the genes encodingfor the enzymes phosphoglucose isomerase and phosphofructokinase,are knocked out or are rendered less functional. More specifically,the present invention relates to the latter organisms wherein theenzyme phosphoglucose isomerase is encoded by the gene pgi andwherein the enzyme phosphofructokinase is encoded by the gene(s)pfkA and/or pfkB.

[0032] The terms `genes which are rendered less-functional ornon-functional` refer to the well-known technologies for a skilledperson such as the usage of siRNA, RNAi, miRNA, asRNA, mutatinggenes, knocking-out genes, transposon mutagenesis, etc. . . . whichare used to change the genes in such a way that they are less able(i.e. statistically significantly `less able` compared to afunctional wild-type gene) or completely unable (such asknocked-out genes) to produce functional final products. The term`(gene) knock out` thus refers to a gene which is renderednon-functional. The term `deleted gene` or `gene deletion` alsorefers to a gene which is rendered non-functional.

[0033] The present invention further relates to a mutated and/ortransformed organism as described in the latter paragraph whereinsaid organism is further transformed with a gene encoding for asucrose phosphorylase.

[0034] The present invention also relates to a mutated and/ortransformed organism as described above wherein, in addition, theactivity and/or expression of the gene encoding for a lactosepermease is made constitutive and/or increased. Said activity canbe increased by over-expressing said gene and/or by transformingsaid organisms with a gene encoding for a lactose permease.

[0035] The present invention further relates to any mutated and/ortransformed organism as described above wherein at least one of thefollowing genes is knocked out or is rendered less functional:

[0036] a gene encoding for a beta-galactosidase, a gene encodingfor a glucose-1-phosphate adenylyltransferase, a gene encoding fora glucose-1-phosphatase, a gene encoding for phosphogluconatedehydratase, a gene encoding for2-keto-3-deoxygluconate-6-phosphate aldolase, a gene encoding for aglucose-1-phosphate uridyltransferase, a gene encoding for anUDP-glucose-4-epimerase, a gene encoding for anUDP-glucose:galactose-1-phosphate uridyltransferase, a geneencoding for an UDP-galactopyranose mutase, a gene encoding for anUDP-galactose:(glucosyl)lipopolysaccharide-1,6-galactosyltransferase,a gene encoding for an UDP-galactosyltransferase, a gene encodingfor an UDP-glucosyltransferase, a gene encoding for anUDP-glucuronate transferase, a gene encoding for an UDP-glucoselipid carrier transferase, a gene encoding for a GDP-mannosehydrolase, a gene encoding for an UDP-sugar hydrolase, a geneencoding for a mannose-6-phosphate isomerase, a gene encoding foran UDP-N-acetylglucosamine enoylpyruvoyl transferase, a geneencoding for an UDP-N-acetylglucosamine acetyltransferase, a geneencoding for an UDP-Nacetylglucosamine-2-epimerase, a gene encodingfor an undecaprenyl-phosphate alfa-N-acetylglucosaminyltransferase, a gene encoding for aglucose-6-phosphate-1-dehydrogenase, and/or, a gene encoding for aL-glutamine:D-fructose-6-phosphate aminotransferase, a geneencoding for a mannose-6-phosphate isomerase, a gene encoding for asorbitol-6-phosphate dehydrogenase, a gene encoding for amannitol-1-phosphate 5-dehydrogenase, a gene encoding for aallulose-6-phosphate 3-epimerase, a gene encoding for an invertase,a gene encoding for a maltase, a gene encoding for a trehalase, agene encoding for a sugar transporting phosphotransferase, a geneencoding for a protease, or a gene encoding for a hexokinase. Theterm `at least one` indicated that at least 1, but also 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32 or all 33 genes is (are) knockedout or is (are) rendered less functional.

[0037] The present invention further relates also to the usage of amutated and/or transformed microorganism such as an Escherichiacoli strain comprising a genetic change leading to a modifiedexpression of the transcriptional regulators the aerobicrespiration control protein ArcA and the isocitrate lyase regulatorIclR to upregulate at least one of the following acid resistancerelated genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadWand/or slp (17, 22). These genes are normally expressed instationary phase conditions; however, the present mutated and/ortransformed microorganism is able to enhance the expression ofthese acid resistance related genes in the exponential growthphase. Hence, the present invention relates to the usage asdescribed above for the synthesis of acids or pH sensitivemolecules such as but not limited to glucosamine which is pHsensitive and should be produced at low pH (12). Organic acids,such as but not limited to pyruvic acid, succinic acid, adipic,sialic acid, sialylated oligosaccharides (e.g. sialyllactose,sialyl Lewis X sugars, . . . ), acetylated oligosaccharides(chitins, chitosans, . . . ), sulfonated oligosaccharides (heparansand heparosans) . . . are preferably produced at low pH fordownstream processing purposes (4). In other words, the presentinvention relates to a process for the synthesis of acids, sialicacid, sialylated oligosaccharides or glucosamine comprisinggenetically changing the transcriptional regulators the aerobicrespiration control protein ArcA and the isocitrate lyase regulatorIclR to upregulate at least one of the following acid resistancerelated genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadWand/or slp.

[0038] The present invention will now be illustrated by thefollowing non-limiting examples.

EXAMPLES

[0039] A high throughput RT-qPCR screening of the microorganisms ofthe present invention has been setup with Biotrove OpenArray.RTM.technology. In this experiment the transcription of 1800 genes weremeasured in 4 strains (wild type, .DELTA.arcA, .DELTA.iclR,.DELTA.arcA .DELTA.iclR) in two conditions (chemostat and batch).The data was processed using a curve fitting toolbox in R (25, 34)and Quantile Normalization, the error on the data was calculatedusing Bayesian statistics (20, 21, 31).

Material and Methods

Strains and Plasmids

[0040] Escherichia coli MG1655 [.sup.-, F.sup.-, rph-1] wasobtained from the Netherlands Culture Collection of Bacteria(NCCB). Escherichia coli BL21(DE3) was obtained from Novagen.Escherichia coli MG1655 ackA-pta, poxB, pppc ppc-p37 (10), thesingle knock-outs E. coli MG1655 arcA and E. coli MG1655 iclR andthe double knock-out E. coli MG1655 arcA, iclR were constructed inthe Laboratory of Genetics and Microbiology (MICR) using the methodof Datsenko & Wanner (9).

Media

[0041] The Luria Broth (LB) medium consisted of 1% tryptone peptone(Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5%sodium chloride (VWR, Leuven, Belgium). Shake flask mediumcontained 2 g/l NH.sub.4Cl, 5 g/l (NH.sub.4).sub.2SO.sub.4, 2.993g/l KH.sub.2PO.sub.4, 7.315 g/l K.sub.2HPO.sub.4, 8.372 g/l MOPS,0.5 g/l NaCl, 0.5 g/l MgSO.sub.4.7H.sub.2O, 16.5 g/lglucose.H.sub.2O, 1 ml/l vitamin solution, 100 .mu.l/l molybdatesolution, and 1 ml/l selenium solution. The medium was set to a pHof 7 with 1M KOH.

[0042] Vitamin solution consisted of 3.6 g/l FeCl.sub.2.4H.sub.2O,5 g/l CaCl.sub.2.2H.sub.2O, 1.3 g/l MnCl.sub.2.2H.sub.2O, 0.38 g/lCuCl.sub.2.2H.sub.2O, 0.5 g/l CoCl.sub.2.6H.sub.2O, 0.94 g/lZnCl.sub.2, 0.0311 g/l H.sub.3BO.sub.4, 0.4 g/lNa.sub.2EDTA.2H.sub.2O and 1.01 g/l thiamine.HCl. The molybdatesolution contained 0.967 g/l Na.sub.2MoO.sub.4.2H.sub.2O. Theselenium solution contained 42 g/l SeO.sub.2.

[0043] The minimal medium for fermentations contained 6.75 g/lNH.sub.4Cl, 1.25 g/l (NH.sub.4).sub.2SO.sub.4, 1.15 g/lKH.sub.2PO.sub.4, 0.5 g/l NaCl, 0.5 g/l MgSO.sub.4.7H.sub.2O, 16.5g/l glucose.H.sub.2O, 1 ml/l vitamin solution, 100 .mu.l/lmolybdate solution, and 1 ml/l selenium solution with the samecomposition as described above.

Cultivation Conditions

[0044] A preculture, from a single colony on a LB-plate, in 5 ml LBmedium was incubated during 8 hours at 37.degree. C. on an orbitalshaker at 200 rpm. From this culture, 2 ml was transferred to 100ml minimal medium in a 500 ml shake flask and incubated for 16hours at 37.degree. C. on an orbital shaker at 200 rpm. 4% inoculumwas used in a 2 l Biostat B Plus culture vessel with 1.5 l workingvolume (Sartorius Stedim Biotech, Melsungen, Germany). The cultureconditions were: 37.degree. C., stirring at 800 rpm, and a gas flowrate of 1.5 l/min. Aerobic conditions were maintained by spargingwith air, anaerobic conditions were obtained by flushing theculture with a mixture of 3% CO.sub.2 and 97% of N.sub.2. The pHwas maintained at 7 with 0.5 M H.sub.2504 and 4 M KOH. The exhaustgas was cooled down to 4.degree. C. by an exhaust cooler (Frigomix1000, Sartorius Stedim Biotech, Melsungen, Germany). 10% solutionof silicone antifoaming agent (BDH 331512K, VWR Int Ltd., Poole,England) was added when foaming raised during the fermentation(approximately 10 .mu.l). The off-gas was measured with an EL3020off-gas analyser (ABB Automation GmbH, 60488 Frankfurt am Main,Germany).

[0045] All data was logged with the Sartorius MFCS/win v3.0 system(Sartorius Stedim Biotech, Melsungen, Germany).

[0046] All strains were cultivated at least twice and the givenstandard deviations on yields and rates are based on at least 10data points taken during the repeated experiments.

Sampling Methodology

[0047] The bioreactor contains in its interior a harvest pipe (BDSpinal Needle, 1.2.times.152 mm (BDMedical Systems, Franklin Lakes,N.J.--USA) connected to a reactor port, linked outside to aMasterflex-14 tubing (Cole-Parmer, Antwerpen, Belgium) followed bya harvest port with a septum for sampling. The other side of thisharvest port is connected back to the reactor vessel with aMasterflex-16 tubing. This system is referred to as rapid samplingloop. During sampling, reactor broth is pumped around in thesampling loop. It has been estimated that, at a flow rate of 150ml/min, the reactor broth needs 0.04 s to reach the harvest portand 3.2 s to re-enter the reactor. At a pO2 level of 50%, there isaround 3 mg/l of oxygen in the liquid at 37.degree. C. The pO2level should never drop below 20% to avoid micro-aerobicconditions. Thus 1.8 mg/l of oxygen may be consumed during transitthrough the harvesting loop. Assuming an oxygen uptake rate of 0.4g oxygen/g biomass/h (the maximal oxygen uptake rate found at.mu..sub.max), this gives for 5 g/l biomass, an oxygen uptake rateof 2 g/l/h or 0.56 mg/l/s, which multiplied by 3.2 s (residencetime in the loop) gives 1.8 mg/l oxygen consumption.

[0048] In order to quench the metabolism of cells during thesampling, reactor broth was sucked through the harvest port in asyringe filled with 62 g stainless steel beads pre-cooled at-20.degree. C., to cool down 5 ml broth immediately to 4.degree. C.Sampling was immediately followed by cold centrifugation (15000 g,5 min, 4.degree. C.). During the batch experiments, a sample forOD.sub.600nm and RT-qPCR measurements was taken using the rapidsampling loop and the cold stainless bead sampling method.

RT-qPCR

[0049] mRNA was extracted with the RNeasy kit (Qiagen, Venlo, TheNetherlands). RNA quality and quantity was checked with a nanodropND-1000 spectrophotometer (Nanodrop technologies, Wilmingto, USA).The ratios 260:280 (nm) and 260:230 (nm) were between 1.8 and 2 andat least 100 ng/.mu.l was needed for further analysis. cDNA wassynthesised with random primers with the RevertAid.TM. H minusfirst strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany).Finally, the gene expression of 1800 genes was measured with theBiotrove OpenArray Real time PCR platform. The primers for theRT-PCR assay were designed with Primer design tools from the Primerdatabase (23).

[0050] The reaction mixture was composed as described in theBiotrove OpenArray.TM. Real-Time qPCR system users' manual. Inshort, a mastermix was made with 26.4 .mu.l LightCycler.RTM. DNAMaster SYBR.RTM. Green I (Roche applied Science), 1.1 .mu.l SYBRGREEN I (100.times. stock solution, Sigma S9430), 8.8 .mu.lglycerol (Sigma G5150), 5.3 .mu.l Pluronic.RTM. F68 (10% stock,Invitrogen), 2.64 .mu.l BSA (Sigma A7906), 26.4 .mu.l magnesiumchloride (25 mM stock solution, supplied in the LightCycler.RTM.kit of Roche applied Science), 21.1 .mu.l HiDi.TM. formamide(Applied biosystems), and 94.66 .mu.l RNase free sterile waterresulting in a 186.4 .mu.l mastermix, which is enough to load 1OpenArray.TM.. For 1 SubArray (each OpenArray is subdivided in 48SubArrays on which 1 sample can be loaded) 1.5 .mu.l sample (with aconcentration of 100 ng/.mu.l) was mixed with 3.5 .mu.l ofmastermind, as a no template control, water was used as blanc. Thesample-mastermix mixture was loaded in a Loader plate(MatriPlate.TM. 384-well black low volume polypropylene plate,Biotrove) in a RNase free hood. A full loader plate was loaded withan AutoLoader (Biotrove) and loader tips onto the OpenArrays. TheseOpenArrays were then submerged in OpenArray.TM. immersion fluid inan OpenArray.TM. Real-Time qPCR case. The case was sealed with Casesealing glue and incubated in the Case Sealing station, whichpolymerizes the glue with UV light.

Analytical Methods

[0051] Cell density of the culture was frequently monitored bymeasuring optical density at 600 nm (Uvikom 922 spectrophotometer,BRS, Brussel, Belgium). Cell dry weight was obtained bycentrifugation (15 min, 5000 g, GSA rotor, Sorvall RC-5B, GoffinMeyvis, Kapellen, Belgium) of 20 g reactor broth in pre-dried andweighted falcons. The pellets were subsequently washed once with 20ml physiological solution (9 g/l NaCl) and dried at 70.degree. C.to a constant weight. To be able to convert OD.sub.600nmmeasurements to biomass concentrations, a correlation curve of theOD.sub.600nm to the biomass concentration was made. Theconcentrations of glucose and organic acids were determined on aVarian Prostar HPLC system (Varian, Sint-Katelijne-Waver, Belgium),using an Aminex HPX-87H column (Bio-Rad, Eke, Belgium) heated at65.degree. C., equipped with a 1 cm precolumn, using 5 mM H2SO4(0.6 ml/min) as mobile phase. A dual-wave UV-VIS (210 nm and 265nm) detector (Varian Prostar 325) and a differential refractiveindex detector (Merck LaChrom L-7490, Merck, Leuven, Belgium) wasused for peak detection. By dividing the absorptions of the peaksin both 265 and 210 nm, the peaks could be identified. The divisionresults in a constant value, typical for a certain compound(formula of Beer-Lambert).

[0052] Glucose, fructose, sucrose, fucosyllactose andglucose-1-phosphate were measured by HPLC with a Hypercarb columnand were detected with an MSMS detector (Antonio et al., 2007;Nielsen et al., 2006).

Genetic Methods

[0053] All mutant strains were constructed via the methodsdescribed below.

[0054] Plasmids were maintained in the host E. coli DH5.alpha.(F.sup.-, .phi.80dlacZ.DELTA.M15, .DELTA.(lacZYA-argF)U169, deoR,recA1, endA1, hsdR17(rk.sup.-, mk.sup.+), phoA, supE44,.lamda..sup.-, thi-1, gyrA96, relA1).

[0055] Plasmids.

[0056] pKD46 (Red helper plasmid, Ampicillin resistance), pKD3(contains an FRT-flanked chloramphenicol resistance (cat) gene),pKD4 (contains an FRT-flanked kanamycin resistance (kan) gene), andpCP20 (expresses FLP recombinase activity) plasmids were used forthe mutant construction. The plasmid pBluescript (Fermentas, St.Leon-Rot, Germany) was used to construct the derivates of pKD3 andpKD4 with a promoter library, or with alleles carrying a pointmutation.

[0057] Mutations.

[0058] The mutations consisted in gene disruption (knock-out, KO).They were introduced using the concept of Datsenko and Wanner (9).The primers for the mutation strategies are described in Table1.

[0059] Transformants carrying a Red helper plasmid were grown in 10ml LB media with ampicillin (100 mg/l) and L-arabinose (10 mM) at30.degree. C. to an OD.sub.600nm, of 0.6. The cells were madeelectrocompetent by washing them with 50 ml of ice-cold water, afirst time, and with 1 ml ice-cold water, a second time. Then, thecells were resuspended in 50 .mu.l of ice-cold water.Electroporation was done with 50 .mu.l of cells and 10-100 ng oflinear double-stranded-DNA product by using a Gene Pulser.TM.(BioRad) (600.OMEGA., 25 .mu.FD, and 250 volts).

[0060] After electroporation, cells were added to 1 ml LB mediaincubated 1 h at 37.degree. C., and finally spread onto LB-agarcontaining 25 mg/l of chloramphenicol or 50 mg/l of kanamycin toselect antibiotic resistant transformants. The selected mutantswere verified by PCR with primers upstream and downstream of themodified region and were grown in LB-agar at 42.degree. C. for theloss of the helper plasmid. The mutants were tested for ampicillinsensitivity.

[0061] Linear Double-Stranded-DNA.

[0062] The linear ds-DNA amplicons were obtained by PCR using pKD3,pKD4 and their derivates as template. The primers used had a partof the sequence complementary to the template and another partcomplementary to the side on the chromosomal DNA where therecombination has to take place (Table 1). For the KO, the regionof homology was designed 50-nt upstream and 50-nt downstream of thestart and stop codon of the gene of interest. For the KI, thetranscriptional starting point (+1) had to be respected. PCRproducts were PCR-purified, digested with Dpnl, repurified from anagarose gel, and suspended in elution buffer (5 mM Tris, pH8.0).

[0063] Elimination of the Antibiotic Resistance Gene.

[0064] The selected mutants (chloramphenicol or kanamycinresistant) were transformed with pCP20 plasmid, which is anampicillin and chloramphenicol resistant plasmid that showstemperature-sensitive replication and thermal induction of FLPsynthesis. The ampicillin-resistant transformants were selected at30.degree. C., after which a few were colony purified in LB at42.degree. C. and then tested for loss of all antibiotic resistanceand of the FLP helper plasmid. The gene knock outs and knock insare checked with control primers (Fw/Rv-gene-out). These primersare given in Table 1.

TABLE-US-00002 TABLE 1 Primers used to create E. coli MG1655 arcA,E. coli MG1655 iclR and the double knock-out E. coil MG1655 arcA,iclR and all other genetic knock outs and knock ins Primer nameSequence lacZ FW_LacZ_P1 CATAATGGATTTCCTTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTAgtgtag gctggagctgcttc (SEQ ID N.degree. 7)RV_LacZ_P2 GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTcatatgaat atcctccttag (SEQ ID N.degree. 8)FW_LacZ_out GCGGTTGGAATAATAGCG (SEQ ID N.degree. 9) RV_LacZ_outCAGGTTTCCCGACTGGAAAG (SEQ ID N.degree. 10) glgC FW-glgC-P1Agaccgccggttttaagcagcgggaacatc tctgaacatacatgtaaaacctgcagtgtaggctggagctgcttc (SEQ ID N.degree. 11) RV-glgC-P2Gtctggcagggacctgcacacggattgtgt gtgttccagagatgataaaaaaggagttagtccatatgaatatcctccttag (SEQ ID N.degree. 12) FW-glgC-outGcgaatatcgggaaatgcagg (SEQ ID N.degree. 13) RV-glgC-outCagagattgttttacctgctgg (SEQ ID N.degree. 14) agp FW_agp_P1CATATTTCTGTCACACTCTTTAGTGATTGA TAACAAAAGAGGTGCCAGGAgtgtaggctggagctgcttc (SEQ ID N.degree. 15) RV_agp_P2TAAAAACGTTTAACCAGCGACTCCCCCGCT TCTCGCGGGGGAGTTTTCTGcatatgaatatcctccttag(SEQ ID N.degree. 16) FW_agp_out GCCACAGGTGCAATTATC (SEQID N.degree. 17) RV_agp_out CATTTTCGAAGTCGCCGGGTACG (SEQ IDN.degree. 18) pgi Fw-pgi-P1 GGCGCTACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCgtgtaggctg gagctgcttc (SEQ ID N.degree. 19)Rv-pgi-P2 GGTTGCCGGATGCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATGcatatgaata tcctccttag (SEQ ID N.degree. 20)Fw_pgi_out(2) GGCTCCTCCAACACCGTTAC (SEQ ID N.degree. 21)Rv_pgi_out(2) TACATATCGGCATCGACCTG (SEQ ID N.degree. 22) pfkAFw-pfkA-out TACCGCCATTTGGCCTGAC (SEQ ID N.degree. 23) Rv-pfkA-outAAAGTGCGCTTTGTCCATGC (SEQ ID N.degree. 24) Fw-pfkA-P1GACTTCCGGCAACAGATTTCATTTTGCATT CCAAAGTTCAGAGGTAGTCgtgtaggctggagctgcttc (SEQ ID N.degree. 25) Rv-pfkA-P2GCTTCTGTCATCGGTTTCAGGGTAAAGGAA TCTGCCTTTTTCCGAAATCcatatgaatatcctccttag (SEQ ID N.degree. 26) pfkB Fw-pfkB-outTAGCGTCCCTGGAAAGGTAAC (SEQ ID N.degree. 27) Rv-pfkB-outTCCCTCATCATCCGTCATAG (SEQ ID N.degree. 28) Fw-pfkB-P1CACTTTCCGCTGATTCGGTGCCAGACTGAA ATCAGCCTATAGGAGGAAATGgtgtaggctggagctgcttc (SEQ ID N.degree. 29) Rv-pfkB-P2GTTGCCGACAGGTTGGTGATGATTCCCCCA ATGCTGGGGGAATGTTTTTGcatatgaatatcctccttag (SEQ ID N.degree. 30) arcA FW-arcA-P1Ggttgaaaaataaaaacggcgctaaaaagc gccgttttttttgacggtggtaaagccgagtgtaggctggagctgcttc (SEQ ID N.degree. 31) RV-arcA-P2Ggtcagggacttttgtacttcctgtttcga tttagttggcaatttaggtagcaaaccatatgaatatcctccttag (SEQ ID N.degree. 32) FW-arcA-outCtgccgaaaatgaaagccagta (SEQ ID N.degree. 33) RV-arcA-outGgaaagtgcatcaagaacgcaa (SEQ ID N.degree. 34) iclR FW-iclR-P1Ttgccactcaggtatgatgggcagaatatt gcctctgcccgccagaaaaaggtgtaggctggagctgcttc (SEQ ID N.degree. 35) RV-iclR-P2Gttcaacattaactcatcggatcagttcag taactattgcattagctaacaataaaacatatgaatatcctccttag (SEQ ID N.degree. 36) FW-iclR-outCggtggaatgagatcttgcga (SEQ ID N.degree. 37) RV-iclR-outActtgctcccgacacgctca (SEQ ID N.degree. 38) FW_iclR_P8TTGCCACTCAGGTATGATGGGCAGAATATT GCCTCTGCCCGCCAGAAAAAGccgcttacagacaagctgtg (SEQ ID N.degree. 39) RV_iclR_P9GTTCAACATTAACTCATCGGATCAGTTCAG TAACTATTGCATTAGCTAACAATAAAAagccatgacccgggaattac (SEQ ID N.degree. 40) Rv-iclR-CTATTGCATTAGCTAACAATAAAACTTTTT scarless KO CTGGCGGGCAGAGG (SEQ IDN.degree. 41) stap 2 Fw-iclR- CCTCTGCCCGCCAGAAAAAGTTTTATTGTTscarless KO AGCTAATGCAATAGTTAC stap 2 (SEQ ID N.degree. 42) wcaJFw_wcaJ_out GCCAGCGCGATAATCACCAG (SEQ ID N.degree. 43) Rv_wcaJ_outTGCGCCTGAATGTGGAATC (SEQ ID N.degree. 44) Fw-wcaJ_2-TTTTGATATCGAACCAGACGCTCCATTCGC P1 GGATGTACTCAAGGTCGAACgtgtaggctggagctgcttc (SEQ ID N.degree. 45) Rv-wcaJ_2-TCTATGGTGCAACGCTTTTCAGATATCACC P2 ATCATGTTTGCCGGACTATGcatatgaatatcctccttag (SEQ ID N.degree. 46) fw_wcaJ_H1'TCAATATGCCGCTTTGTTAACGAAACCTTT GAACACCGTCAGGAAAACGATTTTGATATCGAACCAGACG (SEQ ID N.degree. 47) Rv_wcaJ_H2'TGACAAATCTAAAAAAGCGCGAGCGAGCGA AAACCAATGCATCGTTAATCTCTATGGTGCAACGCTTTTC (SEQ ID N.degree. 48) Fw_wcaJ_H1'_2CGCTTTGTTAACGAAACCTTTGAACACCGT CAGGAAAACGATTTTGATATCGAACCAGACGCTCCATTCG (SEQ ID N.degree. 49) lon FW-lon-P1CAGTCGTGTCATCTGATTACCTGGCGGAAA TTAAACTAAGAGAGAGCTCTgtgtaggctggagctgcttc(SEQ ID N.degree. 50) oMEMO100_RV-CGAATTAGCCTGCCAGCCCTGTTTTTATTA lon-P2GTGCATTTTGCGCGAGGTCAcatatgaata tcctccttag (SEQ ID N.degree. 51)oMEMO101_FW- AGCGCAACAGGCATCTGGTG lon-out (SEQ ID N.degree. 52)oMEMO102_RV- TATATCAGGCCAGCCATCCC lon-out (SEQ ID N.degree. 53)lacZYA:P22- lacY Fw_lacZYA_c GCTGAACTTGTAGGCCTGATAAGCGCAGCG hlTATCAGGCAATTTTTATAATCTTCATTTAA ATGGCGCGC (SEQ ID N.degree. 54)rv_lacZYA_c GCGCAACGCAATTAATGTGAGTTAGCTCAC hlTCATTAGGCACCCCAGGCTTCGCCTACCTG TGACGGAAG (SEQ ID N.degree. 55)fw_P22lacY- GCGCAACGCAATTAATGTGAGTTAGCTCAC KI_P1TCATTAGGCACCCCAGGCTTGTGTAGGCTG GAGCTGCTTC (SEQ ID N.degree. 56)ry_P22lacY- GCTGAACTTGTAGGCCTGATAAGCGCAGCG KITATCAGGCAATTTTTATAATCTTAAGCGAC TTCATTCACC (SEQ ID N.degree. 57)fw_lacZYA_H1' CGACGCTTGTTCCTGCGCTTTGTTCATGCCGGATGCGGCTAATGTAGATCGCTGAACTTG TAGGCCTG (SEQ ID N.degree. 58)rv_lacZYA_H2' CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCA ATTAATGTG (SEQ ID N.degree. 59)pfkA:P22- BaSP Fw-pfkA-P1 GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggctgg agctgcttc (SEQ ID N.degree. 60)Rv-pfkA- GCTTCTGTCATCGGTTTCAGGGTAAAGGAA pCXP22_P2TCTGCCTTTTTCCGAAATCaagcttgcatg cctgcatcc (SEQ ID N.degree. 61)FW_kan AGAGGCTATTCGGCTATGAC (SEQ ID N.degree. 62) Fw_baSP_seqCGCCATGTTGGAATGGGAGG (SEQ ID N.degree. 63) Fw_pfkA_H1_extTGATTGTTATACTATTTGCACATTCGTTGG ATCACTTCGATGTGCAAGAAGACTTCCGGCAACAGATTTC (SEQ ID N.degree. 64) Rv_pfkA_H2_extAATTGCAGAATTCATGTAGGCCTGATAAGC GAAGCGCATCAGGCATTTTTGCTTCTGTCATCGGTTTCAG (SEQ ID N.degree. 65) Fw-pfkA-out TACCGCCATTTGGCCTGAC(SEQ ID N.degree. 66) Rv-pfkA-out AAAGTGCGCTTTGTCCATGC (SEQ IDN.degree. 67) adhE:P22- frk Fw-adhE-ATCGGCATTGCCCAGAAGGGGCCGTTTATG

pCXP22-P1 TTGCCAGACAGCGCTACTGAgtgtaggctg gagctgcttc (SEQ IDN.degree. 68) Rv-adhE- ATTCGAGCAGATGATTTACTAAAAAAGTTT pCXP22-P2AACATTATCAGGAGAGCATTaagcttgcat gcctgcatcc (SEQ ID N.degree. 69)Fw-adhE-H1' AAGCCGTTATAGTGCCTCAGTTTAAGGATCGGTCAACTAATCCTTAACTGATCGGCATTG CCCAGAAG (SEQ ID N.degree. 70)Rv-adhE-H2' TTGATTTTCATAGGTTAAGCAAATCATCACCGCACTGACTATACTCTCGTATTCGAGCAG ATGATTTACTAAAAAAG (SEQ ID N.degree.71) FW_adhE_out GCGTCAGGCAGTGTTGTATC (SEQ ID N.degree. 72)RV_adhE_out CTGGAAGTGACGCATTAGAG (SEQ ID N.degree. 73) ldhA:P14-FT_H. pylori FW_ldhA_out tgtcattacttacacatcccgc (SEQ ID N.degree.74) RV_ldhA_out gcattcaatacgggtattgtgg (SEQ ID N.degree. 75)Fw-ldhA- CATTGGGGATTATCTGAATCAGCTCCCCTG pCXP22_P1GAATGCAGGGGAGCGGCAAGgtgtaggctg gagctgcttc (SEQ ID N.degree. 76)Rv-ldhA- TATTTTTAGTAGCTTAAATGTGATTCAACA pCXP22_P2TCACTGGAGAAAGTCTTATGaagcttgcat gcctgcatcc (SEQ ID N.degree. 77)Fw-ldhA-H1' CAATTACAGTTTCTGACTCAGGACTATTTTAAGAATAGAGGATGAAAGGTCATTGGGGAT TATCTGAATCAG (SEQ ID N.degree. 78)Rv-ldhA-H2' GAATTTTTCAATATCGCCATAGCTTTCAATTAAATTTGAAATTTTGTAAAATATTTTTAG TAGCTTAAATGTGATTCAAC (SEQ IDN.degree. 79) Fw-ldhA- TTCACCGCTAAAGCGGTTAC long homol (SEQ IDN.degree. 80) Rv-ldhA- CGCGTAATGCGTGGGCTTTC long homol (SEQ IDN.degree. 81) promCA:P14 pCXP14_SP_Fw CCGGCATATGGTATAATAGGG (SEQ IDN.degree. 82) yegH_rc_pure_rv ACGGCTTGCTGGCCATCA (SEQ ID N.degree.83) fw_P14- CGAATATAAGGTGACATTATGGTAATTGAA CA_KI_tetATATTGGCTTTCCAATAATGCTACGGCCCCA AGGTCCAA (SEQ ID N.degree. 84)rv_P14- AATATTGTCAACCTAAAGAAACTCCTAAAA CA_KI_tetAACCATATTGAATGACACTTATTGGCTTCAG GGATGAGGCG (SEQ ID N.degree. 85)fw_P14- TCCCGACTACGTGGACCTTG CA_KI_overl (SEQ ID N.degree. 86) apArv_P14- CATATGGTATAATAGGGAAATTTCCATGGC CA_KI_overlGGCCGCTCTAGAAGAAGCTTGGGATCCGTC apA GACCTCGGCATTATTGGAAAGCCAATATTC(SEQ ID N.degree. 87) fw_P14- GCCGCCATGGAAATTTCCCTATTATACCATCA_KI_overl ATGCCGGCCAAGATGTCAAGAAACTTATAG apBAATGAAGTAAGTGTCATTCAATATGG (SEQ ID N.degree. 88) fw_P14-AATATTGTCAACCTAAAGAAACTCCTAAAA CA_KI_H1ACCATATTGAATGACACTTACTTCATTCTA TAAGTTTCTTGAC (SEQ ID N.degree. 89)rv_P14- CGAATATAAGGTGACATTATGGTAATTGAA CA_KI_H2TATTGGCTTTCCAATAATGCCGAGGTCGAC GGATCCCAAGCTTC (SEQ ID N.degree.90)

[0065] Transformation.

[0066] Plasmids were transformed in CaCl.sub.2 competent cellsusing the simplified procedure of Hanahan (16) or viaelectroporation as described above.

Calculation Methods

Introduction

[0067] Different experiments with different strains were performed.In total 8 different conditions were tested. There was variation inthe genetic background (WT, iclR knock-out, arcA knock-out, andcombined iclR-arcA knock-out) and the mode of fermentation (batch,and chemostat). Each experiment was repeated twice.

[0068] When running the samples through the BioTrove apparatus, aqPCR curve (fluorescences in function of cycle number) and a meltcurve (fluorescences in function of the temperature) is obtainedfor each sample. Those data were exported from the BioTrovesoftware and further analysed in R. The analysis was divided in twosteps: first the qPCR curves were fitted and Ct values werecalculated and in the second step the Ct values were converted toexpression data.

Calculating the qPCR Curves

[0069] The raw qPCR curve data were extracted from the BioTrovesoftware and imported in R (1). The curves were fitted to a 5parameter sigmoidal model, with the R package qPCR (25, 34). Themaximum of the second derivative of those curves was used as Ctvalue. No normalisation was applied to the data prior to the curvefitting. However, outliers were removed. The detection of theoutliers was done using the following procedure: [0070] Fit themodel to the data. [0071] Calculate the residuals (defined as themeasured fluorescences minus the model-calculated ones). [0072]Assuming the residuals are normally distributed, calculate the meanand standard deviation of the residuals. [0073] Using this mean andstandard deviation, the 95% interval is calculated. [0074] Alldata-points for which the residuals fall out of this 95% intervalare considered as outliers. [0075] The curve is refitted withoutthe outliers. [0076] This is repeated until no outliers aredetected anymore. Using this procedure, the data do not have to benormalised prior to fitting, neither must the first data-points beremoved.

[0077] Many curves have to be fitted (1800 genes for oneexperiment). Therefore, it is undoable to manually check each curveand automated methods have to be applied to reject bad curves. Forthis different parameters are extracted from the curves: the cyclenumber value at which the maximum of the first derivative occurs(D1), the cycle number value at which the maximum of the secondderivative occurs (D2), the minimal fluorescence (Fmin), and themaximal fluorescence (Fmax). Combining the values of thoseparameters, the validity of the curve and the extent of expressionis assessed. How this is done is explained in the next section.

Filtering the Data

[0078] For some gene-experiment combinations, no amplification isdetected. This can be due to a variety of reasons: [0079]Expression is too low and 32 cycles (the number of cycles for allBioTrove arrays was set to 32) is not enough to detect theexpression. In this case, the real Ct cannot be determined and issomewhere between 32 and infinity. [0080] No expression. In thiscase, the real Ct is infinite. [0081] Technical failures: primersnot suitable, wrong loading (it is very difficult to uniformly loadthe BioTrove arrays, especially the holes at the sides of the arrayare frequently empty), etc. In this case the real Ct can varybetween 0 and infinity.

[0082] Some genes are genuinely not expressed and setting their Ctvalue to something else than infinity is not correct. For genesthat are expressed, but for which the expression value, due totechnical failures or limitations, are not known, setting the Ctvalue to infinity is not correct. Furthermore, using arbitraryvalues that are outside the range of expression complicates thecalculation routines and visualisation routines. Therefore it wasopted to remove the gene-experiment combinations for which nocorrect expression data was detected.

[0083] An obvious case of gene-experiment pairs for which noexpression is detected, are those for which no curve could befitted to the qPCR data. Less obvious cases are detailed below.

[0084] Typically for expressed genes, is that the fluorescencevalues cover a certain range. Data points for which this range wasnot high enough, were discarded, as they pointed to very poorlyfitted curves and generally bad data. The minimal fluorescencerange was set to 400 (thus Fmax-Fmin>400).

[0085] In a good amplification curve, the first (D1) and second(D2) derivative are quite close to each other (see thedocumentation of the SOD function in the qpcR package (25)).Therefore, all data-points for which the difference between D1 andD2 is larger than an arbitrary value (7 was used) werediscarded.

[0086] For each primer-pair, a qPCR experiment was performedwithout adding DNA. Only water was added. Normally no expressionshould be observed in those samples. However, amplification isdetected in water for some primer-pairs. Genes for which the Ctvalue (as mentioned before, D2 was used) is more than the Ct valueof water minus 5, are discarded, as it cannot be excluded that thefluorescence comes from the amplification of the primers and notthe added DNA.

Normalising and Calculating the Contrasts

[0087] Prior to calculating the expression differences, the Ctvalues have to be normalised. As so many genes were measured(1800), quantile normalisation could be used (33). The 1800 genesmeasured, were divided over 3 types of arrays, each containing 600genes. Quantile normalisation was done for each type of arrayseparately. A table was constructed where the rows represent thedifferent genes and the columns the different experiments (T1, seeEquations 1). Each column was sorted independently (T2) and theoriginal position of the elements was saved. The values in this newtable were replaced with the mean value over the different rows(T3). And finally this table was transformed so that the positionsof the values corresponded again to the original positions(T4).

Example of a quantile normalisation T 1 = [ 2 4 6 8 4 12 ] T 2 = [2 4 6 8 4 12 ] T 3 = [ 3 3 6 6 9 9 ] T 4 = [ 3 3 6 6 9 9 ] Equation1 ##EQU00001##

[0088] Differential expressions were calculated with the normaliseddata. This was done with the R package limma, which uses a Bayesianapproach to calculate the statistical relevances of the differences(31, 32). Limma was adapted to be able to cope with missing data:the original limma package discards all expression values from agene over the different experiments, when one value in oneexperiment is not available. This hampers the analysis when one hasmany different conditions, as for each gene for which one of theexperimental conditions produces no expression values, a differentcontrast matrix has to be generated omitting that experimentalcondition. Therefore the function for fitting the contrasts wasadapted to drop data-points with missing data.

[0089] Differential expressions were calculated between Ct valuesand the mean Ct value for a certain gene. Thus, the higher thevalue, the lower the expression. For each gene, plots weregenerated showing those differences. However, in those plots, theCt values were inversed, so that the higher the value, the higherthe expression.

Example 1

Effect of arcA and iclR Gene Deletions on the Gene Expression ofthe Colanic Acid Biosynthesis

[0090] FIGS. 1 and 2 show the expression pattern of genes involvedin colanic acid biosynthesis (35). Single arcA or iclR knock outmutations did not affect the expression of the operon in comparisonof the wild type strain in batch and chemostat conditions. Thedouble mutant strain, .DELTA.arcA.DELTA.iclR, however upregulatesthe genes of the colanic acid operon 6 to 8 times in comparison tothe wild type and the single mutant strains in both chemostat andbatch conditions. Both regulators have thus a surprisinglycooperative effect on the expression of this operon which isindependent from the culturing condition that is applied. Lookingat the regulatory network of this operon, no direct link could befound between both ArcA and IclR and the transcription factor thatcontrols the operon, RcsA (FIG. 5). Only ArcA is connected withRcsA via 3 other transcription factors, which are all upregulatedas well. However the .DELTA.arcA single gene deletion mutant straindid not affect the transcription of the operon.

Example 2

Effect of arcA and iclR Gene Deletions on the Gene Expression ofthe GDP-Fucose Biosynthesis Genes

[0091] FIGS. 4 and 6 show the relationship of the colanic acidoperon with GDP-fucose biosynthesis. In FIG. 6 the upregulation ofGDP-fucose biosynthesis specific genes is shown. These mutationsthus enhance the biosynthesis of GDP-fucose, which is a precursorfor fucosylated oligosaccharides such as fucosyllactose,fucosyllactoNbiose and lewis X oligosaccharide or fucosylatedproteins. These sugars and proteins, as already indicated above,have applications in therapeutics as nutraceutical, as componentsin human mother milk in which they have anti-inflammatory andprebiotic effects (5, 8, 27).

Example 3

Enhancement of GDP-Fucose and Fucosylated OligosaccharideBiosynthesis

[0092] The mutations .DELTA.arcA.DELTA.iclR applied in combinationwith other mutations enhance the production of fucosylatedcompounds. A first, `other` genetic modification that enhances saidproduction is the deletion of wcaJ from the colanic operon,stopping the initiation of the colanic acid biosynthesis and thusthe accumulation of GDP-fucose. Further, a fucosyltransferase hasto be introduced to link fucose with different acceptor moleculessuch as lactose. The metabolism is then engineered further toaccumulate the precursor of the GDP-fucose biosynthetic pathway.These modifications are shown in FIG. 7. Additional to wcaJ, thecolanic acid operon genes that do not code for GDP-fucosebiosynthesis reactions are knocked out, such as gmm, wcaA, wcaB,wcaC, wcaD, wcaE, wcaF, wcaI, wcaK, wcaL and/or, wcaM. For theproduction of fucosyllactose, lacZ coding for .beta.-galactosidase,is knocked out to avoid lactose degradation and the expression oflacY, coding for a lactose permease, is enhanced by means of astrong constitutive promoter.

Example 4

Enhancement of GDP-Fucose and Fucosylated OligosaccharideProduction Via a Split Metabolism with Sucrose as a Substrate

[0093] To accumulate the GDP-fucose precursor fructose andfructose-6-phosphate, a sucrose phosphorylase or invertase isintroduced. Because fructose-6-phosphate is easily degraded in theglycolysis, the glycolysis is interrupted in order to steer allfructose-6-phosphate in the direction of GDP-fucose. The genes pgi,pfkA and pfkB are thus knocked out, coding for glucose-6-phosphateisomerase and phosphofructokinase A and B. Finally afucosyltransferase is introduced to link fucose to an acceptormolecule.

[0094] The growth rate of the wild type strain is somewhat affectedwhen grown on sucrose after introduction of a sucrose phosphorylase(BaSP) (plasmid with sequence SEQ ID No 2) (Table 2), however theintroduction of pgi mutations and pfkA and pfkB double mutationsled to significant reduction of growth rate, the latter wasextremely low (0.02 h.sup.-1). The combination of all mutations(Apgi and ApfkA and ApfkB) led to the lowest growth rate, however,the growth rate on both sucrose and glucose was surprisinglysimilar to that of the pgi single mutant.

TABLE-US-00003 TABLE 2 specific growth rates of the glycolysisknock out strains on a minimal medium with glucose and sucroseGrowth rate on sucrose (h.sup.-1) Growth rate on (strainstransformed with Strain glucose (h.sup.-1) plasmid containing BaSP)Wild type 0.64 0.41 .DELTA.pgi 0.18 0.23 .DELTA.pfkA.DELTA.pfkB0.02 n.d. .DELTA.pgi.DELTA.pfkA.DELTA.pfkB 0.23 0.24

TABLE-US-00004 SEQ ID N.degree. 2: Plasmid sequence with sucrosephosphorylase BaSP AATTCGGAGGAAACAAAGATGGGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGAAAAACAAGGTGCAGCTCATCACTTACGCCGACCGCCTTGGCGACGGCACCATCAAGTCGATGACCGACATTCTGCGCACCCGCTTCGACGGCGTGTACGACGGCGTTCACATCCTGCCGTTCTTCACCCCGTTCGACGGCGCCGACGCAGGCTTCGACCCGATCGACCACACCAAGGTCGACGAACGTCTCGGCAGCTGGGACGACGTCGCCGAACTCTCCAAGACCCACAACATCATGGTCGACGCCATCGTCAACCACATGAGTTGGGAATCCAAGCAGTTCCAGGACGTGCTGGCCAAGGGCGAGGAGTCCGAATACTATCCGATGTTCCTCACCATGAGCTCCGTGTTCCCGAACGGCGCCACCGAAGAGGACCTGGCCGGCATCTACCGTCCGCGTCCGGGCCTGCCGTTCACCCACTACAAGTTCGCCGGCAAGACCCGCCTCGTGTGGGTCAGCTTCACCCCGCAGCAGGTGGACATCGACACCGATTCCGACAAGGGTTGGGAATACCTCATGTCGATTTTCGACCAGATGGCCGCCTCTCACGTCAGCTACATCCGCCTCGACGCCGTCGGCTATGGCGCCAAGGAAGCCGGCACCAGCTGCTTCATGACCCCGAAGACCTTCAAGCTGATCTCCCGTCTGCGTGAGGAAGGCGTCAAGCGCGGTCTGGAAATCCTCATCGAAGTGCACTCCTACTACAAGAAGCAGGTCGAAATCGCATCCAAGGTGGACCGCGTCTACGACTTCGCCCTGCCTCCGCTGCTGCTGCACGCGCTGAGCACCGGCCACGTCGAGCCCGTCGCCCACTGGACCGACATACGCCCGAACAACGCCGTCACCGTGCTCGATACGCACGACGGCATCGGCGTGATCGACATCGGCTCCGACCAGCTCGACCGCTCGCTCAAGGGTCTCGTGCCGGATGAGGACGTGGACAACCTCGTCAACACCATCCACGCCAACACCCACGGCGAATCCCAGGCAGCCACTGGCGCCGCCGCATCCAATCTCGACCTCTACCAGGTCAACAGCACCTACTATTCGGCGCTCGGGTGCAACGACCAGCACTACATCGCCGCCCGCGCGGTGCAGTTCTTCCTGCCGGGCGTGCCGCAAGTCTACTACGTCGGCGCGCTCGCCGGCAAGAACGACATGGAGCTGCTGCGTAAGACGAATAACGGCCGCGACATCAATCGCCATTACTACTCCACCGCGGAAATCGACGAGAACCTCAAGCGTCCGGTCGTCAAGGCCCTGAACGCGCTCGCCAAGTTCCGCAACGAGCTCGACGCGTTCGACGGCACGTTCTCGTACACCACCGATGACGACACGTCCATCAGCTTCACCTGGCGCGGCGAAACCAGCCAGGCCACGCTGACGTTCGAGCCGAAGCGCGGTCTCGGTGTGGACAACGCTACGCCGGTCGCCATGTTGGAATGGGAGGATTCCGCGGGAGACCACCGTTCGGATGATCTGATCGCCAATCCGCCTGTCGTCGCCTGACTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATATCCCGGGCGGCCGCTTCATTTATAAATTTCTTGACATTTTGGAATAGATGTGATATAATGTGTACATATCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTCGACCTCG

[0095] The flux redirections and mutations for GDP-fucose andfucosyllated oligosaccharide biosynthesis in a split metabolism areshown in FIG. 8, both for a strain expressing a heterologousinvertase and sucrose phosphorylase. Additional to wcaJ, thecolanic acid operon genes that do not code for GDP-fucosebiosynthesis reactions are knocked out, such as gmm, wcaA, wcaB,wcaC, wcaD, wcaE, wcaF, wcaI, wcaK, wcaL and/or, wcaM. For theproduction of fucosyllactose, lacZ, coding for.beta.-galactosidase, is knocked out to avoid lactose degradationand the expression of lacY, coding for a lactose permease, isenhanced by means of a strong constitutive promoter.

Example 5

Enhancement of GDP-Fucose and Fucosylated OligosaccharideProduction Via a Split Metabolism with Glucose as Substrate

[0096] When the genes pgi, pfkA, and pfkB are knocked out, carbon,taken up as glucose can only be metabolised via the pentosephosphate pathway. Due to the biochemical properties of thispathway, fructose-6-phosphate is formed (FIGS. 9 and 10). To formbiomass glyceraldehyde-3-phosphate has to be formed, which isformed by the transketolase reactions coded by tktA and tktB in E.coli. This Glyceraldehyde-3-phosphate is formed together withfructose-6-phosphate from xylulose-5-phosphate anderythrose-5-phosphate. The latter is in turn formed together withfructose-6-phosphate from glyceraldehyde-3-phosphate andsedoheptulose-7-phosphate via transaldolase reactions coded by talAand talB. To balance all of these reactions together the flux hasto be distributed between xylulose-5-phosphate andribose-5-phosphate, as such that from 1 mole glucose, 2/3 mole ofxylulose-5-phosphate and 1/3 mole ribose-5-phosphate is formed. Todrive these equilibrium reactions, fructose-6-phosphate is pulledout of the pentose phosphate pathway by the GDP-fucose andfucosyllacted oligosaccharide biosynthesis pathway. Additional towcaJ, the colanic acid operon genes that do not code for GDP-fucosebiosynthesis reactions are knocked out, such as gmm, wcaA, wcaB,wcaC, wcaD, wcaE, wcaF, wcaI, wcaK, wcaL and/or, wcaM. For theproduction of fucosyllactose, lacZ coding for .beta.-galactosidase,is knocked out to avoid lactose degradation and the expression oflacY, coding for a lactose permease, is enhanced by means of astrong constitutive promoter.

Example 6

Fermentative 2-Fucosyllactose Production with a FucosyltransferaseOriginating from Helicobacter pylori

[0097] The mutant strain in which the genes lacZ, glgC, agp, pfkA,pfkB, pgi, arcA, ic/R, wcaJ are knocked out and lacY was expressedvia constitutive expression to ensure expression under allculturing conditions, was transformed further with afucosyltransferase originating from Helicobacter pylori and asucrose phosphorylase originating from Bifidobacteriumadolescentis, which were also constitutively expressed. Theconstitutive promoters originate from the promoter librarydescribed by De Mey et al. 2007. This strain was cultured in amedium as described in the materials and methods, however with 30g/l of sucrose and 50 g/l of lactose. This resulted in theformation of 2-fucosyllactose as shown in FIGS. 13 and 14.

Example 7

Fermentative Fucosyllactose Production with a FucosyltransferaseOriginating from Dictyostellium discoideum

[0098] The mutant strain in which the genes lacZ, glgC, agp, pfkA,pfkB, pgi, arcA, ic/R, wcaJ are knocked out and lacY was expressedvia constitutive expression to ensure expression under allculturing conditions, was transformed further with afucosyltransferase originating from Dictyostellium discoideum and asucrose phosphorylase originating from Bifidobacteriumadolescentis, which were also expressed constitutively. Theconstitutive promoters originate from the promoter librarydescribed by De Mey et al. 2007. This strain was cultured in amedium as described in the materials and methods, however with 30g/l of sucrose and 50 g/l of lactose. This resulted in theformation of 2-fucosyllactose as shown in FIGS. 13 and 14.

Example 8

Enhancement of GDP-Mannose and Mannosylated OligosaccharideProduction Via a Split Metabolism with Sucrose as Substrate

[0099] To accumulate the GDP-mannose precursors fructose andfructose-6-phosphate, a sucrose phosphorylase or invertase isintroduced. Because fructose-6-phosphate is easily degraded in theglycolysis, the glycolysis is interrupted in order to steer allfructose-6-phosphate in the direction of GDP-fucose. The genes pgi,pfkA and pfkB are thus knocked out, coding for glucose-6-phosphateisomerase and phosphofructokinase A and B. Finally amannosyltransferase is introduced to link mannose to an acceptormolecule. To avoid GDP-mannose degradation the genes gmm and gmdhave to be knocked out in the colanic acid operon. In addition, thegenes that do not code for GDP-mannose biosynthesis reactions areknocked out, such as wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI,wcaJ, wcaK, wcaL and/or, wcaM.

Example 9

Upregulation of Acid Resistance Related Genes

[0100] Similar to the colanic acid operon upregulation, acidresistance related genes are also upregulated in a.DELTA.arcA.DELTA.iclR double mutant strain in comparison to thewild type strain and the single mutant strains. These genes make astrain more resistant to low pH, which is beneficial for theproduction of acids (4) or the production of glucosamine (12) whichis not stable at neutral and high pH. FIG. 12 presents the geneexpression pattern of these acid resistance related genes andindicates up to 8 fold expression increase in the double mutantstrain.

Example 10

Fed Batch Production of 2-Fucosyllactose

[0101] A mutant strain was constructed via the genetic engineeringmethodologies described above with the following genotype:

[0102].DELTA.lacZYA::P22-lacY.DELTA.glgC.DELTA.agp.DELTA.pgi.DELTA.pfkA-P-22-baSP.DELTA.pfkB.DELTA.arcA.DELTA.iclR::sl.DELTA.wcaJ.DELTA.Ion.DELTA.ad-hE-P14-frk+pCXP14-FT.sub.--H. pylori (a vector with sequence SEQ IDNo 1). The promoter P22 and P14 originate from the promoter libraryconstructed by De Mey et al (11) and was cloned similar to themethodology described by Aerts et al (2). "::sl" marks a scarlessgene deletion, thus without a FRT site that remains in thechromosome.

[0103] This strain was cultured in a bioreactor as described abovein materials and methods, in the mineral medium with 30 g/l ofsucrose and 50 g/l of lactose. After the batch phase the bioreactorwas fed with 500 g/l of sucrose, 50 g/l lactose and 1 g/l ofmagnesium sulphate heptahydrate. This led to the accumulation of27.5 g/l of fucosyllactose in the supernatant.

TABLE-US-00005 SEQID N.degree. 1: pCXP14-FT_H. pyloriCGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATATCCCGGGCGGCCGCCTTCATTCTATAAGTTTCTTGACATCTTGGCCGGCATATGGTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTCGACCTCGAATTCGGAGGAAACAAAGATGGCCTTTAAAGTTGTTCAGATTTGTGGTGGTCTGGGCAATCAGATGTTTCAGTATGCATTTGCAAAAAGCCTGCAGAAACATAGCAATACACCGGTTCTGCTGGATATTACCAGCTTTGATTGGAGCAATCGTAAAATGCAGCTGGAACTGTTTCCGATTGATCTGCCGTATGCAAGCGAAAAAGAAATTGCAATTGCCAAAATGCAGCATCTGCCGAAACTGGTTCGTAATGTTCTGAAATGCATGGGTTTTGATCGTGTGAGCCAAGAAATCGTGTTTGAATATGAACCGAAACTGCTGAAAACCAGCCGTCTGACCTATTTTTATGGCTATTTTCAGGATCCGCGTTATTTTGATGCAATTAGTCCGCTGATCAAACAGACCTTTACCCTGCCTCCGCCTCCGGAAAATGGTAATAACAAAAAAAAAGAAGAAGAGTATCATCGTAAACTGGCACTGATTCTGGCAGCAAAAAATAGCGTGTTTGTGCATATTCGTCGCGGTGATTATGTTGGTATTGGTTGTCAGCTGGGCATCGATTATCAGAAAAAAGCACTGGAATACATGGCAAAACGTGTTCCGAATATGGAACTGTTTGTGTTTTGCGAGGACCTGGAATTTACCCAGAATCTGGATCTGGGCTATCCGTTTATGGATATGACCACCCGTGATAAAGAGGAAGAGGCATATTGGGATATGCTGCTGATGCAGAGCTGTAAACATGGTATTATTGCCAACAGCACCTATAGTTGGTGGGCAGCATATCTGATTAATAACCCGGAAAAAATCATTATTGGTCCGAAACATTGGCTGTTTGGCCATGAAAACATCCTGTGTAAAGAATGGGTGAAAATCGAAAGCCACTTTGAAGTGAAAAGCCAGAAATATAATGCCTAATAAGAGCTCCCAA

Example 11

Fed Batch Production of 2-Fucosyllactose with a Hybrid Colanic AcidPromoter

[0104] A hybrid colanic acid promoter was constructed based on thegenome information and the sequences from the promoter librarydescribed by De Mey et al (11).

[0105].DELTA.lacZYA::P22-lacY.DELTA.glgC.DELTA.agp.DELTA.pgi.DELTA.pfkA::-P22-BaSP.DELTA.pfkB .DELTA.arcA.DELTA.iclR:sl .DELTA.wcaJ.DELTA.Ion .DELTA.adhE-P14-frk .DELTA.ldhA::P14-FT.sub.--H. pylori.DELTA.promCA:P14

[0106] This strain was cultured in a bioreactor as described abovein materials and methods, in the mineral medium with 30 g/l ofsucrose and 20 g/l of lactose. After the batch phase the bioreactorwas fed with 500 g/l of sucrose, 20 g/l lactose and 1 g/l ofmagnesium sulphate heptahydrate. This led to the accumulation of 26g/l of fucosyllactose in the supernatant with nearly stoichiometricconversion of lactose. Increasing the lactose feed concentrationsleads further to increased final fucosyllactose titers andstoichiometric lactose conversion.

REFERENCES

[0107] 1. 2006. R: A Language and Environment for StatisticalComputing. R Foundation for Statistical Computing. R-DevelopmentCore Team, Vienna, Austria. [0108] 2. Aerts, D., T. Verhaeghe, M.De Mey, T. Desmet, and W. Soetaert. 2010. A constitutive expressionsystem for high throughput screening. Engineering in Life Sciences10:DOI: 10.1002/elsc.201000065. [0109] 3. Alper, H., C. Fischer, E.Nevoigt, and G. Stephanopoulos. 2005. Tuning genetic controlthrough promoter engineering. Proceedings of the national academyof sciences of the United States of America 102:12678-12683. [0110]4. Beauprez, J. 2010. Metabolic modelling and engineering ofEscherichia coli for succinate production. PhD. Ghent University,Ghent. [0111] 5. Bode, L. 2006. Recent Advances on Structure,Metabolism, and Function of Human Milk Oligosaccharides. TheJournal of Nutrition 136:2127-2130. [0112] 6. Canton, B., A. Labno,and D. Endy. 2008. Refinement and standardization of syntheticbiological parts and devices. Nat Biotech 26:787-793. [0113] 7.Cavallaro, G., L. Maniscalco, P. Caliceti, S. Salmaso, A.Semenzato, and G. Giammona. 2004. Glycosilated MacromolecularConjugates of Antiviral Drugs with a Polyaspartamide. Journal ofDrug Targeting 12:593-605. [0114] 8. Coppa, G. V., L. Zampini, T.Galeazzi, and O. Gabrielli. 2006. Prebiotics in human milk: areview. Digestive and Liver Disease 38:S291-S294. [0115] 9.Datsenko, K. A., and B. L. Wanner. 2000. One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products.Proceedings of the national academy of sciences of the UnitedStates of America 97:6640-6645. [0116] 10. De Mey, M., G. J.Lequeux, J. J. Beauprez, J. Maertens, E. Van Horen, W. K. Soetaert,P. A. Vanrolleghem, and E. J. Vandamme. 2007. Comparison ofdifferent strategies to reduce acetate formation in Escherichiacoli. Biotechnology Progress. [0117] 11. De Mey, M., J. Maertens,G. J. Lequeux, W. K. Soetaert, and E. J. Vandamme. 2007.Construction and model-based analysis of a promoter library for E.coli: an indispensable tool for metabolic engineering. BMCBiotechnology 7:34-48. [0118] 12. Deng, M.-D., D. K. Severson, A.D. Grund, S. L. Wassink, R. P. Burlingame, A. Berry, J. A. Running,C. A. Kunesh, L. Song, T. A. Jerrell, and R. A. Rosson. 2005.Metabolic engineering of Escherichia coli for industrial productionof glucosamine and N-acetylglucosamine. Metabolic Engineering7:201-214. [0119] 13. Ebel, W., and J. E. Trempy. 1999. Escherichiacoli RcsA, a Positive Activator of Colanic Acid CapsularPolysaccharide Synthesis, Functions To Activate Its Own Expression.Journal of bacteriology 181:577-584. [0120] 14. Foster, J. W. 2004.Escherichia coli acid resistance: Tales of an amateur acidophile.Nature Reviews Microbiology 2:898-907. [0121] 15. Hammer, K., I.Mijakovic, and P. R. Jensen. 2006. Synthetic promoterlibraries--tuning of gene expression. TRENDS in Biotechnology24:53-55. [0122] 16. Hanahan, D., J. Jessee, and F. R. Bloom. 1991.Plasmid transformation of Escherichia coli and other bacteria.Methods in Enzymology 204:63-113. [0123] 17. Hommais, F., E. Krin,J.-Y. Coppee, C. Lacroix, E. Yeramian, A. Danchin, and P. Bertin.2004. GadE (YhiE): a novel activator involved in the response toacid environment in Escherichia coli. Microbiology 150:61-72.[0124] 18. Jigami, Y. 2008. Yeast Glycobiology and Its Application.Bioscience, Biotechnology, and Biochemistry 72:637-648. [0125] 19.Keasling, J. D. 1999. Gene-expression tools for the metabolicengineering of bacteria. Trends in Biotechnology 17:452-460. [0126]20. Lequeux, G. 2008. Metabolic modelling and analysis for theoptimization of Escherichia coli as a production host. GhentUniversity, Ghent. [0127] 21. Lequeux, G., L. Johansson, J.Maertens, P. Vanrolleghem, and G. Liden. 2005. Metabolic fluxanalysis of C- and P-limited shikimic acid producing E. coli.Journal of Biotechnology 118:S121-S121. [0128] 22. Masuda, N., andG. M. Church. 2003. Regulatory network of acid resistance genes inEscherichia coli. Molecular Microbiology 48:699-712. [0129] 23.Pattyn, F., F. Speleman, A. De Paepe, and J. Vandesompele. 2003.RTPrimerDB: The Real-Time PCR primer and probe database. NucleicAcids Research 31:122-123. [0130] 24. Perrenoud, A., and U. Sauer.2005. Impact of global transcriptional regulation by ArcA, ArcB,Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichiacoli. Journal of Bacteriology 187:3171-3179. [0131] 25. Ritz, C.,and A.-N. Spiess. 2008. qpcR: an R package for sigmoidal modelselection in quantitative real-time polymerase chain reactionanalysis. Bioinformatics 24:1549-1551. [0132] 26. Salis, H. M., E.A. Mirsky, and C. A. Voigt. 2009. Automated design of syntheticribosome binding sites to control protein expression. Nat Biotech27:946-950. [0133] 27. Seed, B., and J. Holgersson. 1999.Fucosyltransferase genes and uses thereof U.S. Pat. No. 5,858,752.[0134] 28. Shalel-Levanon, S., K. Y. San, and G. N. Bennett. 2005.Effect of ArcA and FNR on the expression of genes related to theoxygen regulation and glycolysis pathway in Escherichia coli undergrowth conditions. Biotechnology and Bioengineering 92:147-159.[0135] 29. Shalel-Levanon, S., K. Y. San, and G. N. Bennett. 2005.Effect of oxygen on the Escherichia coli ArcA and FNR regulationsystems and metabolic responses. Biotechnology and Bioengineering89:556-564. [0136] 30. Smith, S., A. D. Elbein, and Y. T. Pan.1999. Inhibition of Pathogenic Bacterial Binding by High MannoseOligosaccharides U.S. Pat. No. 5,939,279. [0137] 31. Smyth, G. K.2005. limma: Linear models for microarray data., p. 397-420. In R.Gentleman, V. Carey, W. Huber, R. A. Irizarry, and S. Dudoit (ed.),Bioinformatics and Computational Biology Solutions Using R andBioconductor. Springer. [0138] 32. Smyth, G. K. 2004. Linear Modelsand Empirical Bayes Methods for Assessing Differential Expressionin Microarray Experiments. Statistical Applications in Genetics andMolecular Biology 3:3. [0139] 33. Smyth, G. K., and T. Speed. 2003.Normalization of cDNA microarray data. Methods 31:265-273. [0140]34. Spiess, A.-N., C. Feig, and C. Ritz. 2008. Highly accuratesigmoidal fitting of real-time PCR data by introducing a parameterfor asymmetry. BMC Bioinformatics 9:221. [0141] 35. Stevenson, G.,K. Andrianopoulos, M. Hobbs, and P. R. Reeves. 1996. Organizationof the Escherichia coli K-12 gene cluster responsible forproduction of the extracellular polysaccharide colanic acid.Journal of Bacteriology 178:4885-4893. [0142] 36. Stout, V., A.Torres-Cabassa, M. R. Maurizi, D. Gutnick, and S. Gottesman. 1991.RcsA, an unstable positive regulator of capsular polysaccharidesynthesis. Journal of bacteriology 173:1738-1747. [0143] 37.Sunnarborg, A., D. Klumpp, T. Chung, and D. C. Laporte. 1990.Regulation of the glyoxylate bypass operon: cloning andcharacterization of IclR. Journal of Bacteriology 172:2642-2649.[0144] 38. Waegeman, H. J., J. Beauprez, H. Moens, J. Maertens, M.De Mey, M. R. Foulquie-Moreno, J. J. Heijnen, D. Charlier, and W.Soetaert. 2010. Effect of iclR and arcA knockouts on biomassformation and metabolic fluxes in Escherichia coli K12 and itsimplications on understanding the metabolism of Escherichia coliBL21 (DE3). BMC microbiology 11:1-17. [0145] 39. Waegeman, H. J.,J. Beauprez, H. Moens, J. Maertens, M. De Mey, M. R.Foulquie-Moreno, J. J. Heijnen, D. Charlier, and W. Soetaert. 2011.Effect of iclR and arcA knockouts on biomass formation andmetabolic fluxes in Escherichia coli K12 and its implications onunderstanding the metabolism of Escherichia coli BL21 (DE3). BMCmicrobiology 11:1-17. [0146] 40. Warnecke, T., and R. T. Gill.2005. Organic acid toxicity, tolerance, and production inEscherichia coli biorefining applications. Microbial Cell Factories4.

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