NZ625199B2 - Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides - Google Patents

Abstract

Disclosed is the use of a modified expression of the transcriptional regulators; the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl that code for a phosphomannomutase, a mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase and GDP-fucose synthase, respectively. Further disclosed is a process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: genetically changing the transcriptional regulators, the aerobic respiration control protein ArcA, and the isocitrate lyase regulator IclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl. Further disclosed is a mutated and/or transformed bacterium in which the regulators ArcA and IclR, in combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional. Further disclosed is the use of a modified expression of the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gada, gadC, gadE, gadX, gadWand/or slp. hat code for a phosphomannomutase, a mannose-1-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase and GDP-fucose synthase, respectively. Further disclosed is a process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: genetically changing the transcriptional regulators, the aerobic respiration control protein ArcA, and the isocitrate lyase regulator IclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl. Further disclosed is a mutated and/or transformed bacterium in which the regulators ArcA and IclR, in combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional. Further disclosed is the use of a modified expression of the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gada, gadC, gadE, gadX, gadWand/or slp.

Description

MUTANT RGANISMS TO SYNTHESIZE COLANIC ACID, MANNOSYLATED AND/OR LATED OLIGOSACCHARIDES.

FIELD OF THE INVENTION The present invention relates to mutated and/or transformed microorganisms for the synthesis of various compounds. More specifically, the present invention discloses microorganisms mutated in the genes encoding for the regulators ArcA and lclR. The latter mutations result in a significant upregulation of the genes that are part of the colanic acid operon. Hence, said microorganisms are useful for the synthesis of any compound being part of the colanic acid pathway such as GDP-fucose, GDP-mannose and colanic acid, and/or, can be further used — starting from GDP—fucose as a precursor — to synthesize fucosylated oligosaccharides or - ng from GDP~mannose as a precursor— to synthesize mannosylated oligosaccharides. In addition, mutations in the genes coding for the transcriptional regulators ArcA and lclR lead to an acid resistance phenotype in the ntial growth phase ng the sis of pH sensitive molecules and organic acids.

OUND OF THE INVENTION The genes arcA encoding for the aerobic respiration control protein and ic/R encoding the 2O rate lyase regulator are known to regulate the central carbon metabolism. ArcA is a global transcriptional regulator that regulates a wide variety of genes, while lclR is a local riptional regulator that regulates the glyoxylate y. ArcA is known to regulate the central carbon metabolism in response to oxygen deprivation and has no connection with lclR other than that it also regulates the glyoxylate pathway (24, 28, 29, 37, 38). In an earlier study the combined effect of AicIRAarcA mutant strains on the central carbon metabolism has been observed. Increased fluxes were shown in the tricarboxylic acid (TCA) cycle and late pathway and an interesting and surprising phenotype appeared when both genes where knocked out, namely the double mutant strain formed biomass with a yield that approached the maximal theoretical yield (4, 39).

Some compounds, such as GDP-fucose, are in high . The latter compound is indeed a precursor of fucosylated oligosaccharides such as llactose, fucosyllactoNbiose and lewis X oligosaccharides, or, of fucosylated proteins. These sugars are components of human mother milk in which they have anti-inflammatory and prebiotic effects and/or have applications in therapeutics as nutraceutical, anti— inflammatory agent or tic, in addition, fucosylated proteins find applications in the pharmaceutics (5, 8, 27). However, an ent method to produce the latter high-value nds is still .

In addition GDP-mannose is also an intermediate of the pathway towards GDP-fucose.

Interrupting the pathway prematurely leads to the accumulation of this compound, which is a precursor of ylated oligosacharides. These oligosaccharides find for example applications in the ent of gram—negative bacterial infections, in addition, GDP- mannose is important for the humanization of protein glycosylations, which is ial for the production of certain therapeutic proteins (18, 30). Mannosylated oligosaccharides and mannosylated glycoconjugates are also used for drug targeting, for instance mannosylated antivirals can specifically target the liver and kidneys (7).

The present invention provides microorganisms which are genetically d in such a manner that they can efficiently produce the latter compounds.

Moreover, the sis of pH sensitive molecules, such as — but not limited to -— glucosamine, and organic acids, such as —— but not limited to — pyruvic acid, succinic acid, adipic, sialic acid, sialylated oligosaccharides... are preferably produced at low pH, either to stabilize the product or for downstream processing reasons (4, 12, 40). Therefore, strains that can grow at low pH are cial for these production processes. E. coli is an organism that can adapt easily to various conditions, for instance it can easily adapt to the 2O harsh pH conditions in the stomach, which is about pH 2 (14). Nonetheless, E. coli does not seem to grow at these conditions, but induces its acid resistance mechanisms in the nary phase (40). During this phase the cell does not multiply anymore and therefore s productivity. Up to now, no solution was found to this problem. However, in the present invention, a genetically engineered microorganism is provided that can induce acid resistance mechanisms in the exponential growth phase, which is the phase that is mostly used for production of organic acids and pH instable products.

Y OF THE INVENTION in one aspect of the present invention, there is provided a use of a modified expression of the transcriptional regulators; the aerobic respiration l protein ArcA and the isocitrate lyase regulator lclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fol that code for a phosphomannomutase, a mannose-l-phosphate guanylyltransferase, GDP-mannose 4,6-dehydratase and GDP-fucose synthase, respectively. [followed by page 2a] In a further aspect of the present invention, there is provided a s for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: genetically changing the transcriptional regulators, the aerobic ation control protein ArcA, and the isocitrate lyase regulator lclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl.

In a further aspect of the present ion, there is provided a mutated and/or transformed bacterium in which the regulators ArcA and lclR, in combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional.

In a r aspect of the present invention, there is provided a use of a modified expression of the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase tor lclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or slp. in a further aspect of the present invention, there is provided a process for the synthesis of acids, sialic acid, sialylated oligosaccharides or glucosamine comprising genetically changing the transcriptional regulators the aerobic respiration control protein ArcA and the rate lyase regulator lclR to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, d/or slp.

BRIEF PTION OF FIGURES Figure 1: Relative gene sion pattern of the wild type, the AicIR and AarcA mutant strain to the AaroAAic/R mutant strain of genes involved in colanic acid thesis in batch fermentation conditions. The genes involved in colanic acid biosynthesis are presented in Figures 3 and 4. [followed by page 3] WO 2013087884 Figure 2: Gene expression pattern of the coianic acid operon of the wild type, the Aic/R and AarcA mutant strain in chemostat fermentation conditions relative to the AaroAAic/R mutant strain.

Figure 3: The gene organisation of the colanic acid operon and an overview of the function of these genes: Gene: Function: Colanic acid polymerase gmm nnose hydroiase wcal Glycosyitransferase Mannose~1—phosphate guanyiyltransferase Glycosyltransterase ted protein in colanic acid biosynthesis Figure 4: The colanic acid biosynthesis pathway.

Figure 5: Regulatory network of the colanic acid . This network was constructed with Pathway tools v 13.0.

Figure 6: Effect of the AarcAAic/R mutations on the GDP—fucose biosynthesis route.

Figure 7: Overview of the genetic modifications needed to enhance fucosyllactose and fucosylated accharides production starting from glucose as a substrate.

Figure 8: Starting from sucrose, fucosylated sugar derivates such as fucosyllactose and more specifically 1,2-fucosyllactose are produced. The strain is ed to force the cell to produce frucose-B-phosphate which is an intermediate in the synthesis of GDP-fucose.

Glucose or glucose-t-phosphate (if the starting enzyme is either a sucrase or a sucrose phosphorylase) is then fed to the central carbon metabolism via the pentose phosphate pathway.

Figure 9: Overview of the c modifications needed to enhance fucosyllactose and fucosylated oligosaccharides production starting from glucose as a substrate in a split metabolism.

Figure 10: Detail of the pentose phosphate y flux in a strain in which the genes coding for phosphoglucose isomerase and phosphofructokinase are d out.

Figure 11: Starting from sucrose, mannosylated sugar derivates are ed. The strain is modified to force the cell to produce frucose-G-phosphate which is an intermediate in the synthesis of GDP—fucose. Glucose or glucose—i—phosphate (if the starting enzyme is either a sucrase or a sucrose phosphorylase) is then fed to the central carbon metabolism via the pentose phosphate pathway.

Figure 12: Gene sion pattern acid resistance related genes of the wild type, the Aid]? and AarcA mutant strain in batch culturing ions relative to the AaroAAic/R mutant .

Figure 13: LC MSMS analysis chromatograms of culture broth and a 2-fucosyllactose standard. A. LC MSMS analysis of the standard, B. LC MSMS analysis of a sample of the culture broth of a mutant strain expressing a H. pylori fucosyltransferase, C. LC MSMS W0 20131087884 PCT/EP2012/O75639 analysis of a sample of the e broth of a mutant strain expressing a H. pylori fucosyltransferase.

Figure 14: LC MSMS analysis mass spectrum from the chromatograms shown in Figure 13 of culture broth and a 2-fucosyllactose standard. A. Mass (m/z) of the rd, B.

Mass (m/z) of the sample of the culturing broth of a mutant strain expressing a H. pylori fucosyltransferase, C. Mass (m/z) of the sample of the ing broth of a mutant strain expressing a H. pylori fucosyltransferase.

Figure 15: The sequence of the artificial hybrid promoter as given by SEQ ID N° 6 (the combination of the native and an artificial promoter) that was cloned in front of the colanic acid operon.

DESCRIPTION OF INVENTION The present ion provides microorganisms such as Enterobacteriaceae which are genetically changed in such a manner that they can ntly produce compounds which are part of the colanic acid pathway. A particular compound of interest is GDP-fucose which is used as a sor to synthesize fucosylated oligosaccharides. The latter have health-promoting effects as indicated above but there is no efficient production method available to produce said compounds.

The present invention thus provides for the usage of a d and/or ormed microorganism comprising a genetic change leading to a modified expression and/or activity of the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator lclR to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fa! that code for a phosphomannomutase, a mannose-t-phosphate guanylyltransferase, GDP-mannose 4,6—dehydratase and GDP-fucose synthase, respectively. The latter operon may also comprise the genes cpsG, cpsB, gmd, fcl and wza. In addition the expression of the gene rcsA is increased. This gene is a transcriptional regulator of the colanic acid operon.

Enhanced expression of this gene increases transcription of the colanic acid operon (13, 36).

Hence the present invention relates to the usage of a mutated and/or transformed microorganism comprising a genetic change leading to a modified sion and/or activity of the transcriptional regulator, the aerobic ation control n, ArcA and the isocitrate lyase regulator lclR to upregulate the riptional regulator of the coianic acid operon, rcsA, which in turn upregulates at least one of the genes of the colanic acid Hence, the present invention relates to a mutated and/or ormed microorganism such as ~but not limited to Enterobacteriaceae such as an Escherichia coli (E. coli) strain comprising a genetic change leading to a ed expression of the transcriptional regulators: the aerobic respiration control protein ArcA and the isocitrate lyase regulator lclR.

A mutated and/or transformed microorganism such as E. coli as used here can be ed by any method known to the person skilled in the art, including but not d to UV mutagenesis and chemical mutagenesis. A preferred manner to obtain the latter microorganism is by disrupting (knocking-out) the genes (arcA and fell?) ng for the proteins ArcA and lclR, or, by replacing the nous promoters of said genes by artificial promoters or replacing the endogenous ribosome binding site by an artificial ribosome binding site. The term ‘artificial promoters’ s to heterologous or non-natural or in siiico designed promoters with known expression th, these promoters can be derived from ies as described by Alper et al. (2005), Hammer 92‘ al. (2006), or De Mey et al. (2007) (3, 11, 15). The term logous promoter refers to any promoter that does not naturally occur in front of the gene. The term ‘artificial promoter’ may also refer to promoters with DNA sequences that are combinations of the native (autologous) promoter sequence with parts of different ogous or heterologous) promoter sequences as for example shown further in the examples. ces of such cial promoters’ can be found in databases such as for example partsregistry.org (6). The term ‘artificial ribosome binding site’ relates to heterologous or non—natural or in silico designed ribosome binding sites with known or measurable translation rates, these libraries can be derived from libraries or designed via algorithms as described by Salis at al (2009) (26). Hence, the present invention specifically relates to a mutated and/or transformed microorganism as indicated above wherein said genetic change is disrupting the genes encoding for ArcA and lclR, or, reducing or eliminating the function of ArcA and lclR via mutations in the coding sequence of the genes coding for ArcA and lclR , or, is replacing the endogenous promoters of the genes encoding for ArcA and lclR by artificial promoters; or, is replacing the endogenous ribosome binding site by an artificial me binding site. It is further clear that the mutant and/or transformant according to the present invention may r comprise additional genetic changes in one or more other genes within its genome as is also described further. The term microorganism specifically relates to a bacterium, more specifically a bacterium belonging to the family of Enterobacteriaceae. The latter bacterium preferably relates to any strain belonging to the species Escherichia coli such W0 20131087884 as but not limited to ichia coli B, Escherichia coli C, Escherichia coli W, Escherichia coli K12, Escherichia coli Nissle. More specifically, the latter term relates to cultivated Escherichia coli strains — designated as E. coli K12 s — which are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their y to thrive in the intestine. Well-known examples of the E. coli K12 strains are K12 Wild type, W3110, MG1655, M182, MC1000, M01060, M01061, MC4100, JM101, NZN111 and AA200.

Hence, the present invention specifically relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said E. coli strain is a K12 strain. More specifically, the present invention relates to a mutated and/or transformed Escherichia coli strain as indicated above wherein said K12 strain is E. coli M61655.

The terms ‘leading to a modified expression or activity’ tes that the above described mutations/transformations affects the transcription and/or translation and/or post- translational modification of said genes (arcA and iciR) into the transcriptional regulator proteins of the t invention (ArcA and IclR) in such a way that the latter transcription has significantly decreased or has even been completely abolished compared to a wild type strain, which has not been mutated or transformed with regard to both particular genes of the t invention. Hence, the present invention relates to a mutated and/or transformed microorganism such as an Escherichia coli strain as indicated above n said modified expression is a decreased expression, and, to a mutated and/or transformed microorganism such as an Escherichia coli strain as indicated above wherein said sed expression is an hed sion.

The terms ‘upregulating at least one of the genes of the colanic acid operon’ indicates that the expression of at least 1, 2, 3, 4,..., or all of the genes belonging to the colanic acid operon are significantly (= P > 0.05) upregulated when compared to the expression of said genes within a corresponding wild type rganism which is cultivated under the same conditions as the mutated and/0r transformed microorganism. The genes which belong to the colanic acid operon are wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcal, cpsB, cpsG, wcaJ, wsz, wcaK, wcaL and wcaM as indicated in Fig. 3 and/or as described in (35). Furthermore, the gene rcsA, coding for the transcriptional regulator of the c acid operon is lated (13, 36). More specifically the terms ‘upregulating at least one of the genes of the colanic acid operon’ or the transcriptional regulator of the colanic acid operon indicates that at least one of the genes of the colanic acid operon is 6 to 8 times upregulated in comparison to the expression of the genes of the colanic acid operon in the corresponding wild type microorganism. in addition the present invention relates to upregulating genes of the colanic acid operon as described above by replacing the native promoter by an ‘artificial er‘. More specifically, the present invention relates to a combination of the sequence of the native promoter with sequences of other artificial promoter sequences. The ation of the sequence of the native promoter with the sequence of other artificial promoter sequences is more cally the replacement of the sigma factor binding site of the native promoter with a stronger sigma factor binding site. Sigma factors, such as but not limited to sigma70, sigmaS, sigma24,..., are described (41) , ts of RNA polymerase that determine the affinity for promoter ces and the transcription rate of genes. The present invention provides microorganisms which are cally changed in such a manner that they can efficiently produce compounds which are part of the colanic acid pathway. The terms ‘compounds which are part of the colanic acid pathway’ refer to all compounds as indicated on Figure 4 ng from fructose-G-P and resulting in extracellular colanic acid.

More specifically the latter terms refer to the nds mannose—6—P, mannose-i-P, GDP-mannose, GDPdehydro-Bdeoxy—mannose, GDP-fucose and colanic acid. Hence the present invention specifically relates to the usage as indicated for the synthesis of colanic acid and/or for the synthesis of cose. As GDP-fucose is a precursor for fucosylated oligosaccharides such as fucosyllactose, fucosyllactoNbiose and lewis X accharide or fucosylated proteins, and as these sugars have therapeutical, nutraceutical, anti-inflammatory and prebiotic effects, the t invention specifically relates to the usage as described above for the sis of fucosylated oligosaccharides.

In other words, the present invention relates to a process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising genetically ng the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator lclR to upregulate at least one of the genes of the c acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fc/ or genes cpsG, cpsB, gmd, fcl and wza. More specifically, the present invention relates to a process as described wherein the mutations for ArcA and lclR are applied in combination with at least one mutation that enhances the production of fucosylated compounds. in order to efficiently produce fucosylated oligosaccharides (see Figures 1, 2 and 5-10), the above described ons in arcA and iclR can be applied in combination with other mutations which further enhance the production of fucosylated compounds. Some of these — non- limiting- other mutations are: a) the deletion of wcaJ from the colanic operon, stopping the initiation of the colanic acid thesis and thus allowing the accumulation of GDP— fucose; b) the introduction of a fucosyltransferase to link fucose with different acceptor molecules such as e; c) for the accumulation of the precursor of the GDP-fucose biosynthetic pathway and additional to the deletion of wcaJ, at least one of the following colanic acid operon genes that do not code for cose biosynthesis is knocked out: gmm, wcaA, wcaB, wcaC, wcaD, wan, wan, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM; d) for the production of fucosyllactose, lacZ coding for [3- galactosidase, is knocked out to avoid lactose degradation; e) to accumulate the precursor fructose and fructose-G-phosphate, a sucrose orylase or invertase is introduced; f) because fructose-S-phosphate is easily degraded in the glycolysis, the glycolysis has to be interrupted in order to steer all fructose-B-phosphate in the direction of GDP—fucose and the genes pgi, pka and pka (coding for glucose—G—phosphate isomerase and phosphofructokinase A and B) are thus d out; g) ng protein ation by knocking out a protease coded by a gene such as Ion; h) By constitutively expressing a lactose permease, subpopulations are avoided in the production process which are common for e induced gene expression systems (19). ln other words, the present invention s to a process as described above for the synthesis of fucosylated oligosaccharides wherein said at least one on that enhance the production of fucosylated compounds is: the deletion of the wcaJ gene, and/or, ng-out the colanic acid operon genes gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM, and/or, knocking-out 1802, and/or, introducing a sucrose phosporylase or invertase, and/or, knocking out the genes pgi, pka and ,0ka, and/or, knocking out the gene Ion, and/or introducing a fucosyltransferase, and/or a lactose permease. The term ducing a fucosyltransferase’ relates to upregulating or heterologous expression of fucosyltransferases which are within, but not limited to the enzymes in enzyme classes classes EC2.4.1.65, 2.4.1.68, 2.4.1.69, 2.4.1.152, 2.4.1.214, and/or 2.4.1.221 and/or the glycosyltransferase families GT1, GT2, GT1O GT11, GT23, GT37, GT65, GT68, and/or GT74 and/or originating from but not limited to Helicobacter pylori, Campylobacterjejuni, ste/Iium discoideum, Mus musculus, Homo sapiens, and these fucosyltransferases catalyse the formation of cr(1,2), a(1,3), d(1,4), or a(1,6) bonds on other sugars such as but not limited to galactose, e, lactoNbiose, lactoNtetraose, lactosamine, lactoNtetraose, lactoses, disialyllactoses, or fucosylated proteins, or fucosylated fatty acids, or fucosylated aglycons such as, but not limited to, antivirals, antibiotics,... .

The present invention provides for the usage of a mutated and/or transformed microorganism comprising a genetic change g to a modified sion and/or activity of the transcriptional tors the aerobic respiration control n ArcA and the isocitrate lyase regulator IclR to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG and cpsB, coding for phosphomannomutase and mannose-1—phosphate guanylyltransferase, which are needed for the biosynthesis of GDP—mannose. As GDP-mannose is a precursor for mannosyllated W0 2013l087884 oligosaccharides and mannosylated glycoconjugates. These oligosaccharides and onjugates find for example applications in the treatment of gram-negative bacterial infections, in addition, GDP-mannose is important for the humanization of protein glycosylations, which is essential for the tion of certain therapeutic proteins (18, ). Mannosylated oligosaccharides and mannosylated glycoconjugates are also used for drug targeting, for instance mannosylated antivirals can specifically target the liver and kidneys (7). In order to efficiently produce mannosylated oligosaccharides (see Figures 1, 2, 5, 6 and 11), the above described mutations in arcA and is”? can be applied in combination with other mutations which r enhance the production of mannosylated compounds. Some of these — non—limiting- other mutations are: a) the gene gmd of the colanic acid operon is d, , b) n the gene gmm coding for GDP- mannose hydrolase is deleted, and/or, c) wherein the colanic acid operon genes that do not code for GDP—mannose biosynthesis reactions, the genes gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, weal, wcaJ, wcaK, wcaL, fol, gmd, wzx, wza, wzb and/or, wcaM, are deleted, and/or, d) wherein a gene encoding for a sucrose phosphorylase or an invertase is introduced, and/or, e) wherein the the genes pgi, pka and pka, coding for oglucose isomerase, phosphofructokinase A and ofructokinase B respectively, are deleted, , f) knocking out the gene Ion encoding for a se, and/or f) wherein a gene encoding for a mannosyltransferase is introduced. In other words, the present invention relates to a process as described above for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides for the synthesis of GDP—mannose and/or for the synthesis of mannosylated oligosaccharides. The present invention further relates to said process wherein the genes cpsG and cpsB of the c acid operon are lated and wherein: a) the gene gmd of the colanic acid operon is d, and/or, b) wherein the gene gmm is deleted, and/or c) wherein the colanic acid operon genes fcl, gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM are knocked out and/or, d) wherein a gene encoding for a sucrose orylase or an invertase is introduced, and/or, 9) wherein the the genes pgi, pka and pka are deleted, and/or, f) knocking out the gene Ion, and/or 9) wherein a gene encoding for a mannosyltransferase is uced. The term ‘introducing a mannosyltransferase’ relates to lating or heterologous expression of mannosyltransferases which are within, but not limited to the enzymes in enzyme classes EC 2.4.1.32, 2.4.1.827, 2.4.1.844, 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, 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 families GT1, GT2, GT4, GT15, GT22, GT32, GT33, GT39, GT50 and/or GT58 and/or originating from but not limited to Helicobacter pylori, Campy/obacter , Dictyostellium disco/deum, Mus musculus, Homo sapiens, and these mannosyltransferases catalyse the formation of d(1,2), a(1,3), , or o(1,6) bonds on other sugars such as but not d to galactose, N- acetylglucosamine, Rhamnose, lactose, biose, lactoNtetraose, iactosamine, lactoNtetraose, sialyllactoses, disialyllactoses, or ylated proteins, or mannosylated fatty acids, or mannosylated aglycons such as, but not limited to, antivirals, antibiotics,... .

The term ‘heterologous expression’ relates to the expression of genes that are not naturally present in the production host, genes which can be synthesized chemically or be picked up from their natural host via PCR, genes which can be codon optimized for the production host or in which point mutation can be added to enhance enzyme activity or expression. Expressing heterologous and/or native genes can either be done on the chromosome, artificial chromosomes or plasmids and transcription can be controlled via inducible, constitutive, native or artificial promoters and translation can be controlled via native or artificial ribosome binding sites.

Consequently, the present invention further relates to mutated and/or transformed organisms in which the tors ArcA and IclR as be above, in combination with the genes encoding for 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 the enzyme phosphoglucose isomerase is d by the gene pgi and wherein the enzyme phosphofructokinase is encoded by the gene(s) pka and/or pka.

The terms ‘genes which are rendered less—functional or nctional’ refer to the well- known technologies for a skilled person such as the usage of siRNA, RNAi, miRNA, asRNA, nggenes, knocking-out genes, oson mutagenesis, etc... which are used to change the genes in such a way that they are less able (i.e. statistically significantly ‘less able’ compared to a functional wild-type gene) or completely unable (such as knocked-out genes) to e functional final products. The term ‘(gene) knock out’ thus refers to a gene which is rendered non~functional. The term ‘deleted gene’ or ‘gene deletion’ also refers to a gene which is rendered non-functional.

The present invention further relates to a mutated and/or transformed organism as described in the latter aph wherein said organism is further transformed with a gene encoding for a sucrose phosphorylase.

The present ion also relates to a mutated and/ortransformed organism as described above wherein, in addition, the activity and/or expression of the gene encoding for a lactose se is made constitutive and/or increased. Said ty can be increased by over-expressing said gene and/or by transforming said organisms with a gene ng for a lactose permease.

The t invention further s to any mutated and/or transformed organism as described above wherein at least one of the following genes is knocked out or is rendered less functional: a gene ng for a beta-galactosidase, a gene encoding for a glucose—1—phosphate yltransferase, a gene encoding for a glucose—1—phosphatase, a gene encoding for phosphogluconate dehydratase, a gene encoding for 2-keto-3~deoxygluconate—6- phosphate aldolase ,a gene encoding for a glucose-i-phosphate uridyltransferase, a gene encoding for an UDP-glucose~4-epimerase, a gene encoding for an UDP» glucosezgalactosephosphate uridyltransferase, a gene encoding for an UDP- galactopyranose mutase, a gene encoding for an UDP- galactose:(glucosyl)lipopolysaccharide—1,6-galactosyltransferase, a gene ng for an UDP—galactosyltransferase, a gene encoding for an UDP-glucosyltransferase, a gene encoding for an UDP-glucuronate transferase, a gene ng for an UDP-glucose lipid carrier transferase, a gene encoding for a GDP-mannose hydrolase, a gene encoding for an UDP-sugar hydrolase, a gene encoding for a mannose-B-phosphate isomerase, a gene encoding for an UDP—N—acetylglucosamine enoylpyruvoyl erase, a gene encoding for an UDP-N—acetylglucosamine acetyltransferase, a gene encoding for an UDP—Nacetylglucosamine—2-epimerase, a gene encoding for an undecaprenyl-phosphate alfa-N—acetylglucosaminyl transferase, a gene encoding for a glucose-G-phosphate—t— dehydrogenase, and/or, a gene encoding for a L-glutamine:D-fructose~6-phosphate aminotransferase, a gene encoding for a mannose-B—phosphate isomerase, a gene encoding for a sorbitol-e-phosphate dehydrogenase, a gene encoding for a mannitoI—1— phosphate 5-dehydrogenase, a gene encoding for a allulose-G-phosphate 3-epimerase, a gene encoding for an invertase, a gene encoding for a maltase, a gene encoding for a ase, a gene ng for a sugar transporting phosphotransferase, a gene encoding for a protease, or a gene encoding for a hexokinase. The term ‘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) knocked out or is (are) ed less functional.

The present invention further relates also to the usage of a mutated and/or transformed rganism such as an Escherichia coli strain comprising a genetic change leading to a modified expression of the transcriptional regulators the aerobic respiration control W0 2013f087884 protein ArcA and the isocitrate lyase regulator lclR to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or slp (17, 22). These genes are normally expressed in stationary phase conditions; however, the present mutated and/or transformed rganism is able to enhance the expression of these acid resistance related genes in the exponential growth phase. Hence, the present invention relates to the usage as described above for the sis of acids or pH sensitive molecules such as but not limited to glucosamine which is pH sensitive and should be produced at low pH (12). c acids, such as but not limited to pyruvic acid, succinic acid, adipic, siaiic acid, sialylated oligosaccharides (e.g. sialyilactose, sialyl Lewis X sugars,... ), acetylated oligosaccharides ns, chitosans,... ), sulfonated oligosaccharides (heparans and sans)... are preferably produced at low pH for ream processing purposes (4). in other words, the present invention relates to a process for the synthesis of acids, sialic acid, sialylated oligosaccharides or glucosamine comprising genetically changing the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase tor chR to upregulate at least one of the following acid resistance d genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or s/p.

The present invention will now be illustrated by the following non—limiting examples.

EXAMPLES A high throughput RT—qPCR screening of the microorganisms of the present invention has been setup with Biotrove OpenArray® technology. in this experiment the transcription of 1800 genes were measured in 4 strains (wild type, AarcA, AicIR, AarcA Aic/R) in two conditions (chemostat and batch). The data was processed using a curve fitting toolbox in R (25, 34) and le Normalization, the error on the data was ated using Bayesian statistics (20, 21, 31).

Material and s Strains and plasmids Escherichia coli M61655 [ ', F”, rph-1] was obtained from the Netherlands Culture Collection of Bacteria (NCCB). Escherichia coli BL21(DE3) was obtained from Novagen.

Escherichia coli M61655 ackA—pta, poxB, pppc ppc-p37 (10), the single knock-outs E. coli MG1655 arcA and E. coii MG1655 ic/R and the double knock-out E coli MG1655 arcA, ic/R were constructed in the Laboratory of Genetics and iology (MICR) using the method of Datsenko & Wanner (9).

Media The Luria Broth (LB) medium ted of 1 % tryptone peptone (Difco, Erembodegem, Belgium), 0.5 % yeast extract (Difco) and 0.5 % sodium chloride (VWR, Leuven, Belgium).

Shake flask medium contained 2 g/I NH4CI, 5 g/l SO4, 2.993 in KH2P04, 7.315 9/! , 8.372 g/l MOPS, 0.5 g/l NaCl, 0.5 g/l MgSO4-7HZO, 16.5 g/l e-H20, 1 ml/I n solution, 100 ul/l ate solution, and 1 ml/I selenium solution. The medium was set to a pH of 7 with 1M KOH. 1O Vitamin solution consisted of 3.6 g/I FeC|2 - 4H20, 5 g/l CaCl2 - 2H20, 1.3 g/l MnClz ' 2H20, 0.38 g/l CuC12-2H20, 0.5 g/I CoCIz - 6H20, 0.94 g/l ZHC12, 0.0311 g/I H3804, 0.4 g/I NagEDTA- 2H20 and 1.01 g/l thiamine - HCl. The molybdate solution contained 0.967 g/I NazMoO4 - 2H20. The selenium solution contained 42 g/I Seog.

The minimal medium for fermentations contained 6.75 g/I NH4CI, 1.25 g/l (NH4)2SO4, 1.15 g/l KH2PO4, 0.5 g/l NaCl, 0.5 g/l MgSO4-7H20, 16.5 9/1 glucose-H20, 1 mill n solution, 100 ul/I molybdate solution, and 1 mill selenium solution with the same composition as described above.

Cultivation conditions A preculture, from a single colony on a LB-plate, in 5 ml LB medium was incubated during 8 hours at 37 °C on an orbital shaker at 200 rpm. From this culture, 2 ml was transferred to 100 ml minimal medium in a 500 ml shake flask and incubated for 16 hours at 37 °C on an orbital shaker at 200 rpm. 4 % inoculum was used in a 2 | Biostat B Plus culture vessel with 1.5 l working volume (Sartorius Stedim Biotech, Melsungen, Germany). The culture conditions were: 37 °C, stirring at 800 rpm, and a gas flow rate of 1.5 l/min. Aerobic conditions were ined by sparging with air, anaerobic conditions were obtained by flushing the culture with a mixture of 3 % 002 and 97 % of N2. The pH was maintained at 7 with 0.5 M H2804 and 4 M KOH. The exhaust gas was cooled down to 4 °C by an exhaust cooler (Frigomix 1000, ius Stedim Biotech, Melsungen, Germany). 10 % solution of silicone antifoaming agent (BDH 331512K, VWR Int Ltd., Poole, England) was added when foaming raised during the fermentation (approximately 10 pl). The off-gas was ed with an EL3020 off-gas analyser (ABB Automation GmbH, 60488 Frankfurt am Main, Germany).

PCT/EP2012l075639 All data was logged with the Sartorius MFCS/win v3.0 system rius Stedim h, Melsungen, Germany).

All strains were cultivated at least twice and the given standard deviations on yields and rates are based on at least 10 data points taken during the repeated experiments.

Sampling methodology The bioreactor contains in its interior a harvest pipe (BD Spinal , 1.2x152 mm (BDMedical Systems, Franklin Lakes, NJ - USA) connected to a r port, linked outside to a Masterflex—14 tubing (Cole-Parmer, Antwerpen, Belgium) followed by a harvest port with a septum for sampling. The other side of this harvest port is connected 1O back to the reactor vessel with a Masterflex-iG tubing. This system is referred to as rapid sampling loop. During sampling, reactor broth is pumped around in the sampling loop. it has been estimated that, at a flow rate of 150 ml/min, the r broth needs 0.04 s to reach the harvest port and 3.2 s to er the reactor. At a p02 level of 50 %, there is around 3 mg/l of oxygen in the liquid at 37 °C. The p02 level should never drop below 20 % to avoid micro—aerobic conditions. Thus 1.8 mg/l of oxygen may be consumed during transit through the harvesting loop. Assuming an oxygen uptake rate of 0.4 g oxygen/g biomass/h (the maximal oxygen uptake rate found at pmax), this gives for 5 g/l biomass, an oxygen uptake rate of 2 g/l/h or 0.56 mg/l/s, which multiplied by 3.2 s ence time in the loop) gives 1.8 mg/I oxygen consumption.

In order to quench the metabolism of cells during the sampling, r broth was sucked through the harvest port in a syringe filled with 62 g stainless steel beads pre—cooled at -20 °C, to cool down 5 ml broth immediately to 4 °C. Sampling was immediately followed by cold centrifugation (15000 g, 5 min, 4 °C). During the batch experiments, a sample for ODSOOnm and RT—qPCR measurements was taken using the rapid sampling loop and the cold stainless bead sampling method.

RT-gPCR mRNA was extracted with the RNeasy kit (Qiagen,Venlo, The Netherlands). RNA quality and quantity was checked with a nanodrop 0 spectrophotometer (Nanodrop technologies, Wilmingto, USA). The ratios 260:280 (nm) and 2602230 (nm) were n 1.8 and 2 and at least 100ng/pl was needed for further analysis. cDNA was synthesised with random primers with the RevertAidTM H minus first strand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany). Finally, the gene expression of 1800 genes was ed with the ve OpenArray Real time PCR platform. The primers for the RT— PCR assay were designed with Primer design tools from the Primer database (23).

W0 20131087884 The on mixture was ed as described in the Biotrove OpenArrayTM Real-Time qPCR system users’ . In short, a mastermix was made with 26.4pl LightCycler® DNA Master SYBR® Green i (Roche applied Science), 1.1 pl SYBR GREEN l (100x stock solution, Sigma , 8.8;” glycerol (Sigma G5150), 5.3ul Pluronic® F68 (10% stock, lnvitrogen), 2.64m BSA (Sigma A7906), 26.4ul magnesium chloride (25mM stock solution, supplied in the LightCycler® kit of Roche applied Science ), 21.1pl HiDiTM formamide (Applied biosystems), and 94.66;.” RNase free sterile water resulting in a 186.4pl mastermix, which is enough to load 1 OpenArrayTM. For 1 SubArray (each ray is subdivided in 48 SubArrays on which 1 sample can be loaded) 1.5pl sample (with a 1O concentration of 100ng/pl) was mixed with 3.5pl of mastermind, as a no template control, water was used as blanc. The sample—mastermix mixture was loaded in a Loader plate (MatriPlateTM 384-well black low volume polypropylene plate, Biotrove) in a RNase free hood. A full loader plate was loaded with an AutoLoader (Biotrove) and loader tips onto the OpenArrays. These OpenArrays were then submerged in OpenArrayTM immersion fluid in an OpenArrayTM ime qPCR case. The case was sealed with Case sealing glue and ted in the Case Sealing station, which polymerizes the glue with UV light.

Analytical methods Cell density of the culture was ntly monitored by measuring optical density at 600 nm (Uvikom 922 spectrophotometer, BRS, Brussel, m). Cell dry weight was obtained by centrifugation (15 min, 5000 g, GSA rotor, l RC—SB, Goffin Meyvis, Kapeilen, Belgium) of 20 g reactor broth in pre-dried and weighted falcons. The pellets were subsequently washed once with 20 ml physioiogical solution (9 9/! NaCl) and dried at 70 °C to a constant weight. To be able to convert ODGOOnm measurements to biomass concentrations, a correlation curve of the ODSOOnm to the biomass concentration was made. The concentrations of glucose and organic acids were ined on a Varian Prostar HPLC system (Varian, Sint—Katelijne-Waver, m), using an Aminex HPX—87H column (Bio-Rad, Eke, Belgium) heated at 65 °C, equipped with a 1 cm precolumn, using mM H2804 (0.6 ml/min) as mobile phase. A dual-wave UV—VIS (210 nm and 265 nm) detector (Varian Prostar 325) and a differential refractive index detector (Merck LaChrom L—7490, Merck, Leuven, m) was used for peak detection. By dividing the absorptions of the peaks in both 265 and 210 nm, the peaks could be identified. The division s in a constant value, typical for a certain compound (formula of Beer- Lambert).

W0 2013f087884 Glucose, se, sucrose, llactose and glucose-l-phosphate were measured by HPLC with a Hypercarb column and were detected with an MSMS detector (Antonio et al., 2007; Nielsen et al., 2006).

Genetic methods All mutant strains were constructed via the methods described below.

Plasmids were maintained in the host E. coli DH50c (F', <p80d/acZAM15, A(lacZYA- argF)U169, deoR, recAl, endAl, hst17(rk', mk+), phoA, supE44, A', thi—l, gyrA96, reIAi).

Plasmids. pKD46 (Red helper plasmid, Ampicillin resistance), pKD3 (contains an FRT— flanked chloramphenicol resistance (cat) gene), pKD4 (contains an FRT—flanked kanamycin resistance (kan) gene), and pCPZO (expresses FLP recombinase activity) plasmids were used for the mutant construction. The plasmid pBluescript (Fermentas, St.

Leon-Rot, Germany) was used to construct the derivates of pKD3 and pKD4 with a promoter library, or with alleles ng a point mutation.

Mutations. The mutations ted in gene disruption (knock-out, KO). They were introduced using the concept of ko and Wanner (9). The s for the mutation strategies are described in Table 1.

Transformants carrying a Red helper plasmid were grown in 10 ml LB media with ampicillin (100 mg/l) and L—arabinose (10 mM) at 30 °C to an ODSOOnm of 0.6. The cells were made electrocompetent by g them with 50 ml of ice-cold water, a first time, and with 1 ml ice-cold water, a second time. Then, the cells were resuspended in 50 pl of ice-cold water. Electroporation was done with 50 ul of cells and 10—100 ng of linear double-stranded-DNA product by using a Gene PulserTM d) (600 Q, 25 uFD, and 250 volts).

After electroporation, cells were added to 1 ml LB media incubated 1 h at 37 °C, and finally spread onto LB—agar containing 25 mg/l of chloramphenicol or 50 mg/l of kanamycin to select antibiotic resistant transformants. The selected mutants were verified by PCR with s upstream and downstream of the modified region and were grown in LB—agar at 42 °C for the loss of the helper plasmid. The mutants were tested for llin ivity.

Linear double-stranded-DNA. The linear ds-DNA amplicons were obtained by PCR using pKD3, pKD4 and their derivates as template. The primers used had a part of the sequence complementary to the template and another part complementary to the side on the chromosomal DNA where the recombination has to take place ( Table 1). For the K0, the region of homology was designed 50-nt upstream and 50-nt downstream of the start and stop codon of the gene of interest. For the KI, the transcriptional starting point (+1) had to be respected. PCR products were PCR-purified, digested with Dpnl, repurified from an agarose gel, and suspended in elution buffer (5 mM Tris, pH 8.0).

Elimination of the antibiotic resistance gene. The selected mutants (Chloramphenicol or kanamycin resistant) were transformed with pCP20 plasmid, which is an ampicillin and 1O chloramphenicol resistant d that shows temperature-sensitive replication and thermal induction of FLP sis. The ampicillin—resistant transformants were selected at 30 °C, after which a few were colony purified in LB at 42 °C and then tested for loss of all antibiotic resistance and of the FLP helper plasmid. The gene knock outs and knock ins are checked with l s (Fw/Rv-gene-out). These primers are given in Table 1.

Table 1: s used to create E. 00/] M61655 arcA E. coli MG1655 iclR and the double knock-out E. coli M61655 arcA felt? and all other enetic knock outs and knock ins Primer name Sequence lacZ FW_LaCZ_Pl CATAATGGATTTCCTTACGCGAAATACGGGCAGACATGGCCTGCCCGGTTATTAgtgta ggctqgagctgcttc (SEQ ID N° 7) RV_LacZ“P2 GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTcatatgaa tatcctccttag (SEQ ID N° 8) FW_LacZ_out GCGGTTGGAATAATAGCG (SEQ ID No 9) RV_LacZ_out CAGGTTTCCCGACTGGAAAG (SEQ ID N° 10) glgC FW—glgC-Pl ccggttttaagcagcgggaacatctctgaacatacatgtaaaacctgcagtgt aggctggagctgcttc (SEQ ID N° 11) RV—glgC—P2 Gtctggcagggacctgcacacggattgtgtgtgttccagagatgataaaaaaggagtta atgaatatcctccttag (SEQ ID N° 12) FW—glgC-out Gcgaatatcgggaaatgcagg (SEQ ID N° 13) RV—glgC—out Cagagattgttttacctgctgg (SEQ ID N° 14) WO 87884 CATATTTCTGTCACACTCTTTAGTGATTGATAACAAAAGAGGTGCCAGGAgtgtaggct ggagctgcttc (SEQ ID N° 15) TAAAAACGTTTAACCAGCGACTCCCCCGCTTCTCGCGGGGGAGTTTTCTGcatatgaat atcctccttag(SEQ ID N° 16) GCCACAGGTGCAATTATC (SEQ ID N° 17) CATTTTCGAAGTCGCCGGGTACG(SEQ ID N° 18) -Pl GGCGCTACAATCTTCCAAAGTCACAATTCTCAAAATCAGAAGAGTATTGCgtgtaggct ggagctgcttc (SEQ ID N° 19) RV-pgi-PZ GGTTGCCGGATGCGGCGTGAACGCCTTATCCGGCCTACATATCGACGATGcatatgaat atcctccttag (SEQ ID N° 20) Fw_pgi*out( GGCTCCTCCAACACCGTTAC (SEQ ID N° 21) TACATATCGGCATCGACCTG (SEQ ID N° 22) Fw pka p1 GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggctg gagctgcttc(SEQ ID N° 25) RV-pka P2 GCTTCTGTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCCatatgaata tcctccttag (SEQ ID N° 26) Fw-pka-out TAGCGTCCCTGGAAAGGTAAC (SEQ ID N° 27) RV-pka—out ATCATCCGTCATAG (SEQ ID N° 28) Fw—pka P1 CACTTTCCGCTGATTCGGTGCCAGACTGAAATCAGCCTATAGGAGGAAATthgtaggc tggagctgcttc (SEQ ID N° 29) RV pka P2 GTTGCCGACAGGTTGGTGATGATTCCCCCAATGCTGGGGGAATGTTTTTGcatatgaat atcctccttag (SEQ ID N° 30) FW—arcA—Pl thtgaaaaataaaaacggcgctaaaaagcgccgttttttttgacggtggtaaagccga gtgtaggctggagctgcttc (SEQ ID N° 31) RV‘—arcA—P2 thcagggacttttgtacttcctgtttcgatttagttggcaatttaggtagcaaaccat atgaatatcctccttag (SEQ ID N° 32) Ctgccgaaaatgaaagccagta (SEQ ID N" 33) Ggaaagtgcatcaagaacgcaa (SEQ ID N° 34) FW—iclR-Pl Ttgccactcaggtatgatgggcagaatattqcctctgcccgccagaaaaaggtgtaggc tggagctgcttc (SEQ ID N° 35) RV—iclR—P2 Gttcaacattaactcatcggatcagttcagtaactattgcattagctaacaataaaaca tatgaatatcctccttag (SEQ ID N° 36) FW-iclR—out ngtggaatgagatcttgcga (SEQ ID N° 37) RV—iclR~out Acttgctcccgacacgctca (SEQ ID N° 38) TTGCCACTCAGGTATGATGGGCAGAATATTGCCTCTGCCCGCCAGAAAAAGCCgCttac gctgtg (SEQ ID N° 39) RV_iclR*P9 CATTAACTCATCGGATCAGTTCAGTAACTATTGCATTAGCTAACAATAAAAag ccatgacccgggaattac (SEQ ID N° 40) Rv—iclR— CTATTGCATTAGCTAACAATAAAACTTTTTCTGGCGGGCAGAGG (SEQ ID N° 41) scarless KO stap 2 Fw—iclR— CCTCTGCCCGCCAGAAAAAGTTTTATTGTTAGCTAATGCAATAGTTAC (SEQ ID N° ~scarless KO 42) stap 2 GCCAGCGCGATAATCACCAG (SEQ ID N° 43) TGAATGTGGAAT‘C (SEQ ID N° 44) Fw—wcaJ_2- TTTTGATATCGAACCAGACGCTCCATTCGCGGATGTACTCAAGGTCGAACgtgtaggct Pl ggagctgcttc (SEQ ID N° 45) Rv—wcaJ_2- TCTATGGTGCAACGCTTTTCAGATATCACCATCATGTTTGCCGGACTATGcatatgaat P2 atcctccttag (SEQ ID N° 46) fwwwcaJ_Hl' TCAATATGCCGCTTTGTTAACGAAACCTTTGAACACCGTCAGGAAAACGATTTTGATAT CGAACCAGACG (SEQ ID N° 47) Rv_wcaJ_H2' TGACAAATCTAAAAAAGCGCGAGCGAGCGAAAACCAATGCATCGTTAATCTCTATGGTG CAACGCTTTTC (SEQ :3 N° 48) Fw_wcaJ_H1' CGCTTTGTTAACGAAACCTTTGAACACCGTCAGGAAAACGATTTTGATATCGAACCAGA CGCTCCATTCG (SEQ ID N° 49) CAGTCGTGTCATCTGATTACCTGGCGGAAATTAAACTAAGAGAGAGCTCTgtgtaggct ggagctgcttc(SEQ ID N° 50) OMEM0100_RV AGCCTGCCAGCCCTGTTTTTATTAGTGCATTTTGCGCGAGGTCAcatatgaat —lon—P2 atcctccttag (SEQ ID N° 51) oMEMOlOl_FW AGCGCAACAGGCATCTGGTG (SEQ ID No 52) —lon—out OMEM0102_RV TATATCAGGCCAGCCATCCC (SEQ ID N° 53) -lon-out lacZXA:P22— lacY Fw_lacZYA_c GCTGAACTTGTAGGCCTGATAAGCGCAGCGTATCAGGCAATTTTTATAATCTTCATTTA hl AATGGCGCGC (SEQ ID N° 54) rv_lacZYA_c GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTCGCCTACCT hl GTGACGGAAG (SEQ ID N° 55) fw_P221acY- GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTGTGTAGGCT KI_P1 GGAGCTGCTTC (SEQ ID N° 56) rv_P221acY— GCTGAACTTGTAGGCCTGATAAGCGCAGCGTATCAGGCAATTTTTATAATCTTAAGCGA KI CTTCATTCACC (SEQ ID N° 57) fw_lacZYA_H CGACGCTTGTTCCTGCGCTTTGTTCATGCCGGATGCGGCTAATGTAGATCGCTGAACTT 1 1 GTAGGCCTG (SEQ ID N° 58) IV lacZYA H CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGC 2" " TGTG (SEQ ID N° 59) pka:P22- BaSP GACTTCCGGCAACAGATTTCATTTTGCATTCCAAAGTTCAGAGGTAGTCgtgtaggCtg gagctgcttc (SEQ ID N° 60) - GTCATCGGTTTCAGGGTAAAGGAATCTGCCTTTTTCCGAAATCaagcttgcat pCXP22_P2 gcctgcatcc (SEQ ID N° 61) AGAGGCTATTCGGCTATGAC (SEQ ID N° CGCCEEEEECGEEEGEGEEG (SEQ ED E TGATTGTTATACTATTTGCACATTCGTTGGATCACTTCGATGTGCAAGAAGACTTCCGG CAACAGATTTC (SEQ ID N° 64) Rv_pka_H2_ AATTGCAGAATTCATGTAGGCCTGATAAGCGAAGCGCATCAGGCATTTTTGCTTCTGTC ext ATCGGTTTCAG (SEQ ID N° 65) FW--pka—out CATTTGGCCTGAC (SEQ ID N° IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiiIIIIIIIIIIIIIIIIIIIIIIIIIEEEGEGCECTEEGECCEEEC SEQ ID E Fw-adhE— ATCGGCATTGCCCAGAAGGGGCCGTTTATGTTGCCAGACAGCGCTACTGAgtgtaggct pCXP22—PI ggagctgcttc (SEQ ID N° 68) ATTCGAGCAGATGATTTACTAAAAAAGTTTAACATTATCAGGAGAGCATTaagcttgca pCXP22—P2 tgcctgcatcc (SEQ ID N° 69) Fw—adhE-Hl' AAGCCGTTATAGTGCCTCAGTTTAAGGATCGGTCAACTAATCCTTAACTGATCGGCATT GCCCAGAAG (SEQ ID N° 70) Rv-ddhE-HZ' TTGATTTTCATAGGTTAAGCAAATCATCACCGCACTGACTATACTCTCGTATTCGAGCA GATGATTTACTAAAAAAG (SEQ ID N° 71) FW_adhE_out GCGTCAGGCAGTGTTGTATC (SEQ ID N° 72) RV_adhE_out CTGGAAGTGACGCATTAGAG (SEQ ID No 73) FW_ldhA_out tgtcattacttacacatcccgc (SEQ ID N° 74) RV_ldhA_out gcattcaatacgggtattgtgg (SEQ ID N° 75) Fw-ldhA- CATTGGGGATTATCTGAATCAGCTCCCCTGGAATGCAGGGGAGCGGCAAGQtgtaggct _Pl gcttc (SEQ ID N° 76) Rv—ldhA- TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGaagCttgca pCXP22_PZ tgcctgcatcc (SEQ ID N° 77) Fw-ldhA-Hl' CAATTACAGTTTCTGACTCAGGACTATTTTAAGAATAGAGGATGAAAGGTCATTGGGGA TTATCTGAATCAG (SEQ ID No 78) RV-ldhA-HZ' GAATTTTTCAATATCGCCATAGCTTTCAATTAAATTTGAAATTTTGTAAAATATTTTTA GTAGCTTAAATGTGATTCAAC (SEQ ID N° 79) Fw—ldhA— TTCACCGCTAAAGCGGTTAC (SEQ ID N° 80) long homol Rv-ldhA— CGCGTAATGCGTGGGCTTTC (SEQ ID N° 81) long homol :Pl4 pCXPl4_SP_I‘ CCGGCATATGGTATAATAGGG (SEQ ID N° 82) yegH_rc_pur ACGGCTTGCTGGCCATCA (SEQ ID N° 83) e rv fw_Pl4- CGAATATAAGGTGACATTATGGTAATTGAATATTGGCTTTCCAATAATGCTACGGCCCC CA_KI_tetA. AAGGTCCAA (SEQ ID N° 84) — AATATTGTCAACCTAAAGAAACTCCTAAAAACCATATTGAATGACACTTATTGGCTTCA CA_KI_tetA GGGATGAGGCG (SEQ ID N° 85) fw_Pl4— TCCCGACTACGTGGACCTTG (SEQ ID N° 86) overl rv_Pl4- CATATGGTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGT CA_KI_overl CGACCTCGGCATTATTGGAAAGCCAATATTC (SEQ ID NO 87) 2012/075639 - GCCGCCATGGAAATTTCCCTATTATACCATATGCCGGCCAAGATGTCAAGAAACTTATA CA_KI_overl GAATGAAGTAAGTGTCATTCAATATGG (SEQ ID N° 88) fw_Pl4— AATATTGTCAACCTAAAGAAACTCCTAAAAACCATATTGAATGACACTTACTTCATTCT CA_KI__H1 ATAAGTTTCTTGAC (SEQ ID N° 89) rv_Pl 4 — CGAATATAAGGTGACATTATGGTAATTGAATATTGGCTTTCCAATAATGCCGAGGTCGA CA__KI_H2 CGGATCCCAAGCTTC (SEQ ID N° 90) ormation. ds were transformed in CaCI2 competent cells using the simplified procedure of Hanahan (16) or via electroporation as described above.

Calculation methods uction Different experiments with ent strains were performed. in total 8 different conditions were tested. There was variation in the genetic background (WT, ic/R knock-out, arcA knock-out, and combined ic/R—arcA knock-out) and the mode of fermentation (batch, and chemostat). Each experiment was repeated twice. to When running the samples through the BioTrove apparatus, a qPCR curve escences in function of cycle number) and a melt curve scences in function of the temperature) is obtained for each sample. Those data were exported from the BioTrove software and further anaiysed in R. The analysis was divided in two steps: first the qPCR curves were fitted and Ct values were calculated and in the second step the Ct values were converted to expression data.

Calculating the qPCR curves The raw qPCR curve data were extracted from the ve software and imported in R (1). The curves were fitted to a 5 parameter sigmoidal model, with the R package qPCR (25, 34). The maximum of the second derivative of those curves was used as Ct value. No normalisation was applied to the data prior to the curve fitting. However, outliers were removed. The detection of the outliers was done using the following procedure: - Fit the model to the data.

- Calculate the residuals (defined as the measured fluorescences minus the modelcalculated ones).

- Assuming the residuals are normally distributed, calculate the mean and standard deviation of the residuals.

- Using this mean and standard deviation, the 95 % interval is calculated.

- All data—points for which the residuals fall out of this 95 % interval are considered as outliers.

- The curve is refitted without the outliers.

- This is repeated until no outliers are detected anymore. Using this procedure, the data do not have to be ised prior to fitting, neither must the first oints be removed.

Many curves have to be fitted (1800 genes for one experiment). ore, it is undoable to manually check each curve and automated methods have to be applied to reject bad curves. For this ent parameters are ted from the curves: the cycle number value at which the maximum of the first derivative occurs (D1), the cycle number value at which the maximum of the second derivative occurs (D2), the minimal fluorescence , and the maximal fluorescence . Combining the values of those parameters, the validity of the curve and the extent of expression is assessed. How this is done is explained in the next section.

Filtering the data For some gene—experiment combinations. no amplification is detected. This can be due to a y of reasons: - Expression is too low and 32 cycles (the number of cycles for all BioTrove arrays was set to 32) is not enough to detect the expression. in this case, the real Ct cannot be determined and is somewhere between 32 and infinity.

- No expression. in this case, the real Ct is infinite.

- Technical failures: primers not suitable, wrong loading (it is very difficult to uniformly load the BioTrove arrays, especially the holes at the sides of the array are frequently empty), etc. in this case the real Ct can vary between 0 and infinity.

Some genes are genuinely not expressed and setting their Ct value to ing else than infinity is not t. For genes that are expressed, but for which the expression value, due to technical es or limitations, are not known, setting the Ct value to infinity is not correct. Furthermore, using arbitrary values that are outside the range of expression complicates the calculation routines and visualisation routines. Therefore it was opted to remove the gene-experiment ations for which no correct expression data was detected.

An obvious case of gene-experiment pairs for which no sion is detected, are those for which no curve could be fitted to the qPCR data. Less s cases are detailed below.

Typically for expressed genes, is that the fluorescence values cover a certain range. Data points for which this range was not high enough, were discarded, as they pointed to very poorly fitted curves and generally bad data. The minimal fluorescence range was set to 400 (thus Fmax - Fmin > 400).

In a good amplification curve, the first (D1) and second (D2) derivative are quite close to each other (see the documentation of the SOD on in the qpcR package(25)). 1O Therefore, all data-points for which the difference between D1 and D2 is larger than an arbitrary value (7 was used) were discarded.

For each primer-pair, a qPCR experiment was performed without adding DNA. Only water was added. Normally no expression should be ed in those samples. However, amplification is detected in water for some primer—pairs. Genes for which the Ct value (as mentioned before, D2 was used) is more than the Ct value of water minus 5, are discarded, as it cannot be excluded that the fluorescence comes from the amplification of the primers and not the added DNA.

Normaiising and calculating the contrasts Prior to calculating the expression differences, the Ct values have to be ised. As so many genes were measured (1800), quantile normalisation could be used (33). The 1800 genes measured, were divided over 3 types of arrays, each containing 600 genes.

Quantile normalisation was done for each type of array separately. A table was constructed where the rows represent the different genes and the columns the different experiments (T1, see ons 1). Each column was sorted independently (T2) and the original position of the elements was saved. The values in this new table were replaced with the mean value over the different rows (T3). And y this table was transformed so that the ons of the values corresponded again to the original positions (T4). 24 33 33 T1=68 72—68 T3=6 2",: 412 412 99 99 Equations 1: Example of quantile normalisation Differential expressions were ated with the normalised data. This was done with the R package limma, which uses a Bayesian approach 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 a gene over the different experiments, when one value in one experiment is not available. This hampers the analysis when one has many different conditions, as for each gene for which one of the experimental ions es no expression values, a different contrast matrix has to 1O be generated ng that experimental condition. Therefore the function for fitting the contrasts was adapted to drop data~points with missing data.

Differential expressions were calculated n Ct values and the mean Ct value for a certain gene. Thus, the higher the value, the lower the expression. For each gene, plots were generated showing those differences. However, in those plots, the Ct values were inversed, so that the higher the value, the higher the expression.

Example 1: Effect of arcA and icIR gene deletions on the gene expression of the colanic acid thesis Figures 1 and 2 show the expression n of genes involved in colanic acid biosynthesis (35). Single arcA or ic/R knock out ons did not affect the expression of the operon in comparison of the wild type strain in batch and chemostat conditions. The double mutant strain, AarcAAiclR, however upregulates the genes of the colanic acid operon 6 to 8 times in ison to the wild type and the single mutant strains in both tat and batch conditions. Both regulators have thus a surprisingly cooperative effect on the expression of this operon which is independent from the culturing condition that is applied. Looking at the regulatory network of this operon, no direct link could be found between both ArcA and lclR and the ription factor that controls the operon, RcsA e 5). Only ArcA is connected with RcsA via 3 other transcription factors, which are all upregulated as well. However the AarcA single gene deletion mutant strain did not affect the transcription of the operon.

Example 2: Effect of arcA and icIR gene deletions on the gene expression of the GDP-fucose biosynthesis genes Figures 4 and 6 show the relationship of the colanic acid operon with GDP-fucose biosynthesis. in Figure 6 the upregulation of GDP-fucose biosynthesis specific genes is shown. These mutations thus enhance the biosynthesis of GDP—fucose, which is a precursor for fucosylated oligosaccharides such as tucosyllactose, fucosyllactoNbiose and lewis X oligosaccharide or fucosylated proteins. These sugars and proteins, as y indicated above, have applications in therapeutics as nutraceutical, as components in human mother milk in which they have anti—inflammatory and prebiotic effects (5, 8, 27).

Example 3: Enhancement of GDP-fucose and fucosylated oligosaccharide biosynthesis The mutations AarcAAic/R applied in combination with other mutations enhance the production of fucosylated compounds. A first, ‘other’ genetic modification that enhances said production is the on of wcaJ from the colanic operon, stopping the initiation of the colanic acid biosynthesis and thus the accumulation of GDP-fucose. Further, a fucosyltransferase has to be introduced to link fucose with ent or molecules such as lactose. The metabolism is then ered further to accumulate the sor of the GDP-fucose biosynthetic pathway. These modifications are shown in Figure 7. onal to wcaJ , the colanic acid operon genes that do not code for cose biosynthesis ons are knocked out, such as gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaK, wcaL and/or, wcaM. For the production of fucosyllactose, lacZ coding for B-galactosidase, is knocked out to avoid lactose degradation and the expression of lach coding for a lactose permease, is enhanced by means of a strong constitutive promoter.

Example 4: Enhancement of GDP-fucose and fucosylated oligosaccharide tion via a split metabolism with sucrose as a substrate To accumulate the cose precursor fructose and fructose—6-phosphate, a sucrose phosphorylase or invertase is uced. Because fructosephosphate is easily degraded in the glycolysis, the glycolysis is interrupted in order to steer all fructose—6- phosphate in the direction of GDP-fucose. The genes pgi, ,0ka and pka are thus knocked out, coding for glucosephosphate isomerase and ofructokinase A and B. Finally a fucosyltransferase is introduced to link fucose to an acceptor molecule.

The growth rate of the wild type strain is somewhat affected when grown on sucrose after introduction of a sucrose phosphorylase (BaSP) (plasmid with sequence SEQ ID N" 2) (Table 2), however the introduction of pgi mutations and pka and pka double mutations led to cant reduction of growth rate, the latter was extremely low (0.02 h"). The combination of all mutations (Apgi and Apka and Apka) led to the lowest growth rate, however, the growth rate on both sucrose and glucose was surprisingly similar to that of the pgi single mutant.

Table 2: sgecific growth rates of the glycolysis knock out strains on a minimal medium with glucose and sucrose Strain Growth rate on glucose (h‘1) Growth rate on sucrose (h'1) (strains transformed with plasmid containing BaSP) Wild type Apgi ApkaApka Apg/‘ApkaApka SEQ ID N° 2: d sequence with sucrose ghosphomlase BaSP AATTCGGAGGAAACAAAGATGGGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGC ATGAAAAACAAGGTGCAGCTCATCACTTACGCCGACCGCCTTGGCGACGGCACCATCAAG TCGATGACCGACATTCTGCGCACCCGCTTCGACGGCGTGTACGACGGCGTTCACATCCTG TTCACCCCGTTCGACGGCGCCGACGCAGGCTTCGACCCGATCGACCACACCAAG GTCGACGAACGTCTCGGCAGCTGGGACGACGTCGCCGAACTCTCCAAGACCCACAACATC ATGGTCGACGCCATCGTCAACCACATGAGTTGGGAATCCAAGCAGTTCCAGGACGTGCTG GGCGAGGAGTCCGAATACTATCCGATGTTCCTCACCATGAGCTCCGTGTTCCCG AACGGCGCCACCGAAGAGGACCTGGCCGGCATCTACCGTCCGCGTCCGGGCCTGCCGTTC ACCCACTACAAGTTCGCCGGCAAGACCCGCCTCGTGTGGGTCAGCTTCACCCCGCAGCAG GTGGACATCGACACCGATTCCGACAAGGGTTGGGAATACCTCATGTCGATTTTCGACCAG ATGGCCGCCTCTCACGTCAGCTACATCCGCCTCGACGCCGTCGGCTATGGCGCCAAGGAA ACCAGCTGCTTCATGACCCCGAAGACCTTCAAGCTGATCTCCCGTCTGCGTGAG GAAGGCGTCAAGCGCGGTCTGGAAATCCTCATCGAAGTGCACTCCTACTACAAGAAGCAG GTCGAAATCGCATCCAAGGTGGACCGCGTCTACGACTTCGCCCTGCCTCCGCTGCTGCTG CACGCGCTGAGCACCGGCCACGTCGAGCCCGTCGCCCACTGGACCGACATACGCCCGAAC AACGCCGTCACCGTGCTCGATACGCACGACGGCATCGGCGTGATCGACATCGGCTCCGAC CAGCTCGACCGCTCGCTCAAGGGTCTCGTGCCGGATGAGGACGTGGACAACCTCGTCAAC ACCATCCACGCCAACACCCACGGCGAATCCCAGGCAGCCACTGGCGCCGCCGCATCCAAT CTCGACCTCTACCAGGTCAACAGCACCTACTATTCGGCGCTCGGGTGCAACGACCAGCAC TACATCGCCGCCCGCGCGGTGCAGTTCTTCCTGCCGGGCGTGCCGCAAGTCTACTACGTC GGCGCGCTCGCCGGCAAGAACGACATGGAGCTGCTGCGTAAGACGAATAACGGCCGCGAC ATCAATCGCCATTACTACTCCACCGCGGAAATCGACGAGAACCTCAAGCGTCCGGTCGTC AAGGCCCTGAACGCGCTCGCCAAGTTCCGCAACGAGCTCGACGCGTTCGACGGCACGTTC TCGTACACCACCGATGACGACACGTCCATCAGCTTCACCTGGCGCGGCGAAACCAGCCAG GCCACGCTGACGTTCGAGCCGAAGCGCGGTCTCGGTGTGGACAACGCTACGCCGGTCGCC ATGTTGGAATGGGAGGATTCCGCGGGAGACCACCGTTCGGATGATCTGATCGCCAATCCG GTCGCCTGACTGCAGGTCGACCATATGGGAGAGCTCCCAACGCGTTGGATGCAG 1O GCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAA TCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTC CCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGG TCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAA GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA TCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACG CCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTT TGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTC ATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATT CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCT CACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT ATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGAC GCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTAC TCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCT GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCG AAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTACAGCA ATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAA CAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTT CCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATC ATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGG _.________________________*__________________________ AGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATT AAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTT CATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC ______________________________________________________________ CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCT TCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTA CCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGC PCT/EP20121075639 TTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACG ACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGA CTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGC AACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCT CGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTG ATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTC AGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTG ACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTT GTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATATC CCGGGCGGCCGCTTCATTTATAAATTTCTTGACATTTTGGAATAGATGTGATATAATGTG TACATATCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTCGACCTCG The flux redirections and mutations for GDP—fucose and fucosyllated oligosaccharide biosynthesis in a split metabolism are shown in Figure 8, both for a strain expressing a heterologous ase and sucrose phosphorylase. onal to wcaJ, the colanic acid operon genes that do not code for GDP-fucose biosynthesis reactions are knocked out, such as gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaK, wcaL andlor, wcaM.

For the production of fucosyllactose, laCZ, coding for B-galactosidase, is knocked out to avoid lactose degradation and the expression of lacY, coding for a lactose permease, is enhanced by means of a strong tutive promoter.

Example 5: Enhancement of cose and fucosylated oligosaccharide production via a split metabolism with e as substrate When the genes pgi, pka, and pka are knocked out, carbon, taken up as glucose can only be metabolised via the pentose ate pathway. Due to the biochemical ties of this pathway, fructose-G-phosphate is formed (Figures 9 and 10). To form biomass aldehyde—3—phosphate has to be formed, which is formed by the transketolase reactions coded by thA and thB in E. coli. This Glyceraldehyde phosphate is formed together with fructose—6—phosphate from xylulose—5—phosphate and erythrose-S—phosphate. The latter is in turn formed together with fructose-S—phosphate from glyceraldehyde—3-phosphate and sedoheptulosephosphate via transaldolase ons coded by taIA and ta/B. To balance all of these reactions together the flux has to be distributed between xylulose-5—phosphate and ribose—5-phosphate, as such that from 1 mole glucose, 2/3 mole of xylulosephosphate and 1/3 mole ribose—5-phosphate is formed. To drive these equilibrium reactions, fructose-S—phosphate is pulled out of the pentose phosphate pathway by the GDP—fucose and fucosyllacted accharide thesis pathway. Additional to wcaJ, the colanic acid operon genes that do not code for GDP—fucose biosynthesis reactions are knocked out, such as gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaK, wcaL and/or, wcaM. For the production of fucosyllactose, lacZ coding for B~galactosidase, is knocked out to avoid lactose degradation and the expression of laCY, coding for a lactose permease, is enhanced by means of a strong constitutive promoter.

Example 6: tative 2-fucosyllactose production with a fucosyltransferase originating from Helicobacter pylori The mutant strain in which the genes lacZ, g/gC, agp, pka, pka, pgi, arcA, iclR, wcaJ are knocked out and lacY was expressed via constitutive expression to ensure sion under all culturing conditions, was transformed r with a fucosyltransferase originating from Helicobacter pylori and a e phosphorylase originating from Bifidobacterium adolescentis, which were also constitutively expressed. The constitutive promoters originate from the promoter library described by De Mey et al. 2007. This strain was cultured in a medium as described in the materials and s, however with 30 g/l of sucrose and 50 g/l of lactose. This resulted in the formation of 2—fucosyllactose as shown in Figure 13 and 14.

Example 7: tative fucosyllactose production with a fucosyltransferase originating from Dictyostellium discoideum The mutant strain in which the genes lacZ, glgC, agp, pka, pka, pgi, arcA, iclR, wcaJ are knocked out and IacY was expressed via constitutive expression to ensure expression under all culturing conditions, was transformed further with a fucosyltransferase originating from Dictyoste/lium deum and a sucrose phosphorylase originating from Bifidobacterium adolescentis, which were also sed constitutively. The tutive promoters originate from the promoter library described by De Mey er al. 2007. This strain was cultured in a medium as described in the materials and methods, however with 30 g/l of sucrose and 50 g/l of lactose. This resulted in the formation of 2-fucosyllactose as shown in Figure 13 and 14. e 8: Enhancement of GDP-mannose and mannosylated oligosaccharide production via a split metabolism with sucrose as substrate To late the GDP-mannose precursors fructose and fructose-S-phosphate, a sucrose phosphorylase or invertase is introduced. Because fructose-G-phosphate is easily ed in the glycolysis, the glycolysis is interrupted in order to steer all fructose—6— phosphate in the direction of GDP-fucose. The genes pgi, pka and pka are thus knocked out, coding for glucosephosphate isomerase and phosphofructokinase A and B. Finally a yltransferase is introduced to link mannose to an acceptor molecule. To avoid GDP-mannose ation the genes gmm and gmd have to be knocked out in the colanic acid operon. in addition, the genes that do not code for GDP—mannose biosynthesis reactions are d out, such as wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL and/or, wcaM.

Example 9: lation of acid resistance related genes Similar to the colanic acid operon upregulation, acid resistance related genes are also upregulated in a AaroAA/c/R double mutant strain in comparison to the wild type strain and the single mutant strains. These genes make a strain more resistant to low pH, which is beneficial for the production of acids (4) or the production of glucosamine (12) which is not stable at neutral and high pH. Figure 12 presents the gene expression pattern of these acid resistance related genes and indicates up to 8 fold expression increase in the double mutant strain.

Example 10: Fed batch production of 2-fucosyllactose A mutant strain was constructed via the genetic engineering methodologies described above with the following genotype: AlacZYA::P22-la0YAglgCAagpApgiApka-P22- kaAarcAAiciR::s/AwcaJA/onAadhE—PM-frk + pCXP14~FT_H. pylori (a vector with sequence SEQ ID N°1). The promoter P22 and P14 originate from the promoter library ucted by De Mey et a/ (11) and was cloned similar to the methodology described by Aerts et al (2). “:zsl” marks a scarless gene deletion, thus without a FRT site that s in the chromosome.

This strain was cultured in a bioreactor as described above in materials and methods, in the mineral medium with 30 g/l of sucrose and 50 g/l of lactose. After the batch phase the bioreactor was fed with 500 g/I of sucrose, 50 g/l lactose and 1 g/I of magnesium sulphate heptahydrate. This led to the accumulation of 27.5 g/l of llactose in the supernatant.

SEQID N° 1: pCXP14-FT_H. pyiori CGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCC TGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCA GTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCG ATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGA AAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC CTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGG TGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTG 1O ACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAA ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGA AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCC TTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGG GTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT TATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAG AATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTC GCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA CGATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTC CCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTG GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTA TCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAG GTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGA TTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGT AGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCC TGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGAC GATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCA GCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTC ACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGT GAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAG CGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCA TATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCC GCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGC GCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGG GAGAGCTCGATATCCCGGGCGGCCGCCTTCATTCTATAAGTTTCTTGACATCTTGGCCGG GTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGCTTGGGATCCGTC GACCTCGAATTCGGAGGAAACAAAGATGGCCTTTAAAGTTGTTCAGATTTGTGGTGGTCT GGGCAATCAGATGTTTCAGTATGCATTTGCAAAAAGCCTGCAGAAACATAGCAATACACC GGTTCTGCTGGATATTACCAGCTTTGATTGGAGCAATCGTAAAATGCAGCTGGAACTGTT TCCGATTGATCTGCCGTATGCAAGCGAAAAAGAAATTGCAATTGCCAAAATGCAGCATCT GCCGAAACTGGTTCGTAATGTTCTGAAATGCATGGGTTTTGATCGTGTGAGCCAAGAAAT TGAATATGAACCGAAACTGCTGAAAACCAGCCGTCTGACCTATTTTTATGGCTA TTTTCAGGATCCGCGTTATTTTGATGCAATTAGTCCGCTGATCAAACAGACCTTTACCCT GCCTCCGCCTCCGGAAAATGGTAATAACAAAAAAAAAGAAGAAGAGTATCATCGTAAACT GGCACTGATTCTGGCAGCAAAAAATAGCGTGTTTGTGCATATTCGTCGCGGTGATTATGT TGGTATTGGTTGTCAGCTGGGCATCGATTATCAGAAAAAAGCACTGGAATACATGGCAAA ACGTGTTCCGAATATGGAACTGTTTGTGTTTTGCGAGGACCTGGAATTTACCCAGAATCT GGATCTGGGCTATCCGTTTATGGATATGACCACCCGTGATAAAGAGGAAGAGGCATATTG GGATATGCTGCTGATGCAGAGCTGTAAACATGGTATTATTGCCAACAGCACCTATAGTTG AGCATATCTGATTAATAACCCGGAAAAAATCATTATTGGTCCGAAACATTGGCT GTTTGGCCATGAAAACATCCTGTGTAAAGAATGGGTGAAAATCGAAAGCCACTTTGAAGT GAAAAGCCAGAAATATAATGCCTAATAAGAGCTCCCAA Example 11: Fed batch production of 2-fucosyllactose with a hybrid colanic acid promoter A hybrid colanic acid promoter was constructed based on the genome information and the sequences from the promoter library described by De Mey eta/(11).

AlacZYA::P22—lacYAglgCAagpApgiApka::P22-BaSPApka AarcAAiclesl chaJ Alon PM—frk AldhA::P14- FT_H. pylori ApromCA:P14 This strain was cultured in a ctor as described above in materials and methods, in the mineral medium with 30 g/I of sucrose and 20 g/l of lactose. After the batch phase the bioreactor was fed with 500 g/l of sucrose, 20 Q]! lactose and 1 g/l of magnesium sulphate heptahydrate. This led to the accumulation of 26 g/l of fucosyllactose in the atant with nearly stoichiometric conversion of lactose. increasing the lactose feed concentrations leads further to increased final fucosyllactose titers and stoichiometric e conversion.

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Claims (20)

1. CLAIMS : Use of a modified expression of the transcriptional regulators; the aerobic respiration control n ArcA and the isocitrate lyase regulator lclR, to upregulate at least one of the genes of the colanic acid operon, wherein said operon ses the genes cpsG, cpsB, gmd and fc/ that code for a phosphomannomutase, a mannosephosphate guanylyltransferase, GDP- mannose 4,6-dehydratase and GDP-fucose synthase, respectively.
2. Use according to claim 1, wherein said upregulation of at least one of the genes of 10 the c acid operon is preceded by the upregulation of the transcriptional regulator of said c acid operon rcsA.
3. Use according to claim 1 or 2, wherein the transcriptional regulators, the aerobic respiration control protein ArcA and the isocitrate lyase regulator lclR are expressed by an Escherichia coli , wherein said E. coli strain is specifically a 15 K12 strain or, wherein said K12 strain is more specifically E. coli M61655.
4. Use according to any one of claims 1-3, wherein said modified expression results from disrupting the genes encoding for ArcA and chR, replacing the endogenous promoters of the genes encoding for ArcA and lclR by artificial promoters, or replacing the endogenous ribosome binding site by an artificial ribosome binding 20 site.
5. Use according to any one of claims 1-4 wherein said modified expression is a decreased expression, or n said sed expression specifically is an hed expression.
6. Use ing to any one of claims 1-5, n at least one of the genes of the 25 colanic acid operon are upregulated 6 to 8 times in comparison to the expression of the colanic acid operon in the corresponding wild type microorganism. .
7. A process for the synthesis of colanic acid and/or GDP-fucose and/or fucosylated oligosaccharides comprising: cally changing the transcriptional regulators, the aerobic respiration control protein ArcA, and the isocitrate lyase regulator lclR, 30 to upregulate at least one of the genes of the colanic acid operon, wherein said operon comprises the genes cpsG, cpsB, gmd and fcl. .
8. A process according to claim 7, wherein said genetically changing comprises at least one mutation that enhances the production of fucosylated compounds, selected from the group consisting of: 35 the on of the wcaJ gene; knocking-out the colanic acid operon genes gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc and/or wcaM; knocking-out lacZ; introducing a sucrose phosporyiase or ase; knocking out the genes pgi, pka and pka; knocking out the gene Ion; introducing a fucosyltransferase and/or a lactose se; or combinations thereof.
9. A s according to claim 7, for the synthesis of GDP-mannose and/or for the synthesis of mannosylated accharides.
10. 10. A process according to claim 9 wherein the genes cpsG and cpsB of the colanic acid operon are upregulated and wherein: a) the gene gmd of the colanic acid operon is deleted, and/or, 1O b) wherein the gene gmm is deleted, and/or c) wherein the colanic acid operon genes fcl, gmd, gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcal, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc, and/or, wcaM are knocked out and/or, d) wherein a gene encoding for a sucrose phosphorylase or an invertase is 15 uced, and/or, e) n the the genes pgi, pka and pka are deleted, , f) knocking out the gene Ion, and/or 9) wherein a gene encoding for a mannosyltransferase is uced.
11. 11. A mutated and/or transformed bacterium in which the regulators ArcA and chR, in 2O combination with the genes encoding for the enzymes phosphoglucose isomerase and phosphofructokinase, are knocked out or are rendered less functional.
12. 12. A mutated and/or transformed bacterium according to claim 11 n the enzyme phosphoglucose isomerase is encoded by the gene pgi and wherein the enzyme phosphofructokinase is encoded by the gene(s) pka and/or pka. 25
13. 13. A mutated and/or transformed bacterium according to claim 11 or 12 wherein said organism is further transformed with a gene encoding for a sucrose phosphorylase or invertase.
14. 14. A mutated and/or transformed bacterium according to any one of claims 11 to 13 wherein the ty of the gene encoding for a lactose permease is increased. 30
15. 15. A mutated and/or ormed bacterium according to any one of claims 11 to 14 wherein at least one of the following genes is knocked out or is rendered less functional: a gene encoding for a beta-galactosidase, a gene encoding for a glucose phosphate adenylyltransferase, a gene encoding for a glucose—1-phosphatase, a 35 gene encoding for phosphogluconate atase, a gene encoding for 2—keto-3— deoxygIuconate-S-phosphate aldoiase ,a gene encoding for a glucose phosphate transferase, a gene encoding for an UDP-glucoseepimerase, a gene encoding for an UDP-glucosezgalactose-t—phosphate uridyltransferase, a gene encoding for an UDPgalactopyranose mutase, a gene ng for an UDP- galactose:(glucosyl)Iipcpolysaccharide-1,6-galactosyltransferase, a gene encoding for an UDP-galactosyltransferase, a gene encoding for an UDP- glucosyltransferase, a gene encoding for an UDP-glucuronate transferase, a gene encoding for an UDP-glucose lipid carrier transferase, a gene encoding for a GDP- mannose hydrolase, a gene encoding for an UDP—sugar hydrolase, a gene encoding for a mannose-B-phosphate isomerase, a gene ng for an UDP-N- 1O acetylglucosamine enoylpyruvoyl transferase, a gene ng for an UDP— Nacetylglucosamine acetyltransferase, a gene encoding for an UDP- Nacetylglucosamine-Z-epimerase, a gene encoding for an undecaprenyl- phosphate alfa—N-acetylglucosaminyl transferase, a gene encoding for a glucose- 6-phosphatedehydrogenase, and/or, a gene encoding for a L-glutaminezD- 15 fructosephosphate aminotransferase, a gene encoding for a mannose-6— phosphate ase, a gene encoding for a ol-G-phosphate dehydrogenase, a gene encoding for a mannitol—‘l-phosphate 5-dehydrogenase, a gene encoding for a allulose-B—phosphate 3-epimerase, a gene encoding for an invertase, a gene incoding for a maltase, a gene encoding for a trehalase, a gene encoding for a 20 sugar transporting phosphotransferase, a gene ng for a protease, or a gene encoding for a hexokinase.
16. 16. Use of a modified expression of the transcriptional regulators the aerobic ation control protein ArcA and the isocitrate lyase regulator lclR, to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, 25 hdeA, hdeD, gadB, gadC, gadE, gadX, gadWand/or slp.
17. 17. Use ing to claim 16 for the synthesis of acids, sialic acid, sialylated oligosaccharides or glucosamine.
18. 18. Use according to claim 16 or 17, wherein said modified expression is a decreased expression, or wherein said decreased expression is an abolished expression. 3O
19. 19. A process for the synthesis of acids, sialic acid, sialylated oligosaccharides or glucosamine comprising cally ng the transcriptional regulators the aerobic respiration control protein ArcA and the isocitrate lyase regulator chR to upregulate at least one of the ing acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, d/or slp. 35
20. 20. A use according to claim 1 or 16, substantially as herein described with reference to any one of the Examples and/or