# Patent

## Title
Mutant microorganisms to synthesize colanic acid, mannosylated and/or fucosylated oligosaccharides

## Abstract

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 IclR. 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 form GDP-fucose as a precursor—to synthesize fucosylated oligosaccharides or—starting from GDP-mannose as a precursor—to synthesize mannosylated oligosaccharides. In addition, mutations in the genes coding for the transcriptional regulators ArcA and IclR lead to an acid resistance phenotype in the exponential growth phase allowing the synthesis of pH sensitive molecules or organic acids.

## Background
This application is a US national phase of International Application No. PCT/EP2012/075639 filed on Dec. 14, 2012, which claims the benefit of European patent application 11194101.5, filed Dec. 16, 2011, the disclosures of which are incorporated herein by reference in their entirety.
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 IclR. 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—starting from GDP-mannose as a precursor—to synthesize mannosylated oligosaccharides. In addition, mutations in the genes coding for the transcriptional regulators ArcA and IclR lead to an acid resistance phenotype in the exponential growth phase allowing the synthesis of pH sensitive molecules and organic acids.
BACKGROUND OF THE INVENTION
The genes arcA encoding for the aerobic respiration control protein and iclR encoding the isocitrate lyase regulator are known to regulate the central carbon metabolism. ArcA is a global transcriptional regulator that regulates a wide variety of genes, while IclR is a local transcriptional regulator that regulates the glyoxylate pathway. ArcA is known to regulate the central carbon metabolism in response to oxygen deprivation and has no connection with IclR other than that it also regulates the glyoxylate pathway (24, 28, 29, 37, 38). In an earlier study the combined effect of ΔiclRΔarcA mutant strains on the central carbon metabolism has been observed. Increased fluxes were shown in the tricarboxylic acid (TCA) cycle and glyoxylate 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 demand. The latter compound is indeed a precursor of fucosylated oligosaccharides such as fucosyllactose, 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 prebiotic, in addition, fucosylated proteins find applications in the pharmaceutics (5, 8, 27). However, an efficient method to produce the latter high-value compounds is still needed.
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 mannosylated oligosaccharides. These oligosaccharides 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 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 changed in such a manner that they can efficiently produce the latter compounds.
Moreover, the synthesis 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 beneficial for these production processes. E. coli is an organism that can adapt easily to various conditions, for instance it can easily adapt to the 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 stationary phase (40). During this phase the cell does not multiply anymore and therefore hampers 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.



BRIEF DESCRIPTION OF FIGURES
FIG. 1: Relative gene expression pattern of the wild type, the ΔiclR and ΔarcA mutant strain to the ΔarcAΔiclR mutant strain of genes involved in colanic acid biosynthesis in batch fermentation conditions. The genes involved in colanic acid biosynthesis are presented in FIGS. 3 and 4.
FIG. 2: Gene expression pattern of the colanic acid operon of the wild type, the ΔiclR and ΔarcA mutant strain in chemostat fermentation conditions relative to the ΔarcAΔiclR mutant strain.
FIG. 3: The gene organisation of the colanic acid operon and an overview of the function of these genes:














Gene:
Function:









wza
Component of capsular polysaccharide export




apparatus



wzb
Tyrosine phosphatase



wzc
Tyrosine kinase



wcaA
Glycosyltransferase



wcaB
Acyltransferase



wcaC
Glycosyltransferase



wcaD
Colanic acid polymerase



wcaE
Glycosyltransferase



wcaF
Acyltransferase



gmd
GDP-mannose-4,6-dehydratase



fcl
GDP-fucose synthase



gmm
GDP-mannose hydrolase



wcal
Glycosyltransferase



cpsB
Mannose-1-phosphate guanylyltransferase



cpsG
Phosphomannomutase



wcaJ
UDP-glucose lipid carrier transferase



wzxC
Putative transporter



wcaK
Pyruvyltransferase



wcaL
Glycosyltransferase



wcaM
Predicted protein in colanic acid biosynthesis










FIG. 4: The colanic acid biosynthesis pathway.
FIG. 5: Regulatory network of the colanic acid operon. This network was constructed with Pathway tools v 13.0.
FIG. 6: Effect of the ΔarcAΔiclR mutations on the GDP-fucose biosynthesis route.
FIG. 7: Overview of the genetic modifications needed to enhance fucosyllactose and fucosylated oligosaccharides production starting from glucose as a substrate.
FIG. 8: Starting from sucrose, fucosylated sugar derivates such as fucosyllactose and more specifically 1,2-fucosyllactose are produced. The strain is modified to force the cell to produce frucose-6-phosphate which is an intermediate in the synthesis of GDP-fucose. Glucose or glucose-1-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.
FIG. 9: Overview of the genetic modifications needed to enhance fucosyllactose and fucosylated oligosaccharides production starting from glucose as a substrate in a split metabolism.
FIG. 10: Detail of the pentose phosphate pathway flux in a strain in which the genes coding for phosphoglucose isomerase and phosphofructokinase are knocked out.
FIG. 11: Starting from sucrose, mannosylated sugar derivates are produced. The strain is modified to force the cell to produce frucose-6-phosphate which is an intermediate in the synthesis of GDP-fucose. Glucose or glucose-1-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.
FIG. 12: Gene expression pattern acid resistance related genes of the wild type, the ΔiclR and ΔarcA mutant strain in batch culturing conditions relative to the ΔarcAΔiclR mutant strain.
FIG. 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 analysis of a sample of the culture broth of a mutant strain expressing a H. pylori fucosyltransferase.
FIG. 14: LC MSMS analysis mass spectrum from the chromatograms shown in FIG. 13 of culture broth and a 2-fucosyllactose standard. A. Mass (m/z) of the standard, 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 culturing broth of a mutant strain expressing a H. pylori fucosyltransferase.
FIG. 15: The sequence of the artificial hybrid promoter as given by SEQ ID No 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 invention provides microorganisms such as Enterobacteriaceae which are genetically changed in such a manner that they can efficiently produce compounds which are part of the colanic acid pathway. A particular compound of interest is GDP-fucose which is used as a precursor 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 mutated and/or transformed 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 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. 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 expression and/or activity of the transcriptional regulator, the aerobic respiration control protein, ArcA and the isocitrate lyase regulator IclR to upregulate the transcriptional regulator of the colanic acid operon, rcsA, which in turn upregulates at least one of the genes of the colanic acid operon.
Hence, the present invention relates to a mutated and/or transformed microorganism such as—but not limited to Enterobacteriaceae such as an Escherichia coli (E. coli) strain comprising a genetic change leading to a modified expression of the transcriptional regulators: the aerobic respiration control protein ArcA and the isocitrate lyase regulator IclR.
A mutated and/or transformed microorganism such as E. coli as used here can be obtained by any method known to the person skilled in the art, including but not limited to UV mutagenesis and chemical mutagenesis. A preferred manner to obtain the latter microorganism is by disrupting (knocking-out) the genes (arcA and iclR) encoding for the proteins ArcA and IclR, or, by replacing the endogenous promoters of said genes by artificial promoters or replacing the endogenous ribosome binding site by an artificial ribosome binding site. The term ‘artificial promoters’ relates to heterologous or non-natural or in silico designed promoters with known expression strength, these promoters can be derived from libraries as described by Alper et al. (2005), Hammer et al. (2006), or De Mey et al. (2007) (3, 11, 15). The term heterologous 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 (autologous or heterologous) promoter sequences as for example shown further in the examples. Sequences of such ‘artificial 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 et 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 IclR, or, reducing or eliminating the function of ArcA and IclR via mutations in the coding sequence of the genes coding for ArcA and IclR, or, is replacing the endogenous promoters of the genes encoding for ArcA and IclR by artificial promoters; or, is replacing the endogenous ribosome binding site by an artificial ribosome binding site. It is further clear that the mutant and/or transformant according to the present invention may further 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 as but not limited to Escherichia 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 strains—which are well-adapted to the laboratory environment, and, unlike wild type strains, 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, 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 MG1655.
The terms ‘leading to a modified expression or activity’ indicates that the above described mutations/transformations affects the transcription and/or translation and/or post-translational modification of said genes (arcA and iclR) into the transcriptional regulator proteins of the present 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 present invention. Hence, the present invention relates to a mutated and/or transformed microorganism such as an Escherichia coli strain as indicated above wherein 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 decreased expression is an abolished expression.
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 microorganism which is cultivated under the same conditions as the mutated and/or transformed microorganism. The genes which belong to the colanic acid operon are wza, wzb, wzc, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, gmd, fcl, gmm, wcaI, cpsB, cpsG, wcaJ, wzxC, 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 colanic acid operon is upregulated (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 promoter’. More specifically, the present invention relates to a combination of the sequence of the native promoter with sequences of other artificial promoter sequences. The combination of the sequence of the native promoter with the sequence of other artificial promoter sequences is more specifically 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), subunits of RNA polymerase that determine the affinity for promoter sequences and the transcription rate of genes. The present invention provides microorganisms which are genetically 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 FIG. 4 starting from fructose-6-P and resulting in extracellular colanic acid. More specifically the latter terms refer to the compounds mannose-6-P, mannose-1-P, GDP-mannose, GDP-4-dehydro-6deoxy-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 GDP-fucose. As GDP-fucose is a precursor for fucosylated oligosaccharides such as fucosyllactose, fucosyllactoNbiose and lewis X oligosaccharide or fucosylated proteins, and as these sugars have therapeutical, nutraceutical, anti-inflammatory and prebiotic effects, the present invention specifically relates to the usage as described above for the synthesis 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 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 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 IclR are applied in combination with at least one mutation that enhances the production of fucosylated compounds. In order to efficiently produce fucosylated oligosaccharides (see FIGS. 1, 2 and 5-10), the above described mutations 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 biosynthesis and thus allowing the accumulation of GDP-fucose; b) the introduction of a fucosyltransferase to link fucose with different acceptor molecules such as lactose; 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 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 β-galactosidase, is knocked out to avoid lactose degradation; e) to accumulate the precursor fructose and fructose-6-phosphate, a sucrose phosphorylase or invertase is introduced; f) because fructose-6-phosphate is easily degraded in the glycolysis, the glycolysis has to be interrupted in order to steer all fructose-6-phosphate in the direction of GDP-fucose and the genes pgi, pfkA and pfkB (coding for glucose-6-phosphate isomerase and phosphofructokinase A and B) are thus knocked out; g) reducing protein degradation 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 lactose induced gene expression systems (19). In other words, the present invention relates to a process as described above for the synthesis of fucosylated oligosaccharides wherein said at least one mutation that enhance the production of fucosylated 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 a sucrose phosporylase or invertase, and/or, knocking out the genes pgi, pfkA and pfkB, and/or, knocking out the gene Ion, and/or introducing a fucosyltransferase, and/or a lactose permease. The term ‘introducing 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, GT10 GT11, GT23, GT37, GT65, GT68, and/or GT74 and/or originating from but not limited to Helicobacter pylori, Campylobacter jejuni, Dictyostellium discoideum, Mus musculus, Homo sapiens, . . . and these fucosyltransferases catalyse the formation of α(1,2), α(1,3), α(1,4), or α(1,6) bonds on other sugars such as but not limited to galactose, lactose, lactoNbiose, lactoNtetraose, lactosamine, lactoNtetraose, sialyllactoses, 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 leading to a modified expression and/or activity 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 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 oligosaccharides and mannosylated glycoconjugates. These oligosaccharides and glycoconjugates 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 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). In order to efficiently produce mannosylated oligosaccharides (see FIGS. 1, 2, 5, 6 and 11), the above described mutations in arcA and iclR can be applied in combination with other mutations which further enhance the production of mannosylated compounds. Some of these—non-limiting—other mutations are: a) the gene gmd of the colanic acid operon is deleted, and/or, b) wherein 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, wcaI, wcaJ, wcaK, wcaL, fcl, 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 genes pgi, pfkA and pfkB, coding for phosphoglucose isomerase, phosphofructokinase A and phosphofructokinase B respectively, are deleted, and/or, f) knocking out the gene Ion encoding for a protease, 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 colanic acid operon are upregulated and wherein: a) the gene gmd of the colanic acid operon is deleted, 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, wcaI, 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 introduced, and/or, e) wherein the genes pgi, pfkA and pfkB are deleted, and/or, f) knocking out the gene Ion, and/or g) wherein a gene encoding for a mannosyltransferase is introduced. The term ‘introducing a mannosyltransferase’ relates to upregulating 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.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 families GT1, GT2, GT4, GT15, GT22, GT32, GT33, GT39, GT50 and/or GT58 and/or originating from but not limited to Helicobacter pylori, Campylobacter jejuni, Dictyostellium discoideum, Mus musculus, Homo sapiens, . . . and these mannosyltransferases catalyse the formation of α(1,2), α(1,3), α(1,4), or α(1,6) bonds on other sugars such as but not limited to galactose, N-acetylglucosamine, Rhamnose, lactose, lactoNbiose, lactoNtetraose, lactosamine, lactoNtetraose, sialyllactoses, disialyllactoses, or mannosylated 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 regulators ArcA and IclR as describe 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 encoded by the gene pgi and wherein the enzyme phosphofructokinase is encoded by the gene(s) pfkA and/or pfkB.
The terms ‘genes which are rendered less-functional or non-functional’ refer to the well-known technologies for a skilled person such as the usage of siRNA, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon 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 produce 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 paragraph wherein said organism is further transformed with a gene encoding for a sucrose phosphorylase.
The present invention also relates to a mutated and/or transformed organism as described above wherein, in addition, the activity and/or expression of the gene encoding for a lactose permease is made constitutive and/or increased. Said activity can be increased by over-expressing said gene and/or by transforming said organisms with a gene encoding for a lactose permease.
The present invention further relates 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 encoding for a beta-galactosidase, a gene encoding for a glucose-1-phosphate adenylyltransferase, 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-1-phosphate uridyltransferase, a gene encoding for an UDP-glucose-4-epimerase, a gene encoding for an UDP-glucose:galactose-1-phosphate uridyltransferase, a gene encoding for an UDP-galactopyranose mutase, a gene encoding for an UDP-galactose:(glucosyl)lipopolysaccharide-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-6-phosphate isomerase, a gene encoding for an UDP-N-acetylglucosamine enoylpyruvoyl transferase, 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-6-phosphate-1-dehydrogenase, and/or, a gene encoding for a L-glutamine:D-fructose-6-phosphate aminotransferase, a gene encoding for a mannose-6-phosphate isomerase, a gene encoding for a sorbitol-6-phosphate dehydrogenase, a gene encoding for a mannitol-1-phosphate 5-dehydrogenase, a gene encoding for a allulose-6-phosphate 3-epimerase, a gene encoding for an invertase, a gene encoding for a maltase, a gene encoding for a trehalase, a gene encoding 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) rendered less functional.
The present invention further relates also to the usage of a mutated and/or transformed microorganism such as an Escherichia coli strain comprising a genetic change leading to 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, 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 microorganism 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 synthesis 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). 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 (heparans and heparosans) . . . are preferably produced at low pH for downstream 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 regulator IclR to upregulate at least one of the following acid resistance related genes: ydeP, ydeO, hdeA, hdeD, gadB, gadC, gadE, gadX, gadW and/or slp.
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, ΔarcA, ΔiclR, ΔarcA ΔiclR) 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 calculated using Bayesian statistics (20, 21, 31).
Material and Methods
Strains and Plasmids
Escherichia coli MG1655 [−, F−, rph-1] was obtained 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), the single knock-outs E. coli MG1655 arcA and E. coli MG1655 iclR and the double knock-out E. coli MG1655 arcA, iclR were constructed in the Laboratory of Genetics and Microbiology (MICR) using the method of Datsenko & Wanner (9).
Media
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 medium contained 2 g/l NH4Cl, 5 g/l (NH4)2SO4, 2.993 g/l KH2PO4, 7.315 g/l K2HPO4, 8.372 g/l MOPS, 0.5 g/l NaCl, 0.5 g/l MgSO4.7H2O, 16.5 g/l glucose.H2O, 1 ml/l vitamin solution, 100 μl/l molybdate solution, and 1 ml/l selenium solution. The medium was set to a pH of 7 with 1M KOH.
Vitamin solution consisted of 3.6 g/l FeCl2.4H2O, 5 g/l CaCl2.2H2O, 1.3 g/l MnCl2.2H2O, 0.38 g/l CuCl2.2H2O, 0.5 g/l CoCl2.6H2O, 0.94 g/l ZnCl2, 0.0311 g/l H3BO4, 0.4 g/l Na2EDTA.2H2O and 1.01 g/l thiamine.HCl. The molybdate solution contained 0.967 g/l Na2MoO4.2H2O. The selenium solution contained 42 g/l SeO2.
The minimal medium for fermentations contained 6.75 g/l NH4Cl, 1.25 g/l (NH4)2SO4, 1.15 g/l KH2PO4, 0.5 g/l NaCl, 0.5 g/l MgSO4.7H2O, 16.5 g/l glucose.H2O, 1 ml/l vitamin solution, 100 μl/l molybdate solution, and 1 ml/l 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 l 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 maintained by sparging with air, anaerobic conditions were obtained by flushing the culture with a mixture of 3% CO2 and 97% of N2. The pH was maintained at 7 with 0.5 M H2504 and 4 M KOH. The exhaust gas was cooled down to 4° C. by an exhaust cooler (Frigomix 1000, Sartorius 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 μl). The off-gas was measured with an EL3020 off-gas analyser (ABB Automation GmbH, 60488 Frankfurt am Main, Germany).
All data was logged with the Sartorius MFCS/win v3.0 system (Sartorius Stedim Biotech, 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 Needle, 1.2×152 mm (BDMedical Systems, Franklin Lakes, N.J.—USA) connected to a reactor 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 back to the reactor vessel with a Masterflex-16 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 reactor broth needs 0.04 s to reach the harvest port and 3.2 s to re-enter the reactor. At a pO2 level of 50%, there is around 3 mg/l of oxygen in the liquid at 37° C. The pO2 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 μmax), 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 (residence time in the loop) gives 1.8 mg/l oxygen consumption.
In order to quench the metabolism of cells during the sampling, reactor 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 OD600nm and RT-qPCR measurements was taken using the rapid sampling loop and the cold stainless bead sampling method.
RT-qPCR
mRNA was extracted with the RNeasy kit (Qiagen, Venlo, The Netherlands). RNA quality and quantity was checked with a nanodrop ND-1000 spectrophotometer (Nanodrop technologies, Wilmingto, USA). The ratios 260:280 (nm) and 260:230 (nm) were between 1.8 and 2 and at least 100 ng/μl 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 measured with the Biotrove OpenArray Real time PCR platform. The primers for the RT-PCR assay were designed with Primer design tools from the Primer database (23).
The reaction mixture was composed as described in the Biotrove OpenArrayTM Real-Time qPCR system users' manual. In short, a mastermix was made with 26.4 μl LightCycler® DNA Master SYBR® Green I (Roche applied Science), 1.1 μl SYBR GREEN I (100× stock solution, Sigma S9430), 8.8 μl glycerol (Sigma G5150), 5.3 μl Pluronic® F68 (10% stock, Invitrogen), 2.64 μl BSA (Sigma A7906), 26.4 μl magnesium chloride (25 mM stock solution, supplied in the LightCycler® kit of Roche applied Science), 21.1 μl HiDiTM formamide (Applied biosystems), and 94.66 μl RNase free sterile water resulting in a 186.4 μl mastermix, which is enough to load 1 OpenArrayTM. For 1 SubArray (each OpenArray is subdivided in 48 SubArrays on which 1 sample can be loaded) 1.5 μl sample (with a concentration of 100 ng/μl) was mixed with 3.5 μl 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 Real-Time qPCR case. The case was sealed with Case sealing glue and incubated in the Case Sealing station, which polymerizes the glue with UV light.
Analytical Methods
Cell density of the culture was frequently monitored by measuring optical density at 600 nm (Uvikom 922 spectrophotometer, BRS, Brussel, Belgium). Cell dry weight was obtained by centrifugation (15 min, 5000 g, GSA rotor, Sorvall RC-5B, Goffin Meyvis, Kapellen, Belgium) of 20 g reactor broth in pre-dried and weighted falcons. The pellets were subsequently washed once with 20 ml physiological solution (9 g/l NaCl) and dried at 70° C. to a constant weight. To be able to convert OD600nm measurements to biomass concentrations, a correlation curve of the OD600nm to the biomass concentration was made. The concentrations of glucose and organic acids were determined on a Varian Prostar HPLC system (Varian, Sint-Katelijne-Waver, Belgium), using an Aminex HPX-87H column (Bio-Rad, Eke, Belgium) heated at 65° 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 265 nm) detector (Varian Prostar 325) and a differential refractive index detector (Merck LaChrom L-7490, Merck, Leuven, Belgium) 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 results in a constant value, typical for a certain compound (formula of Beer-Lambert).
Glucose, fructose, sucrose, fucosyllactose and glucose-1-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 DH5α (F−, φ80dlacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, λ−, thi-1, gyrA96, relA1).
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 pCP20 (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 carrying a point mutation.
Mutations. 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 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 OD600nm, of 0.6. The cells were made electrocompetent by washing 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 μl of ice-cold water. Electroporation was done with 50 μl of cells and 10-100 ng of linear double-stranded-DNA product by using a Gene PulserTM (BioRad) (600Ω, 25 μFD, 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 primers 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 ampicillin sensitivity.
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 KO, 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 chloramphenicol resistant plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. 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 control primers (Fw/Rv-gene-out). These primers are given in Table 1.







TABLE 1







Primers used to create E. coli


MG1655 arcA, E. coli MG1655 iclR and


the double knock-out E. coli MG1655


arcA, iclR and all other genetic


knock outs and knock ins








Primer name
Sequence





lacZ



FW_LacZ_P1
CATAATGGATTTCCTTACGCGAAATACGGG



CAGACATGGCCTGCCCGGTTATTAgtgtag



gctggagctgcttc (SEQ ID N° 7)


 


RV_LacZ_P2
GTATGTTGTGTGGAATTGTGAGCGGATAAC



AATTTCACACAGGAAACAGCTcatatgaat



atcctccttag (SEQ ID N° 8)


 


FW_LacZ_out
GCGGTTGGAATAATAGCG 



(SEQ ID N° 9)


 


RV_LacZ_out
CAGGTTTCCCGACTGGAAAG 



(SEQ ID N° 10)


 


glgC



FW-glgC-P1
Agaccgccggttttaagcagcgggaacatc



tctgaacatacatgtaaaacctgcagtgta



ggctggagctgcttc (SEQ ID N° 11)


 


RV-glgC-P2
Gtctggcagggacctgcacacggattgtgt



gtgttccagagatgataaaaaaggagttag



tccatatgaatatcctccttag 



(SEQ ID N° 12)


 


FW-glgC-out
Gcgaatatcgggaaatgcagg 



(SEQ ID N° 13)


 


RV-glgC-out
Cagagattgttttacctgctgg 



(SEQ ID N° 14)


 


agp



FW_agp_P1
CATATTTCTGTCACACTCTTTAGTGATTGA



TAACAAAAGAGGTGCCAGGAgtgtaggctg



gagctgcttc (SEQ ID N° 15)


 


RV_agp_P2
TAAAAACGTTTAACCAGCGACTCCCCCGCT



TCTCGCGGGGGAGTTTTCTGcatatgaata



tcctccttag(SEQ ID N° 16)


 


FW_agp_out
GCCACAGGTGCAATTATC 



(SEQ ID N° 17)


 


RV_agp_out
CATTTTCGAAGTCGCCGGGTACG



(SEQ ID N° 18)


 


pgi



Fw-pgi-P1
GGCGCTACAATCTTCCAAAGTCACAATTCT



CAAAATCAGAAGAGTATTGCgtgtaggctg



gagctgcttc (SEQ ID N° 19)


 


Rv-pgi-P2
GGTTGCCGGATGCGGCGTGAACGCCTTATC



CGGCCTACATATCGACGATGcatatgaata



tcctccttag (SEQ ID N° 20)


 


Fw_pgi_out(2)
GGCTCCTCCAACACCGTTAC 



(SEQ ID N° 21)


 


Rv_pgi_out(2)
TACATATCGGCATCGACCTG 



(SEQ ID N° 22)


 


pfkA



Fw-pfkA-out
TACCGCCATTTGGCCTGAC 



(SEQ ID N° 23)


 


Rv-pfkA-out
AAAGTGCGCTTTGTCCATGC 



(SEQ ID N° 24)


 


Fw-pfkA-P1
GACTTCCGGCAACAGATTTCATTTTGCATT



CCAAAGTTCAGAGGTAGTCgtgtaggctgg



agctgcttc (SEQ ID N° 25)


 


Rv-pfkA-P2
GCTTCTGTCATCGGTTTCAGGGTAAAGGAA



TCTGCCTTTTTCCGAAATCcatatgaatat



cctccttag (SEQ ID N° 26)


 


pfkB



Fw-pfkB-out
TAGCGTCCCTGGAAAGGTAAC 



(SEQ ID N° 27)


 


Rv-pfkB-out
TCCCTCATCATCCGTCATAG 



(SEQ ID N° 28)


 


Fw-pfkB-P1
CACTTTCCGCTGATTCGGTGCCAGACTGAA



ATCAGCCTATAGGAGGAAATGgtgtaggct



ggagctgcttc (SEQ ID N° 29)


 


Rv-pfkB-P2
GTTGCCGACAGGTTGGTGATGATTCCCCCA



ATGCTGGGGGAATGTTTTTGcatatgaata



tcctccttag (SEQ ID N° 30)


 


arcA



FW-arcA-P1
Ggttgaaaaataaaaacggcgctaaaaagc



gccgttttttttgacggtggtaaagccgag



tgtaggctggagctgcttc 



(SEQ ID N° 31)


 


RV-arcA-P2
Ggtcagggacttttgtacttcctgtttcga



tttagttggcaatttaggtagcaaaccata



tgaatatcctccttag (SEQ ID N° 32)


 


FW-arcA-out
Ctgccgaaaatgaaagccagta 



(SEQ ID N° 33)


 


RV-arcA-out
Ggaaagtgcatcaagaacgcaa 



(SEQ ID N° 34)


 


iclR



FW-iclR-P1
Ttgccactcaggtatgatgggcagaatatt



gcctctgcccgccagaaaaaggtgtaggct



ggagctgcttc (SEQ ID N° 35)


 


RV-iclR-P2
Gttcaacattaactcatcggatcagttcag



taactattgcattagctaacaataaaacat



atgaatatcctccttag 



(SEQ ID N° 36)


 


FW-iclR-out
Cggtggaatgagatcttgcga 



(SEQ ID N° 37)


 


RV-iclR-out
Acttgctcccgacacgctca 



(SEQ ID N° 38)


 


FW_iclR_P8
TTGCCACTCAGGTATGATGGGCAGAATATT



GCCTCTGCCCGCCAGAAAAAGccgcttaca



gacaagctgtg (SEQ ID N° 39)


 


RV_iclR_P9
GTTCAACATTAACTCATCGGATCAGTTCAG



TAACTATTGCATTAGCTAACAATAAAAagc



catgacccgggaattac 



(SEQ ID N° 40)


 


Rv-iclR-
CTATTGCATTAGCTAACAATAAAACTTTTT


scarless KO
CTGGCGGGCAGAGG (SEQ ID N° 41)


stap 2



 


Fw-iclR-
CCTCTGCCCGCCAGAAAAAGTTTTATTGTT


scarless KO
AGCTAATGCAATAGTTAC 


stap 2
(SEQ ID N° 42)


 


wcaJ



Fw_wcaJ_out
GCCAGCGCGATAATCACCAG 



(SEQ ID N° 43)


 


Rv_wcaJ_out
TGCGCCTGAATGTGGAATC 



(SEQ ID N° 44)


 


Fw-wcaJ_2-
TTTTGATATCGAACCAGACGCTCCATTCGC


P1
GGATGTACTCAAGGTCGAACgtgtaggctg



gagctgcttc (SEQ ID N° 45)


 


Rv-wcaJ_2-
TCTATGGTGCAACGCTTTTCAGATATCACC


P2
ATCATGTTTGCCGGACTATGcatatgaata



tcctccttag (SEQ ID N° 46)


 


fw_wcaJ_H1′
TCAATATGCCGCTTTGTTAACGAAACCTTT



GAACACCGTCAGGAAAACGATTTTGATATC



GAACCAGACG (SEQ ID N° 47)


 


Rv_wcaJ_H2′
TGACAAATCTAAAAAAGCGCGAGCGAGCGA



AAACCAATGCATCGTTAATCTCTATGGTGC



AACGCTTTTC (SEQ ID N° 48)


 


Fw_wcaJ_H1′_2
CGCTTTGTTAACGAAACCTTTGAACACCGT



CAGGAAAACGATTTTGATATCGAACCAGAC



GCTCCATTCG (SEQ ID N° 49)


 


lon



FW-lon-P1
CAGTCGTGTCATCTGATTACCTGGCGGAAA



TTAAACTAAGAGAGAGCTCTgtgtaggctg



gagctgcttc(SEQ ID N° 50)


 


oMEMO100_RV-
CGAATTAGCCTGCCAGCCCTGTTTTTATTA


lon-P2
GTGCATTTTGCGCGAGGTCAcatatgaata



tcctccttag (SEQ ID N° 51)


 


oMEMO101_FW-
AGCGCAACAGGCATCTGGTG 


lon-out
(SEQ ID N° 52)


 


oMEMO102_RV-
TATATCAGGCCAGCCATCCC 


lon-out
(SEQ ID N° 53)


 


lacZYA:P22-



lacY



Fw_lacZYA_c
GCTGAACTTGTAGGCCTGATAAGCGCAGCG


hl
TATCAGGCAATTTTTATAATCTTCATTTAA



ATGGCGCGC (SEQ ID N° 54)


 


rv_lacZYA_c
GCGCAACGCAATTAATGTGAGTTAGCTCAC


hl
TCATTAGGCACCCCAGGCTTCGCCTACCTG



TGACGGAAG (SEQ ID N° 55)


 


fw_P22lacY-
GCGCAACGCAATTAATGTGAGTTAGCTCAC


KI_P1
TCATTAGGCACCCCAGGCTTGTGTAGGCTG



GAGCTGCTTC (SEQ ID N° 56)


 


ry_P22lacY-
GCTGAACTTGTAGGCCTGATAAGCGCAGCG


KI
TATCAGGCAATTTTTATAATCTTAAGCGAC



TTCATTCACC (SEQ ID N° 57)


 


fw_lacZYA_H1′
CGACGCTTGTTCCTGCGCTTTGTTCATGCC



GGATGCGGCTAATGTAGATCGCTGAACTTG



TAGGCCTG (SEQ ID N° 58)


 


rv_lacZYA_H2′
CATTAATGCAGCTGGCACGACAGGTTTCCC



GACTGGAAAGCGGGCAGTGAGCGCAACGCA



ATTAATGTG (SEQ ID N° 59)


 


pfkA:P22-



BaSP



Fw-pfkA-P1
GACTTCCGGCAACAGATTTCATTTTGCATT



CCAAAGTTCAGAGGTAGTCgtgtaggctgg



agctgcttc (SEQ ID N° 60)


 


Rv-pfkA-
GCTTCTGTCATCGGTTTCAGGGTAAAGGAA


pCXP22_P2
TCTGCCTTTTTCCGAAATCaagcttgcatg



cctgcatcc (SEQ ID N° 61)


 


FW_kan
AGAGGCTATTCGGCTATGAC 



(SEQ ID N° 62)


 


Fw_baSP_seq
CGCCATGTTGGAATGGGAGG 



(SEQ ID N° 63)


 


Fw_pfkA_H1_ext
TGATTGTTATACTATTTGCACATTCGTTGG



ATCACTTCGATGTGCAAGAAGACTTCCGGC



AACAGATTTC (SEQ ID N° 64)


 


Rv_pfkA_H2_ext
AATTGCAGAATTCATGTAGGCCTGATAAGC



GAAGCGCATCAGGCATTTTTGCTTCTGTCA



TCGGTTTCAG (SEQ ID N° 65)


 


Fw-pfkA-out
TACCGCCATTTGGCCTGAC 



(SEQ ID N° 66)


 


Rv-pfkA-out
AAAGTGCGCTTTGTCCATGC 



(SEQ ID N° 67)


 


adhE:P22-



frk



Fw-adhE-
ATCGGCATTGCCCAGAAGGGGCCGTTTATG


pCXP22-P1
TTGCCAGACAGCGCTACTGAgtgtaggctg



gagctgcttc (SEQ ID N° 68)


 


Rv-adhE-
ATTCGAGCAGATGATTTACTAAAAAAGTTT


pCXP22-P2
AACATTATCAGGAGAGCATTaagcttgcat



gcctgcatcc (SEQ ID N° 69)


 


Fw-adhE-H1′
AAGCCGTTATAGTGCCTCAGTTTAAGGATC



GGTCAACTAATCCTTAACTGATCGGCATTG



CCCAGAAG (SEQ ID N° 70)


 


Rv-adhE-H2′
TTGATTTTCATAGGTTAAGCAAATCATCAC



CGCACTGACTATACTCTCGTATTCGAGCAG



ATGATTTACTAAAAAAG 



(SEQ ID N° 71)


 


FW_adhE_out
GCGTCAGGCAGTGTTGTATC 



(SEQ ID N° 72)


 


RV_adhE_out
CTGGAAGTGACGCATTAGAG 



(SEQ ID N° 73)


 


ldhA:P14-



FT_H.



pylori



FW_ldhA_out
tgtcattacttacacatcccgc 



(SEQ ID N° 74)


 


RV_ldhA_out
gcattcaatacgggtattgtgg 



(SEQ ID N° 75)


 


Fw-ldhA-
CATTGGGGATTATCTGAATCAGCTCCCCTG


pCXP22_P1
GAATGCAGGGGAGCGGCAAGgtgtaggctg



gagctgcttc (SEQ ID N° 76)


 


Rv-ldhA-
TATTTTTAGTAGCTTAAATGTGATTCAACA


pCXP22_P2
TCACTGGAGAAAGTCTTATGaagcttgcat



gcctgcatcc (SEQ ID N° 77)


 


Fw-ldhA-H1′
CAATTACAGTTTCTGACTCAGGACTATTTT



AAGAATAGAGGATGAAAGGTCATTGGGGAT



TATCTGAATCAG (SEQ ID N° 78)


 


Rv-ldhA-H2′
GAATTTTTCAATATCGCCATAGCTTTCAAT



TAAATTTGAAATTTTGTAAAATATTTTTAG



TAGCTTAAATGTGATTCAAC 



(SEQ ID N° 79)


 


Fw-ldhA-
TTCACCGCTAAAGCGGTTAC 


long homol
(SEQ ID N° 80)


 


Rv-ldhA-
CGCGTAATGCGTGGGCTTTC 


long homol
(SEQ ID N° 81)


 


promCA:P14



pCXP14_SP_Fw
CCGGCATATGGTATAATAGGG 



(SEQ ID N° 82)


 


yegH_rc_pure_rv
ACGGCTTGCTGGCCATCA 



(SEQ ID N° 83)


 


fw_P14-
CGAATATAAGGTGACATTATGGTAATTGAA


CA_KI_tetA
TATTGGCTTTCCAATAATGCTACGGCCCCA



AGGTCCAA (SEQ ID N° 84)


 


rv_P14-
AATATTGTCAACCTAAAGAAACTCCTAAAA


CA_KI_tetA
ACCATATTGAATGACACTTATTGGCTTCAG



GGATGAGGCG (SEQ ID N° 85)


 


fw_P14-
TCCCGACTACGTGGACCTTG 


CA_KI_overl
(SEQ ID N° 86)


apA



 


rv_P14-
CATATGGTATAATAGGGAAATTTCCATGGC


CA_KI_overl
GGCCGCTCTAGAAGAAGCTTGGGATCCGTC


apA
GACCTCGGCATTATTGGAAAGCCAATATTC 



(SEQ ID N° 87)


 


fw_P14-
GCCGCCATGGAAATTTCCCTATTATACCAT


CA_KI_overl
ATGCCGGCCAAGATGTCAAGAAACTTATAG


apB
AATGAAGTAAGTGTCATTCAATATGG 



(SEQ ID N° 88)


 


fw_P14-
AATATTGTCAACCTAAAGAAACTCCTAAAA


CA_KI_H1
ACCATATTGAATGACACTTACTTCATTCTA



TAAGTTTCTTGAC (SEQ ID N° 89)


 


rv_P14-
CGAATATAAGGTGACATTATGGTAATTGAA


CA_KI_H2
TATTGGCTTTCCAATAATGCCGAGGTCGAC



GGATCCCAAGCTTC (SEQ ID N° 90)









Transformation. Plasmids were transformed in CaCl2 competent cells using the simplified procedure of Hanahan (16) or via electroporation as described above.
Calculation Methods
Introduction
Different experiments with different strains were performed. In total 8 different conditions were tested. There was variation in the genetic background (WT, iclR knock-out, arcA knock-out, and combined iclR-arcA knock-out) and the mode of fermentation (batch, and chemostat). Each experiment was repeated twice.
When running the samples through the BioTrove apparatus, a qPCR curve (fluorescences in function of cycle number) and a melt curve (fluorescences in function of the temperature) is obtained for each sample. Those data were exported from the BioTrove software and further analysed 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 BioTrove 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 model-calculated 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 normalised prior to fitting, neither must the first data-points be removed.
    
    


Many curves have to be fitted (1800 genes for one experiment). Therefore, it is undoable to manually check each curve and automated methods have to be applied to reject bad curves. For this different parameters are extracted 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 (Fmin), and the maximal fluorescence (Fmax). 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 variety 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 something else than infinity is not correct. For genes that are expressed, but for which the expression value, due to technical failures 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 combinations for which no correct expression data was detected.
An obvious case of gene-experiment pairs for which no expression is detected, are those for which no curve could be fitted to the qPCR data. Less obvious 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 function in the qpcR package (25)). 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 observed 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.
Normalising and Calculating the Contrasts
Prior to calculating the expression differences, the Ct values have to be normalised. 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 Equations 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 finally this table was transformed so that the positions of the values corresponded again to the original positions (T4).




  
    
      
        
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Differential expressions were calculated 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 conditions produces no expression values, a different contrast matrix has to be generated omitting that experimental condition. Therefore the function for fitting the contrasts was adapted to drop data-points with missing data.
Differential expressions were calculated between 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 iclR Gene Deletions on the Gene Expression of the Colanic Acid Biosynthesis
FIGS. 1 and 2 show the expression pattern of genes involved in colanic acid biosynthesis (35). Single arcA or iclR knock out mutations did not affect the expression of the operon in comparison of the wild type strain in batch and chemostat conditions. The double mutant strain, ΔarcAΔiclR, however upregulates the genes of the colanic acid operon 6 to 8 times in comparison to the wild type and the single mutant strains in both chemostat 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 IclR and the transcription factor that controls the operon, RcsA (FIG. 5). Only ArcA is connected with RcsA via 3 other transcription factors, which are all upregulated as well. However the ΔarcA single gene deletion mutant strain did not affect the transcription of the operon.
Example 2
Effect of arcA and iclR Gene Deletions on the Gene Expression of the GDP-Fucose Biosynthesis Genes
FIGS. 4 and 6 show the relationship of the colanic acid operon with GDP-fucose biosynthesis. In FIG. 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 fucosyllactose, fucosyllactoNbiose and lewis X oligosaccharide or fucosylated proteins. These sugars and proteins, as already 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 ΔarcAΔiclR applied in combination with other mutations enhance the production of fucosylated compounds. A first, ‘other’ genetic modification that enhances said production is the deletion 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 different acceptor molecules such as lactose. The metabolism is then engineered further to accumulate the precursor of the GDP-fucose biosynthetic pathway. These modifications are shown in FIG. 7. 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, wcaI, wcaK, wcaL and/or, wcaM. For the production of fucosyllactose, lacZ coding for β-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 4
Enhancement of GDP-Fucose and Fucosylated Oligosaccharide Production Via a Split Metabolism with Sucrose as a Substrate
To accumulate the GDP-fucose precursor fructose and fructose-6-phosphate, a sucrose phosphorylase or invertase is introduced. Because fructose-6-phosphate 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, pfkA and pfkB are thus knocked out, coding for glucose-6-phosphate isomerase and phosphofructokinase 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 No 2) (Table 2), however the introduction of pgi mutations and pfkA and pfkB double mutations led to significant reduction of growth rate, the latter was extremely low (0.02 h−1). The combination of all mutations (Δpgi and ΔpfkA and ΔpfkB) 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







specific growth rates of the glycolysis knock out strains on a


minimal medium with glucose and sucrose











Growth rate on sucrose (h−1)



Growth rate on
(strains transformed with


Strain
glucose (h−1)
plasmid containing BaSP)





Wild type
0.64
0.41


Δpgi
0.18
0.23


ΔpfkAΔpfkB
0.02
n.d.


ΔpgiΔpfkAΔpfkB
0.23
0.24
















SEQ ID N° 2: Plasmid sequence with sucrose 


phosphorylase BaSP


AATTCGGAGGAAACAAAGATGGGGGGTTCTCATCATCATCATCATCAT


 


GGTATGGCTAGCATGAAAAACAAGGTGCAGCTCATCACTTACGCCGAC


 


CGCCTTGGCGACGGCACCATCAAGTCGATGACCGACATTCTGCGCACC


 


CGCTTCGACGGCGTGTACGACGGCGTTCACATCCTGCCGTTCTTCACC


 


CCGTTCGACGGCGCCGACGCAGGCTTCGACCCGATCGACCACACCAAG


 


GTCGACGAACGTCTCGGCAGCTGGGACGACGTCGCCGAACTCTCCAAG


 


ACCCACAACATCATGGTCGACGCCATCGTCAACCACATGAGTTGGGAA


 


TCCAAGCAGTTCCAGGACGTGCTGGCCAAGGGCGAGGAGTCCGAATAC


 


TATCCGATGTTCCTCACCATGAGCTCCGTGTTCCCGAACGGCGCCACC


 


GAAGAGGACCTGGCCGGCATCTACCGTCCGCGTCCGGGCCTGCCGTTC


 


ACCCACTACAAGTTCGCCGGCAAGACCCGCCTCGTGTGGGTCAGCTTC


 


ACCCCGCAGCAGGTGGACATCGACACCGATTCCGACAAGGGTTGGGAA


 


TACCTCATGTCGATTTTCGACCAGATGGCCGCCTCTCACGTCAGCTAC


 


ATCCGCCTCGACGCCGTCGGCTATGGCGCCAAGGAAGCCGGCACCAGC


 


TGCTTCATGACCCCGAAGACCTTCAAGCTGATCTCCCGTCTGCGTGAG


 


GAAGGCGTCAAGCGCGGTCTGGAAATCCTCATCGAAGTGCACTCCTAC


 


TACAAGAAGCAGGTCGAAATCGCATCCAAGGTGGACCGCGTCTACGAC


 


TTCGCCCTGCCTCCGCTGCTGCTGCACGCGCTGAGCACCGGCCACGTC


 


GAGCCCGTCGCCCACTGGACCGACATACGCCCGAACAACGCCGTCACC


 


GTGCTCGATACGCACGACGGCATCGGCGTGATCGACATCGGCTCCGAC


 


CAGCTCGACCGCTCGCTCAAGGGTCTCGTGCCGGATGAGGACGTGGAC


 


AACCTCGTCAACACCATCCACGCCAACACCCACGGCGAATCCCAGGCA


 


GCCACTGGCGCCGCCGCATCCAATCTCGACCTCTACCAGGTCAACAGC


 


ACCTACTATTCGGCGCTCGGGTGCAACGACCAGCACTACATCGCCGCC


 


CGCGCGGTGCAGTTCTTCCTGCCGGGCGTGCCGCAAGTCTACTACGTC


 


GGCGCGCTCGCCGGCAAGAACGACATGGAGCTGCTGCGTAAGACGAAT


 


AACGGCCGCGACATCAATCGCCATTACTACTCCACCGCGGAAATCGAC


 


GAGAACCTCAAGCGTCCGGTCGTCAAGGCCCTGAACGCGCTCGCCAAG


 


TTCCGCAACGAGCTCGACGCGTTCGACGGCACGTTCTCGTACACCACC


 


GATGACGACACGTCCATCAGCTTCACCTGGCGCGGCGAAACCAGCCAG


 


GCCACGCTGACGTTCGAGCCGAAGCGCGGTCTCGGTGTGGACAACGCT


 


ACGCCGGTCGCCATGTTGGAATGGGAGGATTCCGCGGGAGACCACCGT


 


TCGGATGATCTGATCGCCAATCCGCCTGTCGTCGCCTGACTGCAGGTC


 


GACCATATGGGAGAGCTCCCAACGCGTTGGATGCAGGCATGCAAGCTT


 


GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAA


 


TCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGT


 


AGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGC


 


CGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAAC


 


TGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT


 


TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAA


 


TCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTG


 


GCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAA


 


GGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTTGT


 


TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA


 


CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATT


 


CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTT


 


CCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA


 


GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGC


 


GGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATG


 


AGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGAC


 


GCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGAC


 


TTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATG


 


ACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT


 


GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC


 


GCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGG


 


GAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACG


 


ATGCCTACAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAA


 


CTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCG


 


GATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGG


 


TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATC


 


ATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATC


 


TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATC


 


GCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAA


 


GTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTT


 


AAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATC


 


CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAG


 


ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGC


 


TTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGAT


 


CAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG


 


CAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCAC


 


TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTG


 


TTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG


 


GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG


 


GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA


 


CTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAA


 


GGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGA


 


GAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGT


 


CCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGC


 


TCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTT


 


TTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT


 


GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA


 


GCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTG


 


AGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCAT


 


CTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC


 


TCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTG


 


ACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGC


 


CCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGA


 


CCGTCTCCGGGAGAGCTCGATATCCCGGGCGGCCGCTTCATTTATAAA


 


TTTCTTGACATTTTGGAATAGATGTGATATAATGTGTACATATCCATG


 


GCGGCCGCTCTAGAAGAAGCTTGGGATCCGTCGACCTCG






The flux redirections and mutations for GDP-fucose and fucosyllated oligosaccharide biosynthesis in a split metabolism are shown in FIG. 8, both for a strain expressing a heterologous invertase and sucrose phosphorylase. 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, wcaI, wcaK, wcaL and/or, wcaM. For the production of fucosyllactose, lacZ, coding for β-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 5
Enhancement of GDP-Fucose and Fucosylated Oligosaccharide Production Via a Split Metabolism with Glucose as Substrate
When the genes pgi, pfkA, and pfkB are knocked out, carbon, taken up as glucose can only be metabolised via the pentose phosphate pathway. Due to the biochemical properties of this pathway, fructose-6-phosphate is formed (FIGS. 9 and 10). To form biomass glyceraldehyde-3-phosphate has to be formed, which is formed by the transketolase reactions coded by tktA and tktB in E. coli. This Glyceraldehyde-3-phosphate is formed together with fructose-6-phosphate from xylulose-5-phosphate and erythrose-5-phosphate. The latter is in turn formed together with fructose-6-phosphate from glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate via transaldolase reactions coded by talA and talB. 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 xylulose-5-phosphate and 1/3 mole ribose-5-phosphate is formed. To drive these equilibrium reactions, fructose-6-phosphate is pulled out of the pentose phosphate pathway by the GDP-fucose and fucosyllacted oligosaccharide biosynthesis 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, wcaI, wcaK, wcaL and/or, wcaM. For the production of fucosyllactose, lacZ coding for β-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
Fermentative 2-Fucosyllactose Production with a Fucosyltransferase Originating from Helicobacter pylori 
The mutant strain in which the genes lacZ, glgC, agp, pfkA, pfkB, pgi, arcA, ic/R, wcaJ are knocked out and lacY was expressed via constitutive expression to ensure expression under all culturing conditions, was transformed further with a fucosyltransferase originating from Helicobacter pylori and a sucrose 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 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 FIGS. 13 and 14.
Example 7
Fermentative Fucosyllactose Production with a Fucosyltransferase Originating from Dictyostellium discoideum 
The mutant strain in which the genes lacZ, glgC, agp, pfkA, pfkB, pgi, arcA, ic/R, wcaJ are knocked out and lacY was expressed via constitutive expression to ensure expression under all culturing conditions, was transformed further with a fucosyltransferase originating from Dictyostellium discoideum and a sucrose phosphorylase originating from Bifidobacterium adolescentis, which were also expressed constitutively. 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 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 FIGS. 13 and 14.
Example 8
Enhancement of GDP-Mannose and Mannosylated Oligosaccharide Production Via a Split Metabolism with Sucrose as Substrate
To accumulate the GDP-mannose precursors fructose and fructose-6-phosphate, a sucrose phosphorylase or invertase is introduced. Because fructose-6-phosphate 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, pfkA and pfkB are thus knocked out, coding for glucose-6-phosphate isomerase and phosphofructokinase A and B. Finally a mannosyltransferase is introduced to link mannose to an acceptor molecule. To avoid GDP-mannose degradation 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 knocked out, such as wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, wcaI, wcaJ, wcaK, wcaL and/or, wcaM.
Example 9
Upregulation of Acid Resistance Related Genes
Similar to the colanic acid operon upregulation, acid resistance related genes are also upregulated in a ΔarcAΔiclR 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. FIG. 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:
ΔlacZYA::P22-lacYΔglgCΔagpΔpgiΔpfkA-P22-baSPΔpfkBΔarcAΔiclR::slΔwcaJΔIonΔadhE-P14-frk+pCXP14-FT_H. pylori (a vector with sequence SEQ ID No 1). The promoter P22 and P14 originate from the promoter library constructed by De Mey et al (11) and was cloned similar to the methodology described by Aerts et al (2). “::sl” marks a scarless gene deletion, thus without a FRT site that remains 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/l of sucrose, 50 g/l lactose and 1 g/l of magnesium sulphate heptahydrate. This led to the accumulation of 27.5 g/l of fucosyllactose in the supernatant.







SEQID N° 1: pCXP14-FT_H. pylori


CGCGTTGGATGCAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGA


 


AGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGA


 


TAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACC


 


CCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGG


 


GGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGA


 


AAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG


 


GTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC


 


GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAA


 


ACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTT


 


TTTGCGTTTCTACAAACTCTTTTTGTTTATTTTTCTAAATACATTCAA


 


ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAAT


 


ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA


 


TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAA


 


CGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGG


 


GTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC


 


GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTAT


 


GTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTC


 


GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCA


 


CAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG


 


CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA


 


CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG


 


ATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCA


 


TACCAAACGACGAGCGTGACACCACGATGCCTACAGCAATGGCAACAA


 


CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC


 


AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC


 


TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAG


 


CCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG


 


GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA


 


CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA


 


TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGA


 


TTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCC


 


TTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCC


 


ACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATC


 


CTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC


 


TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC


 


CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTC


 


TAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGC


 


CTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTG


 


GCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG


 


ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCA


 


GCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC


 


TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATC


 


CGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG


 


GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT


 


GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT


 


GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT


 


GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGG


 


ATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCC


 


GAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCC


 


TGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCA


 


TATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC


 


AGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCG


 


ACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCC


 


GGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGAGCTCGATA


 


TCCCGGGCGGCCGCCTTCATTCTATAAGTTTCTTGACATCTTGGCCGG


 


CATATGGTATAATAGGGAAATTTCCATGGCGGCCGCTCTAGAAGAAGC


 


TTGGGATCCGTCGACCTCGAATTCGGAGGAAACAAAGATGGCCTTTAA


 


AGTTGTTCAGATTTGTGGTGGTCTGGGCAATCAGATGTTTCAGTATGC


 


ATTTGCAAAAAGCCTGCAGAAACATAGCAATACACCGGTTCTGCTGGA


 


TATTACCAGCTTTGATTGGAGCAATCGTAAAATGCAGCTGGAACTGTT


 


TCCGATTGATCTGCCGTATGCAAGCGAAAAAGAAATTGCAATTGCCAA


 


AATGCAGCATCTGCCGAAACTGGTTCGTAATGTTCTGAAATGCATGGG


 


TTTTGATCGTGTGAGCCAAGAAATCGTGTTTGAATATGAACCGAAACT


 


GCTGAAAACCAGCCGTCTGACCTATTTTTATGGCTATTTTCAGGATCC


 


GCGTTATTTTGATGCAATTAGTCCGCTGATCAAACAGACCTTTACCCT


 


GCCTCCGCCTCCGGAAAATGGTAATAACAAAAAAAAAGAAGAAGAGTA


 


TCATCGTAAACTGGCACTGATTCTGGCAGCAAAAAATAGCGTGTTTGT


 


GCATATTCGTCGCGGTGATTATGTTGGTATTGGTTGTCAGCTGGGCAT


 


CGATTATCAGAAAAAAGCACTGGAATACATGGCAAAACGTGTTCCGAA


 


TATGGAACTGTTTGTGTTTTGCGAGGACCTGGAATTTACCCAGAATCT


 


GGATCTGGGCTATCCGTTTATGGATATGACCACCCGTGATAAAGAGGA


 


AGAGGCATATTGGGATATGCTGCTGATGCAGAGCTGTAAACATGGTAT


 


TATTGCCAACAGCACCTATAGTTGGTGGGCAGCATATCTGATTAATAA


 


CCCGGAAAAAATCATTATTGGTCCGAAACATTGGCTGTTTGGCCATGA


 


AAACATCCTGTGTAAAGAATGGGTGAAAATCGAAAGCCACTTTGAAGT


 


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 et al (11).
ΔlacZYA::P22-lacYΔglgCΔagpΔpgiΔpfkA::P22-BaSPΔpfkB ΔarcAΔiclR:sl ΔwcaJ ΔIon ΔadhE-P14-frk ΔldhA::P14-FT_H. pylori ΔpromCA:P14
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 20 g/l of lactose. After the batch phase the bioreactor was fed with 500 g/l of sucrose, 20 g/l lactose and 1 g/l of magnesium sulphate heptahydrate. This led to the accumulation of 26 g/l of fucosyllactose in the supernatant with nearly stoichiometric conversion of lactose. Increasing the lactose feed concentrations leads further to increased final fucosyllactose titers and stoichiometric lactose conversion.
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## Claims

1. A method for synthesizing GDP-fucose and/or fucosylated oligosaccharides, the method comprising: genetically modifying transcriptional regulators: aerobic respiration control protein ArcA, and isocitrate lyase regulator IcIR, in an Escherichia coli strain, thereby upregulating at least one of the genes of the colanic acid operon, wherein said method further comprises introducing at least one mutation in said E. coli strain that enhances the production of fucosylated compounds.

2. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of deletion of wcaJ gene.

3. A method for synthesizing GDP-mannose and/or mannosylated oligosaccharides, the method comprising: genetically modifying transcriptional regulators: aerobic respiration control protein ArcA, and isocitrate lyase regulator IcIR, in an Escherichia coli strain, thereby upregulating at least one of the genes of the colanic acid operon, said method further comprising introducing at least one mutation in said E. coli strain that enhances the production of mannosylated compounds.

4. The method according to claim 3 wherein the genes cpsG and cpsB of the colanic acid operon are upregulated.

5. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of knocking-out colanic acid operon genes gmm, wcaA, wcaB, wcaC, wcaD, wcaE, wcaF, mai, wcaJ, wcaK, wcaL, wzx, wza, wzb, wzc and/or wcaM.

6. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of knocking-out lacZ.

7. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of introducing a sucrose phosporylase or invertase.

8. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of knocking out genes pgi, pfkA and pfkB.

9. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of knocking out gene Ion.

10. The method according to claim 1, wherein said at least one mutation that enhances the production of fucosylated compounds consists of introducing a fucosyltransferase and/or a lactose permease.

11. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of deletion of the gene gmd of the colanic acid operon.

12. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of deletion of the gene gmm.

13. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of knocking out 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.

14. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of introducing a gene encoding for a sucrose phosphorylase or an invertase is introduced.

15. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of deletion of the genes pgi, pfkA and pfkB.

16. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of knocking out the gene Ion.

17. The method according to claim 3, wherein said at least one mutation that enhances the production of mannosylated compounds consists of introducing a gene encoding for a mannosyltransferase.