Method for the production of amino sugar containing products

Field of Invention

The present invention relates to genetically engineered organisms, especially microorganisms such as bacteria and yeasts, for the production of amino sugar containing products such as specialty saccharides, oligo- and polysaccharides, glycolipids, glycosides, glycoproteins, glycosylphosphates, nucleosides and glycosylsulphates. More specifically, the present invention relates to microorganisms that are metabolically engineered so that these microorganisms can produce said amino sugar (containing) products in large quantities and at a high rate by bypassing classical technical problems that occur in bio-catalytic or fermentative production processes.

Background art

For a long time, saccharides, the most abundant biomolecules in nature, have predominantly been viewed as energy-supplier, backbone of nucleic acids or as main cell wall substituent. However, it is becoming increasingly apparent that this emerging third class of information bearing molecules, poly- and oligosaccharides, are involved in numerous key biological processes as carriers of molecular information (Gabius and Roth 2017; Cocinero and Carcabal 2013). These complex carbohydrates possess a high conformational flexibility, and thus encode for extremely dense information via their structure (Varki 1993; Gabius and Roth 2017; Cocinero and Carcabal 2013; Gabius 2000). Moreover, specific biological activities are increasingly attributed to defined structural groups of molecules, resulting in ground-breaking discoveries (Gabius et al. 2011; Bemardi and Cheshev 2008; Varki 1993; Bertozzi 1995; Boltje, Buskas, and Boone 2009). As such, numerous saccharides and derivatives, i.e. oligo- and polysaccharides, glycolipids, glycosides, glycoproteins, nucleosides, glycosylphosphates and glycosylsulphates have a vast potential in multiple sectors, e.g., the cosmetics, food, agriculture, pharma.

Nowadays, these saccharides and derivatives are typically obtained either via extraction processes from natural producers, via not very efficient or sustainable, low-yielding chemical synthesis or via bioconversion processes. For these bioconversion processes, isolated and purified enzymes (so called in vitro bioconversions) and whole cell biocatalysts are commonly used. In essence these bioconversion processes convert one or more precursors into a desired bio-product making use of one or multiple carbohydrate-active enzymes, such as glycoside hydrolases (GHs), transglycosidases (TGs), glycoside phosphorylases (GPs) and (Leloir) glycosyltransferases (GTs) (Desmet et al., 2012). Each of them has its own characteristics and drawbacks concerning substrate usage, yields and scale-up.

The last type of carbohydrate-active enzymes are GT glycosyltransferases, which can transfer the sugar residue from an activated sugar donor, typically a nucleotide sugar, to various acceptors (Lairson et al., 2008), display superior conversion efficiencies (up to 100%) towards an enormous variety of small molecules. The uridine diphosphate (UDP) sugars form the largest group of nucleotide sugars (Yonekura-Sakakibara and Hanada, 2011) and consequently give rise to the large class of uridine glycosyltransferases (UGTs), which are characterized by a unique carboxy-terminal consensus sequence (Ross et al, 2001).

These UDP-sugars and corresponding UGTs are thus capable of efficiently glycosylating various compounds from diverse chemical classes in a regio- and stereoselective way (Bowles et al., 2005, 2006). In this context, the nucleotide sugar UDP-N-acetylglucosamine (UDP- GlcNAc), and the derived nucleotide sugars UDP-N-acetylmannosamine (UDP-ManNAc), CMP-N-acetylneuramie acid (CMP-Neu5Ac) and UDP-N-acetylgalactosamine (UDP- Gal Ac) (Figure 1), are key building blocks for the synthesis of numerous amino sugar containing products such as specialty saccharides, oligo- and polysaccharides, glycolipids, glycosides, glycoproteins, nucleosides, glycosylphosphates and glycosylsulphates.

With regard to in vitro bioconversions, their application is typically hampered because these require multiple enzymatic steps and/or because additional cofactors are required (NADH, NADPH, UTP, etc.), which are expensive. Other drawbacks of in vitro synthesis are the fact that the expression and purification of many enzymes is laborious and their purification process may result in a decreased enzymatic activity. Furthermore, each enzyme in such a multi enzyme bioconversion process has its own optimal process parameters, resulting in very complicated optimization schemes. In such a process, the reaction equilibria may also play an important role. For instance, when using a phosphorylase, a set substrate/product ratio that limits product yield will be at hand. This may also lead to complicated downstream processing schemes to separate the product from the substrate (Goedl et al. 2007; Graslund et al. 2008).

Alternatively, microbial hosts can be used to synthesize in vivo aforementioned amino sugar containing products. Typically, whole cells have been metabolically engineered to produce saccharides and derivatives by expressing UGTs in a micro-organism; thus making use of their intracellular UDP-sugar pool. This methodology is the basis for in vivo UDP-sugar based glycosylation and eliminates the need for extensive enzyme purification and the addition of expensive cofactors.

However, these UDP-sugars have also an essential role in the host’s metabolism. More specific, UDP-GlcNAc is an essential cell envelope precursor in the cell. The bacterial cell envelope is a complex multi-layered structure that serves to protect these organisms from their unpredictable and often hostile environment. The cell envelopes of most bacteria fall into one of two major groups. Gram-negative bacteria are surrounded by a thin peptidoglycan cell wall, which itself is surrounded by an outer membrane containing lipopolysaccharide. Gram-positive bacteria lack an outer membrane but are surrounded by layers of peptidoglycan many times thicker than is found in the Gram-negatives. Threading through these layers of peptidoglycan are long anionic polymers, called teichoic acids (Silhavy, Kahne, and Walker 2010; Neidhardt and Curtiss 1996). Bacterial peptidoglycan is a major component of the bacterial cell wall, and it provides rigidity and enables bacteria to survive in hypotonic environments. Peptidoglycan forms around 90% of the dry weight of gram-positive bacteria but only 10% of gram-negative strains. For both gram-positive and Gram-negative bacteria, particles of approximately 2 nm can pass through the peptidoglycan (Demchick and Koch 1996).

Peptidoglycan, also known as murein, is a polymer consisting of alternating residues of b-(1 ,4) linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNac). Attached to the N-acetylmuramic acid is a peptide chain of three to five amino acids. The peptide chain can be cross-linked to the peptide chain of another strand forming the 3D mesh-like layer. Peptidoglycan serves a structural role in the bacterial cell wall, giving structural strength, as well as counteracting the osmotic pressure of the cytoplasm. Peptidoglycan is also involved in binary fission during bacterial cell reproduction (Neidhardt and Curtiss 1996; Silhavy, Kahne, and Walker 2010).

The peptidoglycan biosynthesis pathway is one of the best-known processes in bacteria. The Mur enzymes, MurA-F, catalyse the last six steps in the formation of the final cytoplasmic peptidoglycan biosynthesis precursor uridine 5 '-diphosphate (UDP)-N-acetylmuramyl- pentapeptide (Figure 2). MurA and MurB catalyse the formation of UDP-N-acetyl muramic acid (UDP-MurNAc) from UDP-N-acetyl glucosamine (UDP-GlcNAc). First, MurA catalyses the transfer of enolpyruvate from phosphoenolpyruvate to UDP-GlcNAc. The resulting product, UDP-GlcNAc-enolpyruvate then undergoes a reduction that is catalysed by MurB. In the next steps, the Mur ligases (MurC-F) catalyse the sequential addition of l-Ala, d-Glu, and meso-diaminopimelic acid (in Gram-negative bacteria) or l-Lys (in Gram-positive bacteria), and the dipeptide d-Ala-d-Ala to UDP-MurNAc, to form the target UDP-MurNAc- pentapeptide (Neidhardt and Curtiss 1996). However, apart from some fundamental studies on gene stringency (Goh et al. 2009) or on antibacterial agents (Kahan et al. 1974; Hendlin et al. 1967; Chang et al. 2015; Molina-Lopez, Sanschagrin, and Levesque 2006; Hrast et al. 2014; Rozman et al. 2017), the synthesis of these cell envelope precursors and molecules has not yet been targeted for metabolic engineering purposes applications, i.e., due to its complex endogenous regulation system and its essential nature. This is also observed by Westbrook et al. (Westbrook et al. 2018). Attempts to reduce the expression of essential genes involved in the cell wall synthesis in order to obtain equal amounts of the hyaluronic acid (HA) precursors UDP-GlcNAc and UDP-a-D-glucuronic acid (UDP-GlcUA) resulted in poor growth and genetic instability, as certain derived B. subtilis strains lost the mucoid phenotype for HA production (Westbrook et al. 2018).

Pseudopeptidoglycan (also known as pseudomurein) is a major cell wall component of some Archaea that differs from bacterial peptidoglycan in chemical structure, but resembles bacterial peptidoglycan in function and physical structure. The basic components are N- acetylglucosamine and N-acetyltalosaminuronic acid (peptidoglycan has N-acetylmuramic acid instead), which are linked by b- 1 ,3-glycosidic bonds.

The cell wall of yeast and other fungi consists of three main groups of polysaccharides: polymers of mannose (mannoproteins, ca 40% of the cell dry mass), polymers of glucose (b- glucan, ca 60% of the cell wall dry mass) and polymers of N-acetylglucosamine (chitin, ca 2% of the cell wall dry mass). b-Glucan can be divided into two subtypes following the mode of glucose linkages: long chains of ca 1500 b-l, 3-glucose units which represents ca 85% of total cell wall b-glucan, and short chain of ca 150 b-l, 6-glucose units that accounts for ca 15% of the b-glucan (Aguilar-Uscanga and Francois 2003; Lipke and Ovalle 1998; Xie and Lipke 2011). In response to cell wall perturbations or cell wall mutations a cell wall compensatory mechanism is activated which results in a strong increase of chitin that can reach up to 20% of the cell wall dry mass.

The chitin biosynthesis pathway utilizes UDP-GlcNAc in a polymerization reaction to form chitin, catalysed by a polymer chitin synthase (pCHS). The pCHS reaction occurs in specialized microdomains of the plasma membrane. The pCHS is an integral membrane protein complex that polymerizes and extrudes chitin. pCHS encoding genes of fungi may be found in multiple copies and are divided into two families based on amino acid sequence motifs. Each family contains several classes. The yeast Saccharomyces cerevisiae contains three pCHS genes, CHS1, CHS2 and CHS3 which have different roles in the life cycle, with CHS3 producing most of the chitin in this organism including the lateral cell wall. In insects, only two pCHS encoding genes have been identified to date and are divided into class A and class B (Merzendorfer 2011).

To improve these whole cell bio catalyst processes, metabolic engineering efforts have predominantly focused on augmenting the product yield, e.g., by applying two-phase production systems avoiding losses of precursors to sinks, by increasing the flux of the biosynthesis pathway and by supplying direct precursors of these special carbohydrates (De Bruyn, Van Brempt, et al. 2015; De Bruyn, De Paepe, et al. 2015; Fierfort and Samain 2008; Samain et al. 1997; Priem et al. 2002; Antoine et al. 2005; Ruffing and Chen 2006; Kogure et al. 2007; J. Zhang et al. 2003; D. Zhang, Wang, and Qi 2007; Rodriguez-Diaz, Rubio-del- Campo, and Yebra 2012; Byun et al. 2007; Jennewein 2014; Boddy, Christopher et al. 201 1). In addition, only non-essential genes of minor precursor sink pathways, e.g. enterobacterial common antigen biosynthesis, are targeted for modification (Boddy, Christopher et al. 2011) as decreasing or deleting the expression of essential genes would yield lower growth or no growth, respectively (Tweeddale, Robb, and Ferenci 2006). For this reason, the major precursor sink of the nucleotide sugar UDP-GlcNAc pool, i.e (pseudo)peptidoglycan or chitin, is not be targeted yet.

In this context, a first drawback of whole cell production systems for the production of amino sugar containing products and derivatives is that metabolic engineering of the microbial cell to increase the nucleotide sugar UDP-GlcNAc pool and its conversion to amino sugar containing products is not straightforward due to its role as cell wall precursor (i.e. (pseudo)peptidoglycan or chitin) and hence its essential function in the cell. Moreover, the concentration of UDP- GlcNAc (as well as that of (the) other nucleotide sugars), increases with decreasing growth rate (Tweeddale, Robb, and Ferenci 2006). Hence, a second drawback of whole cell production systems for the production of special carbohydrates is that there is typically a need for two phases, a growth phase, in which biomass is formed (or biomass synthesis), followed by a production phase of the envisaged product. This means that the growth phase and the production phase are separated in the time (consecutive phases). This results in very low overall production rates of the desired product(s). In addition, this type of process is hard to optimize. Indeed, fermentation processes have been developed making use of metabolically engineered cells which over-express production pathway genes. A large amount of the substrate is converted into biomass, resulting in only a minor flux of the substrate towards the product (D. Zhang, Wang, and Qi 2007; Byun et al. 2007).

Alternatively, the metabolism of the organism can be split in two parts: 1) a so-called ‘production part’, and 2) a‘biomass formation part’ (Maertens, Beauprez, and De Mey 2010). This is achieved by splitting a saccharide into an activated saccharide and a (non-activated) sugar which are the precursors of either the production part or biomass formation part and, additionally, by rendering genes less-fimctional or non-fimctional that encode for enzymes which convert said intermediates from the production part into intermediates of the biomass formation part of the metabolism. As such, both biomass and product formation are ensured, however, this inherently goes at the expense of yield as one part of the splitted sugar is used for biomass formation and not for production of the specialty sugar. In this context, UDP- GlcNAc, is used either as‘production part’ or‘biomass formation part’, but not both at the same time. Another drawback of this approach is that the fermentation process requires a disaccharide, oligosaccharide, polysaccharide or mixture thereof as carbon-source in combination with a suitable enzyme enabling the splitting of these carbon sources into the required activated sugar, in this context UDP-GlcNAc, and a (non-activated) sugar to fuel either the production of the special carbohydrates and biomass formation. Hence, cheap carbon sources such as the monosaccharides glucose or glycerol cannot be used, as they cannot be split in two parts, one to fuel the biomass formation and one to fuel production.

The present invention overcomes the above-described disadvantages as it provides metabolically engineered organisms which are capable to produce desired products with a high productivity and a guaranteed high yield. This is accomplished by tuning the enzyme activity/activities converting the nucleotide sugar UDP-GlcNAc to essential cell envelope precursors and molecules. Description of the Figures

Figure 1. Overview of the derivatives of UDP-GlcNAc. With GlcNAc= N-acetylglucosamine; paCOS= partially acetylated chito-oligosaccharides; GalNAc= N-acetylgalactosamine; ManNAc= N-acetylmannosamine; Neu5Ac= N-acetylneuraminic acid, lipo-COS= acylated chitooligosaccharides, sulfoCOS= sulfated chitooligosaccharides, glycoCOS= glycosylated chitooligosaccharides.

Figure 2. Peptiodglycan biosynthesis pathway obtained from ECOCYC (Keseler et al. 2013). EC number for each reaction step and corresponding gene in Escherichia coli is given left from the arrow.

Figure 3. Detailed plasmid maps of constructed vectors. Bars indicate special regions (an origin of replication, a specific promoter (e.g. P14), terminator (e.g. rrnB Tl), or something else (e.g. FRT site)). Arrows depict specific coding sequences. TCC stands for translational coupling cassette.

Figure 4. TLC results of fosfomycin inhibition experiment with E. coli s3KO + pCOS. Legend: A. minimal medium (MM)+glucose; B. E. coli sWT grown on MM+glucose; C. chitooligosaccharides (COS)-mixture (15% fully acetylated chitotetramers (A4), 85% fully acetylated chitopentamers (A5)) (10 g/l); D. 0 mM fosfomycin; E. 0.1 mM fosfomycin; F. 0.25 mM fodfomycin; G. 1 mM fosfomycin; H. N-acetylglucosamine (GlcNAc). Bars are a measure for the intensity of the COS spots determined with ImageJ.

Figure 5. The genetic situation of the wild-type Escherichia coli MG 1655. Four predicted promoter (and operator) sites that drive the ibaG-murA operon (Weaver et al. 2014) and the pathway towards the UDP-GlcNAc pool are depicted. The gray arrow represents the branch point where heterologous pathways for complex carbohydrates can be installed.

Figure 6. The genetic situation after knocking in the targeted fragment generated from p_P22RBS-cITCmurA. The expression of cl represses the expression of mKATE2, converting the expression of murA into a measurable signal. Figure 7. The genetic situation after knocking in the targeted fragments generated from p PRND-cITCmurA showing how expression randomization was effectuated and how it generated strain library sRND.

Figure 8. The high-throughput MTP -based fluorescence screening of strain library sRND. Low expression of the cl repressor (and thus low murA expression), correlates with a high fluorescent signal, and vice versa.

Figure 9. Specific fluorescence and specific corrected maximal growth rate measurements for the seven selected mutant strain library (sRND). Bar plots show the average specific fluorescence per strain. Box plots indicate the distribution of the specific corrected maximal growth rate per strain. sWT, sWT+ (E. coli sWT carrying plndicator) and sP22 are also depicted as control.

Figure 10. qPCR data for murA of E. coli sRND 1-7 and sWT. For each strain the mid exponential average calibrated normalized relative quantity (CNRQ) of MurA mRNA and standard deviations of 3 biological and 2 technical replicates are depicted.

Figure 11. Validation of the fluorescent high-throughput screening technique. mRNA levels of the E. coli sRND strains highly correlate with the specific fluorescence (R ² =0.9960)

Figure 12. The average cellular UDP-GlcNAc pools of mid-exponentially grown E. coli sRND 1-7 and sWT, and standard deviations of 2 (3*) biological replicates are depicted. Cellular concentrations were assessed assuming an intracellular volume of lfL and 8* 10 ^s cells per OD600.

Figure 13. Production of chitopentaose in E. coli sCOSl-5, relatively compared to the wild type production titre in sWTCOS. Error bars depict the propagated error.

Figure 14. Production of Neu5Ac in E. coli sSAl-5, relatively compared to the wild type production titre in sWTSA. Error bars depict the propagated error.

Figure 15. Production of COS in E. coli sCOS2 + pHBP, sWTCOS + pHBP, sCOS2 and sWTCOS grown in minimal medium with glucose and varies inducer concentrations: aTc/IPTG [mM/mM] of 5/0.01, 5/0.05, 10/0.01 and 10/0.05. Error bars depict the propagated error. Detailed description of the Invention

The present invention discloses metabolically engineered organisms, especially microorganisms, which are capable to produce amino sugar containing products, especially UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-GlcNAc-derived saccharides, UDP- GlcNAc-derived nucleosides, UDP-GlcNAc-derived glycolipids, UDP-GlcNAc-derived glycosides, UDP-GlcNAc-derived glycoproteins, UDP-GlcNAc-derived glycosylphosphates, UDP-GlcNAc-derived glycosylsulphates, saccharides derived from UDP-GlcNAc-derived nucleosides, glycolipids derived from UDP-GlcNAc-derived nucleosides, glycosides derived from UDP-GlcNac-derived nucleosides, glycoproteins derived from UDP-GlcNAc-derived nucleosides, glycosylphosphates derived from UDP-GlcNAc-derived nucleosides, glycosylsulphates derived from UDP-GlcNAc-derived nucleosides, with a guaranteed high yield and a high productivity.

The term “metabolically engineering” refers to the practice of optimizing genetic and regulatory processes within said organism to increase the organism’s production of a certain desired amino sugar containing product. To this end, any well-known technique which can be used to (genetically) modify an organism’s metabolism and hence phenotype can be used as is described in (Verpoorte et al. 1999; Yadav et al. 2013; De Mey et al. 2007; Tyo, Alper, and Stephanopoulos 2007; Gaj, Sirk, and Barbas 2014; De Bruyn, Van Brempt, et al. 2015; Farmer and Liao 2000; Biggs et al. 2014; Stephanopoulos 2012; Trantas et al. 2015; Pirie et al. 2013; Patil, Akesson, and Nielsen 2004; Alper and Stephanopoulos 2007; Bhan, Xu, and Koffas 2013; Moon et al. 2012; Biggs et al. 2016; Ajikumar et al. 2010; Geert Peters et al. 2015; Coussement et al. 2014).

The microorganisms of the present invention are metabolically engineered so that the flux of UDP-GlcNAc to the biomass component“cell envelope precursors and molecules” is reduced while the microorganisms retain their capacities to grow. This is achieved by altering the enzyme activity and/or activities catalyzing essential reactions converting UDP-GlcNAc to cell envelope precursors and molecules.

Using the engineered organisms of present invention, product formation through the conversion of UDP-GlcNAc to an amino sugar containing product is not impaired by excessive withdrawal of this precursor for the formation of cell envelope precursors and molecules, i.e. biomass production instead of product formation. More specific, the essential reactions converting UDP-GlcNAc to cell envelope precursors and molecules are reduced enabling increased UDP-GlcNAc availability for the formation of an amino sugar containing product. This reduction of essential reactions involved in the formation of cell envelope precursors and molecules is not accompanied with decreased cell fitness, e.g. cell growth, which normally occurs when cognate essential genes are rendered less- functional or non-functional.

This means that the former drawback of having to produce biomass before the actual production of the product can start, is eliminated. This methodology results in high production rates, without the inherent problems that come with multi-enzymes systems and two phase fermentation systems.

The present invention relates to a method to produce at least one amino sugar containing product chosen from the group consisting of especially UDP-N-acetylglucosamine (UDP- GlcNAc), UDP-GlcNAc-derived saccharides, UDP-GlcNAc-derived nucleosides, UDP- GlcNAc-derived glycolipids, UDP-GlcNAc-derived glycosides, UDP-GlcNAc-derived glycoproteins, UDP-GlcNAc-derived glycosylphosphates, UDP-GlcNAc-derived glycosylsulphates, saccharides derived from UDP-GlcNAc-derived nucleosides, glycolipids derived from UDP-GlcNAc-derived nucleosides, glycosides derived from UDP-GlcNac- derived nucleosides, glycoproteins derived from UDP-GlcNAc-derived nucleosides, glycosylphosphates derived from UDP-GlcNAc-derived nucleosides, glycosylsulphates derived from UDP-GlcNAc-derived nucleosides, comprising: a) obtaining a suitable microorganism,

b) decreasing the synthesis or activity of at least one essential enzyme involved in the peptidoglycan biosynthesis, the pseudopeptidoglycan biosynthesis, the UDP-N- acetylmuramoyl-pentapeptide biosynthesis, the lipid IV _A biosynthesis, or the chitin biosynthesis without reducing cell growth of said microorganism,

c) cultivating said microorganism wherein endogenous UDP-GlcNac is the building block of (or fuels) both the production of specialty carbohydrates and biomass formation, and,

d) extracting and purifying said specialty product.

More specifically, the present invention relates to a method as indicated above wherein said ‘decreasing the endogenous conversion of UDP-GlcNAc to at least one cell envelope precursor or component’ is undertaken by genetically modifying said organism or by adding an inhibitor of the enzymes of the peptidoglycan biosynthesis, pseudopeptidoglycan biosynthesis, UDP-N- acetylmuramoyl-pentapeptide biosynthesis, lipid IV _A biosynthesis, or chitin biosynthesis.

The term‘amino sugar’ relates to a sugar molecule in which a hydroxyl group has been replaced with an amine group such as, but not limited to, GlcNac, ManNAc, GalNAc and Neu5Ac. Derivatives of amine containing sugars, such as, but not limited to, GlcNac, ManNAc, GalNAc and Neu5Ac, whose nitrogens are part of more complex functional groups rather than formally being amines, are also considered amino sugars.

The term‘saccharide’ relates to monosaccharides such as, but not limited to, aldoses, ketoses, pentoses, methylpentoses, hexoses, polyols with or without either carbonyl, carboxyl, amino groups or in which a hydroxyl group is replaced by, but not limited to a hydrogen, amino, thiol, phosphate and/or similar group or a derivative of these groups. The term‘saccharide’ also relates to di-, oligo-, and polysaccharide which are made up of one or more monosaccharides as described above, linked to each other by a glycosidic bond.

The term‘nucleoside’ relates to each monosaccharide that is substituted with a nucleotide which is for instance, but not limited to, UDP, GDP, ADP, TDP, CMP, or dTDP.

The term‘glycoside’ relates to a saccharide which forms a glycosidic bond with other chemical compounds, such as, but not limited to sterols, phenols, fatty acids, phosphatidylinositols, vitamine C, cartenoides and artimisinine.

The term‘glycolipid’ relates to a saccharide which forms a glycosidic bond with a fatty acid or lipid.

The term‘glycoprotein’ relates to a saccharide which forms a glycosidic bond with a protein. The term‘glycosylphosphate’ relates to a phosphorylated saccharide.

The term‘glycosylsulphate’ relates to a sulfated saccharide.

More specifically, the present invention relates to amino sugar containing products consisting at least of a homo or hetero-oligosaccharide having one of the following degrees of polymerization: one, two, three, four, five, six, seven, eight, nine or ten.

The term“cell envelope” refers to a complex multilayered structure that serves to protect these organisms from their environment. The term“cell envelope precursors and molecules” refers to all cell envelope components (i.e. proteins, phosphatidylserine, phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, putrescine, spermidine, wall teichoic acid, lipoteichoic acid, (pseudo)peptidoglycan, glycogen, lipopolysaccharide, and/or chitin) and their precursors, i.e. intermediates of the cell wall biosynthesis comprising the peptidoglycan biosynthesis and maturation, peptidoglycan cross- bridge biosynthesis, teichoic acids biosynthesis, UDP-N-acetylmuamoyl-pentapeptide biosynthesis, lipid IV _A biosynthesis, pseudopeptidoglycan biosynthesis and chitin biosynthesis pathway.

More specifically, the present invention relates to a metabolically engineered organism as indicated above wherein said“genetically modifying” meant essential genes rendered less- functional or non- functional.

The terms“essential genes” refer to genes of an organism that are critical for its survival, i.e. required to thrive in a given environment. Rendering these genes less-functional or non functional will result in e.g. less growth or no growth, respectively.

The terms“genes which are rendered less-fimctional or non-functional” refer to well-known technologies for a skilled person (such as siRNA, RNAi, miRNA, asRNA, mutating genes, knocking-out genes, transposon mutagenesis, CRISPR, CRIPRi, promoter engineering, RBS engineering, enzyme engineering, etc.) which are used to change the genes or cognate RNA in such a way that they are less-able (i.e. statistically significantly less-able compared to a functional wild-type enzyme) or completely unable (such as knocked-out gene or inactive enzyme) to produce functional final products, i.e. enzyme (Larson et al. 2013; Perez-Pinera, Kocak, and Vockley 2013; Copeland, Politz, and Pfleger 2014; Maeder et al. 2013; Politz, Copeland, and Pfleger 2013; Farzadfard, Perli, and Lu 2013; Cong et al. 2013; Cheng et al. 2013; Didovyk and Tsimring 2016; Qi et al. 2013; Qi and Arkin 2014; Geert Peters et al. 2015; Tsuda 1998; Cherepanov and Wackemagel 1995; Nevoigt et al. 2006; Palmeros et al. 2000; Bryant et al. 2014; Mutalik et al. 2013; Hoang et al. 1998; Schweizer 2003; Brophy et al. 2016; Kristensen et al. 1995; Hebert, Valdes, and Bentley 2008; Rasmussen, Sperling-Petersen, and Mortensen 2007; Sauer 1987; Agrawal et al. 2003; Datsenko and Wanner 2000; Avihoo et al. 2007; Williams, Luke, and Hodgson 2009; Balbas et al. 1996; Balbas and Gosset 2001; Van Hove et al. 2016; Pitzer et al. 2016; Van Hove et al. 2017; Alper et al. 2005; Alper and Stephanopoulos 2007; Cox, Surette, and Elowitz 2007; Salis 2011; Pirie et al. 2013; Coussement et al. 2014, 2017).

The terms “gene(s) which is/are rendered less-functional or non-functional” refers to a reduction of the activity of the corresponding gene product(s) with 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%. A gene can be rendered non- functional, i.e. reduced activity of the corresponding gene products with 100%, if there are multiple copies of the gene or isoenzymes present all catalyzing the same chemical reaction.

The term“(gene) knockout” thus refers to a gene which is rendered non- functional.

The term“without limiting cell fitness” refers to cells displaying equal or higher cell fitness compared to a cell in which the synthesis or activity of at least one enzyme involved in the endogenous conversion of UDP-GlcNAc to at least one cell envelope precursor or component is not decreased. In other words, wherein said cell fitness is not statistically lower compared to a cell in which the synthesis or activity of at least one enzyme involved in the endogenous conversion of UDP-GlcNAc to at least one cell envelope precursor or component is not decreased. Only when the p-value is <0.01, <0.02, <0.03, <0.04 to <0.05 the hypothesis is rejected that the cell fitness is not lower compared to a cell in which the synthesis or activity of at least one enzyme involved in the endogenous conversion of UDP-GlcNAc to at least one cell envelope precursor or component is not decreased.

The term“cell fitness” refers to the ability of a cell to thrive in a given environment, an ability determined by a number of parameters, such as cellular growth, product profile and genetic stability.

The term“cellular growth” refers to the accumulation of mass by a cell and is typically described by the maximal growth rate, maximal biomass yield and lag phase (Birch 1999).

The term“genetic stability” refers to a zero or low frequency of mutations within the genome or plasmids of a cellular lineage. These mutations can include changes in nucleic acid sequences, chromosomal/plasmid rearrangements or aneuploidy.

The term“product profile” refers to the pattern and amounts of product synthesized by the cell.

Even more specifically, said inhibitors of the enzymes of the (pseudo)peptidoglycan biosynthesis or UDP-N-acetylmuramoyl-pentapeptide biosynthesis, lipid IV _A biosynthesis, or chitin biosynthesis are selected from but not limited to the group consisting of fosfomycin, bacitracin, cycloserine, vancomycin, teicoplanin, ramoplanin, an avenaciolide, a peptide inhibitor pyrazolopyrimidine, tulipaline B, cnicin, benzothioxalone, nitrovinylfuran, b- lactams, penicillins, penems, carbapenems, cephems, cephalosporins, cephamycins, monobactams, b-lactamase inhibitors, cefsulodin, ampicillin, carbenicillin, tyrothricin, teixobactin.

Additionally, said enzymes involved in the (pseudo)peptidoglycan biosynthesis, UDP-N- acetylmuramoyl-pentapeptide biosynthesis, lipid IV _A biosynthesis, or chitin biosynthesis are selected from but not limited to the group consisting of a UDP-N-acetylglucosamine 1- carboxyvinyltransferase, a UDP-N-acetylenolpyruvoylglucosamine reductase, a UDP-N- acetylmuramate-L-alanine ligase, glutamate racemase, UDP-N-acetylmuramoyl-L-alanine-D- glutamate ligase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelat e ligase, phospho-N-acetylmuramoyl-pentapeptide-transferase, N-acetylglucosaminyl transferase, UDP-3-O-acyl-N-acetylglucosamine deacetylase, UDP-N-acetylglucosamine acyltransferase, tetraacyldisaccharide 4'-kinase, lipid A disaccharide synthase, UDP-2,3-diacylglucosamine diphosphatase, UDP-3-0-(3-hydroxymyristoyl)glucosamine N-acyltransferase, or (polymer) chitin synthase.

Additionally, said enzymes involved in the (pseudo)peptidoglycan biosynthesis, UDP-N- acetylmuramoyl-pentapeptide biosynthesis, lipid IV _A biosynthesis, or chitin biosynthesis is encoded by a gene selected from but not limited to the group consisting of murAA, murAB, inel, murZ, murA, murAl, murA2, murA_l, murA_2, murA-l, murA-2, murA3, murA5, murA22, murA.l, murA.2, murA2-l, murAA_l, murAA_2, Cgl0352, Cgl2558, sle_l7l40, sle_43250, nurZ, murB, murBl, murB2, murB-l, murB-2, murB l, murB_2, Cgl0353, murB_[H], XOO2101, sle_29960, murC, murCl, murC2, murC-l, murC-2, murC l, murC_2, XOO3603, murC_[H], sle_l6l70, murC ddlA, murC-ddlA, murC ddl, mpl, mudD, murE l, murD, murDl, murD2, murD l, murD_2, murD_[H], murE, murEl, murE2, murE3, murE-l, murE-2, murE.l, murE.2, murE l, murE_2, murE_[H], mure, XOO3608, ylbD, sle_50520, murT, mur, murF l, murC, murC2, murD2, murF, murFl, murF2, murF l, murF_2, murf, mraY, XOO3607, STY0144, Cgl2l62, sle_50530, alr, murfF, murfEF murf_[H], murE, murE_l, murC ddlA, murC-ddlA, murC, murC ddl, mudD, murB, murF/ mraY, rfe, XOO3606, murX, MRAY, murX mraY, murY, mraYl, mraY2, mraY-l, mraY-2, mraY l, mraY_2, sle_50540, Rfe,/ murG, murGl, murG2, murG3, murG l, murG_2, murG_3, murG_[H], sle_50570, murM, murM. l , murM.2, murMl , murM2, femB, fibA, murM_fibA, femX, murN, murNl , femA, femB fibB, femX, ffnhB, femA, femA_l, femA_2, femB, femB- 2, murl, murll , murI2, murB, murI5, murl l , murl_2, racE, racEl , racE2, yrpC, glr, sle_60800, lpxA, lpxK, ycaH, lpxB, pgsB, lpxH, ybbF, lpxD, omsA, firA, hlpA, ssc, lpxC, asmB, envA, chs2, kkv, Chsl , Chs2, CS-2, CHS, CHS1 , CHS2 CHS3, CHS8, CHS2.2, CHS5, CHS6, or CHS7.

Additionally, the expression of said genes is altered so that the mid-exponential average calibrated normalized relative quantity (CNRQ) varies from -3.50x l0 ^_1 to 2.00x 10 ¹ log(CNRQ). This corresponds with a variance in relative expression from 35% to 95% of the endogenous expression of said genes. The latter variation in expression is obtained by, but not limited to, the use of a constitutive promoter to control transcription and a 5’-UTR to control translation selected from but not limited to the group consisting of:

5’- ATTTATAAATTTCTTGACACAGCATCGGAACTACCCTATAATGTGTACATAAA C AC AAGCT C AAC AT AT ACT AG AC A AAGT C AGGC (SEQ ID N° l),

5’- ATTT AT AA ATTT C TT G AC AAC T AAC ACT AC AG AG ATT AT AAT GT GT AC AT AAA C AC AAGCT C AAC AT AT ACT AG AC A AAGT C AGGC (SEQ ID N°2°),

5’- ATTT AT AAATTT CTT G AC ATTTT GG AAT AG AT GT GAT AT AAT GT GT AC AT AAA CACAAGCTCAACCTATACTAGAGAAGTCAGGC (SEQ ID N°3),

5’- ATTT ATAAATTTCTTGACATATAGTAGATATCACCATATAATGTGTACAT AAA CACAAGCTCATCCTATACTAGAGGAAGTCAGGC (SEQ ID N°4°) and

5’- ATTT ATAAATTTCTTGACAGGACGTCGCCAGCGCGCTATAATGTGTACAT AAA CACAAGCTCATCCTATACTAGAGGAAGTCAGGC (SEQ ID N°5).

The present invention further relates to an organism as indicated above wherein said organism is further genetically modified so that at least one other gene than any of the altered genes of said organism is introduced and wherein said other gene encodes for a carbohydrate synthase, glycosyl transferase and/or epimerase, so that said organism is capable to convert UDP- GLcNAc to a saccharide, nucleoside, glycoside, glycolipid, glycoprotein, glycosylphosphate and/or glycosylsulphate. More specifically, the present invention relates to a metabolically engineered organism as indicated above, wherein said UDP-GlcNAc derived nucleoside are selected from but not limited to the group consisting of UDP-GalNAc, UDP-ManNAc, and CMP-N- acetylneuraminic acid (CMP-Neu5Ac).

More specifically, the present invention relates to a metabolically engineered organism as indicated above wherein said“carbohydrate synthase, glycosyltransferase and/or epimerase” is selected from but not limited to the group consisting of UDP-N-acetylglucosamine 2- epimerase, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, UDP-N- acetylglucosamine 2-epimerase, UDP-N-acetyl-D-glucosamine 6-dehydrogenase, UDP-N- acetylglucosamine 4-epimerase, b-polysaccharide synthases, chitin synthase, N- acetylglucosaminyltransferase, b- 1 ,4-N-acctylglucosaminyltransfcrasc, Nodulation protein C (NodC), NodC-like enzyme, chitooligosaccharide synthase, N-acyltransferase nodulation protein, hyaluronan synthase, glycosyl transferase family 2, N-acylmannosamine kinase, sialic acid synthase, N-acylneuraminate-9-phosphatase, N-acetylneuraminate synthase, N- acylneuraminate/3-deoxy-D-glycero-D-galacto-nononate cytidylyltransferase, hyaluronic acid synthase, b-l,3-galactosyl-N-acetylhexosamine phosphorylase, b-1,3-N- acetylglucosaminyltransferase, sialyltransferase, 2,3-sialyltransferase, 2,6-sialyltransferase, 2,8-sialyltransferase, N-acetylmannosamine transferase, N-acetylmannosaminyltransferase N- acetylgalactosamine transferase, N-acetylgalactosaminyltransferase and b-1,3- galactosyltransferase.

Additionally, said enzymes with carbohydrate synthase, glycosyltransferase and/or epimerase activity is encoded by a gene selected from the group gne, siaA, wecB, rffE, wbpA, udg, tuaD, wecC, vipAl, capL, wblA, wbpP, vipB, tviC, wbgU, strE, galE, wbtF, ispL, CHS, NodC, chs, nodBC, nodCB, nanE, nanK, nanEK, nanS, nanP, neuA, neuB, neuC, manA, GNE, gnal, sir 1975, has A, lnpA, lgtA and wbgO.

More specifically, the present invention relates to a metabolically engineered organism as indicated above wherein said“carbohydrate synthase, glycosyltransferase and/or epimerase” is selected from but not limited to the group consisting of UDP-N-acetylglucosamine 2- epimerase, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, UDP-N- acetylglucosamine 2-epimerase, UDP-N-acetyl-D-glucosamine 6-dehydrogenase, UDP-N- acetylglucosamine 4-epimerase, b-polysaccharide synthases, chitin synthase, N- acetylglucosaminyltransferase, b- 1 ,4-N-acctylglucosaminyltransfcrasc, Nodulation protein C (NodC), NodC-like enzyme, chitooligosaccharide synthase, N-acyltransferase nodulation protein, glycosyl transferase family 2, N-acylmannosamine kinase, sialic acid synthase, N- acylneuraminate-9-phosphatase, N-acetylneuraminate synthase, N-acylneuraminate/3-deoxy- D-glycero-D-galacto-nononate cytidylyltransferase, b- 1 ,3-galactosyl-N-acetylhexosamine phosphorylase, b-l,3-N-acetylglucosaminyltransferase, sialyltransferase, 2,3-sialyltransferase, 2,6-sialyltransferase, 2,8-sialyltransferase, N-acetylmannosamine transferase, N- acetylmannosaminyltransferase N-acetylgalactosamine transferase, N- acetylgalactosaminyltransferase and b- 1 ,3-galactosyltransferase.

Additionally, said enzymes with carbohydrate synthase, glycosyltransferase and/or epimerase activity is encoded by a gene selected from the group gne, siaA, wecB, rffE, wbpA, udg, tuaD, wecC, vipAl, capL, wblA, wbpP, vipB, tviC, wbgU, strE, galE, wbtF, ispL, CHS, NodC, chs, nodBC, nodCB, nanE, nanK, nanEK, nanS, nanP, neuA, neuB, neuC, manA, GNE, gnal, sirl975, lnpA, lgtA and wbgO.

More specifically, the present invention relates to a metabolically engineered organism as indicated above wherein said enzymes with carbohydrate synthase, glycosyltransferase and/or epimerase activity are highly selective towards a single donor sugar or sugar-nucleotide to generate the product (De Bruyn, Maertens, et al. 2015). Additionally, said enzyme has a sugar donor specificity or sugar-nucleotide donor specificity of 70%, 75%, 80%, 85%, 90%, 95% to 100 %.

An example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID No 1 - No5, and wherein a gene encoding for a N-acetylglucosamine transferase possibly (but not solely) originating from Pseudomonas sp., Frankia symbiont, Ensifer sp., Streptomyces sp., or rhizobia such as Rhizobium sp., Azorhizobium sp., Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp., Neorhizobium sp., Rhizobiales sp., Paraburkholderia sp., Methylobacterium sp., and Cupriavidus sp. is expressed having an amino acid sequence given by (but not solely) SEQ ID No 6 - No 8, or, a fragment thereof having a chitooligosaccharide synthase activity, or, a variant thereof having a sequence identity of at least 75% and having a chitooligosaccharide synthase activity to produce chitooligosaccharides (COS).

Another example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID Nol - No5, and wherein a gene encoding for a UDP-N-acetylglucosamine 2- epimerase and N-acetylneuraminic acid synthase, respectively, possibly (but not solely) originating from Campylobacter jejuni and having an amino acid sequence given by (but not solely) SEQ ID No 9 and No 10, respectively, or, a fragment thereof having a UDP-N- acetylglucosamine 2-epimerase or N-acetylneuraminic acid synthase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a UDP-N- acetylglucosamine 2-epimerase or N-acetylneuraminic acid synthase activity, respectively, to produce N-acetylneuraminic acid (Neu5Ac).

The present invention further relates to an organism as indicated above wherein said organism is further genetically modified so that at least one other gene than any of the altered genes of said organism is rendered less- functional or non- functional and wherein said other gene encodes for an enzyme with hydrolase or lyase activity.

More specifically, the present invention relates to a metabolically engineered organism as indicated above wherein said“hydrolase or lyase” is selected from but not limited to the group consisting of b-D-galactoside galactohydrolase, b-D-galactosidase, lactase, N-acetyl-b- neuraminate lyase, N-acetylneuraminate lyase, N-acetylneuraminic acid aldolase, acetylneuraminate lyase, sialic aldolase, sialic acid aldolase, sialate lyase, N-acetylneuraminic aldolase, neuraminic aldolase, N-acetylneuraminate aldolase, neuraminic acid aldolase, N- acetylneuraminic acid aldolase, neuraminate aldolase, N-acetylneuraminic lyase, N- acetylneuraminic acid lyase, NPL, NALase, NANA lyase, acetylneuraminate pyruvate- lyase, N-acetylneuraminate pyruvate-lyase, chitinase, endochitinase. exo-chitinase, chitinase A, (1- >4)-2-acetamido-2-deoxy-beta-D-glucan diacetylchitobio hydrolase, b-N- acetylgalactosaminidase, N-acetyl^-galactosaminidase; N-acetyl^-D-galactosaminidase; b- acetylgalactosaminidase; b-D-N-acetylgalactosaminidase; N-acetylgalactosaminidase, b-N- acetyl-D-galactosaminide N-acetylgalactosaminohydrolase, b-N-acetylhexosaminidase, hexosaminidase; b-acetylaminodeoxyhexosidase; N-acetyl-P-D-hexosaminidase; N-acetyl-b- hexosaminidase; b-hexosaminidase; b-acetylhexosaminidinase; b-D-N-acetylhexosaminidase; b-N-acetyl-D-hexosaminidase; b-N-acetylglucosaminidase; hexosaminidase A; N- acetylhexosaminidase; b-D-hexosaminidase, N-acetyl mannosidase, mannosidase.

Additionally, said enzymes with activity is encoded by a gene selected from but not limited to the group consisting of lacZ, lacZI, lacZl, lacZ2, lacZ3, lacZ-l, lacZ-2, lacZ_l, lacZ_2, lacZ_3, lacZ_4, lacZ_5, lacZ_6, lacZ_7, lacZ_8, lacZ_9, lacZ_l0, lacZ_l l, lacZ_l2, lacZ_l3, lacZ_l4, lacZ_l5, lacZ_l6, lacZ_l7, lacZ_l8, lacZ_l9, lacZ_20, lacZ_25, lacZ_26, lacZ_28, lacA, LacA2, lacL, lacH, lacM, lacS, LAC4, bga, bgaA, bga2A, bga35A, bgaB, bgaC, bgaE, bgaH, bgaL, bgaM, bgaS, bgaT, bga_l, bga_2, bgal, bga2, bga3, bga4, bga5, bga6, bga7, bga8, bgalO, bgal l, bgal2, bgal3, bgal4, bgal5, bgal6, bgal7, bgal8, bgal9, bga20, bgal-l , BGAL 17, BGAL2, bbgll, GLB1, GLB1L ,Glbl, glbl, glbll, glbll.L, glbl .L, glb2, Ect3, ebgA, ebgA_3, ebgA_6, Gal, ganA, ganAl, ganA2, ganB, gh2-3, galO, bglY, MgLAC2, MgLAC4, pbg, yesZ, gh2C, nanA, nanAl, nanA2, nanA3, nanA_l, nanA_2, dapA, dapAl, dapA_3, NPL, Npl, npl, npl.L, npl.S, nanH, chiA, chiB, CHIC, NgaP, HEXA, HEXB, HEXDC, CELF6.

An example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID No 1 - No 5, and wherein a gene encoding for a N-acetylglucosamine transferase possibly (but not solely) originating from Pseudomonas sp., Frankia symbiont, Ensifer sp., Streptomyces sp., or rhizobia such as Rhizobium sp., Azorhizobium sp., Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp., Neorhizobium sp., Rhizobiales sp., Paraburkholderia sp., Methylobacterium sp., and Cupriavidus sp. is expressed having an amino acid sequence given by (but not solely) SEQ ID No 6 - No 8, or, a fragment thereof having a chitooligosaccharide synthase activity, or, a variant thereof having a sequence identity of at least 75% and having a chitooligosaccharide synthase activity to produce chitooligosaccharides (COS). Additionally, in said organism, a gene encoding for a chitinase activity given by (but not solely) SEQ ID No 11 - No 12, is deleted.

Another example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID Nol - No5, and wherein a gene encoding for a UDP-N-acetylglucosamine 2- epimerase and N-acetylneuraminic acid synthase, respectively, possibly (but not solely) originating from Campylobacter jejuni and having an amino acid sequence given by (but not solely) SEQ ID No 9 and No 10, respectively, or, a fragment thereof having a UDP-N- acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a UDP-N- acetylglucosamine 2-epimerase or N-acetylneuraminic acid synthase activity, respectively, to produce N-acetylneuraminic acid (Neu5Ac). Additionally, in said organism, a gene encoding for a b-D-galactoside galactohydrolase activity given by (but not solely) SEQ ID No 13, and/or a gene encoding for a N-acetylneuraminate lyase activity given by (but not solely) SEQ ID No 14 is deleted.

The present invention further relates to an organism as indicated above wherein said organism is further genetically modified so that at least one other gene than any of the altered genes of said organism is introduced and wherein said other gene encodes for enzymes involved in the UDP-GlcNAc synthesis.

More specifically, the present invention relates to a metabolically engineered organism as indicated above wherein said“enzymes involved in the UDP-GlcNAc synthesis” is selected from but not limited to the group consisting of glutamine-fructose-6-phosphate aminotransferase, phosphoglucosamine, glucosamine- 1 -phosphate acetyltransferase/N- acetylglucosamine- 1 -phosphate uridyltransferase, N-acetylglucosamine-6-phosphate deacetylase, bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l- phosphate N-acetyltransferase, UDP-N-acetylglucosamine pyrophosphorylase, a glucosamine- phosphate N-acetyltransferase, a phosphoacetylglucosamine mutase, and a UDP-N- acetylglucosamine diphosphorylase, UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine diphosphorylase.

Additionally, said enzymes with activity is encoded by a gene selected from but not limited to the group consisting of glmS, glmSl, glmS2, glmS3, glmS4, glmS-l, glmS-2, glmS_l, glmS_2, glmS_3, glmS_4, GLMS, GLMS 1 , glmSJH], ybcM, Cgl227l, sle_25030, sle_29260, sle_445 l0, glmD, glmS/GFPT, frlB, agaS, nagB, nagBII, nagB-II, nagBl, nagB2, nodM, gfptl, gfpt2, GFPT1, GFPT2, Gfptl, Gfpt2, gfpt2.L, gfptl .S, Gfatl, Gfat2, gfat-l, gfat-2, GFAT, gfal, GFA1, GfaA, GFA l, GFA 2, ATF1, Dsim_GDl8034, Dsim_GDl9703, Dsim_GD28973, Ljlg3v2838l00. l, Ljlg3v2838100.2, Ljlg3v2838 l00.3,

PORTDRAFT 249106, OSl2gOl3 l 100, NCAS0A05750, NDAI0K02700, TPHA0G03180, TBLA0H01620, TBLA0I00790, TDEL0A02530, KAFR0D03180, NEUTE 1 DRAFT_ 149837, A0090003001475, A0090003000003, An03g05940, Anl8g06820,

AGABI1DRAFT 115602, AGABI2DRAFT 194113, 248400008, PC000162.03.0, 2l .m02906, Tb07.l0C21.470, XOO0678, X003696, S3959, TVG0861800, glmM, glmMl, glmM2, glmM_l, glmM_2, glmM_3, glmM-l, glmM-2, glmM_[H], glmM(femD), glmM#femD, femD, msrA, mrsA, mrsa, mrsAl, mrsA_2, MrsA, MRSA, XOO3077, ECS4055, sle_29290, PH1210, ureC, cpsG, cpsG2, cpsG l, ybbT, manB, manBl, manB2, manB3, pmml, pmm_l, pmmB, pmmC, pgm-l, pgm-2, glmU, glmUl, glmU2, glmU3, glmU_l, glmU_2, glmU_3, glmU-2, glmul, glmu3, glmU_[H], GlmU, gluM, gcaD, rfbA, rfbA-4, gcdl, hddC_4, graD2, graD3, graD4, graD6, graD-2, rffHl, rffH2, PH1925, aglF, uap, UAP1, UAP1L1, Uapl, Uaplll, uapl, uaplll, uapl .L, uapl .S, uaplll .L, QRI1, mmy, Dsim_GD22574, C36A4.4, GlcNAclpUTl, GlcNAclpUT2, Lj4g3v0243980.l,

POPTRDRAFT 712364, OJ1119 C05.25, 0s08g0206900, pcol44375b, NCAS0B05930, NDAI0B03240, TPHA0C03700, TBLA0B07300, TDEL0G02780, KAFR0K02470,

NEUTE 1 DRAFT 70531 , A0090038000595, Anl2g00480, PAAG 06885,

AGABI1DRAFT 110647, AGABI2DRAFT 189451, 30400023, 138400017,

PC000356.03.0, l9.m02866, symbB.vl .2.00112841, symbB.vl .2.002l97.tl, symbB.vl .2.00673041, galU, CPj0856, GNA1, gnal, gna-l, Cbr-gna-l, GNPNAT1 , gnpnatl, gnpnatl .L, Gnpnatl, GNAT3, NAT2, Dsim_GD2l459, Lj lg3v4717300.1, Lj lg3v4753330.l, Ljlg3v4753340.l, POPTRDRAFT 669373, sJ_08l56, 0s02g07l7700, 0s09g0488000, NCAS0C03940, NDAI0G03270, TPHA0D00540, TBLA0D02580, TDEL0C00840,

KAFR0C03360, NEUTE 1 DRAFT 92433 , A0090120000132, Anl2g07840,

AGABI 1 DRAFT 61620, AGABI2DRAFT 229877, 405400007, 34400022, symbB.vl .2.03439441, PGM3, Pgm3, pgm3.L, pgm3, nst, Dsim_GDl2708, F21D5.1, DRT101, Lj2g3vl 986460.1, AGM1, PCM1, PAGM1, 0s07g0l95400, NCAS0F00200, NDAI0K02890, TPHA0M00210, TBLA0G00980, TDEL0G04600, KAFR0L00340,

NEUTE1DRAFT 118413, A0090001000429, Anl8g05 l70, SNOG 08065,

AGABI1DRAFT 117388, AGABI2DRAFT 214180, PC301892.00.0, symbB.vl .2.02163841, Tb08.25L8.80. An example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID No 1 - No 5, and wherein a gene encoding for a N-acetylglucosamine transferase possibly (but not solely) originating from Pseudomonas sp., Frankia symbiont, Ensifer sp., Streptomyces sp., or rhizobia such as Rhizobium sp., Azorhizobium sp., Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp., Neorhizobium sp., Rhizobiales sp., Paraburkholderia sp., Methylobacterium sp., and Cupriavidus sp. is expressed having an amino acid sequence given by (but not solely) SEQ ID No 6 - No 8, or, a fragment thereof having a chitooligosaccharide synthase activity, or, a variant thereof having a sequence identity of at least 75% and having a chitooligosaccharide synthase activity to produce chitooligosaccharides (COS). Furthermore, in said organism a gene encoding for phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase, respectively, possibly (but not solely) originating from Escherichia coli and Corynebacterium glutamicum is expressed having an amino acid sequence given by (but not solely) SEQ ID No 15 - No 16 and SEQ ID No 17 - No 19, respectively, or, a fragment thereof having phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N- acetyltransferase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a phosphoglucosamine mutase and bifunctional UDP-N- acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase activity, respectively.

Another example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID No 1 - No 5, and wherein a gene encoding for a N-acetylglucosamine transferase possibly (but not solely) originating from Pseudomonas sp., Frankia symbiont, Ensifer sp., Streptomyces sp., or rhizobia such as Rhizobium sp., Azorhizobium sp., Mesorhizobium sp., Sinorhizobium sp., Bradyrhizobium sp., Neorhizobium sp., Rhizobiales sp., Paraburkholderia sp., Methylobacterium sp., and Cupriavidus sp. is expressed having an amino acid sequence given by (but not solely) SEQ ID No 6 - No 8, or, a fragment thereof having a chitooligosaccharide synthase activity, or, a variant thereof having a sequence identity of at least 75% and having a chitooligosaccharide synthase activity to produce chitooligosaccharides (COS). Furthermore, in said organism a gene encoding for phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase, respectively, possibly (but not solely) originating from Escherichia coli and Corynebacterium glutamicum is expressed having an amino acid sequence given by (but not solely) SEQ ID No 15 - No 16 and SEQ ID No 17 - No 19, respectively, or, a fragment thereof having phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N- acetyltransferase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a phosphoglucosamine mutase and bifunctional UDP-N- acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase activity, respectively. Additionally, in said organism, a gene encoding for a chitinase activity given by (but not solely) SEQ ID No 11 - No 12, is deleted.

Another example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID No 1 - No 5, and wherein a gene encoding for a UDP-N-acetylglucosamine 2- epimerase and N-acetylneuraminic acid synthase, respectively, possibly (but not solely) originating from Campylobacter jejuni and having an amino acid sequence given by (but not solely) SEQ ID No 9 and No 10, respectively, or, a fragment thereof having a UDP-N- acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a UDP-N- acetylglucosamine 2-epimerase or N-acetylneuraminic acid synthase activity, respectively, to produce N-acetylneuraminic acid (Neu5Ac). Furthermore, in said organism a gene encoding for phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase, respectively, possibly (but not solely) originating from Escherichia coli and Corynebacterium glutamicum is expressed having an amino acid sequence given by (but not solely) SEQ ID No 15 - No 16 and SEQ ID No 17 - No 19, respectively, or, a fragment thereof having phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N- acetyltransferase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a phosphoglucosamine mutase and bifunctional UDP-N- acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase activity, respectively.

Another example of the latter metabolically engineered organism is an organism wherein the endogenous UDP-N-acetylglucosamine l-carboxyvinyltransferase expression is decreased with 5% to 65% by altering the endogenous promoter and 5’-UTR sequence with (but not solely) SEQ ID No 1 - No 5, and wherein a gene encoding for a UDP-N-acetylglucosamine 2- epimerase and N-acetylneuraminic acid synthase, respectively, possibly (but not solely) originating from Campylobacter jejuni and having an amino acid sequence given by (but not solely) SEQ ID No 9 and No 10, respectively, or, a fragment thereof having a UDP-N- acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a UDP-N- acetylglucosamine 2-epimerase or N-acetylneuraminic acid synthase activity, respectively, to produce N-acetylneuraminic acid (Neu5Ac). Furthermore, in said organism a gene encoding for phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase, respectively, possibly (but not solely) originating from Escherichia coli and Corynebacterium glutamicum is expressed having an amino acid sequence given by (but not solely) SEQ ID No 15 - No 16 and SEQ ID No 17 - No 19, respectively, or, a fragment thereof having phosphoglucosamine mutase and bifunctional UDP-N-acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N- acetyltransferase activity, respectively, or, a variant thereof having a sequence identity of at least 75% and having a phosphoglucosamine mutase and bifunctional UDP-N- acetylglucosamine pyrophosphorylase/Glucosamine-l -phosphate N-acetyltransferase activity, respectively. Additionally, in said organism, a gene encoding for a b-D-galactoside galacto hydrolase activity given by (but not solely) SEQ ID No 13, and/or a gene encoding for a N-acetylneuraminate lyase activity given by (but not solely) SEQ ID No 14 is deleted.

The term“organism” as indicated above refers to a microorganism chosen from the list consisting of a bacterium, a yeast, fungus cell or archaea, or, refers to a plant or animal cell. The latter bacterium preferably belongs to the species Escherichia coli, Lactobacillus sp., Corynebacterium sp. or Bacillus sp. The latter yeast preferably belongs to the species Saccharomyces cerevisiae or Pichia sp. The latter archaea preferably belong to the species Sulfolobus sp. or Methanobacter sp.

The latter engineered organisms can, for example but not limited to, be used to produce UDP- N-acetylglucosamine, chitin, chitosan, chitooligosaccharide, glycosylated chitooligosaccharide, acylated chitooligosaccharide , sulfated chitooligosaccharide, neomycin, butirosin, an -O-GlcNAcylated molecule, N-acetylglucosamine, heparin, heparin sulfate, heparosan, chondroitin, lacto-N-biose, lacto-N-triose, lacto-N-tetraose, lacto-N-neotetraose, N-acetylmannosamine, N-acetylneuramic acid, a -Neu5Acylated molecule, UDP-N- acetylmannosamine, a -ManAcylated molecule, UDP-N-acetylgalactosamine, a - GalNAcylated molecule, CMP-N-acetylneuraminic acid, 3’-sialyllactose, 6’-sialyllactose, sialyl Lewis X, Sialyl Lewis A, polysialic acid, gangloside, hyaluronic acid, disialyllacto-n- tetraose, 3 '-sialyl-3-fucosyl lactose, sialyllacto-N-tetraoses 6'-sialyllactosaminc, 3'- sialyllactose, 2', 3- difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, 3'-sialyl-3-fucosyllactose, sialylated lacto-N-triose, sialylated lacto-N-tetraose, sialylated lacto-N-neotetraose, lacto-N- fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b, sialyl-lacto-N-tetraose c, fucosyl- sialyllacto-N-neotetraose a, fucosyl-sialyllacto-N-neotetraose b, fucosyl-sialyllacto-N- neotetraose c, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-difucohexaose III, disialyllacto-N-tetraose, fucosyl-disialyllacto-N-tetraose I, disialyl-fucosyllacto-N-tetraose II, monofucosyllactose, monosialyllactose, sialyl-lacto-N-pentaose I, sialyl-lacto-N-pentaose II, sialyl-lacto-N-fucopentaose I, sialyl-lacto-N-fucopentaose II, difucosyllacto-N-hexaose, trifucosyllacto-N-hexaose, difucosyl-p-lacto-N-neohexaose, difucosyl-p-lacto-N-hexaose, difucosyllacto-N-hexaose, monofucosyllacto-N-hexaose II, lacto-N-hexaose, lacto-N- neohexaose, fucosyl-sialyllacto-N-neohexaose, sialylated molecules as amino sugar containing product.

It is clear that any methodology known in the art to cultivate micro-organisms, and, to extract and purify specialty products from said cultivation can be employed in the present invention.

The following specific sequences, as indicated above, are part of the present invention:

Examples

Material and Methods

1. Chemicals, oligonucleotides and molecular biology

All reagents were purchased from Sigma- Aldrich (Bomem, Belgium), unless otherwise stated. Agarose and ethidium bromide were purchased from Thermo Fisher Scientific (Erembodegem, Belgium). Standard molecular biology procedures were conducted as described by Sambrook et al. (Sambrook and Russell 2001). Qiagen kits (Hilden, Germany) were used for all DNA preparations. Oligonucleotides were purchased from Integrated DNA Technologies (Leuven, Belgium), genes were purchased from Geneart (Thermo Fisher Scientific, Erembodegem, Belgium). Sequencing services were conducted by Macrogen (Amsterdam, The Netherlands).

The sequence of the E. coli murA operon was obtained from E. coli K-12 MG1655 complete genome (Genbank access code: NC 000913; MurA, Genbank accession code: NP 417656.1, Table 1). The sequence of the Corynebacterium glutamicum murA operons was obtained from C. glutamicum ATCC 13032 complete genome (Genbank access code: NC 003450).

The sequence of L-glutamine:D-fructose-6-phosphate aminotransferase was obtained from E. coli (EcGlmS, Genbank accession code: NP418185, Table 1). A mutant GlmS was used, GlmS*54, which contains 3 non-silent mutations, i.e., A38T, R249C and G471S (Deng et al. 2006). The sequence of a fused N-acetylglucosamine-l -phosphate uridyltransferase and glucosamine- 1 -phosphate acetyltransferase was obtained from Corynebacterium glutamicum (GlmU, Genbank accession code: WP038583267, Table 1).

The sequence of the oligomer chitin synthase (oCHS) was obtained from Rhizobium sp. GRH2 (NodC, Genbank access code: AJW76243, Table 1).

The sequence of the UDP-N-acetylglucosamine 2-epimerase, N-acetylneuraminic acid synthase and the CMP-Neu5Ac synthetase were obtained from Campylobacter jejuni strain ATCC 43438 (NeuC, NeuBl, andNeuA, respectively, Genbank access code: AF400048, Table 1) (Fierfort and Samain 2008). The sequence for the a-2,3-sialyltransferase was obtained from Neisseria meningitidis (NST, Genbank accession code: U60660, Table 1).

The sequence of the b- 1 ,3-N-acctylglucosaminyltransfcrasc was obtained from Neisseria meningitidis (LgtA, Genbank accession code: U25839, Table 1) and b-1,3- galactosyltransferase was obtained from Escherichia cob 055 :H7 (WgbO, Genbank accession code: AF461121, Table 1).

Table 1. Sequences of used enzymes

SEQ No Enzyme Protein sequence (Genbank accession code)

^~ 35 MurA NP 417656.1

mdkfrvqgpt klqgevtisg aknaalpilf aallaeepve iqnvpklkdv dtsmkllsql gakvemgsv hidardvnvf capydlvktm rasiwalgpl varfgqgqvs lpggctigar pvdlhisgle qlgatiklee gyvkasvdgr lkgahivmdk vsvgatvtim caatlaegtt iienaarepe ivdtanflit lgakisgqgt driviegver lgggvyrvlp drietgtflv aaaisrgkii cmaqpdtld avlaklrdag adievgedwi sldmhgkrpk avnvrtaphp afptdmqaqf tllnlvaegt gfitetvfen rfmhvpelsr mgahaeiesn tvichgvekl sgaqvmatdl rasaslvlag ciaegttwd riyhidrgye riedklralg aniervkge

ΊΪ6 MurAl NCgl0345

myaeinggfipegtvrvsgaknsatrllaaalltdewhlgnfptklvdvehkirfieelg gkvhvdhdeqilwd akdlaaremttdelnipirttyllaaaqigrgeiarvpfpggcaigggpaggrgydlhlm vweqlgckilekddhi evtapqgfiggvidfpistvggtenallcasiasgdtkianayitpeitdliellrrmga eitvygtsrihvkgragllq gaymdvmpdriealtwivygiisggritvegvpfssmevpfihlekagvdlfrnsssvyi tpeclpsgsvqpfel acgthpgvisdmqalfvllglkgagtsrvydyryperiafveeltnlvsgdklsaeagki tiqgdatfrpgyanstdl rgsmawlaalcadgkstinnvhmalrgyneldkklrllgadltiregevpsp

^~ 37 MurA2 NCgl2470

vkdkflvtggaqlqgavkvygaknsvlklmaaallaegtttltncpeildvplmrdvlvg lgcdvtidgstvtittp aelssnadfpavtqfrasvcvlgpltarcgrawslpggdaigsrpldmhqsgleklgatt rishgawaeaeklvg anitldfpsvgateniltasvmaegrtvldnaarepeivdlcrmlrsmganiegegspti tiegvekltptqhevigd rivagtwayaaamtrgditvggiaprylhlpleklkiagakvetyengfrvqmdkqpeat dyqtlpfpgfptdlq pmaiginavsngtsvitenvfesrfrfvdemlrlgadanvdghhwirgieqlsstsvwss diragaglvlaalcad gvtevhdvfhidrgypnfvenlqklgatiervss NodC AJW76243

mdllntigigavscyallstahksmqtlyaqpkdqssasedfaflpsvdiivpcynenph tfseclasianqdyag klrvywddgsanreklervhhtyagdprfdfillrenvgkrkaqiaairgssgdlvlnvd sdstlasdwtklalk mqnpeigaamgqltasnmdtwltrlidmeywlacneeraaqarfgavmcccgpcamyrrs allslldqyesq ffrgkpsdfgedrhltilmlkagfrtdyvpdaiaatwpdrmgpylrqqlrwarstfrdtl lalrllpgldhyitldvig qnlgplllalavltgvlqvaltatvplwtvmmiasmtmircavaavrarqlrflvfslht pinlffllpmkayalctls nsdwlsrsspanktsaggehptteasaggtsgnatplrrlnlardsstvtpagvysdd

LgtA U25839

mqplvsvlicaynvekyfaqslaawnqtwcnldilivddgstdgtlaiakdfqkrdsrik ilaqaqnsglipslni gldelaksggeyiartdaddiaapdwiekivgemekdrsiiamgawlevlseekdgnrla rhhrhgkiwkkptr hediadffpfgnpihnntmimrrsvidgglrynterdwaedyqfwydvsklgrlayypea lvkyrlhanqvssk ysvrqheiaqgiqktamdflqsmgfktrfdsleyrqikavayellekhlpeedferarrf lyqcfkrtdtlpagawl dfaadgrmrrlftlrqyfgilhrllknr

WbgO AF461121

miideaesaesthpwsvilpvnkknpfldeainsilsqtfssfeiiivancctddfynel khkvndkiklirtniayl pyslnkaidlsngefiarmdsddishpdrftkqvdflknnpyvdwgtnaifiddkgrein ktklpeenldivknl pykccivhpsvmfrkkviasiggymfsnysedyelwnrlslakikfqnlpeylfyyrlhe gqstakknlymvm vndlvikmkcffltgninylfggirtiasfiyckyik

AF400048

mslaiiparggskgiknknlvllnnkpliyytikaalnaksiskvwssdsdeilnyaksq nvdilkrpislaqddtt

NeuA

sdkvllhalkfykdyedwflqptsplrtnihineafhlyknsnanalisvsecdnkilka fvcndcgdlagicnde ypfmprqklpktymsngaiyilkikeflnnpsflqsktkhflmdesssldidcledlkkv eqiwkk

U60660

mglkkacltvlclivfcfgifytfdrvnqgemavsllkeklfheegepvnlifcytilqm kvaerimaqhpgerfy wlmsenmekydyyfnqikdkaerayffhlpyglnksfhfiptmaelkvksmllpkvkriy laslekvsiaafls

IN 1

typdaeiktfddgtgnliqsssylgdefsvngtikmfarmmigdwsiaktmasdehytif kglknimddgrrk mtylplfdaselktgdetggtvrillgspdkemkeisekaaknfkiqyvaphprqtygls gvttlnspyviedyilr eikknphtryeiytffsgaaltmkdfpnvhvyalkpaslpedywlkpvyalftqsgipil tfddkn

NeuBl AF400048

mkeikiqniiiseekaplwpeiginhngslelakimvdaafsagakiikhqthivedems kaakkvipgnakis iyeimqkcaldykdelalkeyteklglvylstpfsraganrledmgvsafkigsgecnny plikhiaafkkpmiv stgmnsiesikptvkilldneipfvlmhttnlyptphnlvrlnamlelkkefscmvglsd httdnlaclgawlgac vlerhftdsmhrsgpdivcsmdtkalkeliiqseqmaiirgnneskkaakqeqvtidfaf aswsikdikkgevls mdniwvkrpglggisaaefenilgkkalrdiendaqlsyedfa

10 NeuC AF400048

mvkkilfitgsradyskikslmyrvqnssefelyifatgmhlsknfgytvkelykngfkn iyefinydkyyqtdk alattidgfsryanelkpdliwhgdrieplaaaivgalnnilvahieggeisgtiddslr haisklahihlvndefakr rlmqlgedeksifiigspdlellndnkislseakkyydinyenyallmfhpvtteitsik nqadnlvkaliqsnknyi viypnndlgfelilqsyeefknnprfklfpslrfeyfitllknadfngnsscilkealyl ktagilvgsrqngrlgnentl kvnansdeilkaintihkkqdlfsakleildssklffeylqsgdffklstqkvfkdik

17 C I m S NP418185

mcgivgaiaqrdvaeilleglrrleyrgydsaglawdaeghmtrlrrlgkvqmlaqaaee hplhggtgiahtrw athgepsevnahphvsehivwhngiienheplreelkargytfvsetdteviahlvnwel kqggtlreavlraip qlrgaygtvimdsrhpdtllaarsgsplviglgmgenfiasdqlallpvtrrfifleegd iaeitrrsvnifdktgaevk rqdiesnlqydagdkgiyrhymqkeiyeqpnaikntltgrishgqvdlselgpnadells kvehiqilacgtsyns gmvsrywfeslagipcdveiasefryrksavrmslmitlsqsgetadtlaglrlskelgy lgslaicnvpgsslvres dlalmtnagteigvastkafttqltvllmlvaklsrlkgldasiehdivhglqalpsrie qmlsqdkriealaedfsdk hhalflgrgdqypialegalklkeisyihaeayaagelkhgplalidadmpviwapnnel leklksnieevrarg gqlyvfadqdagfvssdnmhiiemphveeviapifytvplqllayhvalikgtdvdqpml aksvtve

16 GlmU WP038583267

msasdfssavwlaagagtrmksdlqktlhsiggrslishslhaaaglnpehivavighrr dqvgpavsqvaeeld revliaiqeeqngtghavqcamdqlegfegtiivtngdvplltddtlsalldahvevpta vtvltmrlddptgygriv meegevtaiveqkdasaeiqaidevnsgvfafdaailrsalaelksdnaqgelyltdvlg iargeghpvrahtaad arelagvndrvqlaeagaelnrrtviaamrggativdpattwidvevsigrdviihpgtq lkgetvigdrvevgpdt tltnmtigdgaswrthgfdstigenatvgpftyirpgttlgpegklggfvetkkatigrg skvphltyvgdatigees nigassvfvnydgenkhhttigshvrtgsdtmfiapvtvgdgaysgagtvikddvppgal avsggrqmiegwv qkkrpgtaaaqaaeaaqnvhnqeg

2. Strains

Escherichia coli TOP 10 cells (Invitrogen) were used for the construction of all plasmids. Escherichia coli K12 MG1655 (code: E. coli sWT) was used as the parent for all strain engineering experiments and was obtained from ATCC. Escherichia coli K12 MG1655 ArccA AcndA DE3 (code: E. coli sDE3) was used in experiments with pT7 and was carried from Ajikumar et al. (Ajikumar et al. 2010). Site directed chromosomal alterations in E. coli was accomplished by homologous recombination mediated by l-Red recombinase (induced from pKD46) as described by Datsenko and Wanner (Datsenko and Wanner 2000). Linear DNA for homologous recombination was generated by amplifying the FRT flanked antibiotic resistance cassette from the appropriate template (pKD3 or pKD4 for gene deletion, p_P22RBS-cITCmurA for knocking in the translational coupled cassete cITCmurA and p_P22-layY for knocking in lacY under control of P22). Knocking in the translational coupled library was performed identically, with the exception that the linear fragment was amplified directly from the single stranded assembly (SSA) mix. Positive transformants were cured from the antibiotic resistance cassette using FLP recombinase (induced from pCP20). Successful chromosomal integration/deletion was confirmed by colony PCR and subsequent sequencing. All oligonucleotides used are listed in Table 2.

Chromosomal alteration in C. glutamicum was established using the CRISPR/Cpfl system as described by (Jiang et al. 2017). This system, based on the CRISPR mechanism of Francisella novicida, uses two plasmids (pjYSl and pJYS2) to perform genomic alterations.

A list of all used strains is given in Table 3. Genomic sequences of promoter and 5’-UTR regions of the P22RBS-cITCmurA knock-in (sP22) and seven selected mutants from the library (sRNDl-sRND7) is given in Table 4.

Table 2. List of used oligonucleotides

Table 3. List of all used strains Table 4. Genomic sequences of promoter and 5’-UTR regions of the P22RBS-cITCmurA knock-in (sP22) and seven selected mutants from the derived library (sRNDl-sRND7)

3. Plasmids

All plasmids used in this study are listed in Table 5. All plasmids were constructed using Circular Polymerase Extension Cloning (CPEC) assembly (Quan and Tian 2009). DNA oligonucleotides were purchased from IDT and are listed in Table 2. All E. cob expression vectors contained a pBR322 origin of replication (Prentki and Krisch 1982) except for the plndicator plasmid that contained a pSClOl origin of replication (Kazuo and Mitsuyo 1984) and for the pHBP plasmid that contained the pl5A origin of replication (Selzer et al. 1983). The plndicator plasmid was provided with a kanamycin resistance marker (Pridmore 1987), p_P22RBS-cITCmurA, p PRND-cITCmurA and p_P22LacY, with a chloramphenicol resistance marker (Alton and Vapnek 1979), pHBP with a spectinomycin resistance marker (Bose, Fey, and Bayles 2013) and the production plasmids pCOS, pSA, and pLNT with an ampicillin resistance marker (Hedges and Jacob 1974). The pCOS production plasmid was based on the pCXhPl4-mKATE2 expression vector (origin, antibiotic resistance and P14 promoter and RBS (De Mey et al. 2007; Aerts et al. 2011; Shcherbo et al. 2009)). The sequence of the chitin synthase was obtained from Rhizobium sp. GRH2 (NodC, Genbank accesion code: AJW7624371 (Hamer et al. 2015)). The pSA production plasmid used was constructed as described by Peters et al. (Gert Peters et al. 2018). The pLNT production plasmid was based on the pSA vector (Gert Peters et al. 2018). The CMP-Neu5Ac synthetase was obtained from Campylobacter jejuni strain ATCC 43438 (NeuA, Genbank access code: AF400048) (Fierfort and Samain 2008). The sequence for the a-2,3- sialyltransferase was obtained from Neisseria meningitidis (NST, Genbank accession code: U60660).

The plndicator plasmid was provided with a lambda-promoter PR (Ptashne 2004), derived from the pDAWN plasmid (Ohlendorf et al. 2012) that drove the fluorescent mKATE2 reporter (Shcherbo et al. 2009) derived from the in-house pCXhP22-mKATE2 plasmid. The p P22RBS- cITCmurA plasmid was composed of the cl repressor gene with EVA tag, derived from the pDAWN plasmid (Ohlendorf et al. 2012), translationally coupled to the first 1000 bp of the murA coding sequence derived from the E. coli genome. An FRT-site flanked chloramphenicol cassette was cloned from the pKD3 plasmid (Datsenko and Wanner 2000) and the P22 promoter and RBS (De Mey et al. 2007) were derived from the in-house pCXhP22-mKATE2 plasmid. Detailed maps of all plasmids are provided in Figure 3.

The introduction of degenerated DNA sequences, resulting in the p PRND-cITCmurA plasmid, was performed using single strand assembly (SSA), as described in the 2-P CPEC protocol of Coussement et al. (Coussement et al. 2017). Oligonucleotidess used for randomization are summarized in Table 2. Sequencing confirmed the full and partial randomization of the promoter and 5’-UTR region, respectively.

The pHBP plasmid was constructed by golden gate assembly (Coussement et al. 2017) whereby codon optimized glmU from C. glutamicum is controlled by a PTrc promoter (Nielsen and Voigt 2014), with IPTG as inducer, codon optimized glmS*54 is controlled by a PTet promoter, with anhydrinetetracycline (aTc) as inducer (Nielsen and Voigt 2014) and nodC is controlled by the constitutive promoter P14 (De Mey et al. 2007).

The pCOSCg production plasmid for Corynebacterium glutamicum is based on the pEKEx3 E. coli/C. glutamicum shuttle vector (Stansen et al. 2005). The constitutive P14 promoter (De Mey et al. 2007) controlled the nodC expression. The sequence of the chitin synthase was obtained from Rhizobium sp. GRH2 (NodC, Genbank accesion code: AJW7624371 (Hamer et al. 2015)). The pSACg production plasmid for Corynebacterium glutamicum is based on the pEKEx3 E. coli/C. glutamicum shuttle vector (Stansen et al. 2005). The constitutive P14 promoter (De Mey et al. 2007) controlled the NeuC and NeuBl expression. The sequence of the UDP-N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid synthase were obtained from Campylobacter jejuni strain ATCC 43438 (NeuC and NeuBl, respectively, Genbank access code: AF400048).

Table 5. Overview of the used, constructed and characterized plasmids

4. Media

Lysogeni broth (LB) medium consisted of 10 g/L tryptone peptone (Difco, Belgium), 5 g/L yeast extract (Difco) and 10 g/L NaCl and was autoclaved for 21 min at 121 °C. Luria Bertani Agar (LBA) is similarly composed to LB, be it for the addition of 10 g/L agar. Minimal medium contained 2 g/L NH ₄ CL, 5 g/L (NH ₄ ) ₂ S0 ₄ , 3 g/L KH ₂ P0 ₄ , 7.3 g/L K ₂ HP0 ₄ , 8.4 g/L MOPS, 0.5 g/L NaCl, 0.5 g/L MgS0 ₄ .7H ₂ 0, and 16.5 g/L glucose.LLO or 15.3 g/L glycerol as carbon source, 1 mL/L trace element solution and 100 LIL/L molybdate solution. Trace element solution consisted of 3.6 g/L FeCl ₂ .4H ₂ 0, 5 g/L CaCl ₂ .2H ₂ 0, 1.3 g/L MnCl ₂ .2H ₂ 0, 0.38 g/L CuCl ₂ . 2H ₂ 0, 0.5 g/L CoCl ₂ . 6H ₂ 0, 0.94 g/L ZnCl ₂ , 0.0311 g/L H ₃ B0 ₄ , 0.4 g/L Na ₂ EDTA. 2H ₂ 0, 1.01 g/L thiamine.HCl. The molybdate solution contained 0.967 g/L Na ₂ Mo0 ₄ .2H ₂ 0. To avoid Maillard reaction and precipitation during sterilization of the shake flask medium, the glucose and magnesium sulphate were autoclaved separately from the remaining salts. Glucose and magnesium sulphate were autoclaved in a 200 mL solution, the remaining salts in an 800 mL solution. Prior to autoclaving, the latter was set to a pH of 7 with 1 M KOH. After autoclaving, these two solutions were cooled down and mixed. Subsequently, the trace element and molybdate solutions were added filter- sterilized with a bottle top filter (Coming PTFE filter, 0.22um). If required, the culture medium was supplemented with appropriate antibiotics. Stock concentrations for antibiotics were 100 mg/mL for spectinomycin, 100 mg/mL for ampicillin, 25 mg/mL for chloramphenicol, and 50 mg/L for kanamycin. Antibiotic stocks were diluted lOOOx for cell culture experiments. If required, the culture medium was supplemented with inducers.

Brain Heart Infusion (BHI) medium is used as liquid medium for growth as well as basis for agar medium used when working with C. glutamicum strains. The liquid medium consists of 12.5 g/L brain infusion solids, 5.0 g/L beef heart infusion solids, 10.0 g/L proteose peptone,

2.0 g/L glucose, 5 g/L NaCl and 2.5 g/L disodium phosphate. The mixture is bought ready- made from Sigma-Aldrigh (USA). In case BHI agar is needed 12 g/L agar is added before autoclaving. If required, the culture medium was supplemented with appropriate antibiotics. Stock concentrations for antibiotics were 250 mg/mL for spectinomycin, 5 mg/mL for chloramphenicol, and 25 mg/L for kanamycin. Antibiotic stocks were diluted lOOOx for cell culture experiments. If required, the culture medium was supplemented with inducers.

CGXII medium is used as synthetic medium for C. glutamicum. To make CGXII agar plates l2g/L agar is added. CGXII medium contained 20 g/L (NH ₄ ) ₂ SO, 1 g/L KH2PO4, 1 g/L K2HPO4, 0.25 g/L MgS0 ₄ .7H ₂ 0, 10 mg/L CaCl ₂ , 42 g/L MOPS, 0.2 mg/L Biotin, 1 mL/L Trace elements solution, 1 mL/L 3% Protocatechuic acid solution, 100 mL/L 40% glucose solution. The trace elements solution consisted of 10 g/L FeS0 ₄ .7H ₂ 0, 10 g/L MnS0 ₄ .H ₂ 0, 1 g/L ZnS04.7H20, 0.2 g/L Q1SO4.5H2O, 20 mg/L N1CI2.6H2O. The glucose solution is made separately and autoclaved. The trace elements solution is made and components are dissolved by adding concentrated HC1 until a final pH of about 1 is reached. This solution is sterilized via filter sterilization. The protocatechuatic acid is dissolved in diluted NaOH in H2O, sterilized via filtration and stored at 4°C. The CGXII medium is made by dissolving all components except the trace elements, glucose and protocatchuate in 798 mL. The solution is brought to pH 7 and sterilized by autoclaving at l2l°C for 21 minutes (1 atm overpressure). After autoclaving the three remaining components are added once the solution is lukewarm.

5. Culture conditions, optical density (ΌR) measurements

For strain engineering and plasmid construction strains were grown in lysogeny broth (LB) at 30°C with shaking (200 rpm, LS-X AppliTek orbital shaker, Nazareth, Belgium).

In vivo library evaluation was performed in 96-well flat-bottomed microtiter plates (MTP, Greiner) at 30°C with shaking (200 rpm, LS-X AppliTek orbital shaker, Nazareth, Belgium).

For growth experiments, E. coli and C. glutamicum strains were plated on LBA or BHI agar medium, respectively, with appropriate antibiotics for maintenance and selection of the various plasmids used, incubated for 16 h at 30 °C and a single colony was used for a preculture. For flask experiments, precultures were grown in 50ml centrifuge tubes containing 10 ml LB or BHI medium, respectively, with the necessary antibiotic for selection pressure. Pre-cultures were grown overnight (l6h) at 30 °C and 200 rpm (LS-X AppliTek orbital shaker, Nazareth, Belgium) and subsequently, used for 1 % inoculation of 100 ml glucose defined medium, i.e. minimal medium or CGXII medium respectively, in 500 ml shake flasks and grown at 30 °C and 200 rpm (LS-X AppliTek orbital shaker, Nazareth, Belgium). At regular intervals, samples for extracellular metabolites analysis were collected and optical density (OD) at 600 nm is determined. For 24- well deep well plates (DWP) experiments, precultures were grown in 50 ml centrifuge tubes containing 10 ml LB or BHI medium, respectively, with the necessary antibiotic for selection pressure. Pre-cultures were grown overnight (l6h) at 30 °C and 200 rpm (LS-X AppliTek orbital shaker, Nazareth, Belgium) and subsequently, used for 1 % inoculation of 3 ml glucose defined medium, i.e. minimal medium or CGXII, respectively, in 24-well DWP plates with sandwich covers (EnzyScreen, Heemstede, The Netherlands) and grown at 30 °C and 200 rpm (LS-X AppliTek orbital shaker, Nazareth, Belgium). In 24-well DWPs, cultures were sampled at regular intervals for extracellular metabolite analysis and OD measurement. OD was measured at 600 nm using a Jasco V-630Bio spectrophotometer (Easton, UK).

6. Cell fitness and in vivo fluorescence measurements for library evaluation and strain characterization

For screening purposes 276 colonies were picked randomly with an automated colony-picker (QPix2, Genetix) and inoculated into sterile 96-well flat-bottomed microtiter plates (Greiner) enclosed by a sandwich cover (Enzyscreen, Leiden, Netherlands) containing 150 pL minimal medium per well, supplemented with appropriate antibiotics and grown overnight on a Compact Digital Microplate Shaker (Thermo Scientific) at 800 rpm and 30°C. Subsequently, these cultures were 1 :200 diluted in 150 pL of fresh minimal medium containing the appropriate antibiotics and were cultured for 24h at 30°C and measured every 20 minutes for fluorescence and optical density using a Tecan M200 infinite PRO (Tecan, Mechelen, Belgium). Excitation and emission wavelengths were 588 and 633 nm respectively. Optical density was measured for biomass correction and for maximal growth rate calculations at a wavelength of 600 nm. Data collection was based on single colony measurements, except for sWT and sWT+ strains which were analyzed in triplicate.

For strain characterization, a similar protocol is adopted with the exception that strains are not randomly picked and 3 biological replicates were analysed (n=3). Strains were analysed using a Biotek Synergy Hl (Biotek, Vermont, USA), excitation, emission and optical density wavelengths were identical. 7. Culture conditions for glycan biosynthesis

Chitopentaose biosynthesis experiments were performed in defined medium (minimal medium for E. coli and CGXII medium for C. glutamicum) supplemented with the appropriate antibiotics. Experiments were performed for 24 h, in triplicate (n=3), in pyramide-bottem square 24-deepwell microplates (0.5 ml) (Enzyscreen, Heemstede, The Netherlands) at 30°C with shaking (250 rpm/50 mm).

Sialic acid biosynthesis experiments were performed in defined medium (minimal medium for E. coli and CGXII medium for C. glutamicum) supplemented with the appropriate antibiotics. Experiments were performed for 48 h, in duplicate (n=2), in 250 ml shake flasks (25 ml) at 30°C with shaking (250 rpm/50 mm).

8. UDP-GlcNAc pool determination for strain characterization

UDP-GlcNAc was extracted from the wild type strain (sWT) and the seven library strains (sRNDl-7) that were cured from their plndicator plasmid, in 3 biological replicates (n=3) unless stated otherwise. Strains were cultivated in minimal medium until the mid-exponential phase in pyramide-bottem square 24-deepwell microplates (0.5 ml) (Enzyscreen, 150 Heemstede, The Netherlands) at 30°C with shaking (250 rpm/50 mm).

9. Sample preparation

For chitopentaose biosynthesis, first 0.1 mL broth was diluted 10 times in physiological water for OD600 measurements in a Jasco V-630Bio spectrophotometer (Easton, UK). Subsequently, 0.3 mL broth was centrifuged at 14000 rpm for 10 min. The supernatant was stored at -80°C for the analysis of extracellular metabolites. Pellets were stored at -80°C until further use. Pellets were resuspended in 100 pL 60% ACN, vortexed and centrifuged at 14000 rpm for 10 min. The supernatant was subsequently applied for COS analysis. UDP-GlcNAc samples were prepared identically, but were resuspended in 200 pL 60% ACN before analysis.

Sialic acid synthesis samples were collected by collecting 2 mL broth, measuring OD600 as described above, and centrifuged at 14000 rpm for 10 min. The supernatant was subsequently stored at -80°C for SA analysis. 10. HPLC-ELSD/ESI-MS analysis

The applied HPLC-ELSD/ESI-MS method was developed based on the methods described (Leijdekkers et ah, 2011; Remoroza et al, 2012). Analyses of COS were performed using a Shimadzu HPLC system (Shimadzu, Jette, Belgium) coupled to an evaporative light scattering detector or/and an ESI-MS -detector. COS were separated by hydrophilic interaction chromatography (HILIC) using a Kinetix 2.6 _ HILIC 100 A column (2.6 _m, 4.6 mm x 150 mm; Phenomenex, Utrecht, The Netherlands) in combination with an appropriate SecurityGuard ULTRA Cartridge.

Glycan molecules were analyzed on a Waters ACQUITY UPLC system (Waters, Milford, MA, USA). Chitopentaose was separated by hydrophilic interaction chromatography (HILIC) using an ACQUITY UPLC BEH Amide 1.7 pm column (2.1 x 100 mm, Waters) connected to a ELSD detector. Sialic acid was separated by ion exclusion chromatography using a Rezex ROA-Organic Acid H+ 8 pm column (7.8 x 300 mm, Phenomenex) connected to a UV- detector. Detailed information is summarized in table 6. Sialyllactose was separated using a ACQUITY UPLC BEH Amide, 130 A, 1.7 pm column (2.1 mm x 50 mm) connected to a ELSD detector. A mixture of 75/25 acetonitrile/water solution with 1% formic acid was used as mobile phase. The flow rate was set to 0.130 mL/min and the column temperature to 35 °C. Lacto-N-tetraose was separated using an XBridge UPLC BEH Amide 1.8 pm 2.1x100 mm Column (Waters). Chromatographic conditions involved 1 pL sample injection, gradient elution of acetonitrile/water with 0.1% formic acid at 50 °C and at a flow rate of 0.3 ml/min.

UDP-GlcNAc analysis was performed on a Dionex ICS-3000 (Thermo Scientific) using a Carbopac PA20 column (Thermo Scientific) and a ICS-5000 electrochemical detector cell (Thermo Scientific). Llow rate was set to 0.5 ml/min, column temperature at 30°C. 5 pL sample was injected, the elution profile was isocratical and eluent consisted of 500 mM acetic acid and 100 mM NaOH. Table 6. UPLC details and elution profile for glycan analysis

Chit opent a* »v

Del ail.' _' L (ft hod

Sialic acid

1 )et ai!s Met hod

11. TLC analysis

The different chitooligosaccharides (fully acetylated chitopentamers, fully acetylated chitotetramers) are analyzed using thin layer chromatography (TLC). The TLC plates used are HPTLC silica gel 6OF254 plates (Merck). The eluent comprises butanol, methanol, 25% NLL and H ₂ 0 with ratio 5 :4:2: 1. Spots of 3 L sample (supernatant) or standard solution are applied to the TLC plate. Next, the TLC plate are placed in the equilibrium tank and the eluent allowed to develop. The plates are then removed from the tank and quickly dried. Subsequently the TLC plate is stained with 30% [w/v] NH4HSO4 and heat up till 500 °C to visualize the components under UV-light. The standard solutions are 10 g/L glucose, 10 g/L GlcNAc, and 10 g/L COS mixture (15% fully acetylated chitotetramers (A4), 85% fully acetylated chitopentamers (A5)).

12. Quantitative PCR

Quantitative PCR was performed on the wild type strain (sWT) and the seven library strains (sRNDl-7) that were cured from their plndicator plasmid. Measuring the expression levels, RNA was isolated from 3 biological replications (n=3); mid-exponentially growing E. coli cells using RNA later in combination with a RNeasy Mini Kit (QIAGEN, Hilden, Germany). mRNA was subsequently stored at -80°C until further use.

DNA was synthesized using a First Strand cDNA synthesis kit with random hexamers (Thermo Scientific). For the amplification of the three E. coli reference genes (cysG, hcaT and idnT) oligos were ordered as described in Zhou et al. (Zhou et al. 201 1). QPCR oligos for murA are charted in Table 2.

QPCR was performed as a technical duplicate (n=2) using a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and data was analysed using CFX manager Version 3.1.1517.0823 (Bio-Rad).

13. Sequencing

Every constructed plasmid was verified by sequencing. Genetic parts of interest were sequenced upon alteration (Knock-out, Knock-in). All sequencing was performed via sequencing services (Macrogen Inc.).

14. Data and statistical analysis

All data analysis was performed using pandas unless stated otherwise. Fibrary evaluation and strain characterization consisted of maximal growth rate and fluorescent measurements. Maximal growth rates were determined by plotting the OD600 values in function of time and fitting Richards growth-model (Birch 1999). A fluorescent parameter was calculated by correcting each individual fluorescent data-point for its concurrent biomass (OD600), plotting them in function of time and scanning for a typical maximum.

The cellular volumetric determination of the UDP-GlcNAc was determined based on a calibration curve and was calculated assuming an OD600 of 1.0 = 8 x 108 cells/ml, and assuming lfF volume per cell. Chromatogram analysis was performed using the Chromeleon 7.2 software package (Thermo Scientific).

Final glycan concentrations were determined based on a calibration curve, and were corrected for biomass by OD600 measurements in order to overcome influences that were caused by the differences in culturing methods. Chromatogram analysis was performed using the Openchrom 1.1.0 software package. Example 1 - Influence of cell envelope synthesis on COS production

To evaluate the potential of increasing UDP-GlcNAc supply for a UDP-derived product, i.e. COS titer, by decreasing the conversion of UDP-GlcNAc to at least one cell envelope precursors or component, Escherichia coli s3KO transformed with pCOS is grown on minimal medium with glucose until OD600 of 3 is reached. At that point, all cells are collected through centrifugation and resuspended in fresh minimal medium with glucose. This batch is divided in four sub-batches and various concentrations of fosfomycin are added, i.e. 0 mM, 0.1 mM, 0.25 mM and lmM, respectively. Subsequently, samples are taken for COS analysis. Figure 4 gives the result of the TEC analysis of the corresponding cultures. Addition of fosfomycin to a E. coli culture has a positive effect on COS production. Hence, lowering the peptidoglycan synthesis results in an increased conversion of UDP-GlcNAc to COS.

Example 2 - Engineering murA in Escherichia coli

In order to explore the expression profile of MurA, which catalyzes the first committed step in the peptidoglycan synthesis pathway, its expression must be varied as widely as possible and therefore a high-throughput screening method is required (see Figure 5-8). To this end, E. coli sP22 is generated in which the endogenous ibaG-murA operon (Figure 5) is replaced by cl gene translational coupled to murA under control of the P22RBS (Figure 6). To convert the expression of MurA into a measurable signal, i.e. fluorescence, plndicator is constructed. Next, the p PRND cITCmurA plasmid is constructed randomizing the promoter and 5’-UTR regions. The translationally coupled randomized conformations are installed in a plndicator- mKATE2 containing E. coli sP22, yielding a phenotypically diverse strain library (E. coli sRND, Figure 7) in terms of fluorescence levels (Figure 8). From the library seven mutants are randomly selected (displaying a range of red intensity) and characterized in depth. Figure 9 depicts the specific fluorescence and specific corrected maximal growth rate measurements. Analysis of variance indicated that the maximal growth rate of all the selected strains were comparable to one another (one-way ANOVA, p= 0.5436, F= 0.8906), despite the substantial variation in murA expression we anticipated. The ability of the selected strains to maintain their fitness, despite the diverse murA expression is further substantiated by qPCR (Figure 10). Furthermore, the data clearly showed that the fluorescence is inversely correlated to the amount of MurA mRNA (R ² = 0.996), validating the translationally coupled screening technique (Figure 11). Two out of the seven selected strains (sRND6 and sRND7, with the lowest fluorescence) showed an increased MurA expression compared to the wild type strain (E. coli sWT) (Figure 10). Investigating the peptidoglycan synthesis metabolic network and quantifying mutant UDP-GlcNAc pools, surprisingly resulted in comparable titers (Figure 12). Analysis of variance confirmed that altering MurA expression does not affect UDP-GlcNAc levels (one-way ANOVA, p= 0.4065, F= 1.1384).

Example 3 - Production of fully acetylated chitopentaose in Escherichia coli

For the production of fully acetylated chitopentaose E. coli sRNDl-5 and sWT are transformed with pCOS yielding sCOSl-5 and sWTCOS, respectively. These metabolically engineered strains are grown in minimal medium with glucose. Production of chitopentaose is depicted in Figure 13. Production titers for fully acetylated chitopentaose ranged from 1.58 up to 5.43 times that of sWTCOS.

Example 4 - Production of N-acetylneuramic acid (NeuSAc) in Escherichia coli

For the production of Neu5Ac, E. coli sRNDl-5 and sWT were first made deficient in E. coli’s native catabolic sialic acid pathway, yielding sRNDAnanRATEK 1 -5 and sWTAnanRATEK, respectively. Next these metabolically engineered strains are transformed with pSA yielding sSAl-5 and sWTSA, respectively, and grown in minimal medium with glucose. Production of Neu5Ac is depicted in Figure 14. Specific production showed that the engineered MurA expression levels have a major impact on Neu5Ac synthesis. Upon decreasing MurA levels, E. coli sSAl-5 strains were able to produce up to 10.99 times that of E. coli sWTSA.

Example 5 - Production of Lacto-N-tetraose (LNT) in Escherichia coli

For the production of Facto-N-tetraose, lacZ, coding for b-galactosidase, is knocked out to avoid lactose degradation and the expression of lacY, coding for a lactose permease, is ensured by means of a medium strong constitutive promoter in E. coli sRNDl-5. Further, the genes lgtA and wbgO encoding b- 1 ,3-N-acctylglucosaminyltransfcrasc and b-1,3- galactosyltransferase, respectively, are expressed under control of the artificial promoter P14 from production plasmid pBR322 (pLNT). These metabolically engineered strains are grown in minimal medium with glucose, which is supplemented with lOg/L lactose. Strains are cultivated in shake-flask and yielded mg amounts of LNT.

Example 6 - Production of 3'-sialyllactose

For the production of 3'-sialyllactose, lacZ, coding for b-galactosidase, is additionally knocked out to avoid lactose degradation and the expression of lacY, coding for a lactose permease, is ensured by means of a medium strong constitutive promoter in E. coli sRNDl-5. The metabolically engineered E. coli strains additionally expresses UDP-N-acetylglucosamine 2- epimerase (NeuC), N-acetylneuraminic acid synthase (NeuBl), CMP-NeuAc synthetase (NeuA) obtained from Campylobacter jejuni and a a-2,3-sialyltransferase (NST) obtained from Neisseria meningitidis. These metabolically engineered strains are grown in minimal medium with glucose, which is supplemented with lOg/L lactose. This system yielded mg amounts of 3'-sialyllactose.

Example 7 - Engineering of murA in Corynebacterium glutamicum

For the engineering of MurA in C. glutamicum the gene murA2, coding for a UDP-N- acetylglucosamine l-carboxyvinyltransferase (NCgl2470), is deleted yielding strain C. glutamicum sCgl . Additionally, the epression of murAl, coding for a UDP-N- acetylglucosamine l-carboxyvinyltransferase (NCgl0345), is altered by replacing the endogeneous promoter and 5’-UTR sequence with the cognate promoter and 5’-UTR sequence of sRND2 in C. glutamicum sWT and sCgl yielding C. glutamicum sCg2 and C. glutamicum sCg3, respectively.

Example 8 - Production of N-acetylneuramic acid (NeuSAc) in Corynebacterium glutamicum

For the production of N-acetylneuramic acid (Neu5Ac) in C. glutamicum, C. glutamicum sCgl-l and sWT are first made deficient in C. glutamicum’ s native catabolic sialic acid pathway, yielding C. glutamicum sCgAnanA 1 -3 and sWTAnanA, respectively. The metabolically engineered C. glutamicum strains additionally expresses UDP-N- acetylglucosamine 2-epimerase (NeuC) and N-acetylneuraminic acid synthase (NeuBl) obtained from Campylobacter jejuni. These metabolically engineered strains are grown in CGXII medium with glucose, which is supplemented with lOg/L lactose. This system yielded mg amounts of Neu5Ac.

Example 9 - Production of fully acetylated chitopentaose in Corynebacterium glutamicum

For the production of fully acetylated chitopentaose C. glutamicum sCgl-l and sWT additionally expresses the chitin synthase obtained from Rhizobium sp. GRH2 under the control of the artificial promoter P14 from plasmid pEXK3. These metabolically engineered strains are grown in CGXII medium with glucose.

Example 10 - Production of fully acetylated chitopentaose in Escherichia coli with optimized hexosmanine biosynthesis pathway

For COS production in E. coli with optimized hexosmanine biosynthesis pathway strain sWT and sRND2 additionally expresses a chitin synthase obtained from Rhizobium sp. GRH2 (NodC) under control of the constitutive promoter P14, a fused N-acetylglucosamine-l- phosphate uridyltransferase and glucosamine- 1 -phosphate acetyltransferase (GlmU) obtained from Corynebacterium glutamicum under control of a PTrc promoter and a L-glutamine:D- fructose-6-phosphate aminotransferase (GlmS) obtained from E. coli with three mutations A38T, R249C and G471 S under control of a PTet promoter (pHBP) yielding sWTCOS+pHBP and sCOS2+pHBP, respectively. These metabolically engineered strains together with sCOS2 and sWTCOS are grown in minimal medium with varies inducer concentrations (aTc/IPTG [mM/mM]: 5/0.01, 5/0.05, 10/0.01 and 10/0.05] Figure 15 depicts the COS production. The sWTCOS+pHBP strain and sCOS2+pHBP strain were able to produce up to 2.2 and 6.8 times that of E. coli sWTCOS, respectively. List of abbreviations used in the text

5’-UTR: 5’ untranslated region

ADP: adenosine diphosphate

aTc: anhydrinetetracycline

CmR: chloramphenicol resistance

CDW: cell dry weight

CMP: cytidine-5’ -mo nophosphate

CMP-Neu5Ac: CMP-N-acetylneuramie acid CNRQ: calibrated normalized relative quantity COS: chitooligosaccharide

CPEC: Circular Polymerase Extension Cloning DWP: deep well plate

Gal Ac: N-acetylgalactosamine

GDP: guanosine diphosphate

GG: Golden Gate

GH: glycoside hydrolase

Glc: glucose

GlcN: N-glucosamine

GlcNAc: N-acetylglucosamine

GlcUA: D-glucuronic acid

Gly: glycerol

GP: glycoside phosphorylase

GT : glycosyltransferase

HA: hyaleuronic acid IPTG: isopropyl b-D-l-thiogalactopyranoside

KanR: kanamycin resistance

KO: knock-out

KI: knock-in

LB: lysogeni broth

LNT: lacto-N-tetraose

ManNAc: N-acetylmannosamine

MurNac: N-acetylmuramic acid

Neu5Ac: N-acetyl-neuramic acid

oCHS: oligomer chitin synthase

OD: optical density

P14: promoter 14 of the promoter library of De Mey et al (De Mey et al. 2007)

P22: promoter 22 of the promoter library of De Mey et al (De Mey et al. 2007)

Pyr: Pyruvate

pCHS: polymer chitin synthase

RBS: ribosome binding site

rpm: rotations per minute

SSA: single stranded assembly

TC: translational coupling

TCC: translational coupling cassette

TDP: thymidine diphosphate

TG: transglycosidase

UDP: uridine diphosphate

UDP-Gal Ac: UDP-N-acetylgalactosamine

UDP-GlcNAc: UDP-N-acetylglucosamine UDP-GlcUA: UDP-a-D-glucuronic acid UDP-ManNAc: UDP-N-acetylmannosamine UDP-MurNAc: N-acetylmuramic acid (MurNac) WT : wild type

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