Production of Food and Feed Additives From Non-food-competing Feedstocks: Valorizing N-acetylmuramic Acid for Amino Acid and Carotenoid Fermentation With Corynebacterium glutamicum

Corynebacterium glutamicum is used for the million-ton-scale production of food and feed amino acids such as L-glutamate and L-lysine and has been engineered for production of carotenoids such as lycopene. These fermentation processes are based on sugars present in molasses and starch hydrolysates. Due to competing uses of starch and sugars in human nutrition, this bacterium has been engineered for utilization of alternative feedstocks, for example, pentose sugars present in lignocellulosic and hexosamines such as glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc). This study describes strain engineering and fermentation using N-acetyl-D-muramic acid (MurNAc) as non-food-competing feedstock. To this end, the genes encoding the MurNAc-specific PTS subunits MurP and Crr and the etherase MurQ from Escherichia coli K-12 were expressed in C. glutamicumΔnanR. While MurP and MurQ were required to allow growth of C. glutamicumΔnanR with MurNAc, heterologous Crr was not, but it increased the growth rate in MurNAc minimal medium from 0.15 h-1 to 0.20 h-1. When in addition to murP-murQ-crr the GlcNAc-specific PTS gene nagE from C. glycinophilum was expressed in C. glutamicumΔnanR, the resulting strain could utilize blends of GlcNAc and MurNAc. Fermentative production of the amino acids L-glutamate and L-lysine, the carotenoid lycopene, and the L-lysine derived chemicals 1,5-diaminopentane and L-pipecolic acid either from MurNAc alone or from MurNAc-GlcNAc blends was shown. MurNAc and GlcNAc are the major components of the bacterial cell wall and bacterial biomass is an underutilized side product of large-scale bacterial production of organic acids, amino acids or enzymes. The proof-of-concept for valorization of MurNAc reached here has potential for biorefinery applications to convert non-food-competing feedstocks or side-streams to valuable products such as food and feed additives.


INTRODUCTION
Corynebacterium glutamicum is a predominantly aerobic, rodshaped, Gram-positive soil bacterium which is generally recognized as safe (GRAS). Since the 1960s, C. glutamicum was first used for the production of the flavor enhancer (Kinoshita et al., 1957) under biotin limiting conditions (Shiio et al., 1962). C. glutamicum was developed into an important organism for the biotechnological industry, producing amino acids on a million-ton scale (Wendisch, 2014). C. glutamicum has also been engineered to produce diamines, organic acids, carotenoids, proteins and biopolymers . Recently, metabolic engineering of C. glutamicum to expand its substrate scope allowed to use alternative carbon sources that do not have competing uses in the food industry (Zahoor et al., 2012). Access to the hexosamines GlcN (Uhde et al., 2013) and GlcNAc (Matano et al., 2014) has been reported, but utilization of the hexosamine MurNAc as alternative carbon source by C. glutamicum has not been described (Dominguez et al., 1997;Cramer and Eikmanns, 2007).
GlcN and GlcNAc can be gained by hydrolysis of chitin and chitosan that make up the arthropod exoskeleton and are present in fungal cell walls. Every year, circa 100 billion tons of chitin are produced in Nature and GlcNAc and GlcN can be obtained by acid hydrolysis (Chen et al., 2010;Zhang and Yan, 2017) and are available, e.g., from shrimp shell waste, an abundant side stream of the fishery industry. MurNAc and GlcNAc are the hexosamine constituents of peptidoglycan which makes up about 5% of the cell mass of Gram-negative bacteria and up to 20% of the cell mass of Gram-positive bacteria (Munoz et al., 1967;Reith and Mayer, 2011). The peptidoglycan constituents that can be found in all bacterial habitats have been used as indicators of bacterial biomass content in soils (Domsch, 1982). The Gram-positive C. glutamicum is the main producing organism for the annual production of 5 million tons of amino acids . Under the assumptions that (a) the same amount of cell dry weight is produced, (b) 20% of the cell dry weight is peptidoglycan and (c) about half of peptidoglycan is GlcNAc and MurNAc, about 500.000 tons of MurNAc and GlcNAc would be available from the amino acid fermentation industry. Biotechnological processes with bacterial hosts are used at the million-ton scale to produce secreted compounds such as organic acids, amino acids and enzymes. The spent biomass may be used in waste-toenergy applications either by thermal (e.g., incineration), thermochemical (e.g., torrefaction) or by biochemical treatments (e.g., anaerobic digestion). However, it is desirable to make use of the carbon and nitrogen containing hexosamine fraction of peptidoglycan in food and feed fermentation processes. The hexosamines may function both as carbon and nitrogen source for bacterial fermentations.
In chemical hydrolysis, the hexosamine fraction of peptidoglycan is accessible via enzymes of bacterial cell wall recycling. The degradation of the own cell wall by autolytic enzymes as a part of cell wall recycling is a common pathway in bacteria. When an Escherichia coli lys dap mutant was labeled with [ 3 H]diaminopimelate for two generations and then chased, about 45% of its cell wall peptidoglycan was recycled per generation (Goodell and Schwarz, 1985;Uehara et al., 2006;Uehara and Park, 2008). The Gram-positive Bacillus subtilis can degrade, uptake and metabolize the cell wall component MurNAc in the stationary phase (Borisova et al., 2016). While all bacteria require cell wall peptidoglycan remodeling during growth and cell division, not all can utilize the monomeric components as carbon or nitrogen sources for growth. C. glutamicum may possess a minimal set of autolytic enzymes, however, many orthologs of the peptidoglycan degradation machinery from E. coli are absent (Dahl et al., 2004;Reith and Mayer, 2011). Catabolism of MurNAc in E. coli involves uptake and phosphorylation of MurNAc and GlcNAc via the phosphoenolpyruvate dependent phosphotransferase system (PTS). The MurNAc-specific PTS subunits are MurP and Crr (Figure 1). MurP, a two-domain protein that lacks a PTS-EIIA domain, is phosphorylated by EIIA Glc , a kinase encoded by the crr gene (carbohydrate repression resistance), which interacts with several members of the glucose PTS family (Nuoffer et al., 1988;Tchieu et al., 2001;Dahl et al., 2004). MurNAc-6-phosphate is further catabolized by the etherase MurQ (Figure 1) that cleaves the lactyl ether bond yielding GlcNAc-6-phosphate and D-lactate (Jaeger et al., 2005;Hadi et al., 2008). GlcNAc and GlcN are also taken up via the PTS with the specific subunits NagE (Plumbridge, 2009) and PTS Man (Curtis and Epstein, 1975). NagA deacetylates GlcNAc-6-phosphate to GlcN-6-phosphate which is deaminated by NagB to the glycolytic intermediate fructose-6-phosphate (Figure 1).
Corynebacterium glutamicum is able to take up GlcN (Figure 1) by using its glucose specific PTS PtsG (Arndt and Eikmanns, 2008;Uhde et al., 2013). Efficient growth with GlcN required high levels of the endogenous NagB, e.g., in the absence of the repressor protein NanR (Matano et al., 2016). By contrast, high levels of NagA and NagB were not sufficient to support growth with GlcNAc unless nagE from C. glycinophilum was expressed heterologously (Matano et al., 2014). Recombinant C. glutamicum strains carrying a nanR deletion and expressing nagE from C. glycinophilum produced several value-added products from GlcN or GlcNAc.
Here, C. glutamicum strains were developed for utilization of MurNAc as carbon, nitrogen and energy source and for MurNAcbased production of the food amino acid L-glutamate, the feed amino acid L-lysine, L-lysine-derived chemicals as well as the carotenoid lycopene.

Bacterial Strains and Growth Conditions
The strains and plasmids used in this work are listed in Table 1. Pre-cultivation of C. glutamicum strains was carried out at 30 • C in baffled shake flasks using BHI supplemented with 45.5 g/L D-sorbitol. E. coli was grown at 37 • C in LB (Lysogeny Broth) medium. Kanamycin (50 µg/mL), chloramphenicol (4.5 and 25 µg/mL for C. glutamicum and E. coli, respectively) or tetracycline (5 µg/mL) were added, if necessary. To adjust both cultures to growth conditions in the Biolector R system (m2pLabs, Baesweiler), precultures were washed after 24 h with TN-buffer and transferred to CGXII medium (Eggeling and Bott, 2005) with 100 mM GlcN and antibiotics, if necessary. After 24 h, these were transferred to 1 mL cultures in the Biolector R system (1100 rpm) with CGXII medium containing and, if not otherwise stated, 25 mM MurNAc (BACHEM, Bubendorf, Switzerland) as sole carbon source or a combination of 25 mM MurNAc and 25 mM GlcNAc. To trigger glutamate production, penicillin G (10 µM) was added in the main culture. The initial OD 600 was 1 and gene expression from plasmids pVWEx1 and pEC-XT99A was induced by addition of 25 µM IPTG, if not otherwise stated. Correlation factors for light scattering in the Biolector system, OD 600 and biomass concentrations were determined.

Construction of Expression Vectors
Escherichia coli DH5α was used for cloning. Codon usage of murP, crr and murQ from E. coli for C. glutamicum was examined using the graphical codon usage analyzer 1 . The analysis showed, that the codon ATA occurred twice in the sequence of murP. This codon is rarely used in C. glutamicum and was changed to the more frequently used codon of ATC via site directed mutagenesis (SDM). The mutated variation of murP was called murP opt . The genes of murP, crr and murQ were amplified via PCR from genomic DNA of E. coli K-12, while nagE from C. glycinophilum, was amplified from 1 http://gcua.schoedl.de/ pVWEx1_nagE (Matano et al., 2016). The primers used in this study (see Supplementary Table S1) were obtained from Metabion international AG, Planegg. Using Gibson assembly (Gibson, 2011), the vectors pVWEx1_murP, pVWEx1_murP opt , pVWEx1_murPcrr, pVWEx1_murP opt crr, pCXE50_murQ and pEC-XT99A_nagE were constructed. The vectors pVWEx1 and pEC-XT99A are IPTG inducible while pCXE50 has a constitutive EF tu promotor. E. coli was transformed by the CaCl 2 method while transformation through electroporation was applied for C. glutamicum at 2500 V, 25 µF and 200 .

Carotenoid Extraction
Lycopene was extracted as described before (Heider et al., 2012). In short, 5 wells each containing 1 ml cell suspension were combined and pelleted in safe lock micro reaction tubes by centrifugation at 10,000 g for 15 min and resuspended in 800 µL of a 7:3 methanol/acetone mixture and incubated for 15 min at 60 • C and 750 rpm in a thermomixer (Eppendorf). The cell debris was removed by centrifugation and the supernatant used for HPLC analysis. The procedure was repeated to ensure complete extraction until white pellets were obtained.

Quantitation of Fermentation Products
The quantification of MurNAc, GlcNAc, lycopene, L-glutamate, and L-lysine was conducted by HPLC analysis (1200 series Applying OPA derivatisation, L-lysine and L-glutamate were analyzed using an RP8 column with a sodium acetate (0.25 v/v %) buffer at pH 6 and a 1:50 dilution with an internal L-asparagine standard. Using the RP18 column with a Methanol-Milli-Q-water mixture (9:1), lycopene was quantified (Heider et al., 2012).

Metabolic Engineering of C. glutamicum for Growth With MurNAc as Carbon Source
Corynebacterium glutamicum, which has been engineered to utilize GlcN and GlcNAc (Matano et al., 2014(Matano et al., , 2016, cannot utilize MurNAc since no growth was observed in minimal medium with 25 mM MurNAc and 25 ± 0.1 mM MurNAc remained in the growth medium after 25 h of incubation (Figure 2A). As expected, the C. glutamicum genome lacks genes encoding a MurNAc PTS and MurNAc-6-phosphate etherase for uptake and conversion of MurNAc to GlcNAc-6-phosphate, an endogenous intermediate of C. glutamicum metabolism. As described in Section "Materials and Methods, " the genes for the MurNAc PTS murP from E. coli or codon optimized allele murP opt (Nuoffer et al., 1988;Tchieu et al., 2001;Dahl et al., 2004) were cloned into the IPTG-inducible plasmid pVWEx1 alone or as operon with crr. The gene for the MurNAc-6-phosphate etherase murQ from E. coli (Jaeger et al., 2005) was cloned into the constitutive expression vector pCXE50 (Lee, 2014). Functional expression of murQ from pCXE50_murQ was tested by complementation of the E. coli murQ mutant E. coli JW2421-1. While E. coli JW2421-1(pCXE50_murQ) utilized MurNAc as sole carbon source ( OD 600 of 3.2 ± 0.1 and µ max of 0.07 ± 0.01 h −1 ), E. coli JW2421-1 murQ showed no growth (see Supplementary Figure S1). Only murQ was tested by complementation of a E. coli mutant. We neither tested murP nor crr because we expected perturbations due to overexpression since MurP is a membrane protein and Crr serves a regulatory function. C. glutamicum nanR was transformed with the constructed pVWEx1 plasmids and with the pCXE50_murQ. The respective strains were named C. glutamicum nanR PQ, POQ, PCQ, POCQ ( Table 1).
Strains expressing crr from E. coli grew faster in minimal medium containing 25 mM MurNAc as sole source of carbon and energy than strains lacking crr ( Figure 2B). Strains with native murP grew better than strains expressing codon optimized murP ( Figure 2B). IPTG was used at a low concentration (25 µM) to induce heterologous gene expression, since higher concentrations slowed growth ( Table 2). This is not unexpected and presumably due to too high expression of transport protein genes as seen previously for dccT (Youn et al., 2008) and dctA (Youn et al., 2009), coding for dicarboxylate transporters. With 25 µM IPTG, strain nanR PCQ expressing native murP, crr and murQ grew in minimal medium containing 25 mM MurNAc to a biomass concentration of 1.2 ± 0.3 gCDW/L and with 50 mM MurNAc to a biomass concentration of 2.0 ± 0.2 gCDW/L ( Table 2). Biphasic exponential growth was observed: faster growth between 0 and 6 h and slower growth between 6 and 27 h (Figure 2A). The curves appear linear as the Y axis has been logarithmized (Figure 2). During the transition from the first to the second growth phase the medium contained 3.10 ± 0.12 mM lactate. Thus, lactate released by MurQ from MurNAc-6-phosphate may not have been utilized as fast as GlcNAc-6-phosphate, the other product of the MurQ reaction. In consequence, lactate accumulated in the culture medium in the first exponential growth phase and presumably slowed growth in the second exponential growth phase. Transient accumulation of lactate to growth inhibitory concentrations has been observed during growth of C. glutamicum with various carbon sources .
As PTS systems typically support growth on their cognate substrates with high affinity, the dependence of the growth rate on the initial MurNAc concentration in the growth medium was determined using strain nanR PCQ. Different concentrations of MurNAc (1, 2.5, 5, 10, and 20 mM) were used and the maximal growth rates were plotted against the MurNAc concentration to derive the maximal growth rate of 0.22 h −1 and the Monod constant of 0.9 ± 0.1 mM as shown in Figure 3. The final biomass concentrations reached [given as g of cell dry weight (CDW) per L], maximal specific growth rates of the first (µ max1 ) and second growth phase (µ max2 ), and the biomass yield coefficients of cell dry weight formed per used substrate used (Y X/S ) are given as means with standard deviations. A sub-millimolar Monod constant is typical for PTS mediated uptake.

Comparative Analysis of Growth With MurNAc and/or GlcNAc
Growth of recombinant C. glutamicum with MurNAc and/or GlcNAc as sole carbon sources was compared (Figure 4 and Table 3). With 25 mM MurNAc C. glutamicum nanR PCQnE grew to a biomass concentration of 3.0 ± 0.1 gCDW/L, while the maximal biomass concentration was only 2.4 ± 0.1 gCDW/L with GlcNAc. The higher biomass concentration observed with MurNAc in comparison to GlcNAc indicated that lactate released from MurNAc by MurQ contributed to biomass formation. However, the biomass yield was higher with GlcNAc (0.44 ± 0.01 g·g −1 ) than with MurNAc (0.39 ± 0.02 g·g −1 ). GlcNAc catabolism was faster than MurNAc catabolism as the maximal growth rates and the specific substrate uptake rates were lower with MurNAc (0.22 ± 0.10 h −1 and 1.80 ± 0.10 mmol·g −1 ·h −1 ) than with GlcNAc (0.30 ± 0.01 h −1 and 3.00 ± 0.10 mmol·g −1 ·h −1 ) as shown in Table 3. Unlike E. coli and B. subtilis, it is typical for C. glutamicum to simultaneously co-utilize carbon substrates present in blends (Blombach and Seibold, 2010). Therefore, C. glutamicum strain nanR PCQnE was constructed by transforming strain nanR PCQnE with plasmid pEC-XT99A-nagE for expression of the gene for the GlcNAc-specific PTS uptake system to establish whether MurNAc and GlcNAc are co-utilized or utilized sequentially. C. glutamicum strain nanR PCQnE were grown with 25 mM MurNAc and/or 25 mM GlcNAc (Figure 4). With the blend of MurNAc and GlcNAc C. glutamicum nanR PCQnE grew to a biomass concentration of 3.8 ± 0.1 gCDW/L, while a biomass concentration of only 2.1 ± 0.1 g/L was reached in the absence of nagE. Determination of the residual substrate concentrations revealed sequential utilization of GlcNAc before MurNAc ( Figure 4C). Thus, unlike many growth substrates MurNAc and GlcNAc were not co-utilized.  The parameters given are OD 600 for biomass formed and S for substrate used. Monophasic growth was observed with GlcNAc. With MurNAc, two phases were observed, and biomass yield coefficients of cell dry weight formed per used substrate used (Y X/S ), maximal specific growth rates (µmax) and specific substrate uptake rates (qS) are given for the phase 1 and phase 2. With MurNAc+GlcNAc, Y X/S , µmax and qS are reported for the phase where GlcNAc was utilized exclusively (phase 1; 0-8 h) as well as for the phase when MurNAc was utilized exclusively (phase 2; 12-18 h).

MurNAc-Based Production of Food and Feed Additives and Derived Chemicals
MurNAc was expected not only to support growth of recombinant C. glutamicum strains, but also production of value-added compounds. Therefore, MurNAc was tested as sole carbon source or in blends with GlcNAc for production of the amino acids L-lysine and L-glutamate, the diamine 1,5-diaminopentane, the cyclic non-proteinogenic amino acid L-pipecolic acid, and the carotenoid lycopene. The lycopene accumulating strain C. glutamicum crtYEb nanR (Matano et al., 2014) was transformed with the plasmids pVWEx1_murP_crr, pEC-XT99A_nagE and pCXE50_murQ as described above and the resulting strains were named crtYEb nanR PCQ and crtYEb nanR PCQnE. Cells of both strains accumulated lycopene when grown in MurNAc containing minimal medium. Strain crtYEb nanR PCQ showed a lycopene content of 0.04 mg ± 0.01 (g CDW) −1 in MurNAc minimal medium. Growth of C. glutamicum crtYEb nanR PCQnE with a MurNAc/GlcNAc blend led to a lycopene content of 0.10 ± 0.01 mg (g CDW) −1 .

DISCUSSION
In this work, production of food and feed additives by C. glutamicum from MurNAc, an alternative carbon source without competing use in human and animal nutrition, has been established. The food amino acid L-glutamate, the feed amino acid L-lysine and the feed additive lycopene were produced from MurNAc, GlcNAc and blends of both hexosamines.
Metabolic engineering of C. glutamicum for access to MurNAc relied on E. coli genes. Its MurNAc PTS system was active in C. glutamicum and strains expressing crr in addition were able to grow faster and yield more biomass than the strains without heterologous Crr (Figure 2). Crr is a glucose-family specific EIIA component, but it can interact with the PTS-EIIBC components of several members of the glucose PTS family (Barabote and Saier, 2005). C. glutamicum has two complete PTS Glc systems (Barabote and Saier, 2005). The finding that MurNAc could be utilized without heterologous Crr indicated that a PTS component of C. glutamicum made up for its absence. Moreover, the low Monod constant found for growth of the recombinant with MurNAc (Figure 3) indicated that the MurNAc PTS catalyzed high-affinity MurNAc uptake in C. glutamicum, although the MurNAc PTS seems to have a lower affinity for its substrate than, e.g., the heterologous expressed GlcNAc specific PTS system NagE from Corynebacterium glycinophilum which showed a KM value 3.8 ± 0.6 µM (Ferenci, 1996;Matano et al., 2014). The K M for the etherase MurQ in E. coli found in literature had a similar range of value (1.2 mM) (Hadi et al., 2008) with the K M value found experimentally in this study for the MurNAc PTS (0.9 ± 0.1 mM) system, making the two enzymatic steps of up taking, phosphorylation and esterification balanced.
Growth of recombinant C. glutamicum with MurNAc was biphasic and lactate accumulated during the interphase ( Figure 4A). MurNAc differs from GlcNAc only by one additional lactoyl group that is hydrolysed to lactate by etherase MurQ. Although C. glutamicum can utilize lactate as sole carbon source (Eikmanns, 2005), lactate accumulated. Utilization of D-lactate requires dld encoding quinone-dependent D-lactate dehydrogenase (Stansen et al., 2005;Kato et al., 2010). Utilization of L-lactate requires quinone-dependent L-lactate dehydrogenase which is encoded in the LldR repressed operon cg3227-lldD Georgi et al., 2008). L-Lactate is secreted by C. glutamicum under certain conditions, e.g., during growth with glucose when oxygen is limiting, but is quickly re-utilized once cg3227-lldD is derepressed (Eggeling and Bott, 2005;Stansen et al., 2005;Georgi et al., 2008).
Corynebacterium glutamicum co-utilizes glucose simultaneously with many different carbon sources including those that required introduction of heterologous pathways, for example, xylose (requiring xylose isomerase gene from E. coli (Kawaguchi et al., 2006), arabinose (requiring the araBAD operon from E. coli (Kawaguchi et al., 2008;Schneider et al., 2011), cellobiose (requiring β-glucosidase) or glycerol (requiring E. coli glycerol kinase and glycerol-3-phosphate dehydrogenase), (Rittmann et al., 2008;Sasaki et al., 2008;Zahoor et al., 2012;Meiswinkel et al., 2013;Wendisch et al., 2016). Only rarely, glucose repression has been observed, for example, during sequential utilization of glucose before ethanol (due to catabolite repression of the alcohol dehydrogenase gene adhA) (Gerstmeir et al., 2004) or before glutamate (due to catabolite repression of the operon gluABCD encoding the glutamate uptake system) (Wendisch et al., 2000;Blombach and Seibold, 2010). The preferential utilization of GlcNAc before MurNAc by C. glutamicum nanR PCQnE may be explained by an offset between fast uptake and hydrolysis of MurNAc yielding GlcNac-6-P and D-lactate followed by fast utilization of GlcNAc-6-P, but accumulation of D-lactate. It is conceivable that overexpression of dld would accelerate D-lactate catabolism precluding transient D-lactate accumulation during growth with MurNAc.
A proof-of-concept for MurNAc-based fermentative production of food and feed additives was reached. The engineered strain DM1729 nanR PCQ and DM1729 nanR PCQnE showed comparable L-lysine production as observed previously for GlcN and GlcNAc (Matano et al., 2014). Similarly, lycopene production from MurNAc by C. glutamicum crtYEb nanR PCQ was comparable to that observed by similar strains with 100 mM glucose [30 ± 10 µg·g (CDW) −1 ] and 100 mM GlcNAc [29.6 ± 4.5 µg·g (CDW) −1 ] (Heider et al., 2012;Matano et al., 2014). To establish viable production processes with MurNac as sole or combined carbon source, more work to increase titres, yields and volumetric productivities is needed. Conceptually, however, this work laid the foundation for recycling the cell wall fraction of bacterial biomass from large-scale production processes as substrate for fermentative production of food and feed additives.

CONCLUSION
Corynebacterium glutamicum was successfully metabolically engineered for utilization of the amino sugar MurNAc as alternative carbon source for growth and production of relevant value-added compounds, specifically L-lysine, L-glutamate and lycopene, from this carbon source lacking competing uses in human and animal nutrition.