Abstract
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.
Highlights
Corynebacterium glutamicum is a predominantly aerobic, rodshaped, Gram-positive soil bacterium which is generally recognized as safe (GRAS)
Corynebacterium glutamicum, which has been engineered to utilize GlcN and GlcNAc (Matano et al, 2014, 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)
MurQ was tested by complementation of a E. coli mutant
Summary
Corynebacterium glutamicum is a predominantly aerobic, rodshaped, Gram-positive soil bacterium which is generally recognized as safe (GRAS). 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 been engineered to produce diamines, organic acids, carotenoids, proteins and biopolymers (Wendisch et al, 2016). 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)
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