Engineering of Corynebacterium glutamicum for growth and l-lysine and lycopene production from N-acetyl-glucosamine

Abstract

Sustainable supply of feedstock has become a key issue in process development in microbial biotechnology. The workhorse of industrial amino acid production Corynebacterium glutamicum has been engineered towards utilization of alternative carbon sources. Utilization of the chitin-derived aminosugar N-acetyl-glucosamine (GlcNAc) for both cultivation and production with C. glutamicum has hitherto not been investigated. Albeit this organism harbors the enzymes N-acetylglucosamine-6-phosphatedeacetylase and glucosamine-6P deaminase of GlcNAc metabolism (encoded by nagA and nagB, respectively) growth of C. glutamicum with GlcNAc as substrate was not observed. This was attributed to the lack of a functional system for GlcNAc uptake. Of the 17 type strains of the genus Corynebacterium tested here for their ability to grow with GlcNAc, only Corynebacterium glycinophilum DSM45794 was able to utilize this substrate. Complementation studies with a GlcNAc-uptake deficient Escherichia coli strain revealed that C. glycinophilum possesses a nagE-encoded EII permease for GlcNAc uptake. Heterologous expression of the C. glycinophilum nagE in C. glutamicum indeed enabled uptake of GlcNAc. For efficient GlcNac utilization in C. glutamicum, improved expression of nagE with concurrent overexpression of the endogenous nagA and nagB genes was found to be necessary. Based on this strategy, C. glutamicum strains for the efficient production of the amino acid L-lysine as well as the carotenoid lycopene from GlcNAc as sole substrate were constructed.

Transcription of Sialic Acid Catabolism Genes in Corynebacterium glutamicum Is Subject to Catabolite Repression and Control by the Transcriptional Repressor NanR

“…In the related C. glycinophilum, GlcNAc uptake and phosphorylation are accomplished by the nagE-encoded EII permease of the PTS (10). Alongside heterologous expression of nagE from C. glycinophilum, plasmid-encoded overexpression of endogenous nagA and nagB was shown to be necessary for efficient GlcNAc utilization in C. glutamicum (10). Moreover, plasmid-encoded overexpression of nagE from C. glycinophilum in C. glutamicum M4 did not lead to efficient growth on GlcNAc (10), despite the fact that this strain possesses high levels of NagA and NagB due to a point mutation within the promoter of nagAB and is thus able to efficiently utilize GlcN (24).…”

“…Thus, NanR of C. glutamicum is the first transcriptional regulator of sialic acid metabolism depending on the inducer GlcNAc-6P and the first of the GntR-type regulators independent of Neu5Ac (see Table S1). The use of GlcNAc-6P as a signal molecule for the control of Neu5Ac degradation in C. glutamicum is plausible, as this organism cannot utilize GlcNAc as the substrate for growth (10). In E. coli, for which the pathway for sialic acid utilization is regarded as an addition to the pathway for the utilization of the amino sugar GlcNAc (74), distinct regulatory circuits exist for differential control of the genes for Neu5Ac utilization and genes for GlcNAc utilization.…”

“…C. glutamicum utilizes a large variety of sugars and organic acids as sources of carbon and energy (6,7) and additionally has been genetically engineered for the utilization of alternative feedstocks such as starch, glycerol, xylose, glucuronic acid, and N-acetylglucosamine (8)(9)(10)(11)(12). In contrast to many other bacterial species, C. glutamicum prefers to use multiple carbon sources simultaneously.…”

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

“…C. glutamicum grows aerobically on a wide variety of carbon sources, including the sugars glucose, fructose, and sucrose, as well as organic acids, such as citrate, acetate, pyruvate, D-lactate, and L-lactate. Significant efforts have been focused on engineering C. glutamicum to utilize starch (17), glucans (18), crude glycerol (19), amino sugars (20,21), and pentose sugars present in lignocellulosic hydrolysates (22), and the disaccharide cellobiose (23). However, glucose, fructose, and sucrose present in molasses or derived from starch hydrolysis are the main substrates used in industrial fermentations (24).…”

Engineering of Corynebacterium glutamicum for growth and production of L-ornithine, L-lysine, and lycopene from hexuronic acids

“…As yet, feed in the industry relies mainly on glucose and fructose [8], which also have competing uses in the food industry. So far, alternative carbon source utilization in C. glutamicum has been successfully established, among others, for xylose [27,28], galactose [29], arabinose [14,30], glucosamine [31], Nacetyl-glucosamine [32], and glycerol [33]. Plant cell wall materials such as lignocellulose and pectin are promising alternatives as carbon source.…”