NADPH Supply for L-Lysine Production

NADPH supply was also increased based on the membrane-integral nicotinamide nucleotide transhydrogenase PntAB from E. coli. PntAB reduces NADP to NADPH in an NADH-dependent reaction driven by the electrochemical proton gradient across the cytoplasmic membrane. Accordingly, the expression of pntAB from E. coli in C. glutamicum L-lysine producers increased L-lysine production by 10% on glucose, 70% on fructose, and 300% on sucrose [66]. 

The intermediate of L-lysine biosynthesis, L-aspartyl-semialdehyde, is also a precursor for the biosynthesis of L-threonine, L-isoleucine, and L-methionine. Homoserine dehydrogenase catalyses NADPH-dependent reduction of L-aspartyl-semialdehyde to L-homoserine. Flux from L-aspartyl-semialdehyde toward L-homoserine could be reduced by introducing alleles for less active homoserine dehydrogenase variants. In addition, L-lysine production increased as the prevailing threonine concentrations in such horn mutants were too low for feedback inhibition of aspartokinase [67, 68]. 

Trehalose accumulates as a byproduct during glucose-based L-lysine production by C. glutamicum. Overexpression of wild-type or mutated zwf decreased trehalose formation as consequence of an increased flux through the PPP [60]. The use of sucrose can also prevent the formation of certain byproducts at a L-lysine yield comparable to that on glucose, in contrast to fructose where the formation of lactate reduces L-lysine production by 30% [69]. Accumulation of lactate can be avoided by the deletion of the NAD-dependent L-lactate dehydrogenase gene IdhA [70]. 

The glycolytic intermediate pyruvate and the TCA cycle intermediate oxaloac-etate are direct precursors of the aspartate-derived amino acids. Thus, to improve L-lysine production, their supply has to be increased in a balanced manner. Withdrawal of oxaloacetate from the TCA cycle requires replenishment of the 

For better control of fermentation and to reduce production costs, complex media components are avoided and mostly refined carbon sources are used for the industrial production of L-lysine. Sucrose can be obtained from cane or beet molasses, and glucose is provided in hydrolysates of corn, cassava, or wheat starch [30, 83]. Ammonia, as nitrogen source, can be added pure or as salts [32]. Further media components are vitamins, in particular biotin, as well as salts and trace elements. Amino acids for auxotrophic production strains can be provided by peptones, corn steep liquors, or soybean meal hydrolysates [30]. Preferably, media are sterilized continuously, whereas carbon sources and nitrogen sources are typically sterilized separately to avoid Maillard-type reactions [32]. Sterility is important for processes with the mesophilic and neutrophilic C. glutamicum with bacilli as main contamination risk [84], while phage infection is hardly a problem. 

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