Abstract
BACKGROUND: Oxaloacetate (OAA) and L-glutamate are essential precursors for the biosynthesis of L-lysine. Reasonable control of all potentially rate-limiting steps, including the precursors supply rate, is of vital importance to maximize the efficiency of L-lysine fermentation process. RESULTS: In this paper, we have rationally engineered the tricarboxylic acid (TCA) cycle that increased the carbon yield (from 36.18 to 59.65%), final titer (from 14.47 ± 0.41 to 23.86 ± 2.16 g L(-1)) and productivity (from 0.30 to 0.50 g L(-1) h(-1)) of L-lysine by Corynebacterium glutamicum in shake-flask fermentation because of improving the OAA and L-glutamate availability. To do this, the phosphoenolpyruvate-pyruvate-oxaloacetate (PEP-pyruvate-OAA) node's genes ppc and pyc were inserted in the genes pck and odx loci, the P1 promoter of the TCA cycle's gene gltA was deleted, and the nature promoter of glutamate dehydrogenase-coding gene gdh was replaced by P(tac-M) promoter that resulted in the final engineered strain C. glutamicum JL-69P(tac-M) gdh. Furthermore, the suitable addition of biotin accelerates the L-lysine production in strain JL-69P(tac-M) gdh because it elastically adjusts the carbon flux for cell growth and precursor supply. The final strain JL-69P(tac-M) gdh could produce 181.5 ± 11.74 g L(-1) of L-lysine with a productivity of 3.78 g L(-1) h(-1) and maximal specific production rate (q(Lys, max.)) of 0.73 ± 0.16 g g(-1) h(-1) in fed-batch culture during adding 2.4 mg L(-1) biotin with four times. CONCLUSIONS: Our results reveal that sufficient biomass, OAA and L-glutamate are equally important in the development of L-lysine high-yielding strain, and it is the first time to verify that fed-batch biotin plays a positive role in improving L-lysine production.