Abstract
BackgroundOxaloacetate (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.ResultsIn 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 Ptac-M promoter that resulted in the final engineered strain C. glutamicum JL-69Ptac-Mgdh. Furthermore, the suitable addition of biotin accelerates the l-lysine production in strain JL-69Ptac-Mgdh because it elastically adjusts the carbon flux for cell growth and precursor supply. The final strain JL-69Ptac-Mgdh 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 (qLys, 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.ConclusionsOur 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.
Highlights
Oxaloacetate (OAA) and l-glutamate are essential precursors for the biosynthesis of l-lysine
A various genes involved in l-lysine production were characterized at the molecular level, and subsequently, the l-lysine producers were achieved by genetic engineering of l-lysine biosynthetic pathway, central metabolic pathways as well as sugar uptake system in C. glutamicum [2, 4, 6,7,8]
Metabolic engineering PEP–pyruvate–OAA node to increase processor OAA supply Previous reports indicated that PEP–pyruvate–OAA node play an important role in cell growth and metabolites production, because it interconnects four central metabolic pathways of carbon metabolism, such as glycolytic pathway, anaplerotic pathway, gluconeogenesis and tricarboxylic acid (TCA) cycle [18, 19]
Summary
Oxaloacetate (OAA) and l-glutamate are essential precursors for the biosynthesis of l-lysine. One of the most prominent pathways in central metabolic pathways is the tricarboxylic acid (TCA) cycle, which provides several metabolic precursors and cofactors for cell growth and amino acids production [9]. OAA is a key intermediate in TCA cycle, which provides metabolites and energy for cell growth, and for amino acid biosynthesis [10]. Properly adjusting CS activity to balance the cell growth and precursor supply is the wisest choice to increase the l-lysine yield and productivity
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