Dataset Information

Rational modification of tricarboxylic acid cycle for improving L-lysine production in Corynebacterium glutamicum.

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.

SUBMITTER: Xu JZ

PROVIDER: S-EPMC6035423 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

Rational modification of tricarboxylic acid cycle for improving L-lysine production in Corynebacterium glutamicum.

Xu Jian-Zhong JZ Wu Ze-Hua ZH Gao Shi-Jun SJ Zhang Weiguo W

Microbial cell factories 20180707 1

<h4>Background</h4>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.<h4>Results</h4>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<sup> ...[more]

PMID: 29981572

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Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 Â± 0.03 gg-1 and a volumetric productivity of 0.04 Â± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.

Dataset Information

Rational modification of tricarboxylic acid cycle for improving L-lysine production in Corynebacterium glutamicum.

Background

Results

Conclusions

Publications

Rational modification of tricarboxylic acid cycle for improving L-lysine production in Corynebacterium glutamicum.

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