Abstract
2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l−1) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l−1 h−1. Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol.
Highlights
Corynebacterium glutamicum is the major host for biotechnological production of amino acids, the most important ones being the flavor enhancer L-glutamate and the feed additive L-lysine
The spectrum of amino acids produced with C. glutamicum includes the essential branched-chain amino acids (BCAAs) L-valine, L-isoleucine and L-leucine, which are produced in quantities of up to 5000 tons per year in a steadily growing market (Becker and Wittmann, 2012)
In a first series of experiments, we deleted ilvE in the wild-type C. glutamicum ATCC 13032 and transformed the ΔilvE mutant with plasmid pAN6-leuA_B018, carrying an IPTGinducible leuA allele encoding a feedback-resistant 2-isopropylmalate synthase (IPMS) (Vogt et al, 2014). 2-Isopropylmalate synthase of C. glutamicum is strongly inhibited by L-leucine with a Ki of 0.4 mM (Pátek et al, 1994) and the presence of a feedback-resistant variant is the key for L-leucine overproduction (Vogt et al, 2014)
Summary
Corynebacterium glutamicum is the major host for biotechnological production of amino acids, the most important ones being the flavor enhancer L-glutamate and the feed additive L-lysine. Besides a biotransformation process with Rhodococcus opacus using L-leucine as substrate for KIC formation (Zhu et al, 2011), fermentative processes with glucose as substrate have recently been described for the production of KIV (Krause et al, 2010) and KIC (Bückle-Vallant et al, 2014), showing that deletion of ilvE in certain engineered C. glutamicum strains results in KIC formation.
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