Abstract
This work aimed to develop an efficient L-leucine industrial production strain of Corynebacterium glutamicum by using metabolic engineering. A recombinant C. glutamicum strain was constructed by expressing a feedback-resistant leuA-encoded 2-isopropylmalate synthase (IPMS) that carries three amino acid exchanges (R529H, G532D and L535V) from the mutant strain C. glutamicum ML1-9 which was obtained by screening for structural analogues. In order to improve the expression of IPMS, a strong promoter (tac promoter) was used to ensure efficient expression of the rate-limiting enzyme. In addition, reasonable metabolic modifications on the central carbon metabolic pathway and competitive metabolic pathways to optimize the L-leucine biosynthesis pathway by redistribution of various types of precursors and repression of negative regulation were used aimed for increased L-leucine production. The modifications involved (1) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (2) deletion of the gene encoding the AlaT to decrease the concentration of extracellular L-alanine, and increased availability of pyruvate for L-leucine formation, (3) deletion of the gene encoding the threonine dehydratase to abolish L-isoleucine synthesis and to eliminate the intermediate precursor of L-isoleucine biosynthesis competing with L-leucine biosynthesis, (4) inactivation of the pantothenate synthetase to increase α- ketoisovalerate formation, and to enable its further conversion to L-leucine, and (5) inactivation of lactate dehydrogenase to decrease lactate production and its pyruvate consumption, concomitant to decreased glucose consumption rates and prevention of lactic acid to restrict cell growth. The production performance of the engineered strain MDLeu-19/pZ8-1leuAr was characterized with cultivations in a bioreactor. Under fed-batch conditions in a 50-L automated fermentor, the best producer strain accumulated 38.1 g L-1 of L-leucine; the molar product yield being 0.42 mol L-leucine per mole of glucose (glucose conversion rate attained 26.4%). Moreover, during large-scale fermentation using a 150-m3 fermentor, this strain produced more than 37.5 g L-1 L-leucine and the glucose conversion rate was 25.8%, making this process potentially viable for industrial production. Key words: Corynebacterium glutamicum, L-leucine, metabolic engineering, fermentation, industrial production.
Highlights
Corynebacterium glutamicum is a gram-positive, facultatively anaerobic, non-spore-forming, soil bacterium, which was originally discovered as a L-glutamic acidsecreting microorganism in the 1950s (Kinoshita et al., 2004; Nakayama et al, 1961; Udaka, 1960) and initially known as Micrococcus glutamicus; later various isolates were identified (Liebl et al, 1991)
The modifications involved (1) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (2) deletion of the gene encoding the AlaT to decrease the concentration of extracellular L-alanine, and increased availability of pyruvate for L-leucine formation, (3) deletion of the gene encoding the threonine dehydratase to abolish L-isoleucine synthesis and to eliminate the intermediate precursor of L-isoleucine biosynthesis competing with L-leucine biosynthesis, (4) inactivation of the pantothenate synthetase to increase α- ketoisovalerate formation, and to enable its further conversion to L-leucine, and (5) inactivation of lactate dehydrogenase to decrease lactate production and its pyruvate consumption, concomitant to decreased glucose consumption rates and prevention of lactic acid to restrict cell growth
The deletions of ilvA, ldh, alaT, panBC and ltbR in C. glutamicum ML1-9 were confirmed by colony polymerase chain reaction (PCR) using relevant primers P1 and P4
Summary
Corynebacterium glutamicum is a gram-positive, facultatively anaerobic, non-spore-forming, soil bacterium, which was originally discovered as a L-glutamic acidsecreting microorganism in the 1950s (Kinoshita et al., 2004; Nakayama et al, 1961; Udaka, 1960) and initially known as Micrococcus glutamicus; later various isolates were identified (for example Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, and Corynebacterium lilium) (Liebl et al, 1991). C. glutamicum is used in industrial biotechnology to produce several million tons of L-amino acids, such as the flavor enhancer L-glutamate (2,300,000 t/year) and the feed additive L-lysine (1,600,000 t/year) annually (Becker et al, 2011; Chen et al, 2014; Woo and Park, 2014). C. glutamicum has become a platform organism in industrial biotechnology (Becker et al, 2012). Based on the increasing knowledge about this organism, C. glutamicum, is a highly efficient host for the expression of heterologous proteins (Scheele et al, 2013), and gradually is being developed into an efficient industrial producer of branched chain amino acids (BCAAs) (Hasegawa et al, 2013; Vogt et al, 2014; Yin et al, 2014). The annual demand of BCAAs is more than 2500 tons, out of which demand of 1000 ton is for L-leucine alone and constantly increasing
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