Metabolic engineering of Corynebacterium glutamicum to enhance L-leucine production

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/pZ8-1leuA r 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-m 3 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.

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux

“…The regular strategies to improve the l -leucine yield have largely focused on the metabolic engineering of removing feedback inhibition [18,40,41], upstream central carbon flux [3], and downstream by-product synthesis pathways [8]. This is the first report on improvement in redox flux, to enhance the production of l -leucine by C. glutamicum .…”

“…Corynebacterium glutamicum is widely used in industrial fermentation to produce several million tons of amino acids annually, in particular the flavor enhancer l -glutamate and the feed additive l -lysine [6,7]. Besides l -glutamate and l -lysine, C. glutamicum can also be used for production of a variety of other amino acids, including the BCAAs [8,9,10]. To date, C. glutamicum mutant strains remain the dominant industrial producers of l -leucine [11].…”

“…l -leucine is synthesized from pyruvate in a pathway comprising seven reactions (Figure 1), catalyzed by acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase (AHAIR), dihydroxyacid dehydratase (DHAD), α-isopropylmalate synthase (IPMS), α-isopropylmalate isomerase, β-isopropylmalate dehydrogenase (IPMD), and branched-chain amino acid transaminase (TA) [16]. The l -leucine biosynthetic pathway is complex and tightly regulated because the overlapping biosynthesis pathways of the three BCAAs partly share the same precursors and enzymes [8]. l -valine and l -isoleucine are synthesized in parallel pathways in which the reaction steps are catalyzed by the same enzymes, whereas l -leucine is formed in a specific series of four reactions, starting with the last intermediate of l -valine biosynthesis [17].…”

“…Additionally, the gene leuB together with the genes leuCD is repressed by the transcriptional regulator LtbR. Therefore, the regular strategies for the construction of l -leucine producing strains are, removing the l -leucine inhibition and transcriptional attenuation ( leuA , ilvBNC ) [3,18], enhancing the metabolic influx ( leuACDB , ilvBNCE ) to precursor [8], altering substrate and enzyme specificity [19], and so on. However, improvement of the redox cofactor requirement for the l -leucine production has not been investigated.…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…For example, overexpression of a mutated leuA variant (amino acid exchanges R529H, G532D) was combined with deletion of ltbR (leuBCD), PTS-independent glucose uptake via IolR deletion, and an attenuated flux through citrate synthase (gltA), strategies yielding a strain that achieved 24 g.l −1 l-leucine within 72 h in a fed-batch process [68]. The best performance was achieved by metabolic engineering of a classically generated mutant strain (ML1-9), obtained by screening of structural l-leucine analogues [69]. The producer overexpressed a feedback resistant leuA gene variant with three amino acid exchanges (R529H, G532D, and L535V) but lacked ltbR to increase expression of leuBCD and lacked alaT, panBC, and ilvA to increase precursor availability (Table 1).…”