Identification of α-isopropylmalate synthase mutants capable of overproducing L-leucine in Corynebacterium glutamicum

Abstract

The enzyme α-isopropylmalate synthase (α-IPMS) catalyzes the first step of L-leucine biosynthesis and is regulated via feedback inhibition by L-leucine. The gene of α-IPMS variant from strain ARTP-L04 was cloned and sequenced. Interestingly, amino acid mutations of Gly92Asp, Ile162Val, Arg494His and Gly526Asp occurred in the α-IPMS variant. The enzyme of α-IPMS variant was characterized, and exhibited higher resistance to feedback inhibition by L-leucine. In the presence of 20 mM L-leucine, the activity of the α-IPMS variant was still retained at over 50%. The α-IPMS variant almost removed feedback inhibition by L-leucine, while there was no significant difference in specific activities of the α-IPMS variant and wild-type α-IPMS. For the α-IPMS variant, the capacity to overproduce L-leucine was evaluated by recombinant Corynebacterium glutamicum strains. Interestingly, expression of the α-IPMS variant could significantly enhance L-leucine production, especially co-expressed with acetohydroxyacid synthase (accumulating 7.79 g/L L-leucine). Collectively, our findings provided valuable insights into further development in genetic engineering for L-leucine production.

KEYWORDS:

• L-leucine
• α-isopropylmalate synthase
• feedback inhibition
• Corynebacterium glutamicum

Acknowledgments

This work has been published as a preprint by Research Square (https://www.researchsquare.com/article/rs-1717587/v1). This work was supported by the Qingchuang Science and Technology Support Program of Shandong Provincial College with regard to use of laboratory and equipment.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Methodology, Y.-F.G. and Y.-L. W.; investigation, Y.-F.G., Y. L. and. L. S.; data curation, L. S. and J.-H. L.; writing-original draft preparation, Y.-F.G. and Y.-L. W; writing-review and editing, G.-Q. M. and J.-H. L.; supervision, G.-Q. M. and Y.-L. W.; project administration, G.-Q. M.; funding acquisition, G.-Q. M. and Y.-L. W. All authors have read and agreed to the published version of the manuscript.

Data availability statement

The authors confirm that raw data supporting the findings of this study are available at https://www.scidb.cn/anonymous/UXJlWWpp.

Additional information

Funding

This research was funded by the Project of Shandong Province Higher Educational Science and Technology Program (J17KA125) and the Key R & D Project of Shandong Province (2021SFGC1205).