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PLoS One. 2017 Jun 29;12(6):e0179229. doi: 10.1371/journal.pone.0179229. eCollection 2017.

Tuning the transcription and translation of L-amino acid deaminase in Escherichia coli improves α-ketoisocaproate production from L-leucine.

Song Y1,2, Li J1,2, Shin HD3, Liu L1,2, Du G1,2, Chen J1,2.

Author information

1
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
2
Synergetic Innovation of Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China.
3
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, United States of America.

Abstract

α-Ketoisocaproate (KIC) is used widely in the pharmaceutical and nutraceutical industries. In previous studies, we achieved a one-step biosynthesis of KIC from l-leucine, using an Escherichia coli whole-cell biocatalyst expressing an l-amino acid deaminase (l-AAD) from Proteus vulgaris. Herein, we report the fine-tuning of l-AAD gene expression in E. coli BL21 (DE3) at the transcriptional and translational levels to improve the KIC titer. By optimizing the plasmid origin with different copy numbers, modulating messenger RNA structure downstream of the initiation codon, and designing the sequences at the ribosome binding site, we increased biocatalyst activity to 31.77%, 24.89%, and 30.20%, respectively, above that achieved with BL21/pet28a-lad. The highest KIC titers reached 76.47 g·L-1, 80.29 g·L-1, and 81.41 g·L-1, respectively. Additionally, the integration of these three engineering strategies achieved an even higher KIC production of 86.55 g·L-1 and a higher l-leucine conversion rate of 94.25%. The enzyme-engineering strategies proposed herein may be generally applicable to the construction of other biocatalysts.

PMID:
28662040
PMCID:
PMC5491005
DOI:
10.1371/journal.pone.0179229
[Indexed for MEDLINE]
Free PMC Article

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