Optimization of n -butanol synthesis in Lactobacillus brevis via the functional expression of thl , hbd , crt and ter
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METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY - ORIGINAL PAPER
Optimization of n‑butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter Qi Li1,2 · Meixian Wu2 · Zhiqiang Wen2 · Yuan Jiang2 · Xin Wang2 · Yawei Zhao2 · Jinle Liu2 · Junjie Yang2 · Yu Jiang3 · Sheng Yang2,3 Received: 22 July 2020 / Accepted: 2 November 2020 © Society for Industrial Microbiology and Biotechnology 2020
Abstract N-butanol is an important chemical and can be naturally synthesized by Clostridium species; however, the poor n-butanol tolerance of Clostridium impedes the further improvement in titer. In this study, Lactobacillus brevis, which possesses a higher butanol tolerance, was selected as host for heterologous butanol production. The Clostridium acetobutylicum genes thl, hbd, and crt which encode thiolase, β-hydroxybutyryl-CoA dehydrogenase, and crotonase, and the Treponema denticola gene ter, which encodes trans-enoyl-CoA reductase were cloned into a single plasmid to express the butanol synthesis pathway in L. brevis. A titer of 40 mg/L n-butanol was initially achieved with plasmid pLY15-opt, in which all pathway genes are codon-optimized. A titer of 450 mg/L of n-butanol was then synthesized when ter was further overexpressed in this pathway. The role of metabolic flux was reinforced with pLY15, in which only the ter gene was codon-optimized, which greatly increased the n-butanol titer to 817 mg/L. Our strategy significantly improved n-butanol synthesis in L. brevis and the final titer is the highest achieved amongst butanol-tolerant lactic acid bacteria.
Qi Li and Meixian Wu contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10295-020-02331-2) contains supplementary material, which is available to authorized users. * Sheng Yang [email protected] 1
College of Life Sciences, Sichuan Normal University, Chengdu 610101, China
2
Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China
3
Huzhou Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Huzhou 313000, China
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Vol.:(0123456789)
Journal of Industrial Microbiology & Biotechnology
DNA transformation efficiency (CFU/μg DNA)
Graphic abstract 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
W1S1R1 W1S1R2 W1S2R1 W1S2R2 W2S1R1 W2S1R2 W2S2R1 W2S2R2
Keywords Lactic acid bacteria · Lactobacillus brevis · Clostridium · Butanol · Tolerance Abbreviations LC–MS Liquid chromatography-mass spectrometry CFU Colony forming unit
Introduction N-butanol is a bulk chemical, expected to become one of the new generation of biofuels [1–3]. Its production by microbial fermentation has received much recent attention [4]; however, when compared with butanol produced from raw petroleum materials, biobutanol’s production cost is much higher [4–6]. A microorganism’s butanol tolerance limits the amount that can be produced, and the low concentrat
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