The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA.
Microb Cell Fact. 2019 Aug 6;18(1):128. doi: 10.1186/s12934-019-1183-0.
Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively.
In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L of AC and 17.08 g L of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L AC in one-step biocatalysis with the yield of 0.67 mol mol. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L of 2,3-BD with the yield of 0.80 mol mol.
The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.
乙酰丙酮（AC）和 2,3-丁二醇（2,3-BD）作为极具前景的生物基平台化学品，由于其广泛的应用而受到更多关注。然而，在其自然产生菌中，AC 和 2,3-BD 的非高效底物转化和相互转化不仅导致选择性低，而且增加了下游纯化的难度。因此，构建更合适的工程菌株应该是分别选择性生产 AC 和 2,3-BD 的可靠策略。
在本研究中，成功地在非天然 AC 和 2,3-BD 产生菌 Corynebacterium crenatum 中表达了来源于枯草芽孢杆菌 168 的分别的 AC（alsS 和 alsD）和 2,3-BD 生物合成途径基因（alsS、alsD 和 bdhA），并生成了重组菌株 C. crenatum SD 和 C. crenatum SDA，分别证实它们可以产生 9.86 g/L 的 AC 和 17.08 g/L 的 2,3-BD。为了进一步提高 AC 和 2,3-BD 的选择性，然后敲除了 C. crenatum 中的 AC 还原基因（butA）和乳酸脱氢酶基因（ldh）。最终，C. crenatumΔbutAΔldh SD 在一步生物催化中产生了 76.93 g/L 的 AC，产率为 0.67 mol/mol。同时，消除乳酸生成并增强 2,3-丁二醇脱氢酶活性后，C. crenatumΔldh SDA 合成了 88.83 g/L 的 2,3-BD，产率为 0.80 mol/mol。
本研究中构建的合成工程菌 C. crenatumΔbutAΔldh SD 和 C. crenatumΔldh SDA 被证明是一种高效的微生物细胞工厂，可用于选择性地生产 AC 和 2,3-BD。基于本研究的见解，建议对 C. crenatum 进行进一步的合成工程改造，以生产 AC 和 2,3-BD。
