Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis

doi:10.1186/s12934-022-01823-3

	Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis
	Ding,Xiumin 1,2; Zheng,Zhiming 1; Zhao,Genhai 1; Wang,Li 1; Wang,Han 1; Yang,Qiang 1,2; Zhang,Mengxue 1,2; Li,Luyao 1,2; Wang,Peng 1
刊名	Microbial Cell Factories
	2022-05-28
卷号	21
关键词	Menaquinone-7 Bacillus subtilis Synthetic biology Metabolic engineering Cofactor engineering NADH kinase
DOI	10.1186/s12934-022-01823-3
通讯作者	Zheng,Zhiming(zhengzhiming2014@163.com) ; Wang,Peng(pengwang@ipp.ac.cn)
英文摘要	AbstractBackgroundMenaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation.ResultsWe used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01?mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07?mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion.ConclusionsIn summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.
语种	英语
出版者	BioMed Central
WOS记录号	BMC:10.1186/S12934-022-01823-3
内容类型	期刊论文
源URL	[http://ir.hfcas.ac.cn:8080/handle/334002/129494]
专题	中国科学院合肥物质科学研究院
通讯作者	Zheng,Zhiming; Wang,Peng
作者单位	1.Chinese Academy of Sciences; Institute of Intelligent Machines, Hefei Institutes of Physical Science 2.University of Science and Technology of China
推荐引用方式 GB/T 7714	Ding,Xiumin,Zheng,Zhiming,Zhao,Genhai,et al. Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis[J]. Microbial Cell Factories,2022,21.
APA	Ding,Xiumin.,Zheng,Zhiming.,Zhao,Genhai.,Wang,Li.,Wang,Han.,...&Wang,Peng.(2022).Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis.Microbial Cell Factories,21.
MLA	Ding,Xiumin,et al."Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis".Microbial Cell Factories 21(2022).