Engineering of a genome-reduced strain Bacillus amyloliquefaciens for enhancing surfactin production
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Microbial Cell Factories Open Access
RESEARCH
Engineering of a genome‑reduced strain Bacillus amyloliquefaciens for enhancing surfactin production Fang Zhang1, Kaiyue Huo1, Xingyi Song1, Yufen Quan1, Shufang Wang1, Zhiling Zhang2,3*, Weixia Gao4* and Chao Yang1*
Abstract Background: Genome reduction and metabolic engineering have emerged as intensive research hotspots for constructing the promising functional chassis and various microbial cell factories. Surfactin, a lipopeptide-type biosurfactant with broad spectrum antibiotic activity, has wide application prospects in anticancer therapy, biocontrol and bioremediation. Bacillus amyloliquefaciens LL3, previously isolated by our lab, contains an intact srfA operon in the genome for surfactin biosynthesis. Results: In this study, a genome-reduced strain GR167 lacking ~ 4.18% of the B. amyloliquefaciens LL3 genome was constructed by deleting some unnecessary genomic regions. Compared with the strain NK-1 (LL3 derivative, ΔuppΔpMC1), GR167 exhibited faster growth rate, higher transformation efficiency, increased intracellular reducing power level and higher heterologous protein expression capacity. Furthermore, the chassis strain GR167 was engineered for enhanced surfactin production. Firstly, the iturin and fengycin biosynthetic gene clusters were deleted from GR167 to generate GR167ID. Subsequently, two promoters PRsuc and PRtpxi from LL3 were obtained by RNA-seq and promoter strength characterization, and then they were individually substituted for the native srfA promoter in GR167ID to generate GR167IDS and GR167IDT. The best mutant GR167IDS showed a 678-fold improvement in the transcriptional level of the srfA operon relative to GR167ID, and it produced 311.35 mg/L surfactin, with a 10.4-fold increase relative to GR167. Conclusions: The genome-reduced strain GR167 was advantageous over the parental strain in several industrially relevant physiological traits assessed and it was highlighted as a chassis strain for further genetic modification. In future studies, further reduction of the LL3 genome can be expected to create high-performance chassis for synthetic biology applications. Keywords: Bacillus amyloliquefaciens, Genome reduction, Promoter engineering, Surfactin production
*Correspondence: [email protected]; [email protected]; [email protected] 1 Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Key Laboratory of Bioactive Materials for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China 2 Department of Oral and Maxillofacial Radiology, Tianjin Stomatological Hospital, School of Medicine, Nankai University, Tianjin 300041, China 4 MOE Key Laboratory of Industrial Fermentation Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, China Full list of author information is available at the end of the article
Background With the development of systems and synthetic biology, numerous studies have focused on the design and constru
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