Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A

• Published in: Applied microbiology and biotechnology Vol. 108; no. 1; p. 311
• Main Authors: She, Menglin, Zhou, Huijuan, Dong, Wanrong, Xu, Yuxiang, Gao, Lin, Gao, Jiaming, Yang, Yong, Yang, Zhifan, Cai, Dongbo, Chen, Shouwen
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.12.2024; Springer Nature B.V
• Subjects: 5' Untranslated Regions; Applied Genetics and Molecular Biotechnology; Bacillus amyloliquefaciens; Bacillus amyloliquefaciens - genetics; Bacillus amyloliquefaciens - metabolism; Biodegradability; Biodegradation; Biomedical and Life Sciences; Biosurfactants; Biotechnology; Clusters; Corn; Genes; Glucosidase; Glutamic acid; Industrial production; Iturin A; Life Sciences; Ligases - genetics; Ligases - metabolism; Metabolic engineering; Metabolic Engineering - methods; Metabolism; Metabolites; Microbial Genetics and Genomics; Microbiology; Modular engineering; mRNA stability; Peptides, Cyclic - biosynthesis; Peptides, Cyclic - genetics; Peptides, Cyclic - metabolism; poly-γ-Glutamic acid; Polysaccharides; Production costs; Promoter Regions, Genetic; Starch; Surface-Active Agents - metabolism; Surfactin; Synthesis; α-Glucosidase; Iturin A; Metabolic engineering; Synthetase gene cluster; Precursor supply; Bacillus amyloliquefaciens
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-024-13083-9

As a kind of biosurfactants, iturin A has attracted people’s wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens . Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5′-UTRs of downstream genes ( ituA , ituB , and ituC ) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH , serC , and introducing ocD . Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. Key points • Optimizing 5′-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.