Enhanced production of iturin A by strengthening fatty acid synthesis modules in Bacillus amyloliquefaciens

Abstract

Iturin A is a biosurfactant with various applications, and its low synthesis capability limits its production and application development. Fatty acids play a critical role in cellular metabolism and target product syntheses, and the relationship between fatty acid supplies and iturin A synthesis is unclear. In this study, we attempted to increase iturin A production via strengthening fatty acid synthesis pathways in Bacillus amyloliquefaciens. First, acetyl-CoA carboxylase AccAD and ACP S-malonyltransferase fabD were overexpressed via promoter replacement, and iturin A yield was increased to 1.36 g/L by 2.78-fold in the resultant strain HZ-ADF1. Then, soluble acyl-ACP thioesterase derived from Escherichia coli showed the best performance for iturin A synthesis, as compared to those derived from B. amyloliquefaciens and Corynebacterium glutamicum, the introduction of which in HZ-ADF1 further led to a 57.35% increase of iturin A yield, reaching 2.14 g/L. Finally, long-chain fatty acid-CoA ligase LcfA was overexpressed in HZ-ADFT to attain the final strain HZ-ADFTL2, and iturin A yield reached 2.96 g/L, increasing by 6.59-fold, and the contents of fatty acids were enhanced significantly in HZ-ADFTL2, as compared to the original strain HZ-12. Taken together, our results implied that strengthening fatty acid supplies was an efficient approach for iturin A production, and this research provided a promising strain for industrial production of iturin A.

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

“…Plasmid pHY300PLK was applied for gene overexpression strain construction, and construction procedure of gene ycxA overexpression strain was served as an example (Gao et al 2022 ). Briefly, promoter P bacA (Genebank ID 16053260), gene ycxA (14,767,903), and amyL terminator (16,055,532) were amplified and fused by SOE-PCR; the fusion fragment was inserted into pHY300PLK at Eco RI/ Sac I sites; and diagnostic PCR and DNA sequence confirmed that YcxA overexpression vector pHY-YcxA was successfully constructed.…”

“…As for iturin A, the promoter of iturin A synthase operon was replaced by promoter P bacA in B. amyloliquefaciens HZ-12, which led to 1.3-fold increases of iturin A yield in the previous work of our group (Xu et al 2020 ). Meanwhile, strengthening fatty acid synthesis via overexpression of acetyl-CoA carboxylase AccAD, ACP S-malonyltransferase FabD, soluble acyl ACP thioesterase TesA, and long-chain fatty acid-CoA ligase LcfA benefited iturin A synthesis, which yield was increased to 2.96 g/L by 2.95-fold (Gao et al 2022 ). Although various works have been done for efficient synthesis of iturin A, its yield (2 ~ 4 g/L) was still significantly lower than that of surfactin (> 10 g/L) (Table S1 ), which limits iturin A application development.…”

“…B. amyloliquefaciens HZ-12 is a non-pathogenic strain for α-glucosidase inhibitor 1-deoxynojirimycin (1-DNJ) production, and it also owns the synthesis capability of iturin A (Cai et al 2017 ). In the previous research of our group, strengthening iturin A synthetase cluster and fatty acids supplies were conducted for iturin A production (Gao et al 2022 ; Xu et al 2020 ). In this research, to increase iturin A production, the modules of iturin A synthetase gene cluster, fatty acid and precursor amino acid supplies, substrate corn starch utilization, by-product (poly-γ-glutamic acid (γ-PGA), surfactin, extracellular polysaccharide) synthesis, and iturin A transport were engineered for strain optimization (Fig.…”

The metabolic and physiological responses to spaceflight of a lipopeptide- producing Bacillus subtilis

“…Surfactin synthesis was accomplished by non-ribosomal peptide synthetase (NRPSs), which mainly involves three steps: the synthesis of fatty acid chain, the activation and loading of fatty acids, and the extension of the peptide chain [51]. Fatty acid synthesis plays as a critical role in lipopeptide synthesis and the gene expression levels are positively correlated with the lipopeptide yield [52,53]. Previous studies have proved that strengthening branched-chain fatty acid biosynthetic pathways was an e cient approach to enhance lipopeptide yield [54].…”

Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes

“…13 A remarkable 6.59-fold increase in iturin A yield was attained through enhanced fatty acid supply. 14 The suppression of the spoIIIE gene, which is involved in the development of spores and biofilms, led to a 37% enhancement in the production of fengycin. 15 Increased expression of the yngH gene, which encodes for biotin carboxylase II, improves the production of fatty acids and increases surfactin yield by 43%.…”