Search

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., (© 2024. The Author(s).)

1-Deoxynojirimycin (DNJ) is an efficient α-glucosidase inhibitor (α-GI) with potential applications in the prevention and treatment of diabetes. In this study, 16 Bacillus strains were screened for α-GI rate, and the strain HZ-12 with the highest α-GI rate was identified as Bacillus amyloliquefaciens through the analysis of physiological biochemical characteristics and 16S rDNA sequence. By LC-MS/Q-TOF analysis, the α-GI component produced by B. amyloliquefaciens HZ-12 was identified as DNJ. Soybean was used as the substrate for the solid-state fermentation; 870 mg/kg DNJ was produced by B. amyloliquefaciens HZ-12 after optimizing the fermentation conditions and media, which was 3.83-fold higher than the initial yield. Also, evaluations of nutraceutical enrichment in the form of anticoagulant activity, antioxidant activity, total nitrogen (TN), and total reducing sugars (TRS) of the B. amyloliquefaciens HZ-12 fermented soybeans were substantially higher than unfermented soybeans. This study provided a promising strain for high-level production of DNJ and produced nutraceutical enriched soybeans by fermentation.

• The promoter PbacA was proven as the most efficient promoter for iturin A synthesis. • Deletion of regulator gene abrB improved iturin A synthetase gene cluster transcription and iturin A production. • The titer of 2013.43 mg/L iturin A was produced by optimizing fermentation medium formulas, increased by 392.15 %. • Enhancing the synthetic capability of iturin A was beneficial for the suppression of A. alternate. Brown spot disease, caused by Alternaria alternate , is one of the most destructive leaf spot diseases that made a major impact on tobacco growing. Biocontrol has attracted increasing attentions as its features of environmental friendship, promoting plant growth, etc. Iturin A, with broad spectrum antifungal activity, is a kind of biosurfactant that mainly produced by Bacillus. In this study, the promoter of iturin A synthetase cluster of Bacillus amyloliquefaciens HZ-12 was respectively replaced by promoters P43, P bacA , P srfA and P ylb , our results implied that transcriptional level of gene ituD and iturin A titer showed consistent change trends, and P bacA was proven as the most efficient promoter, iturin A titer of which reached 950.08 ± 19.43 mg/L. Furthermore, regulator gene abrB was deleted to release the repression effect of AbrB on P bacA , ituD transcriptional level and iturin A titer were increased by 133.25 % and 20.88 %, respectively. Then, the maximum iturin A titer reached 2013.43 ± 32.86 mg/L by optimizing fermentation medium, increased by 392.15 % compared to the original (408.97 ± 21.35 mg/L). Finally, our results demonstrated that enhancing iturin A synthetic capability benefited the suppression of A. alternate. Collectively, this study provided a promising strain with an efficient fermentation medium for large-scale industrial production of iturin A. [ABSTRACT FROM AUTHOR]