Improvement of macrolactins production by the genetic adaptation of Bacillus siamensis A72 to saline stress via adaptive laboratory evolution

Background Macrolactins, a type of macrolide antibiotic, are toxic to the producer strains. As such, its level is usually maintained below the lethal concentration during the fermentation process. To improve the production of macrolactins, we applied adaptive laboratory evolution technology to engineer a saline-resistant mutant strain. The hypothesis that strains with saline resistance show improved macrolactins production was investigated. Results Using saline stress as a selective pressure, we engineered a mutant strain with saline resistance coupled with enhanced macrolactins production within 60 days using a self-made device. As compared with the parental strain, the evolved strain produced macrolactins with 11.93% improvement in non-saline stress fermentation medium containing 50 g/L glucose, when the glucose concentration increased to 70 g/L, the evolved strain produced macrolactins with 71.04% improvement. RNA sequencing and metabolomics results revealed that amino acid metabolism was involved in the production of macrolactins in the evolved strain. Furthermore, genome sequencing of the evolved strain revealed a candidate mutation, hisD(D41Y), that was causal for the improved MLNs production, it was 3.42 times higher than the control in the overexpression hisD(D41Y) strain. Results revealed that saline resistance protected the producer strain from feedback inhibition of end-product (macrolide antibiotic), resulting in enhanced MLNs production. Conclusions In the present work, we successfully engineered a mutant strain with enhanced macrolactins production by adaptive laboratory evolution using saline stress as a selective pressure. Based on physiological, transcriptomic and genetic analysis, amino acid metabolism was found to benefit macrolactins production improvement. Our strategy might be applicable to improve the production of other kinds of macrolide antibiotics and other toxic compounds. The identification of the hisD mutation will allow for the deduction of metabolic engineering strategies in future research.

Automated summary

In this study, we successfully engineered a mutant strain with enhanced macrolactins production using a self-made device and adaptive laboratory evolution technology. The mutant strain showed saline resistance and increased macrolactins production within 60 days. Amino acid metabolism was found to be involved in the production of macrolactins in the evolved strain. The identified hisD mutation played a crucial role in the improvement of macrolactins production. This research provides insights for future metabolic engineering strategies to improve the production of other antibiotics and toxic compounds.