Structure and Isoforms of Surfactin Produced by Mutant Strain Bacillus subtilis PY79sfp+

Objective: To boost surfactin (Sur) production, a novel Bacillus subtilis PY79 strain was engineered by expressing the sfp gene. The resulting PY79sfp+ strain yielded 300 mg/L, doubling the output of the native NCIB 3610 strain. While 6S-1 and 6S-2 RNA gene deletions had no effect, the main C16 surfactin isoform was consistently predominant across all strains, confirming its industrial relevance. Methods: qPCR was performed to evaluate the effect of 6S RNAs on transcription of srfA operon, colorimetric test was used for quantitative analysis of surfactin produced, HPLC, MALDI-TOF and amino acid analysis were performed for isoforms analysis. Results and Discussion: Surfactin-producing activity in B. subtilis PY79 cells was restored using the pHT01 plasmid with sfp gene, that resulted with 2-fold increase of surfactin yield using the wild-type cells. However, the transcription level was increased in cells with 6S-1 RNA knockout, there was no effect of 6S RNAs on the amount of Sur produced. All the Sur produced by mutant cells consists of the conservative amino acid sequence Glu-Leu-Leu-Val-Asp-Leu-Leu. On the other hand, the fatty acid length consists of 14-16 carbon atoms. The main isoform for all the cell lines (wild-type and knockout) is C16. Conclusions: Research shows that the B. subtilis PY79 is promising for engineering a Sur superproducer. Restoring the native sfp gene sequence doubled Sur production compared to the natural NCIB 3610 strain. Deleting the 6S-1 RNA gene did not increase yield, despite raising surfactin synthetase mRNA levels. Both strains produced three Sur isoforms, with the C16 variant (containing a 16-carbon fatty acid chain) being predominant. This is the most commercially and medically valuable isoform due to its beneficial properties. The obtained results can be applied to the development of a superproducing strain based on the PY79sfp+ strain, described for the first time in this study. This can be achieved by optimizing cultivation conditions, implementing genetic engineering modifications, and utilizing other tools. Future work will focus on further increasing surfactin yield to enable its potential production for various applications.