Closing Fundamental Knowledge Gaps en Route to Efficient Surfactin Production

点击翻译按钮获取中文摘要

The lipopeptide Surfactin, which is formed by Bacillus subtilis non-ribosomally, is considered to be one of the most promising biosurfactants due to its exceptionally strong surface activity and various bioactive properties. In order to ensure a knowledge-based and efficient biotechnological production, basic knowledge gaps remain open, which must be closed. Some of the most urgent issues to be resolved are (1) surfactin biosynthesis regulation, (2) secretion and the (3) putative surfactin-mediated autoinhibition of B. subtilis. To achieve this, the genome of B. subtilis DSM 10T is to be sequenced before further genetic manipulation. Subsequently, the competence formation is separated from the surfactant production. In order to decouple the expression from the regulation, inducible promoters will be applied. The arrest of growth due to competence formation will be prevented by mutation of the comS ORF inside srfAB. A sporulation-deficient B. subtilis will be attended by deleting the spo0A gene. Dispensable regions including antibiotic production genes and phages will be deleted. The over-expression of yerP and ycxA is tested to clarify whether increased surfactin secretion facilitates the growth of cells with high concentrations of surfactin and has a positive effect on the resulting yields.The putative surfactin-mediated autoinhibition of B. subtilis will be elucidated prior to optimizing the fermentation procedure. Surfactin is produced and purified in adequate amounts to perform several antibiotic assays to quantify the inhibitory effect of surfactin on B. subtilis growth. Finally, the effect of some critical deletions in bioreactor cultivation experiments is analyzed.Two basic alternatives are planned for bioprocess development, depending on the prior results. In the case of a strong autoinhibitory effect of surfactin on the B. subtilis growth, the foam generated by the cells during fermentation will be removed using foam fractionation method. On the contrary, in the case of the tolerance of cells to high concentration of surfactin, fermentation will be carried out using antifoam agent. Preliminary experiments will determine whether cell growth is significantly altered in comparison to our standard fermentation process. For further optimization, fed-batch fermentations are performed to further evaluate the improvement in surfactin biosynthesis in the genetically optimized strains.