Closing Fundamental Knowledge Gaps en Route to Efficient Surfactin Production

枯草芽孢杆菌（Bacillus subtilis）非核糖体合成的脂肽表面活性素（Surfactin）具有极强的表面活性和多种生物活性，被认为是最有前途的生物表面活性剂之一。为了确保以知识为基础的高效率生物技术生产，基本知识差距仍然存在，必须加以弥补。一些最迫切需要解决的问题是（1）表面活性素生物合成调节，（2）分泌和（3）推定的表面活性素介导的B自身抑制。枯草杆菌。为了实现这一点，我们将B.枯草芽孢杆菌DSM 10T在进一步的遗传操作之前被测序。随后，从表面活性剂生产中分离出能力形成。为了使表达与调节解耦，将应用诱导型启动子。由于感受态形成导致的生长停滞将通过srfAB内的comS ORF的突变来防止。孢子形成缺陷型B。枯草芽孢杆菌将通过删除spo0A基因来参与。包括抗生素产生基因和p53的可分配区域将被删除。测试yerP和ycxA的过表达以阐明增加的表面活性素分泌是否促进具有高浓度表面活性素的细胞的生长，并且对所得产量具有积极影响。将在优化发酵程序之前阐明枯草芽孢杆菌。产生并纯化足够量的表面活性素，以进行几种抗生素测定来定量表面活性素对B的抑制作用。枯草杆菌生长最后，分析了生物反应器培养实验中某些关键删除的影响，并根据先前的结果，提出了两种生物过程开发的基本方案。在表面活性素对B具有强的自抑制作用的情况下。为了抑制枯草芽孢杆菌的生长，将使用泡沫分离方法除去发酵期间由细胞产生的泡沫。相反，在细胞对高浓度表面活性素耐受的情况下，会使用消泡剂进行发酵。初步实验将确定与我们的标准发酵过程相比，细胞生长是否发生了显著变化。为了进一步优化，进行补料分批发酵以进一步评估遗传优化的菌株中表面活性素生物合成的改善。

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.