Closing Fundamental Knowledge Gaps en Route to Efficient Surfactin Production

由枯草芽孢杆菌非核糖体形成的脂肽表面活性蛋白因其极强的表面活性和多种生物活性而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和高效的生物技术生产，基本知识差距仍然存在，必须弥合。最迫切需要解决的问题是(1)表面素生物合成的调节，(2)分泌和(3)表面素介导枯草杆菌的自身抑制。为了实现这一点，枯草杆菌DSM 10T的基因组将在进一步的遗传操作之前进行测序。随后，将能力形成从表面活性剂生产中分离出来。为了使表达与调控脱钩，将使用诱导型启动子。SrfAB内部coms ORF的突变将阻止由于能力形成而导致的生长停滞。芽胞形成缺陷的枯草芽孢杆菌将通过删除sp0A基因来参与。包括抗生素生产基因和噬菌体在内的可有可无的区域将被删除。通过检测yerp和ycxA的过表达来阐明表面素分泌的增加是否促进了含有高浓度表面素的细胞的生长，并对所产生的产量有积极的影响。在优化发酵工艺之前，将首先阐明表面素介导的枯草杆菌的自抑制作用。生产和提纯足够量的表面活性物质，以进行多种抗生素检测，以量化表面活性物质对枯草杆菌生长的抑制作用。最后，对生物反应器培养实验中的一些关键缺失的影响进行了分析。根据先前的结果，规划了两个基本的生物过程开发方案。在表面素对枯草杆菌生长有很强的自抑制作用的情况下，用泡沫分级法去除发酵过程中细胞产生的泡沫。相反，在细胞对高浓度表面素有耐受性的情况下，将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着变化。为了进一步优化，进行了补料分批发酵，以进一步评估遗传优化菌株在表面素生物合成方面的改善。

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.