Insights into the availability of surfactin-forming nonribosomal peptide synthetase in Bacillus subtilis

枯草芽孢杆菌能够产生大量的表面素，这是一种脂肽，被描述为最强大的生物表面活性剂之一。目前，为了提高细菌的产量，研究小组的重点是加强前体分子的提供以及培养参数和下游加工的适应性。然而，细胞内非核糖体肽合成酶（NRPS）亚基对表面素形成的可用性仍然未知。因此，缺乏对每个细胞在各自条件下NRPS数量的了解。这方面的信息将有助于计算不同生长阶段和条件下NRPS酶复合物的生产速率。这些知识应该用来调整培养参数，以达到最高的NRPS表达，并增强表面素形成的活性。在这种情况下，本提案中提到的第二点是NRPS表达与表面素形成前体分子可用性之间的关系。一些研究表明，代谢途径的改善导致前体的可用性增强后，表面素的产生增加。然而，关于每个细胞内前体分子和NRPS酶复合物浓度之间的平衡，以及在培养过程中哪个因素受到限制，是一个悬而未决的问题。NRPS的翻译率不宜太高，以免造成过高的代谢负担，这是一个合理的假设，也得到了实验的支持。另一方面，NRPS复合体是生物合成表面素所必需的。因此，为了灵活地优化不同菌株NRPS的表达水平，首先必须建立确定每个细胞NRPS量的方法，并确定实际NRPS蛋白量。

Bacillus subtilis is capable to produce notable amounts of surfactin, a lipopeptide described as one of the most powerful biosurfactants. Currently, to improve bacterial production, research groups are focused on enhanced provision of precursor molecules as well as adaptations in cultivation parameters and downstream processing. However, the intracellular availability of nonribosomal peptide synthetase (NRPS) subunits for surfactin formation is still unknown. Hence, there is a lack of knowledge about the number of NRPS per cell under respective conditions. Information about this issue will help to calculate a production rate of NRPS enzyme complex during different growth phases and conditions.This knowledge should then be used to adapt cultivation parameters for highest possible NRPS expression combined with enhanced activity for surfactin formation.In this context, a second point addressed in this proposal is the relationship between NRPS expression and availability of precursor molecules for surfactin formation. Several studies showed an increase in surfactin production after improvement of metabolic pathways leading to enhanced availability of precursors. Nevertheless, the open question is about the balance between intracellular concentration of precursor molecules and NRPS enzyme complexes per cell and which factor is limiting during cultivation.It is a reasonable assumption, which is also experimentally supported, that the amount of NRPS translational rate should not be too high so as not to cause too high a metabolic burden. On the other hand, the NRPS complex is needed for biosynthesis of surfactin. Therefore, in order to flexibly optimize the expression level of NRPS for different strains, methods to determine the amount of NRPS per cell must first be established and the actual NRPS protein amount must be determined.