Data_Sheet_3_Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches.CSV

The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

在日常生活中产品中应用天然生物大分子，以取代传统的合成制造，这一市场正日益增长。此现象之例证为相容溶质ectoine，该物质广泛存在于众多药用产品和化妆品的治疗配方中。迄今为止，ectoine的年产量已达吨级，由天然生产者Halomonas elongata提供。在本研究中，我们探讨了两种互补的方法以获得基因改良的ectoine生产菌株。我们研究了增加前体供应（草酰乙酸）对ectoine生产的影响，以及通过过表达转运蛋白来提升ectoine需求。这两种方法均应用于已进行基因改造的ectoine分泌菌株H. elongata KB2.13（ΔteaABC ΔdoeA），并且均导致了ectoine排泄量更高的新菌株的产生。供给驱动的方法使得两种不同菌株中的ectoine浓度提高了45%。这种增加归因于磷酸烯醇式丙酮酸羧激酶（PEPCK）的去除，这使得17.9%的葡萄糖底物得以转化为ectoine。对于需求驱动的方法，我们研究了ectoine特异性三联体ATP独立周质（TRAP）转运蛋白中的TeaBC跨膜蛋白作为外排通道的潜力，以改善ectoine的排泄。在没有底物结合蛋白TeaA的情况下，TeaBC两个亚基的过表达促使ectoine的排泄率提高了三倍。单独来看，大亚基TeaC在表达诱导后不久，与TeaBC相比，其细胞外ectoine浓度按干重计大约高五倍。然而，在过程结束时对生长和ectoine浓度的负面影响暗示了TeaC过表达对膜完整性的不良影响，并可能导致细胞裂解。通过采用任一策略，均可显著提升H. elongata中的ectoine合成和排泄。这些方法的内在互补性表明，将两者协调实施是未来代谢工程项目中具有前景的策略。此外，观察到菌株间细胞内ectoine水平存在广泛差异，这表明在菌株KB2.13中，负责ectoine调节的机制发生了重大破坏。