Data_Sheet_2_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.

天然生物大分子在日常生活用品中的应用，以替代传统合成制造，正日益扩大其市场份额。其中，兼容溶剂ectoine便是这一趋势的例证，它广泛应用于医疗产品和化妆品的治疗配方中。截至目前，ectoine的年产量已达吨级，由天然生产者Halomonas elongata所生产。在本研究中，我们探讨了两种互补的方法以获得ectoine生产的遗传改良生产菌株。我们研究了增加前体供应（草酰乙酸）对ectoine生产的影响，以及通过转运蛋白过表达提高ectoine需求的实施。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）上，并均导致具有更高ectoine排泄能力的新菌株的产生。供给驱动的方法使两种不同菌株的ectoine浓度提高了45%。这一增长归因于磷烯醇丙酮酸羧激酶（PEPCK）的去除，使得17.9%的葡萄糖底物转化为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跨膜蛋白作为出口通道以改善ectoine排泄的潜力。在缺乏底物结合蛋白TeaA的情况下，TeaBC亚基的过表达使得ectoine的排泄率增加了三倍。单独来看，TeaC大亚基在表达诱导后不久，其每干重细胞外ectoine浓度比TeaBC高约五倍。然而，在过程结束时对生长和ectoine浓度的负面影响表明，TeaC过表达可能对膜完整性产生不利影响，甚至可能导致细胞裂解。通过采用这两种策略中的任何一种，均能显著提高H. elongata中的ectoine合成和排泄。这些方法的固有互补性预示着在代谢工程领域未来项目中，这两种策略的协调实施将是一条有前景的策略。此外，观察到菌株间细胞内ectoine水平存在广泛差异，这表明菌株KB2.13中负责ectoine调控的机制发生了重大破坏。