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.

天然来源生物分子替代传统合成工艺应用于日用产品的市场规模正持续扩大。其中典型代表为相容溶质四氢嘧啶（ectoine），其被广泛应用于各类医药与化妆品配方中。目前，四氢嘧啶的工业化生产均由天然宿主延长盐单胞菌（Halomonas elongata）完成，年产量可达数吨级。
本研究旨在通过两种互补策略，选育用于四氢嘧啶生产的基因工程改良菌株。我们一方面探究了前体物质草酰乙酸（oxaloacetate）供给提升对四氢嘧啶合成的影响，另一方面通过过表达转运蛋白强化四氢嘧啶的胞内需求，以此实现产量提升。
上述两种策略均以已完成基因改造的四氢嘧啶分泌菌株延长盐单胞菌KB2.13（ΔteaABC ΔdoeA）为底盘菌株，改造后均获得了四氢嘧啶分泌能力更强的工程菌株。供给强化策略可使两株工程菌的四氢嘧啶效价提升45%，该效果源于磷酸烯醇式丙酮酸羧激酶（PEPCK）基因的敲除，该改造可使17.9%的葡萄糖底物转化为四氢嘧啶。
针对需求强化策略，本研究探究了四氢嘧啶特异性三组分不依赖ATP的周质（TRAP）转运蛋白中TeaBC跨膜亚基作为分泌通道对提升四氢嘧啶分泌能力的潜力。在底物结合蛋白TeaA缺失的背景下，同时过表达TeaBC两个亚基可使四氢嘧啶的分泌速率提升至原来的三倍。单独过表达大亚基TeaC时，在诱导表达后短期内，每克干细胞重对应的胞外四氢嘧啶浓度较TeaBC组合提升约五倍。但该策略对菌体生长及最终四氢嘧啶效价存在不利影响，这表明过表达TeaC会破坏细胞膜完整性，甚至可能引发细胞裂解。
采用上述任一策略均可大幅提升延长盐单胞菌的四氢嘧啶合成与分泌能力。由于两种策略具有天然互补性，因此协同应用二者有望成为未来代谢工程（Metabolic Engineering）领域极具前景的研究方向。此外，各工程菌株的胞内四氢嘧啶水平存在显著差异，这表明KB2.13菌株中四氢嘧啶调控机制已发生重大紊乱。