Royce Liam A, Yoon Jong Moon, Chen Yingxi, Rickenbach Emily, Shanks Jacqueline V, Jarboe Laura R
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA.
Honors Program, Iowa State University, Ames, IA 50011, USA.
Metab Eng. 2015 May;29:180-188. doi: 10.1016/j.ymben.2015.03.014. Epub 2015 Mar 31.
Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals.
羧酸是一种具有吸引力的生物可再生化学品，但与许多生物可再生资源一样，它们对微生物生物催化剂的毒性限制了其发酵生产。虽然人们普遍认为膜损伤是脂肪酸毒性的主要机制，但之前提高膜完整性的代谢工程努力并未使羧酸产量增加。在这里，我们采用进化方法来提高对外源辛酸的耐受性，目的是从这种进化菌株中学习设计策略。在基本培养基中于中性pH条件下对大肠杆菌MG1655衍生物进行进化，产生了一种不仅对辛酸耐受性增强，而且对己酸、癸酸、正丁醇和异丁醇耐受性也增强的菌株。当表达嗜热栖热放线菌硫酯酶时，这种进化菌株产生羧酸的滴度也比其亲本菌株高5倍。虽然之前有人提出细胞内酸化可能导致羧酸毒性，但我们没有发现证据表明进化菌株对这种酸化的抗性增强。对进化菌株膜的表征表明，相对于其亲本，它的膜极化（流动性）、完整性（渗漏）和组成有显著改变。膜组成的变化包括在多种生长条件下，如30°C、42°C、羧酸挑战和乙醇挑战下，平均脂质长度显著增加。进化菌株的膜组成更具动态性，在膜脂相对丰度方面显示出更多显著变化和更大的倍数变化。这些结果突出了细胞膜在提高微生物对生物可再生燃料和化学品的耐受性及产量方面的重要性。
