Zhang Sai, He Yaodong, Sen Biswarup, Chen Xiaohong, Xie Yunxuan, Keasling Jay D, Wang Guangyi
Center for Marine Environmental Ecology, School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China.
State Key Laboratory of Systems Engines, Tianjin University, Tianjin 300072, China.
Metab Eng Commun. 2018 Mar 27;6:39-48. doi: 10.1016/j.meteno.2018.03.002. eCollection 2018 Jun.
The unicellular heterotrophic thraustochytrids are attractive candidates for commercial polyunsaturated fatty acids (PUFA) production. However, the reactive oxygen species (ROS) generated in their aerobic fermentation process often limits their PUFA titer. Yet, the specific mechanisms of ROS involvement in the crosstalk between oxidative stress and intracellular lipid synthesis remain poorly described. Metabolic engineering to improve the PUFA yield in thraustochytrids without compromising growth is an important aspect of economic feasibility. To fill this gap, we overexpressed the antioxidative gene superoxide dismutase (SOD1) by integrating it into the genome of thraustochytrid sp. PKU#Mn4 using a novel genetic transformation system. This study reports the ROS alleviation, enhanced PUFA production and transcriptome changes resulting from the SOD1 overexpression. SOD1 activity in the recombinant improved by 5.2-71.6% along with 7.8-38.5% decline in ROS during the fermentation process. Interestingly, the total antioxidant capacity in the recombinant remained higher than wild-type and above zero in the entire process. Although lipid profile was similar to that of wild-type, the concentrations of major fatty acids in the recombinant were significantly (p ≤ 0.05) higher. The PUFA titer increased up to 1232 ± 41 mg/L, which was 32.9% higher (p ≤ 0.001) than the wild type. Transcriptome analysis revealed strong downregulation of genes potentially involved in β-oxidation of fatty acids in peroxisome and upregulation of genes catalyzing lipid biosynthesis. Our results enrich the knowledge on stress-induced PUFA biosynthesis and the putative role of ROS in the regulation of lipid metabolism in oleaginous thraustochytrids. This study provides a new and alternate strategy for cost-effective industrial fermentation of PUFA.
单细胞异养型破囊壶菌是商业生产多不饱和脂肪酸（PUFA）的理想候选菌株。然而，其好氧发酵过程中产生的活性氧（ROS）常常限制了PUFA的产量。然而，ROS参与氧化应激与细胞内脂质合成之间相互作用的具体机制仍鲜为人知。在不影响破囊壶菌生长的前提下，通过代谢工程提高其PUFA产量是经济可行性的一个重要方面。为填补这一空白，我们利用一种新型遗传转化系统，将抗氧化基因超氧化物歧化酶（SOD1）整合到破囊壶菌属PKU#Mn4的基因组中，实现了该基因的过表达。本研究报道了SOD1过表达导致的ROS减轻、PUFA产量提高以及转录组变化。重组菌株中SOD1活性提高了5.2 - 71.6%，发酵过程中ROS下降了7.8 - 38.5%。有趣的是，重组菌株的总抗氧化能力在整个过程中始终高于野生型且大于零。虽然脂质谱与野生型相似，但重组菌株中主要脂肪酸的浓度显著更高（p≤0.05）。PUFA产量提高到1232±41 mg/L，比野生型高32.9%（p≤0.001）。转录组分析显示，过氧化物酶体中参与脂肪酸β-氧化的基因强烈下调，而催化脂质生物合成的基因上调。我们的研究结果丰富了关于应激诱导的PUFA生物合成以及ROS在产油破囊壶菌脂质代谢调控中假定作用的知识。本研究为PUFA的经济高效工业发酵提供了一种新的替代策略。
