Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China.
Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China.
Int J Biol Macromol. 2021 Nov 30;191:608-617. doi: 10.1016/j.ijbiomac.2021.09.142. Epub 2021 Sep 25.
Polyhydroxyalkanoate (PHA), a class of biopolyester synthesized by various bacteria, is considered as an alternative to petroleum-based plastics because of its excellent physochemical and material properties. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from glucose, fatty acid and glycerol, and its whole-genome sequences and cellular metabolic networks have been intensively researched. In this study, we aim to improve the PHA yield of P. putida KT2440 using a novel promoter engineering-based strategy. Unlike previous studies, endogenous strong promoters screening from P. putida KT2440 instead of synthetic or exogenous promoters was applied to the optimization of PHA biosynthesis pathway. Based on RNA-seq and promoter prediction, 30 putative strong promoters from P. putida KT2440 were identified. Subsequently, the strengths of these promoters were characterized by reporter gene assays. Furthermore, each of 10 strong promoters screened by transcriptional level and GFP fluorescence was independently inserted into upstream of PHA synthase gene (phaC1) on chromosome. As a result, the transcriptional levels of the phaC1 and phaC2 genes in almost all of the promoter-substituted strains were improved, and the relative PHA yields of the three promoter-substituted strains KTU-P1C1, KTU-P46C1 and KTU-P51C1 were improved obviously, reaching 30.62 wt%, 33.24 wt% and 33.29 wt% [the ratio of PHA weight to cell dry weight (CDW)], respectively. By further deletion of the glucose dehydrogenase gene in KTU-P1C1, KTU-P46C1 and KTU-P51C1, the relative PHA yield of the resulting mutant strain KTU-P46C1-∆gcd increased by 5.29% from 33.24% to 38.53%. Finally, by inserting P46 into upstream of pyruvate dehydrogenase gene in the genome of KTU-P46C1-∆gcd, the relative PHA yield and CDW of the resulting strain KTU-P46C1A-∆gcd reached nearly 42 wt% and 4.06 g/l, respectively, which increased by 90% and 40%, respectively, compared with the starting strain KTU. In particular, the absolute PHA yield of KTU-P46C1A-∆gcd reached 1.7 g/l, with a 165% improvement compared with the strain KTU. Herein, we report the highest PHA yield obtained by P. putida KT2440 in shake-flask fermentation to date. We demonstrate for the first time the effectiveness of endogenous strong promoters for improving the PHA yield and biomass of P. putida KT2440. More importantly, our findings highlight great potential of this strategy for enhanced production of secondary metabolites and heterologous proteins in P. putida KT2440.
聚羟基脂肪酸酯（PHA）是一类由各种细菌合成的生物聚酯，由于其优异的物理化学和材料性能，被认为是石油基塑料的替代品。恶臭假单胞菌 KT2440 可以从葡萄糖、脂肪酸和甘油中生产中链长度 PHA（mcl-PHA），其全基因组序列和细胞代谢网络已得到深入研究。在本研究中，我们旨在使用新型启动子工程策略来提高恶臭假单胞菌 KT2440 的 PHA 产量。与以往的研究不同，我们应用源于恶臭假单胞菌 KT2440 的内源性强启动子筛选，而不是合成或外源性启动子，来优化 PHA 生物合成途径。基于 RNA-seq 和启动子预测，从恶臭假单胞菌 KT2440 中鉴定出 30 个假定的强启动子。随后，通过报告基因检测来表征这些启动子的强度。此外，通过转录水平和 GFP 荧光筛选的 10 个强启动子中的每一个都独立插入染色体上的 PHA 合酶基因（phaC1）的上游。结果，几乎所有启动子替换菌株的 phaC1 和 phaC2 基因的转录水平都得到了提高，并且三个启动子替换菌株 KTU-P1C1、KTU-P46C1 和 KTU-P51C1 的相对 PHA 产量明显提高，分别达到 30.62wt%、33.24wt%和 33.29wt%[PHA 重量与细胞干重（CDW）的比值]。通过进一步删除 KTU-P1C1、KTU-P46C1 和 KTU-P51C1 中的葡萄糖脱氢酶基因，所得突变株 KTU-P46C1-∆gcd 的相对 PHA 产量从 33.24%增加到 38.53%，增加了 5.29%。最后，通过将 P46 插入 KTU-P46C1-∆gcd 基因组中丙酮酸脱氢酶基因的上游，所得菌株 KTU-P46C1A-∆gcd 的相对 PHA 产量和 CDW 分别达到近 42wt%和 4.06g/l，与起始菌株 KTU 相比，分别提高了 90%和 40%。特别是，KTU-P46C1A-∆gcd 的绝对 PHA 产量达到 1.7g/l，比菌株 KTU 提高了 165%。在此，我们报告了迄今为止恶臭假单胞菌 KT2440 在摇瓶发酵中获得的最高 PHA 产量。我们首次证明了内源性强启动子在提高恶臭假单胞菌 KT2440 的 PHA 产量和生物量方面的有效性。更重要的是，我们的发现突出了该策略在增强恶臭假单胞菌 KT2440 中次生代谢物和异源蛋白生产方面的巨大潜力。
