筛选内源性强启动子以提高门多萨假单胞菌 NK-01 中中链长度聚羟基烷酸酯的产量。
Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina NK-01.
机构信息
Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin, 300071, China.
State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, 300071, China.
出版信息
Sci Rep. 2019 Feb 12;9(1):1798. doi: 10.1038/s41598-019-39321-z.
Polyhydroxyalkanoate (PHA) can be produced by microorganisms from renewable resources and is regarded as a promising bioplastic to replace petroleum-based plastics. Pseudomonas mendocina NK-01 is a medium-chain-length PHA (mcl-PHA)-producing strain and its whole-genome sequence is currently available. The yield of mcl-PHA in P. mendocina NK-01 is expected to be improved by applying a promoter engineering strategy. However, a limited number of well-characterized promoters has greatly restricted the application of promoter engineering for increasing the yield of mcl-PHA in P. mendocina NK-01. In this work, 10 endogenous promoters from P. mendocina NK-01 were identified based on RNA-seq and promoter prediction results. Subsequently, 10 putative promoters were characterized for their strength through the expression of a reporter gene gfp. As a result, five strong promoters designated as P4, P6, P9, P16 and P25 were identified based on transcriptional level and GFP fluorescence intensity measurements. To evaluate whether the screened promoters can be used to enhance transcription of PHA synthase gene (phaC), the three promoters P4, P6 and P16 were separately integrated into upstream of the phaC operon in the genome of P. mendocina NK-01, resulting in the recombinant strains NKU-4C1, NKU-6C1 and NKU-16C1. As expected, the transcriptional levels of phaC1 and phaC2 in the recombinant strains were increased as shown by real-time quantitative RT-PCR. The phaZ gene encoding PHA depolymerase was further deleted to construct the recombinant strains NKU-∆phaZ-4C1, NKU-∆phaZ-6C1 and NKU-∆phaZ-16C1. The results from shake-flask fermentation indicated that the mcl-PHA titer of recombinant strain NKU-∆phaZ-16C1 was increased from 17 to 23 wt% compared with strain NKU-∆phaZ. This work provides a feasible method to discover strong promoters in P. mendocina NK-01 and highlights the potential of the screened endogenous strong promoters for metabolic engineering of P. mendocina NK-01 to increase the yield of mcl-PHA.
聚羟基烷酸酯（PHA）可以由微生物从可再生资源中产生，被认为是一种有前途的生物塑料，可以替代基于石油的塑料。门多西纳假单胞菌 NK-01 是一种中链长聚羟基烷酸酯（mcl-PHA）生产菌株，其全基因组序列现已可用。通过应用启动子工程策略，预计门多西纳假单胞菌 NK-01 中的 mcl-PHA 的产量将得到提高。然而，数量有限的经过良好表征的启动子极大地限制了启动子工程在提高门多西纳假单胞菌 NK-01 中 mcl-PHA 产量方面的应用。在这项工作中，基于 RNA-seq 和启动子预测结果，从门多西纳假单胞菌 NK-01 中鉴定了 10 个内源启动子。随后，通过表达报告基因 gfp 对 10 个推定启动子的强度进行了表征。结果，根据转录水平和 GFP 荧光强度测量，鉴定了 5 个强启动子，分别命名为 P4、P6、P9、P16 和 P25。为了评估筛选出的启动子是否可用于增强 PHA 合酶基因（phaC）的转录，将三个启动子 P4、P6 和 P16 分别整合到门多西纳假单胞菌 NK-01 基因组的 phaC 操纵子上游，得到重组菌株 NKU-4C1、NKU-6C1 和 NKU-16C1。正如预期的那样，通过实时定量 RT-PCR 显示，重组菌株中 phaC1 和 phaC2 的转录水平均升高。进一步删除编码 PHA 解聚酶的 phaZ 基因，构建重组菌株 NKU-∆phaZ-4C1、NKU-∆phaZ-6C1 和 NKU-∆phaZ-16C1。摇瓶发酵结果表明，与 NKU-∆phaZ 相比，重组菌株 NKU-∆phaZ-16C1 的 mcl-PHA 产量从 17wt%增加到 23wt%。这项工作提供了一种在门多西纳假单胞菌 NK-01 中发现强启动子的可行方法，并强调了筛选出的内源强启动子在门多西纳假单胞菌 NK-01 的代谢工程中的潜力，以提高 mcl-PHA 的产量。
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