College of Energy and Environmental Engineering, Hebei University of Engineering, Handan, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, Wuhan, China.
Appl Environ Microbiol. 2023 Apr 26;89(4):e0002123. doi: 10.1128/aem.00021-23. Epub 2023 Mar 14.
The biosynthetic pathway of eicosapentaenoic acid (EPA) has previously been reported in marine bacteria, while the regulatory mechanism remains poorly understood. In this study, a putative transcriptional regulator PfaR encoded adjacent to the PFA biosynthesis gene cluster () was computationally and experimentally characterized. Comparative analyses on the wild type (WT) strain, in-frame deletion, and overexpression mutants revealed that PfaR positively regulated EPA synthesis at low temperature. RNA-Seq and real-time quantitative PCR analyses demonstrated that PfaR stimulated the transcription of . The transcription start site of was mapped by using primer extension and highly conserved promoter motifs bound by the housekeeping Sigma 70 factor that were identified in the upstream of . Moreover, overexpression of PfaR in WT strain W3-18-1 at low temperature could improve EPA productivity from 0.07% to 0.13% (percentage of EPA to dry weight, mg/mg) of dry weight. Taken together, these findings could provide important implications into the transcriptional control and metabolic engineering in terms of EPA productivity for industrial strains. We have experimentally confirmed that PfaR is a positive transcription regulator that promotes EPA synthesis at low temperature in Shewanella putrefaciens W3-18-1. Overexpression of PfaR in WT strain W3-18-1 could lead to a 1.8-fold increase in EPA productivity at low temperature. It is further shown that PfaR may be regulated by housekeeping Sigma 70 factor at low temperature.
先前已经报道过，在海洋细菌中存在二十碳五烯酸（EPA）的生物合成途径，然而，其调控机制仍知之甚少。在本研究中，我们对编码于 PFA 生物合成基因簇（）旁侧的假定转录调控因子 PfaR 进行了计算和实验分析。通过对野生型（WT）菌株、无义突变和过表达突变体的比较分析，发现 PfaR 在低温下正向调控 EPA 的合成。RNA-Seq 和实时定量 PCR 分析表明，PfaR 刺激了基因的转录。通过引物延伸和高度保守的启动子基序分析，确定了基因的转录起始位点，这些基序由在基因上游发现的组成型 Sigma 70 因子结合。此外，在低温下，过表达 PfaR 可以将 WT 菌株 W3-18-1 的 EPA 产量从 0.07%提高到 0.13%（EPA 相对于干重的百分比，mg/mg）。综上所述，这些发现为工业菌株 EPA 生产的转录调控和代谢工程提供了重要启示。我们已经通过实验证实，PfaR 是 Shewanella putrefaciens W3-18-1 中在低温下促进 EPA 合成的正转录调控因子。在 WT 菌株 W3-18-1 中过表达 PfaR 可以使 EPA 的产量在低温下提高 1.8 倍。进一步表明，PfaR 可能受低温下的组成型 Sigma 70 因子调控。
