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Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-07-19 00:00:00 , DOI: 10.1021/acssynbio.8b00102 Rui Shen 1 , Jin Yin 1 , Jian-Wen Ye 1 , Rui-Juan Xiang 2 , Zhi-Yu Ning 1 , Wu-Zhe Huang 1 , Guo-Qiang Chen 1, 3
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2018-07-19 00:00:00 , DOI: 10.1021/acssynbio.8b00102 Rui Shen 1 , Jin Yin 1 , Jian-Wen Ye 1 , Rui-Juan Xiang 2 , Zhi-Yu Ning 1 , Wu-Zhe Huang 1 , Guo-Qiang Chen 1, 3
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Promoters for the expression of heterologous genes in Halomonas bluephagenesis are quite limited, and many heterologous promoters function abnormally in this strain. Pporin, a promoter of the strongest expressed protein porin in H. bluephagenesis, is one of the few promoters available for heterologous expression in H. bluephagenesis, yet it has a fixed transcriptional activity that cannot be tuned. A stable promoter library with a wide range of activities is urgently needed. This study reports an approach to construct a promoter library based on the Pporin core region, namely, from the −35 box to the transcription start site, a spacer and an insulator. Saturation mutagenesis was conducted inside the promoter core region to significantly increase the diversity within the promoter library. The promoter library worked in both E. coli and H. bluephagenesis, covering a wide range of relative transcriptional strengths from 40 to 140 000. The library is therefore suitable for the transcription of many different heterologous genes, serving as a platform for protein expression and fine-tuned metabolic engineering of H. bluephagenesis TD01 and its derivative strains. H. bluephagenesis strains harboring the orfZ gene encoding 4HB-CoA transferase driven by selected promoters from the library were constructed, the best one produced over 100 g/L cell dry weight containing 80% poly(3-hydroxybutyrate-co-11 mol % 4-hydroxybutyrate) with a productivity of 1.59 g/L/h after 50 h growth under nonsterile fed-batch conditions. This strain was found the best for P(3HB-co-4HB) production in the laboratory scale.
中文翻译：
蓝藻嗜盐菌增强P（3HB- co -4HB）生产的启动子工程
在Haloomonas bluephagenesis中表达异源基因的启动子非常有限，并且许多异源启动子在该菌株中异常起作用。P孔蛋白，最强烈表达的蛋白质孔蛋白的启动子H. bluephagenesis，是可用于在异源表达的少数启动子之一H. bluephagenesis，但它有一个不能被调谐固定转录活性。迫切需要一个稳定的，具有广泛活性的启动子文库。这项研究报告了一种基于P孔蛋白构建启动子文库的方法核心区域，即从-35框到转录起始位点，一个间隔子和一个绝缘子。在启动子核心区域内进行饱和诱变，以显着增加启动子文库内的多样性。该启动子文库在大肠杆菌和蓝细菌中都起作用，涵盖了从40到140 000的广泛的相对转录强度。因此，该文库适用于许多不同异源基因的转录，可作为蛋白质表达和表达的平台。H. bluephagenesis TD01及其衍生菌株的微调代谢工程。携带orfZ的H.bluephagenesis菌株编码4HB -CoA转移酶通过从库中选择的启动子驱动的基因构建，最好的一个生产了含有80％的聚（3-羟基丁酸酯为100g / L细胞干重共同的生产率-11％（摩尔）4-羟基丁酸酯）在非无菌补料分批条件下生长50小时后为1.59 g / L / h。在实验室规模中，发现该菌株最适合生产P（3HB- co -4HB）。
更新日期：2018-07-19
中文翻译：
蓝藻嗜盐菌增强P（3HB- co -4HB）生产的启动子工程
在Haloomonas bluephagenesis中表达异源基因的启动子非常有限，并且许多异源启动子在该菌株中异常起作用。P孔蛋白，最强烈表达的蛋白质孔蛋白的启动子H. bluephagenesis，是可用于在异源表达的少数启动子之一H. bluephagenesis，但它有一个不能被调谐固定转录活性。迫切需要一个稳定的，具有广泛活性的启动子文库。这项研究报告了一种基于P孔蛋白构建启动子文库的方法核心区域，即从-35框到转录起始位点，一个间隔子和一个绝缘子。在启动子核心区域内进行饱和诱变，以显着增加启动子文库内的多样性。该启动子文库在大肠杆菌和蓝细菌中都起作用，涵盖了从40到140 000的广泛的相对转录强度。因此，该文库适用于许多不同异源基因的转录，可作为蛋白质表达和表达的平台。H. bluephagenesis TD01及其衍生菌株的微调代谢工程。携带orfZ的H.bluephagenesis菌株编码4HB -CoA转移酶通过从库中选择的启动子驱动的基因构建，最好的一个生产了含有80％的聚（3-羟基丁酸酯为100g / L细胞干重共同的生产率-11％（摩尔）4-羟基丁酸酯）在非无菌补料分批条件下生长50小时后为1.59 g / L / h。在实验室规模中，发现该菌株最适合生产P（3HB- co -4HB）。
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