Department of Laboratory Medicine and Biotechnology, College of Medicine, Tzu Chi University, 701 Section 3 Zhongyang Road, Hualien, Taiwan, R.O.C.
Lett Appl Microbiol. 2011 May;52(5):475-83. doi: 10.1111/j.1472-765X.2011.03029.x. Epub 2011 Mar 1.
To develop a microbial strain producing poly(3-hydroxybutyrate) [P(3HB)], in the absence of antibiotic supplementation (normally required to stabilize a recombinant plasmid), by constructing a recombinant Escherichia coli strain with phaCAB and vgb integrated into the chromosome.
The polyhydroxyalkanoate (PHA) synthesis operon (phaCAB) and the bacterial haemoglobin gene (vgb) were integrated downstream of nlpB (novel lipoprotein B) in E. coli K12, via homologous recombination, to form a recombinant strain, termed YH100. VHb encoded by the vgb gene was successfully expressed in YH100, as confirmed by Western blotting. P(3HB) synthesis by the YH100 strain grown in the absence of antibiotic was analysed by transmission electron microscopy. The yield of P(3HB) is 208 mg g(-1) . The thermal stability of P(3HB) produced from YH100 was similar to that of commercial P(3HB). Further, the polydispersity index (PDI) of the P(3HB) polymer derived from YH100 was 1·37, indicating that polymer uniformity was greater than that of commercial P(3HB), which had a PDI of 1·47.
We successfully constructed a recombinant E. coli strain expressing exogenous genes, specifically phaCAB from Cupriavidus necator and vgb from Vitreoscilla stercoraria, integrated into the downstream of chromosomal dapA-nlpB locus. P(3HB) was stably produced by this strain, without any need for antibiotic supplementation to stabilize a recombinant plasmid at least for 48h.
We report a genetic locus, the downstream of the nlpB locus in E. coli, in which the transcription of the exogenous genes is driven by the dapA-nlpB promoter without the need for the addition of inducer and antibiotic.
通过构建将 phaCAB 和 vgb 整合到染色体中的重组大肠杆菌菌株，开发一种在不存在抗生素补充（通常需要稳定重组质粒）的情况下生产聚（3-羟基丁酸酯）[P（3HB）]的微生物菌株。
聚羟基烷酸酯（PHA）合成操纵子（phaCAB）和细菌血红蛋白基因（vgb）通过同源重组整合到大肠杆菌 K12 中的 nlpB（新型脂蛋白 B）下游，形成重组菌株，称为 YH100。通过 Western 印迹证实，vgb 基因编码的 VHb 在 YH100 中成功表达。通过透射电子显微镜分析在不存在抗生素的情况下生长的 YH100 菌株的 P（3HB）合成。P（3HB）的产量为 208mg g(-1)。YH100 产生的 P（3HB）的热稳定性与商业 P（3HB）相似。此外，YH100 衍生的 P（3HB）聚合物的多分散指数（PDI）为 1.37，表明聚合物均匀性大于商业 P（3HB）的 PDI，商业 P（3HB）的 PDI 为 1.47。
我们成功构建了一种表达外源性基因的重组大肠杆菌菌株，特别是来自 Cupriavidus necator 的 phaCAB 和来自 Vitreoscilla stercoraria 的 vgb，整合到染色体 dapA-nlpB 基因座的下游。该菌株稳定生产 P（3HB），至少在 48 小时内不需要抗生素补充来稳定重组质粒。
我们报告了大肠杆菌中 nlpB 基因座下游的一个遗传基因座，在外源基因的转录由 dapA-nlpB 启动子驱动，而无需添加诱导剂和抗生素。
