Lin Ji-Hong, Lee Ming-Chieh, Sue You-Sheng, Liu Yung-Chuan, Li Si-Yu
Department of Chemical Engineering, National Chung Hsing University, Taichung, 402, Taiwan.
Appl Microbiol Biotechnol. 2017 Aug;101(16):6419-6430. doi: 10.1007/s00253-017-8386-2. Epub 2017 Jun 30.
PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA , 1179 bp), acetoacetyl-CoA reductase (phaB , 738 bp), and PHA synthase (phaC , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.
已鉴定出嗜锰嗜热栖热菌的聚羟基丁酸酯（PHB）生物合成途径，该途径由三个开放阅读框（ORF）组成，分别编码β-酮硫解酶（phaA，1179 bp）、乙酰乙酰辅酶A还原酶（phaB，738 bp）和PHA合酶（phaC，1694 bp）。通过在大肠杆菌中成功重建嗜锰嗜热栖热菌的PHB生物合成途径，证明了PhaA、PhaB和PhaC的功能，其中通过OD、气相色谱、尼罗蓝染色和透射电子显微镜（TEM）确认了PHB的产生。PHB合酶的蛋白质序列比对显示，phaC与I类PHB合酶的序列至少有60%的同一性。研究了PhaA、PhaB和PhaC表达水平对PHB产量的影响。虽然发现PhaB的过表达在重组大肠杆菌中很重要，但PHB生产性能可量化如下：PHB浓度为16.8±0.6 g/L，产率为0.28 g/g葡萄糖，含量为74%，生产率为0.28 g/L/h，分子量为1.41 MDa。
