代谢工程法强化恶臭假单胞菌利用木质素积累PHA的能力

doi:10.13560/j.cnki.biotech.bull.1985.2018-0244

生物技术通报 ›› 2018, Vol. 34 ›› Issue (10): 92-99.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0244

代谢工程法强化恶臭假单胞菌利用木质素积累PHA的能力

高庆龙¹, 陈升宝¹, 田文佳¹, 张学铭², 马玉超¹

1 北京林业大学生物科学与技术学院,北京 100083;
2 北京林业大学材料科学与技术学院,北京 100083

收稿日期:2018-03-20 出版日期:2018-10-26 发布日期:2018-11-07
作者简介:高庆龙,男,硕士研究生,研究方向：木质素生物转化;E-mail：gaoqinglong@bjfu.edu.cn
基金资助:
中央高校基本科研业务费（2016JX03）

Strengthening the Ability of Pseudomonas putida to Accumulate PHA from Lignin by Metabolic Engineering

GAO Qing-long¹, CHEN Sheng-bao¹, TIAN Wen-jia¹, ZHANG Xue-ming², MAYu-chao¹

1.College of Biological Sciences and Biotechnology,Beijing Forestry University,Beijing 100083;
2.College of Materials Science and Technology,Beijing Forestry University,Beijing 100083

Received:2018-03-20 Published:2018-10-26 Online:2018-11-07

摘要/Abstract

摘要：

采用代谢工程方法强化恶臭假单胞菌利用木质素积累PHA的能力,为提高木质素转化效率和PHA的高效、低成本生产提供研究思路和技术基础。将PHA解旋酶编码基因phaZ的上下游片段插入pK18mobsacB,构建了敲除载体pK18PHAC1C2;将来源于R. jostii RHA1的过氧化物酶编码基因dypB插入pVLT33,构建了表达载体pVLTpelBDYPB。以P. putida QSR1为出发菌株,利用同源重组的方法敲除了木质素解旋酶编码基因phaZ;进而通过异源表达的方法在该突变株中引入过氧化物酶DyPB;分析各基因修饰菌株的生长情况和PHA积累量。成功构建了phaZ基因缺失突变株P. putidaQSRZ6和DyPB表达菌株P. putida QSRZ6B。在限制氮源的情况下,以葡萄糖为碳源培养48 h,与出发菌株QSR1相比,QSRZ6的生长量和PHA积累量分别提高了29%和80%。在以木质素为碳源积累PHA的过程中,与QSR1相比,QSRZ6的生长量和PHA积累量分别提高了48%和182%;与QSRZ6和QSR1相比,QSRZ6B的PHA积累量分别提高了13%和218%,达到了140 mg/L。phaZ基因的缺失和异源表达过氧化物酶使菌株利用木质素积累PHA的能力得到增强,表明代谢工程是一种有效的调控木质素转化和PHA积累的有效方法。

关键词: phaZ基因失活, 木质素生物转化, PHA积累, 过氧化物酶表达, 代谢工程

Abstract:

The aim of this study is to strengthen the ability of Pseudomonas putida accumulating PHA from lignin by metabolic engineering,and to provide technical basis and research directions for improving the conversion efficiency of lignin and the production of PHA at low-cost but high-efficiency. The upstream and downstream DNA fragments of PHA helicase-encoding gene phaZ were inserted into pK18mobsacB to construct the knockout vector pK18PHAC1C2. The peroxidase-encoding gene dypB derived from R. jostii RHA1 were inserted into pVLT33 to construct the expression vector pVLTpelBDYPB. The phaZ gene was knocked out from original P. putida QSR1 with the homologous recombination,and mutant P. putida QSRZ6 was obtained. Then the peroxidase DyPB was transformed into the P. putida QSRZ6 with the heterologous recombination,P. putida QSRZ6B with DyPB expressed was constructed. The growth and PHA accumulation of mutant strains were analyzed. Cultured for 48 hours with glucose as carbon source under the N limited situation,the dry cell weight and PHA accumulation of QSRZ6 increased by 29% and 80% compared with QSR1. Cultured for 48 hours with lignin,the dry cell weight and PHA accumulation of QSRZ6 increased by 48% and 182%,as well as the PHA accumulation of P. putida QSRZ6B increased by 13% and 218% compared with the control strain P. putida QSRZ6 and P. putida QSR1,respectively,and the yield of PHA reached 140 mg/L. The ability of the strains utilizing lignin to accumulate PHA was enhanced by the phaZ-inactivation and the expression heterologous peroxidase,suggesting that metabolic engineering is an effective approach for the regulation of lignin conversion and PHA accumulation.

Key words: phaZ inactivation, lignin bioconversion, PHA accumulation, peroxidase expression, metabolic engineering

高庆龙, 陈升宝, 田文佳, 张学铭, 马玉超. 代谢工程法强化恶臭假单胞菌利用木质素积累PHA的能力[J]. 生物技术通报, 2018, 34(10): 92-99.

GAO Qing-long, CHEN Sheng-bao, TIAN Wen-jia, ZHANG Xue-ming, MAYu-chao. Strengthening the Ability of Pseudomonas putida to Accumulate PHA from Lignin by Metabolic Engineering[J]. Biotechnology Bulletin, 2018, 34(10): 92-99.

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代谢工程法强化恶臭假单胞菌利用木质素积累PHA的能力

Strengthening the Ability of Pseudomonas putida to Accumulate PHA from Lignin by Metabolic Engineering

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