CIESC Journal ›› 2023, Vol. 74 ›› Issue (5): 2111-2122.DOI: 10.11949/0438-1157.20221582
• Biochemical engineering and technology • Previous Articles Next Articles
Chunlei ZHAO1,2(), Liang GUO1,2, Cong GAO1,2, Wei SONG3, Jing WU3, Jia LIU1,2, Liming LIU1,2, Xiulai CHEN1,2()
Received:
2022-12-08
Revised:
2023-02-17
Online:
2023-06-29
Published:
2023-05-05
Contact:
Xiulai CHEN
赵春雷1,2(), 郭亮1,2, 高聪1,2, 宋伟3, 吴静3, 刘佳1,2, 刘立明1,2, 陈修来1,2()
通讯作者:
陈修来
作者简介:
赵春雷(1997—),男,硕士研究生,1972630117@qq.com
基金资助:
CLC Number:
Chunlei ZHAO, Liang GUO, Cong GAO, Wei SONG, Jing WU, Jia LIU, Liming LIU, Xiulai CHEN. Metabolic engineering of Escherichia coli for chondroitin production[J]. CIESC Journal, 2023, 74(5): 2111-2122.
赵春雷, 郭亮, 高聪, 宋伟, 吴静, 刘佳, 刘立明, 陈修来. 代谢工程改造大肠杆菌生产软骨素[J]. 化工学报, 2023, 74(5): 2111-2122.
质粒 | 描述 | 来源 |
---|---|---|
pEM | T5 promoter, AmpR, f1 ori | 实验室储存 |
pCDR | Trc promoter, StrR, CloDF13 ori | 实验室储存 |
pEM-kfoA | pEM连接基因kfoA | 本研究构建 |
pCDR-kfoF | pCDR连接基因kfoF | 本研究构建 |
pEM-RBSL | pEM使用低表达强度RBS | 本研究构建 |
pEM-RBSM | pEM使用中表达强度RBS | 本研究构建 |
pEM-RBSH | pEM使用高表达强度RBS | 本研究构建 |
pCDR-kfoF-kfoC-kfoA | pCDR连接基因kfoA、kfoC、kfoF | 本研究构建 |
pEM-RBSL-glmS | pEM连接低表达强度的glmS基因 | 本研究构建 |
pEM-RBSM-glmS | pEM连接中表达强度的glmS基因 | 本研究构建 |
pEM-RBSH-glmS | pEM连接高表达强度的glmS基因 | 本研究构建 |
pEM-RBSL-glmM | pEM连接低表达强度的glmM基因 | 本研究构建 |
pEM-RBSM-glmM | pEM连接中表达强度的glmM基因 | 本研究构建 |
pEM-RBSH-glmM | pEM连接高表达强度的glmM基因 | 本研究构建 |
pEM-RBSL-kfoF | pEM连接低表达强度的kfoF基因 | 本研究构建 |
pEM-RBSM-kfoF | pEM连接中表达强度的kfoF基因 | 本研究构建 |
pEM-RBSH-kfoF | pEM连接高表达强度的kfoF基因 | 本研究构建 |
Table 1 Plasmids used in the study
质粒 | 描述 | 来源 |
---|---|---|
pEM | T5 promoter, AmpR, f1 ori | 实验室储存 |
pCDR | Trc promoter, StrR, CloDF13 ori | 实验室储存 |
pEM-kfoA | pEM连接基因kfoA | 本研究构建 |
pCDR-kfoF | pCDR连接基因kfoF | 本研究构建 |
pEM-RBSL | pEM使用低表达强度RBS | 本研究构建 |
pEM-RBSM | pEM使用中表达强度RBS | 本研究构建 |
pEM-RBSH | pEM使用高表达强度RBS | 本研究构建 |
pCDR-kfoF-kfoC-kfoA | pCDR连接基因kfoA、kfoC、kfoF | 本研究构建 |
pEM-RBSL-glmS | pEM连接低表达强度的glmS基因 | 本研究构建 |
pEM-RBSM-glmS | pEM连接中表达强度的glmS基因 | 本研究构建 |
pEM-RBSH-glmS | pEM连接高表达强度的glmS基因 | 本研究构建 |
pEM-RBSL-glmM | pEM连接低表达强度的glmM基因 | 本研究构建 |
pEM-RBSM-glmM | pEM连接中表达强度的glmM基因 | 本研究构建 |
pEM-RBSH-glmM | pEM连接高表达强度的glmM基因 | 本研究构建 |
pEM-RBSL-kfoF | pEM连接低表达强度的kfoF基因 | 本研究构建 |
pEM-RBSM-kfoF | pEM连接中表达强度的kfoF基因 | 本研究构建 |
pEM-RBSH-kfoF | pEM连接高表达强度的kfoF基因 | 本研究构建 |
菌株 | 描述 | 来源 |
---|---|---|
E. coli JM109 | Wild-type E. coli strain | 实验室储存 |
E. coli BL21 STAR (DE3)(简称STAR) | Wild-type E. coli strain | Addgene公司购买 |
E. coli GZ00 | STAR带有重组质粒pCDR-kfoF-kfoC-kfoA | 本研究构建 |
E. coli GZ01 | STAR基因组整合kfoF-kfoC-kfoA | 本研究构建 |
E. coli GZ02 | GZ00带有重组质粒pEM-RBSL-glmS | 本研究构建 |
E. coli GZ03 | GZ00带有重组质粒pEM-RBSM-glmS | 本研究构建 |
E. coli GZ04 | GZ00带有重组质粒pEM-RBSH-glmS | 本研究构建 |
E. coli GZ05 | GZ01增加一个拷贝glmS | 本研究构建 |
E. coli GZ06 | GZ01增加两个拷贝glmS | 本研究构建 |
E. coli GZ07 | GZ01增加三个拷贝glmS | 本研究构建 |
E. coli GZ08 | GZ00带有重组质粒pEM-RBSL-glmM | 本研究构建 |
E. coli GZ09 | GZ00带有重组质粒pEM-RBSM-glmM | 本研究构建 |
E. coli GZ10 | GZ00带有重组质粒pEM-RBSH-glmM | 本研究构建 |
E. coli GZ11 | GZ06增加一个拷贝glmM | 本研究构建 |
E. coli GZ12 | GZ06增加两个拷贝glmM | 本研究构建 |
E. coli GZ13 | GZ06增加三个拷贝glmM | 本研究构建 |
E. coli GZ14 | GZ00带有重组质粒pEM-RBSL-kfoF | 本研究构建 |
E. coli GZ15 | GZ00带有重组质粒pEM-RBSM-kfoF | 本研究构建 |
E. coli GZ16 | GZ00带有重组质粒pEM-RBSH-kfoF | 本研究构建 |
E. coli GZ17 | GZ12增加一个拷贝kfoF | 本研究构建 |
E. coli GZ18 | GZ12增加两个拷贝kfoF | 本研究构建 |
Table 2 Strains used in the study
菌株 | 描述 | 来源 |
---|---|---|
E. coli JM109 | Wild-type E. coli strain | 实验室储存 |
E. coli BL21 STAR (DE3)(简称STAR) | Wild-type E. coli strain | Addgene公司购买 |
E. coli GZ00 | STAR带有重组质粒pCDR-kfoF-kfoC-kfoA | 本研究构建 |
E. coli GZ01 | STAR基因组整合kfoF-kfoC-kfoA | 本研究构建 |
E. coli GZ02 | GZ00带有重组质粒pEM-RBSL-glmS | 本研究构建 |
E. coli GZ03 | GZ00带有重组质粒pEM-RBSM-glmS | 本研究构建 |
E. coli GZ04 | GZ00带有重组质粒pEM-RBSH-glmS | 本研究构建 |
E. coli GZ05 | GZ01增加一个拷贝glmS | 本研究构建 |
E. coli GZ06 | GZ01增加两个拷贝glmS | 本研究构建 |
E. coli GZ07 | GZ01增加三个拷贝glmS | 本研究构建 |
E. coli GZ08 | GZ00带有重组质粒pEM-RBSL-glmM | 本研究构建 |
E. coli GZ09 | GZ00带有重组质粒pEM-RBSM-glmM | 本研究构建 |
E. coli GZ10 | GZ00带有重组质粒pEM-RBSH-glmM | 本研究构建 |
E. coli GZ11 | GZ06增加一个拷贝glmM | 本研究构建 |
E. coli GZ12 | GZ06增加两个拷贝glmM | 本研究构建 |
E. coli GZ13 | GZ06增加三个拷贝glmM | 本研究构建 |
E. coli GZ14 | GZ00带有重组质粒pEM-RBSL-kfoF | 本研究构建 |
E. coli GZ15 | GZ00带有重组质粒pEM-RBSM-kfoF | 本研究构建 |
E. coli GZ16 | GZ00带有重组质粒pEM-RBSH-kfoF | 本研究构建 |
E. coli GZ17 | GZ12增加一个拷贝kfoF | 本研究构建 |
E. coli GZ18 | GZ12增加两个拷贝kfoF | 本研究构建 |
菌株 | OD600 | CH/ (mg/L) | CH对细胞得率/ (mg/g) | CH对甘油得率/ (mg/g) |
---|---|---|---|---|
GZ01 | 11.0 | 7.1 | 1.6 | 0.5 |
GZ05 | 11.2 | 29.9 | 6.5 | 2.0 |
GZ06 | 11.8 | 46.2 | 9.5 | 3.1 |
GZ07 | 11.2 | 45.1 | 9.8 | 3.0 |
GZ11 | 12.2 | 54.4 | 10.8 | 3.6 |
GZ12 | 12.8 | 80.1 | 15.2 | 5.3 |
GZ13 | 12.2 | 79.9 | 15.9 | 5.3 |
GZ17 | 12.1 | 108.8 | 21.8 | 7.3 |
GZ18 | 11.6 | 70.7 | 14.8 | 4.7 |
Table 3 Comparison of fermentation parameters of chondroitin synthesis strains
菌株 | OD600 | CH/ (mg/L) | CH对细胞得率/ (mg/g) | CH对甘油得率/ (mg/g) |
---|---|---|---|---|
GZ01 | 11.0 | 7.1 | 1.6 | 0.5 |
GZ05 | 11.2 | 29.9 | 6.5 | 2.0 |
GZ06 | 11.8 | 46.2 | 9.5 | 3.1 |
GZ07 | 11.2 | 45.1 | 9.8 | 3.0 |
GZ11 | 12.2 | 54.4 | 10.8 | 3.6 |
GZ12 | 12.8 | 80.1 | 15.2 | 5.3 |
GZ13 | 12.2 | 79.9 | 15.9 | 5.3 |
GZ17 | 12.1 | 108.8 | 21.8 | 7.3 |
GZ18 | 11.6 | 70.7 | 14.8 | 4.7 |
发酵参数 | E. coli GZ01 | E. coli GZ17 |
---|---|---|
起始OD600 | 0.47 | 0.45 |
最终OD600 | 57.00 | 76.00 |
软骨素产量/(g/L) | 0.31 | 2.95 |
软骨素对细胞得率/(mg/g) | 13.20 | 94.20 |
软骨素对甘油得率/(mg/g) | 2.25 | 13.41 |
生产强度/(mg/(L·h)) | 6.20 | 59.00 |
Table 4 Analysis of fermentation results of E. coli GZ01 and E. coli GZ17
发酵参数 | E. coli GZ01 | E. coli GZ17 |
---|---|---|
起始OD600 | 0.47 | 0.45 |
最终OD600 | 57.00 | 76.00 |
软骨素产量/(g/L) | 0.31 | 2.95 |
软骨素对细胞得率/(mg/g) | 13.20 | 94.20 |
软骨素对甘油得率/(mg/g) | 2.25 | 13.41 |
生产强度/(mg/(L·h)) | 6.20 | 59.00 |
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