Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli.
10.1016/S1875-5364(20)60005-X
- Author:
Di LI
1
,
2
,
3
,
4
;
Yang LI
3
,
5
,
6
;
Jiao-Yang XU
3
,
5
,
6
;
Qing-Yan LI
3
,
4
,
5
,
7
;
Jin-Lei TANG
3
,
4
,
5
,
7
;
Shi-Ru JIA
8
;
Chang-Hao BI
3
,
4
,
5
,
7
;
Zhu-Bo DAI
3
,
4
,
5
,
7
;
Xin-Na ZHU
3
,
5
,
7
,
9
;
Xue-Li ZHANG
3
,
5
,
7
,
10
Author Information
1. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 310018, China
2. Tianjin Institute of Industrial Biotechnology, Chinese of Academy of Sciences, Tianjin 300308, China
3. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
4. National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China.
5. Tianjin Institute of Industrial Biotechnology, Chinese of Academy of Sciences, Tianjin 300308, China
6. University of Chinese Academy of Sciences, Beijing 100071, China.
7. University of Chinese Academy of Sciences, Beijing 100071, China
8. College of Biotechnology, Tianjin University of Science and Technology, Tianjin 310018, China.
9. National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China. Electronic address: zhu_xn@tib.cas.cn.
10. National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China. Electronic address: zhang_xl@tib.cas.cn.
- Publication Type:Journal Article
- Keywords:
Astaxanthin;
Cre-loxP;
Escherichia coli;
Metabolic engineering;
RBS library;
β-Carotene ketolase
- From:
Chinese Journal of Natural Medicines (English Ed.)
2020;18(9):666-676
- CountryChina
- Language:English
-
Abstract:
This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.
