Intermediate-sensor assisted push-pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli.
Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push-pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an L-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with L-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & System Biology, Tsinghua University, Beijing 10084, China.
Mingyue
M
Fang
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & System Biology, Tsinghua University, Beijing 10084, China.
Tianmin
T
Wang
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & System Biology, Tsinghua University, Beijing 10084, China. Electronic address: chongzhang@tsinghua.edu.cn.
Chong
C
Zhang
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & System Biology, Tsinghua University, Beijing 10084, China.
Jili
J
Bai
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & System Biology, Tsinghua University, Beijing 10084, China.
Xiang
X
Zheng
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Xuejin
X
Zhao
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Chunbo
C
Lou
Key Laboratory for Industrial Biocatalysis, Ministry of Education, Institute of Biochemical Engineering, Department of Chemical Engineering, Center for Synthetic & System Biology, Tsinghua University, Beijing 10084, China.
Xin-Hui
XH
Xing
D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods; D007211:Indoles / Q000302:isolation & purification / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis / Q000379:methods*; D015398:Signal Transduction / Q000502:physiology; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D007211:Indoles; D014364:Tryptophan; C063155:violacein
Biosensor; Deoxyviolacein biosynthesis; High-throughput screening; Pathway engineering; Sequential optimization