Effect of flexible linker length on the activity of fusion protein 4-coumaroyl-CoA ligase::stilbene synthase.
In order to elucidate the effect of flexible linker length on the catalytic efficiency of fusion proteins, two short flexible peptide linkers of various lengths were fused between Arabidopsis thaliana 4-coumaroyl-CoA ligase (4CL) and Polygonum cuspidatum stilbene synthase (STS) to generate fusion proteins 4CL-(GSG)n-STS (n ≤ 5) and 4CL-(GGGGS)n-STS (n ≤ 4). The fusion proteins were expressed in both Escherichia coli and Saccharomyces cerevisiae, and their bioactivities were tested in vitro and in vivo using purified proteins and engineered strains, respectively. The catalytic efficiency of the fusions decreased gradually with the increase of GSG or GGGGS repeats. In both engineered S. cerevisiae and E. coli in vivo experiments, the capacity of resveratrol production decreased gradually with increasing linker length. In silico analysis showed that the prediction of homology models of fusion proteins was consistent with the in vitro and in vivo results.
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Huili
H
Guo
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Yadong
Y
Yang
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Feiyan
F
Xue
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Hong
H
Zhang
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Tiran
T
Huang
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Wenbin
W
Liu
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Huan
H
Liu
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Fenqiang
F
Zhang
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Mingfeng
M
Yang
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Chunmei
C
Liu
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn.
Heshu
H
Lu
CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, Hubei, China.
Yansheng
Y
Zhang
Key Laboratory of Urban Agriculture (North) of Ministry of Agriculture, Beijing University of Agriculture, Beijing 102206, China. lqma@bua.edu.cn and Beijing Collaborative Innovation Center for Eco-Environmental Improvement with Forestry and Fruit Trees, Beijing 102206, China.
Lanqing
http://orcid.org/0000-0002-5578-6958
L
Ma
D000214:Acyl Coenzyme A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013329:Structure-Activity Relationship
D000214:Acyl Coenzyme A; D011993:Recombinant Fusion Proteins; C058644:4-coumaroyl-coenzyme A; D000217:Acyltransferases; C022674:stilbene synthase