Dissection of malonyl-coenzyme A reductase of Chloroflexus aurantiacus results in enzyme activity improvement.
The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems.
Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China ; University of Chinese Academy of Sciences, Beijing, China.
Changshui
C
Liu
Qi
Q
Wang
Mo
M
Xian
Yamei
Y
Ding
Guang
G
Zhao
D015153:Blotting, Western; D041861:Chloroflexus / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D019344:Lactic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D019344:Lactic Acid; D008316:Malonyl Coenzyme A; D009249:NADP; C031601:hydracrylic acid; D010088:Oxidoreductases; C454539:malonyl-Coa reductase