Overexpression of artificially fused bifunctional enzyme 4CL1-CCR: a method for production of secreted 4-hydroxycinnamaldehydes in Escherichia coli.
BACKGROUND
4-Hydroxycinnamaldehydes are important intermediates in several secondary metabolism pathways, including those involved in the biosynthesis of phenolic acids, flavonoids, terpenoids and monolignols. They are also involved in the biosynthesis and degradation of lignins, which are important limiting factors during the processes of papermaking and biofuel production. Access to these aromatic polymers is necessary to explore the secondary biometabolic pathways they are involved in. Coniferaldehyde, sinapaldehyde, p-coumaraldehyde and caffealdehyde are members of the 4-hydroxycinnamaldehyde family. Although coniferaldehyde and sinapaldehyde can be purchased from commercial sources, p-coumaraldehyde and caffealdehyde are not commercially available. Therefore, there is increasing interest in producing 4-hydroxycinnamaldehydes. Here, we attempted to produce 4-hydroxycinnamaldehydes using engineered Escherichia coli.


RESULTS
4-Coumaric acid: coenzyme A ligase (4CL1) and cinnamoyl coenzyme A reductase (CCR) were fused by means of genetic engineering to generate an artificial bifunctional enzyme, 4CL1-CCR, which was overexpressed in cultured E. coli supplemented with phenylpropanoic acids. Three 4-hydroxycinnamaldehydes, p-coumaraldehyde, caffealdehyde and coniferaldehyde, were thereby biosynthesized and secreted into the culture medium. The products were extracted and purified from the culture medium, and identically characterized by the HPLC-PDA-ESI-MSn. The productivity of this new metabolic system were 49 mg/L for p-coumaraldehyde, 19 mg/L for caffealdehyde and 35 mg/L for coniferaldehyde. Extracellular hydroxycinnamoyl-coenzyme A thioesters were not detected, indicating that these thioesters could not pass freely through the cellular membrane. The fusion enzyme 4CL1-CCR can catalyze sequential multistep reactions, thereby avoiding the permeability problem of intermediates, which reveals its superiority over a mixture of individual native enzymes. Moreover, we have described a highly sensitive and selective method for separation and identification of phenylpropanoic acids and their corresponding cinnamaldehydes in the present paper. The feasibility of this method has been proven in the application of the method to the analysis of the metabolites of whole-cell catalysts.


CONCLUSIONS
We have established a bioconversion pathway for the microbial production of valuable 4-hydroxycinnamaldehydes from phenylpropanoic acids. This biotransformation method is both convenient and environmentally friendly, and provides new insights into the biosynthesis of natural plant secondary products.
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. liushuxin@bjfu.edu.cn.
Shuxin
S
Liu
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. heizhishi8468@126.com.
Qi
Q
Qi
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. chaonan1989@126.com.
Nan
N
Chao
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. houjiayin1989@163.com.
Jiayin
J
Hou
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. raoguodong02@163.com.|Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, People's Republic of China. raoguodong02@163.com.
Guodong
G
Rao
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. jin321108@126.com.|Institute of Agricultural Bio-Resources, Hunan Academy of Agricultural Sciences, Changsha, 410125, People's Republic of China. jin321108@126.com.
Jin
J
Xie
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. luhai1974@163.com.
Hai
H
Lu
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. jiangxn@bjfu.edu.cn.|The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration, National Engineering Laboratory for Tree Breeding, Beijing, 100083, People's Republic of China. jiangxn@bjfu.edu.cn.
Xiangning
X
Jiang
College of Biological Science and Technology, Beijing Forestry University, Beijing, 100083, People's Republic of China. gaiying@bjfu.edu.cn.|The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry Administration, National Engineering Laboratory for Tree Breeding, Beijing, 100083, People's Republic of China. gaiying@bjfu.edu.cn.
Ying
Y
Gai
D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D019149:Bioreactors; D002934:Cinnamates / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000096:biosynthesis / Q000235:genetics; D003373:Coumaric Acids / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D064251:Multifunctional Enzymes / Q000096:biosynthesis / Q000235:genetics; D011422:Propionates / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*
D002934:Cinnamates; D003373:Coumaric Acids; D064251:Multifunctional Enzymes; D011422:Propionates; C117045:p-hydroxycinnamaldehyde; D000445:Aldehyde Oxidoreductases; C019904:cinnamoyl CoA reductase; D003066:Coenzyme A Ligases; C495469:p-coumaric acid