Constructing E. coli Co‐Cultures for De Novo Biosynthesis of Natural Product Acacetin

Abstract

Modular co‐culture engineering is an emerging approach for biosynthesis of complex natural products. In this study, microbial co‐cultures composed of two and three Escherichia coli strains, respectively, are constructed for de novo biosynthesis of flavonoid acacetin, a value‐added natural compound possessing numerous demonstrated biological activities, from simple carbon substrate glucose. To this end, the heterologous biosynthetic pathway is divided into different modules, each of which is accommodated in a dedicated E. coli strain for functional expression. After the optimization of the inoculation ratio between the constituent strains, the engineered co‐cultures show a 4.83‐fold improvement in production comparing to the mono‐culture controls. Importantly, cultivation of the three‐strain co‐culture in shake flasks result in the production of 20.3 mg L−1 acacetin after 48 h. To the authors' knowledge, this is the first report on acacetin de novo biosynthesis in a heterologous microbial host. The results of this work confirm the effectiveness of modular co‐culture engineering for complex flavonoid biosynthesis.

A fungal NRPS-PKS enzyme catalyses the formation of the flavonoid naringenin

“…14 and 15 ). In addition, 9 -producing strain TYHJ49, termed as QL35-ACA hereafter, was constructed by introducing the flavone synthase I gene EbFNSI 13 from Erigeron breviscapus and the flavone 4’- O -methyltransferase gene PaCOMT 33 from Plagiochasma appendiculatum into QL35-NAR (Fig. 6a and Supplementary Fig.…”

“…Although the yield of naringenin is relatively low in comparison with 1,184 mg L −1 from optimized S. cerevisiae strain by expression of 4CL, CHS, and CHI 46 , the refactoring isorhamnetin biosynthetic pathway has not been reported prior to this study. Furthermore, according to Wang et al 33 , the acacetin product yield achieved to 20.3 mg L −1 under co-cultivation of three engineered strains, but merely up to 2.7 mg L −1 under mono-cultivation 47 . In our case, the titer of acacetin attained 10.4 mg L −1 under our cultural condition.…”

Biosynthesis of resveratrol using metabolically engineered Escherichia coli

“…Since C4H is a member of the P450 family, it is difficult to express it functionally in E. coli. For this reason, many studies related to the biosynthesis of phenolic compounds have used TAL, which converts tyrosine to p-coumaric acid [18,35]. In previous studies, TAL cloned from S. espanaensis was used to biosynthesize various phenolic compounds in E. coli based on its excellent enzyme activity [18,36].…”

“…Recently, various methods, such as the co-culture methods, introduction of promoters of different strengths, and gene integration into the genome, have been attempted to resolve the metabolic imbalances. Among them, a co-culture method that divides the metabolic pathway necessary for the biosynthesis of phytochemicals into two or three strains has been frequently used as a method to overcome metabolic imbalance [35,39]. To increase resveratrol biosynthesis, subsequent research such as a co-culture method should be conducted.…”

“…In addition, the synthesized phenolic compounds can be easily purified using simple organic solvents such as ethyl acetate [18,25]. Using E. coli, a number of medicinally important compounds, such as hydroxycinnamic acids, coumarins, stilbenoids, flavonoids, terpenoids, and alkaloids, have been successfully synthesized [18,[27][28][29]35].…”