title: A systematic optimization of medium chain fatty acid biosynthesis via the reverse beta-oxidation cycle in Escherichia coli.
abstract: Medium-chain fatty acids (MCFAs, 6-10 carbons) are valuable precursors to many industrial biofuels and chemicals, recently engineered reversal of the β-oxidation (r-BOX) cycle has been proposed as a potential platform for efficient synthesis of MCFAs. Previous studies have made many exciting achievements on functionally characterizing four core enzymes of this r-BOX cycle. However, the information about bottleneck nodes in this cycle is elusive. Here, a quantitative assessment of the inherent limitations of this cycle was conducted to capitalize on its potential. The selection of the core β-oxidation reversal enzymes in conjunction with acetyl-CoA synthetase endowed the ability to synthesize about 1g/L MCFAs. Furthermore, a gene dosage experiment was developed to identify two rate-limiting enzymes (acetyl-CoA synthetase and thiolase). The de novo pathway was then separated into two modules at thiolase and MCFA production titer increased to 2.8g/L after evaluating different construct environments. Additionally, the metabolism of host organism was reprogrammed to the desired biochemical product by the clustered regularly interspaced short palindromic repeats interference system, resulted in a final MCFA production of 3.8g/L. These findings described here identified the inherent limitations of r-BOX cycle and further unleashed the lipogenic potential of this cycle, thus paving the way for the development of a bacterial platform for microbial production of high-value oleo-chemicals from low-value carbons in a sustainable and environmentally friendly manner.
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing, Jiangsu 210095, China.
authors.forename: Junjun
authors.initials: J
authors.lastname: Wu
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing, Jiangsu 210095, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210095, China.
authors.forename: Xiudong
authors.initials: X
authors.lastname: Xia
authors.affiliation: College of Food Science and Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing, Jiangsu 210095, China. Electronic address: dongms@njau.edu.cn.
authors.forename: Mingsheng
authors.initials: M
authors.lastname: Dong
mesh_terms: D000106:Acetate-CoA Ligase / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D005227:Fatty Acids / Q000096:biosynthesis / Q000235:genetics; D060847:Metabolic Engineering; D010084:Oxidation-Reduction
chemical_list: D029968:Escherichia coli Proteins; D005227:Fatty Acids; D000106:Acetate-CoA Ligase
keywords: Biochemical; Biofuels; Metabolic engineering; Synthetic biology; β-oxidation reversal