Journal
METABOLIC ENGINEERING
Volume 41, Issue -, Pages 115-124Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2017.03.012
Keywords
beta-oxidation reversal; Biofuels; Biochemical; Synthetic biology; Metabolic engineering
Categories
Funding
- National Natural Science Foundation of China [31601448]
- Natural Science Foundation of Jiangsu Province [BK20160718]
- Fundamental Research Funds for the Central Universities [KYZ201654, KJQN201728]
- Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) [201512]
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Medium-chain fatty acids (MCFAs, 6-10 carbons) are valuable precursors to many industrial biofuels and chemicals, recently engineered reversal of the beta-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 beta-oxidation reversal enzymes in conjunction with acetyl-CoA synthetase endowed the ability to synthesize about 1 g/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 2.8 g/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 3.8 g/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 oleochemicals from low-value carbons in a sustainable and environmentally friendly manner.
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