The fatty acid is a carboxylic acid with an aliphatic chain. The majority of fatty acids that are found in nature have an unbranched chain with an even number of carbon atoms, ranging from 4 to 28. There are several ways to categorize fatty acids, including length or saturation vs. unsaturation. Depending on the length of their chains, they can be divided into short-chain, medium-chain, and long-chain fatty acids.
Medium-chain fatty acids (MCFAs) are fatty acids with aliphatic tails of 6 to 10 carbons, which can form medium-chain triglycerides. They are valuable as precursors to industrial chemicals and biofuels.
Figure 1. Medium-chain triglyceride, containing three medium-chain fatty acids
MCFAs are mainly obtained through extraction from natural sources or synthesis in petrochemical-based industrial chains. For natural sources, MCFAs are only present in limited sources, such as coconut and palm kernel, and are usually present in very low concentrations, ranging from 7.9% to 15% of the total fatty acids in coconut or palm kernel oil. Shortages of fossil fuels and environmental concerns have increased the production costs of petrochemical-based MCFAs. Meanwhile, the resulting products are unsuitable for use in the food and pharmaceutical industries due to food safety concerns. Therefore, a scalable, stable, and sustainable process for obtaining MCFAs from renewable sources are urgently needed.
Escherichia coli is an attractive host organism for fatty acid production because of its rapid replication rate, availability of multiple carbon sources, and ease of genetic manipulation. Although studies have been performed to generate thioesterases by expressing substrate specificity for MCFAs, but the yield is much lower than that of long-chain fatty acids (240 mg/L), because the MCFAs produced by the FAB pathway have only a low abundance of acyl-ACPs as precursors. Therefore, there is a great need to optimize fatty acid chain lengths in microbial fatty acid synthesis pathways. specificity.
Functional reversal of the β-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities. In other research, researchers demonstrate the utility of this platform for the synthesis of medium-chain length (C6–C10) products through the manipulation of key components of the pathway.
However, this pathway is currently limited by the low concentrations of acetyl-CoA and NADH produced by the natural central carbon metabolism of E. coli, which poses a challenge for the large-scale production of MCFAs.
In this work, we presented a new approach to producing MCFAs using an engineered reversal of the β-oxidation (r-BOX) cycle (Figure 2), and we use the Escherichia coli strain to set up the platform.
Figure 2. the fatty acid β-oxidation cycle
First, the four genes bktB, ydiI, fadB and ter (r-BOX) were amplified from corresponding templates, and then the amplicons were applied to DNA gel electrophoresis to obtain our target genes.
Next, we extracted plasmids pCDFDuet-1 and pCDFDuet-1vector from E. coli DH5α. Then we inserted genes fadB and ter into pCDFDuet-1vector, and inserted genes bktB and ydiI into pCDFDuet-1vector. After verifying the sequences of the recombinant plasmids by Sanger sequencing, we transferred the recombinant plasmids into BL21(DE3).
Then, we added IPTG to induce the expression of r-BOX-related proteins and detected them through SDS-PAGE. What count is that we measured the yield of MCFAs by HPLC.
Finally, in order to optimize this system to improve the yield of MCFAs, we change the speed we cultured the strain and the concentration of sodium acetate in the medium. What’s more, we also test 4 kinds of mutant T7 promoters and developed a more efficient promoter, the T7-C4 promoter.
Overall, we aimed to intensify the MCFAs synthesis pathway by overexpressing key enzymes in the reversal of the β-oxidation cycle in E. coli and optimize the T7 promoter to increase MCFAs production from 1.3g/L.
In addition, the esterification products of MCFAs have emulsion stabilization and antioxidant properties, which can make cosmetics more delicate and prolong shelf life, so they are widely used in the daily chemical industry.
Medium-chain fatty acids have weight-control benefits that are less likely to be deposited as body fat, allowing them to be used as important food ingredients. Medium-chain fatty acids help reduce the risk of two diseases, atherosclerosis and heart disease, and help prevent heart disease. It can be used as a food for people suffering from fat malabsorption, AIDS, cancer, and diabetes. It can be widely used to treat prostatic hyperplasia, lower cholesterol, prevent hyperlipidemia, etc.
