Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides

Abstract

Increasing energy demands and health-related concerns worldwide have motivated researchers to adopt diverse strategies to improve medium-chain fatty acid (MCFA) biosynthesis for use in the functional food and aviation industries. The abundance of naturally produced MCFAs from botanical sources (i.e., coconut fruit/seeds and palm tree) has been observed to be insufficient compared with the various microorganisms used to cope with industrial demands. Mucor circinelloides is one of many promising microorganisms; it exhibits diverse biotechnological importance ranging from the production of functional lipids to applications in the manufacture of bio-fuel. Thus, research was conducted to acquire the desired elevated amounts of MCFAs (i.e., C8⁻C12) from metabolically engineered strains of M. circinelloides M65. To achieve this goal, four different acyl-acyl carrier protein (ACP) thioesterase (TE)-encoding genes exhibiting a substrate preference for medium-chain acyl-ACP molecules were expressed in M. circinelloides M65, resulting in the generation of C8⁻C12 fatty acids. Among all the engineered strains, M65-TE-03 and M65-TE-04 demonstrated the highest production of non-native C8⁻C10 and C12 fatty acids, respectively, in comparison to the control. These recombinant strains biosynthesized MCFAs de novo within the range from 28 to 46% (i.e., 1.14 to 2.77 g/L) of total cell lipids. Moreover, the reduction in chain length eventually resulted in a 1.5⁻1.75-fold increase in total lipid productivity in the engineered strains. The MCFAs were also found to be integrated into all lipid classes. This work illustrates how the integration of heterologous enzymes in M. circinelloides can offer a novel opportunity to edit the fatty acid synthases (FAS) complex, resulting in increased production of microbial MFCAs.

Keywords: Mucor circinelloides; bio-fuels; medium-chain fatty acids; metabolic engineering; microbial lipids.

Figure 1

Design and strategy for engineering the fungal fatty acid synthases (FASs) to produce medium-chain fatty acids (MCFAs). Reaction cycles catalyzed by different domains of engineered fungal FASs. A heterologous medium-chain thioesterase (TE) was integrated into FASs (as shown in grey box) to release MCFAs. ER: enoyl reductase; DH: dehydratase; MPT: malonyl/palmitoyl transferase; ACP: acyl carrier protein; KS: ketoacyl synthase; KR: ketoacyl reductase; PPT: phosphopantetheinyl transferase; AT: acetyl transferase.

Figure 2

(A,B) Expression of TE-01, TE-02, TE-03, and TE-04 genes. (A) Structure of plasmids pMAT1552-pyrF-TE-01, pMAT1552-pyrF-TE-02, pMAT1552-pyrF-TE-03, pMAT1552-pyrF-TE-04 for TE-01, TE-02, TE-03, TE-04 genes over-expressing in M. circinelloides M65 are demonstrated. Green boxes indicate the coding region of thioesterase genes. (B) Polymerase chain reaction (PCR) amplification of genome of control strain (A) and thioesterase gene (TE) over-expressing strains i.e., M65-TE-01, M65-TE-02, M65-TE-03, and M65-TE-04 was demonstrated to as B–E, respectively with the primers (Supplementary materials: Table S2). M, Gene Ruler DNA Ladder Mix. Sizes in kb of the relevant maker fragments are indicated.

Figure 3

(A–F) Cell growth, lipid accumulation and substrate consumption of control and engineered strains cultivated in 3-L fermenter with 1.5 L modified K and R medium for 96 h. (A) Dry cell weight (DCW), (B) specific growth rate, (C) percentage of total lipid content from total DCW, (D) specific lipid yield, (E) residual glucose concentration, (F) ammonium sulphate concentration. Values were mean of three independent experiments. Error bars represent the standard error of the mean.

Figure 4

(A) Fatty Acid (FA) profiles of the control (M65) and the engineered strains, (B) Abundance of a medium-chain lipids, (C) 16-carbon lipids, and (D) 18-carbon lipids from Mucor circinelloides strains (M65) expressing different heterologous acyl-ACP thioesterase enzymes in different lipid classes, i.e., free fatty acids (FFA), triacylglycerol (TAG), diacylglycerol (DAG), monoacylglycerol (MAG), sterol and steryl ester (S. ester).