Tuning of acyl-ACP thioesterase activity directed for tailored fatty acid synthesis

Abstract

Medium-chain fatty acids have attracted significant attention as sources of biofuels in recent years. Acyl-ACP thioesterase, which is considered as the key enzyme to determine the carbon chain length, catalyzes the termination of de novo fatty acid synthesis. Although recombinant medium-chain acyl-ACP thioesterase (TE) affects the fatty acid profile in heterologous cells, tailoring of the fatty acid composition merely by engineering a specific TE is still intractable. In this study, the activity of a C8-C10-specific thioesterase FatB2 from Cuphea hookeriana on C10-ACP was quantified twice as high as that on C8-ACP based on a synthetic C8-C16 acyl-ACP pool in vitro. Whereas in vivo, it was demonstrated that ChFatB2 preferred to accumulate C8 fatty acids with 84.9% composition in the ChFatB2-engineered E. coli strain. To achieve C10 fatty acid production, ChFatB2 was rationally tuned based on structural investigation and enzymatic analysis. An I198E mutant was identified to redistribute the C8-ACP flow, resulting in C10 fatty acid being produced as the principal component at 57.6% of total fatty acids in vivo. It was demonstrated that the activity of TE relative to β-ketoacyl-ACP synthases (KAS) directly determined the fatty acid composition. Our results provide a prospective strategy in tailoring fatty acid synthesis by tuning of TE activities based on TE-ACP interaction.

Fatty Acid Biosynthesis: Chain‐Length Regulation and Control

“…When plant TEs are overexpressed in E. coli , their substrate specificities determine the product spectrum of fatty acid synthesis . As shown by mutagenesis studies, altering TE activity relative to KAS activity can influence the product spectrum of fatty acid synthesis …”

“…[2a] As shown by mutagenesiss tudies, altering TE activity relativet oK AS activity can influencet he product spectrum of fatty acid synthesis. [134] Directed Engineering for Modulation of Fatty Acid Chain Length Fatty acids are considered strategically important platform chemicals accessible throughs ustainable microbial approaches. [135] The recently acquired structurali nformation on FASs provides an excellent understandingo ft he molecular basis of fatty acids ynthesis.…”

“…[133c] In spite of engineering successes, the impact of mutationso np lant TEs seems unpredictable. [134,156] Although plant TEs overall share high sequence identity (35-60%), they exhibit diverses ubstrate specificities, and only limited structural information has been gathered thus far. In general,a pproaches to produce short-chain fatty acids in bacteria,p lants,a nd algae have been marginally successful.…”

New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution

“…This thioesterase acts as a dimer, its monomers establishing two tandem hot dog folds that are joined by a flexible linker, one close to the C-terminal domain and other close to the N-terminal one. The N-terminal hot dog domain defines the substrate binding pocket, which can be engineered to change the substrate specificity of the enzyme ( Feng et al, 2018 ). This Uc FatB1 is more closely related to other plant thioesterases than to any bacterial isoforms and thus, the structure of this enzyme is likely to be a better reference to model FatA and FatB2 enzymes.…”

Identification and analysis of oil candidate genes revealed the molecular basis of oil accumulation in Gossypium hirsutum L. for cottonseed biofuels development

“…Moreover, HSL1dependent histone methylation plays critical roles in regulation of seed dormancy during seed germination and early seedling growth, which will affect the oil content accumulated in seed [53]. Similarly, AtLPEAT2 [54], AtLACS2 [55] and ChACP4 [56] were found to be associated with PI (34:3), linolenic acid (P = 2.63e-07), and pyruvate (LOD = 14.68) in oil synthesis process, respectively. Ma et al [39] also found that the cotton genes GhHSL1, GhACP2 and GhLPEAT2 were differentially expressed between the upland cotton (G. hirsutum L.) high oil content genotype (3008) and low oil content genotype (3012) in 10, 20 and 30 DPA ovules.…”