Producing Cyclopropane Fatty Acid in Plant Leafy Biomass via Expression of Bacterial and Plant Cyclopropane Fatty Acid Synthases

Abstract

Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA via hydrogenation, and therefore oils rich in DHSA are a potential feedstock for SMCBFA. Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were coexpressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.

Genetics, Breeding and Genetic Engineering to Improve Cottonseed Oil and Protein: A Review

“…The key genes encoding for cyclopropane fatty acid synthases converting oleic acid to DHSA have been identified in Arabidopsis ( Bao et al, 2002 ) and cotton ( Yu et al, 2011 ). Although the proof of concept has been made in producing DHSA in transgenic model plants ( Yu et al, 2018 ; Okada et al, 2020 ), associated genes have yet to be modified in the cotton genome to raise DHSA production in cottonseeds.…”

Toward sustainable production of value‐added bioenergy and industrial oils in oilseed and biomass feedstocks

“…Building on this, current research has focused mainly on the 3P strategy to elevate oil accumulation in leaves and stems (Parajuli et al, 2020;Vanhercke et al, 2019;Zale et al, 2016). By contrast, only limited research has explored the production of unusual fatty acids in vegetative organs, and research to date has largely been limited to model systems such as Arabidopsis and Nicotiana benthamiana (Okada et al, 2020;Reynolds et al, 2015;Yurchenko et al, 2017). Early studies on the production of unusual fatty acids in vegetative organs included research into the constitutive expression of divergent FAD2 hydroxylases in N. tabacum and Arabidopsis Vandeloo et al, 1995).…”