Biosynthesis of Triacylglycerol Molecules with a Tailored PUFA Profile in Industrial Microalgae

Abstract

The composition of polyunsaturated fatty acids (PUFAs) in triacylglycerols (TAGs) is key to health benefits and for oil applications, yet the underlying genetic mechanism remains poorly understood. In this study, by in silico, ex vivo, and in vivo profiling of type-2 diacylglycerol acyltransferases (DGAT2s) in Nannochloropsis oceanica we revealed two novel PUFA-preferring enzymes that discriminate individual PUFA species in TAG assembly, with NoDGAT2J for linoleic acid (LA) and NoDGAT2K for eicosapentaenoic acid (EPA). The LA and EPA composition of TAG molecules is mediated in vivo via the functional partitioning between NoDGAT2J and 2K, both of which are localized in the chloroplast envelope. By modulating transcript abundance of the DGAT2s, an N. oceanica strain bank was created, where proportions of LA and EPA in TAG vary by 18.7-fold (between 0.21% and 3.92% dry weight) and 34.7-fold (between 0.09% and 3.12% dry weight), respectively. These findings lay the foundation for producing designer TAG molecules with tailored health benefits or for biofuel applications in industrial microalgae and higher-plant crops.

The Microalga Nannochloropsis during Transition from Quiescence to Autotrophy in Response to Nitrogen Availability

“…Consequently, to fully utilize algae as a biofactory for TAG production, it is necessary to continue elucidating the mechanisms and optimal conditions for TAG accumulation. In algae, there are multiple TAG synthesis pathways (Liu et al, 2016a;Xin et al, 2017Xin et al, , 2019. In the chloroplast, de novo synthesized fatty acids (FAs) can be incorporated into chloroplast diacylglycerol (DAG), an essential precursor in the synthesis of photosynthetic membrane glycerolipids, or possibly plastidic TAG as reported for Chlamydomonas (Goodson et al, 2011;Goold et al, 2016).…”

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein

“…including Chlamydomonas reinhardtii, Phaeodactylum tricornutum, and Nannochloropsis 91 oceanica (Zulu et al, 2017;Xin et al, 2017;Xin et al, 2018;Mao et al, 2019;Iwai et al, 2014). 92 DGAT1 is an integral membrane protein with multiple transmembrane domains (TMD), 93 the three-dimensional structure of which has not yet been elucidated (Xu, Caldo, et al, 2018).…”

Designing Microalgal Oils

“…By ex vivo feeding assays in yeast, in vitro and in vivo functional characterizations, they first found that NoDGAT2A and NoDGAT2D prefer SFA and MUFA CoAs, respectively, whereas NoDGAT2C shows the highest activity toward PUFA CoAs (Xin et al, 2017). In a new study (Xin et al, 2019), they identified two novel DGAT2s with substrate preference down to the single PUFA CoA species, namely NoDGAT2J prefers linoleic acid (LA) CoA, whereas NoDGAT2K prefers EPA CoA for TAG synthesis. By overexpression or knockdown of each of the five NoDGAT2s, they obtained a bank of engineered N. oceanica strains producing a wide array of designer TAGs with various compositions of FAs.…”

“…By overexpression or knockdown of each of the five NoDGAT2s, they obtained a bank of engineered N. oceanica strains producing a wide array of designer TAGs with various compositions of FAs. The SFA/MUFA/PUFA ratio in TAGs varied in these N. oceanica strains but could be adjusted by modulating the expression level of the individual NoDGAT2 gene (Xin et al, 2017(Xin et al, , 2019. Interestingly, they found that TAG-associated EPA could be greatly enriched by NoDGAT2K overexpression in N. oceanica (Xin et al, 2019), providing promising candidates for rare LC-PUFA production in microalgae.…”

“…The studies by Xin et al (2017Xin et al ( , 2019 discovered the distinct yet hierarchical substrate preferences of NoDGAT2A, 2C, 2D, 2J, and 2K, unveiled a sophisticated mechanism of TAG synthesis in N. oceanica, and provided useful tools for metabolic manipulation of oil properties and productivity in industrial microalgae. Sequence analysis suggests that the amino acid…”