Cloning and characterization of a GPAT-like gene from the microalga Lobosphaera incisa (Trebouxiophyceae): overexpression in Chlamydomonas reinhardtii enhances TAG production

Abstract

The oleaginous green microalga Lobosphaera (formerly Parietochloris) incisa accumulates high amounts of arachidonic-acid-rich triacylglycerols (TAG), in particular under conditions of nitrogen starvation. This photosynthetic organism is of great interest for studying the mechanisms responsible for storage lipid biosynthesis and the deposition of long-chain polyunsaturated fatty acids (LC-PUFA) in TAG. In this work, we report on cloning a complementary DNA (cDNA) for the putative L. incisa glycerol-3-phosphate acyltransferase (GPAT), whose deduced amino acid sequence features distinctive motives found in those mammalian and Arabidopsis GPAT isoforms that have been implicated in TAG biosynthesis. Temporal analysis of LiGPAT expression in the course of nitrogen starvation showed a positive relationship between changes in the transcript level and patterns of fatty acid production. When expressed in Arabidopsis leaf mesophyll protoplasts, the green fluorescent protein (GFP) fused to the C-terminus of LiGPAT localized outside the chloroplasts in agreement with its predicted extraplastidial localization. Based on an in silico analysis of a deduced amino acid sequence and on similarity to other GPATs participating in TAG biosynthesis, LiGPAT was expressed in the green model microalga Chlamydomonas reinhardtii in order to confirm the predicted function in a heterologous microalgal system. Overexpression of LiGPAT resulted in an up to 50 % increase in the content of TAG on a cell dry weight basis as compared to the control in the stationary phase culture without negative impact on growth parameters. Total fatty acids and TAG of the transformant lines featured an elevated level of oleic acid (18:1 n-9) and a concurrent decrease in C18 PUFA. Additional studies on the acyl substrate preference of LiGPAT are required.

Current advances in molecular, biochemical, and computational modeling analysis of microalgal triacylglycerol biosynthesis

“…This strategy increased TAG content by two-fold (Hsieh et al, 2012). Interestingly, channelizing the carbon precursor (Acyl-ACP) towards TAG by overexpression of a Lobosphaera incisa GPAT in C. reinhardtii led to 50% increase in TAG content without compromising growth (Iskandarov et al, 2015), indicating this is a promising strategy for enhanced TAG production. In aggregate, these results generally indicate that the Kennedy pathway genes exert a larger degree of regulation on TAG biosynthesis than FA synthetic genes, suggesting that manipulations that "pull" substrates through the TAG biosynthesis pathway are more effective at increasing TAG production than those that "push" substrates into it.…”

“…Nannochloropsis oceanica and N. gaditana possess nine putative GPAT and LPAAT isoforms of which only plastidic GPATs are unambiguously annotated (Vieler et al, 2012). Interestingly, overexpression of the GPAT gene from the oleaginous green microalga Lobosphaera incisa in Chlamydomonas results in up to 50% increase in TAG without compromising growth (Iskandarov et al, 2015). LPAAT transfers the second acyl group into the sn-2 position of LPA and converts it into phosphatidic acid (PA).…”

“…Catalyzes the first committed step of the Kennedy pathway C. reinhardtii 50% increase in the TAG content without compromising growth (Iskandarov et al, 2015) TAG storage and lipid body formation MLDP (major lipid droplet protein) Implicated in lipid droplet formation or maintenance…”

Plant sn‐Glycerol‐3‐Phosphate Acyltransferases: Biocatalysts Involved in the Biosynthesis of Intracellular and Extracellular Lipids

“…In addition to their role in the biosynthesis of extracellular‐lipid polyesters, ER‐bound GPAT also contribute to TAG biosynthesis by catalyzing the first acyl‐CoA‐dependent acylation reaction in the Kennedy pathway. GPAT9 is very closely related phylogenetically to the mammalian ER‐bound GPAT3, which is known to play a crucial role in storage‐lipid biosynthesis in humans (Gidda et al, ), Furthermore, various algal species possess homologs of AtGPAT9 that are responsible for producing an abundance of TAG (Iskandarov et al, ; Niu et al, ). Hence, studies have been focused on unraveling the function of GPAT9 in plants for some time (Singer et al, ).…”

“…Unlike plant GPAT, the majority of GPAT from green algae species are predicted to localize within plastids (Misra & Panda, ), and many appear to be involved in TAG biosynthesis (Iskandarov et al, ). Sequence similarity between algal and plant GPAT has been found to be low in general; however, there is a highly conserved topology arrangement in these two GPAT types (Misra & Panda, ).…”