Microalgal glycerol-3-phosphate acyltransferase role in galactolipids and high-value storage lipid biosynthesis

Abstract Glycerolipids are the most abundant lipids in microalgae, and glycerol-3-phosphate:acyl-CoA acyltransferase (GPAT) plays an important role in their biosynthesis. However, the biochemical and biological functions of algal GPAT remain poorly characterized. Here, we characterized the endoplasmic reticulum (ER)-associated GPAT of the model unicellular green alga Chlamydomonas reinhardtii (CrGPATer). Enzymatic assays indicated that CrGPATer is an sn-1 acyltransferase using a variety of acyl-CoAs as the acyl donor. Subcellular localization revealed that CrGPATer was associated with ER membranes and lipid droplets. We constructed overexpression (OE) and knockdown (KD) transgenic C. reinhardtii lines to investigate the in vivo function of CrGPATer. Lipidomic analysis indicated that CrGPATer OE enhanced the cellular content of galactolipids, especially monogalactosyldiacylglycerol, under nitrogen deficiency stress. Correspondingly, CrGPATer KD lines contained lower contents of galactolipids than the control. Feeding experiments with labeled phosphatidic acid revealed that the intermediate of the eukaryotic Kennedy pathway could be used for galactolipid biosynthesis in the chloroplasts. These results provided multiple lines of evidence that the eukaryotic Kennedy pathway mediated by CrGPATer may be involved in galactolipid biosynthesis in C. reinhardtii. OE of CrGPATer significantly increased the content of triacylglycerol and the yield of biomass. Moreover, the content and yield of 1, 3-olein-2-palmitin, a high-value lipid that can be used as an alternative for human milk fat in infant formula, were significantly enhanced in the OE transgenic lines. Taken together, this study provided insights into the biochemical and biological functions of CrGPATer and its potential as a genetic engineering target in functional lipid manufacturing.


Heterologous expression of CrGPATer in yeast, yeast cultivation, and protein preparation
The resulting PCR product of CrGPATer was inserted downstream of the GAL1 promoter in the pYES2.1 TOPO ® TA yeast expression vector (Thermo Fisher Scientific, USA) according to the manufacturer's instructions and was confirmed by sequencing.
S. cerevisiae Δgat1 (BY4742,Matα,his3Δ1,leu2Δ0,lys2Δ0,ura3Δ0,YKR067w::kanMX4) (Zheng et al., 2003)  The cells were then broken with a Mini-Beadbeater (BioSpec Products USA) and the crude homogenates were centrifuged at 12,000 g at 4°C for 10 min to remove the cell debris. The supernatant was further centrifuged at 100,000 g at 4°C for 70 min to separate microsomal and cytosolic fractions. The microsomal pellets were washed and resuspended in 50 mM Tris·HCl (pH 7.9) buffer containing 20% (v/v) glycerol and 1mM DTT, flash frozen with liquid nitrogen, and stored at −80°C for CrGPATer assay.

Overexpression and knock-down recombinant plasmid construction
The putative fragment, ble, in target gene OE plasmid,pChlamy4 (Invitrogen,USA) was exchanged by aphVIII which shows paromomycin resistance. To integrate the CrGPATer into the modified pChlamy4 vector, firstly, the full-length cDNA of CrGPATer was amplified from the homemade cDNA library by using the primer sets CrGPATer-F and CrGPATer-R (Supplemental Table S1) and the sequence of lowercase letters were homologous arms of empty vector. Secondly, empty vector was linearized by PCR via P4-F and P4-R (Supplemental Table S1). Lastly, purified empty vector mixed with CrGPATer CDS fragment maintained homologous arms sequence of vector and achieved homologous recombination followed by pEASY®-Basic Seamless Cloning and Assembly Kit protocols (Transgene, China). All of above mentioned PCR reactions were carried out by using Phanta Master Mix high-fidelity DNA polymerase (Vazyme, China) according to the manufacturer's instructions.
And in order to screen the knock down transformants rapidly, followed by Hu's method (Hu et al., 2014), the luciferase gene from the marine copepod Gaussia princeps (G-Luc) was amplified from the plasmid pHK226 (kindly provided by Kaiyao Huang's Lab) using primers PsaD-Luc F and RBSC2-Luc R (Supplemental Table S1), which included homologous flanking sequence of PsaD promoter 3' ends and RBCS2 intron1 5' end. Similarly, the RBCS2 fragment come from pChlamiRNA3int was amplified by using primers Luc-RBCS2 F and MIR1157-RBCS2 R (Supplemental Table S1), which are flanked by an homologous sequence at G-Luc 3' terminus and cre-MIR1157 5' terminus. The linearized pChlamiRNA3int plasmid by NdeI (Invitrogen,USA) mixed with above two remain homologous flaking sequence and followed by pEASY®-Basic Seamless Cloning and Assembly Kit protocols (Transgene, China), yielding new plasmid, pChlamiRNA3int: Luc. It had been sequenced by primers of PsaD F2 and MIR1157 R (Supplemental Table S1). The amiRNA targeting the CrGPATer and CrGPATcp in C. reinhardtii was designed using WMD3 software (wmd3.weigelworld.org) (Ossowski et al., 2008). The output oligonucleotide was a 96 bp amiRNA precursor sequence (Supplemental Table S1), remaining SpeI sticky ends and these DNA sequence synthesized by GenScript Company (China). Next, the precursor of amiRNA was inserted in linearized pChlamiRNA3int: Luc with SpeI, and the correct orientation of the insertion was selected by sequencing result derived by primers of PsaD F2 and MIR1157 R (Supplemental Table S1).

Transmission electron microscopy
C. reinhardtii cells were harvested at 1,500 g for 10 min and fixed overnight at 4°C with a PBS buffer (pH 7.4) containing 2% (v/v) glutaraldehyde. After rinsing three times with PBS, cell samples were post-fixed with 1% (w/v) osmium tetroxide in PBS for 2 hours at room temperature. After post-fixation, the cells were dehydrated as Wayama's research described (Wayama et al., 2013). Then the cell samples were embedded and polymerized in Spurr's epoxy resin at 60°C for 48 hours. A 65 nm thin section were cut using a Leica Ultracut-R microtome and stained with 2% (v/v) uranyl acetate and Sato's lead citrate (Hanaichi et al., 1986) and examined with a Philips CM12 transmission electron microscope.

Organelle preparation
Cells grown in 500 mL TAP for 4 days and TAP-N medium for 6, 12, 24, and 48 hours were centrifuged at 1000 g for 10 min. Cells were washed in ice-cold PBS buffer and resuspended in 5 mL of isolation buffer (10 mM Tris·HCl (pH 7.5), 1 mM EDTA, 0.5 M sucrose) with 1×cocktail protease inhibitors (Sigma-Aldrich, USA). Cells were disrupted with the aid of a homogenizer, using 30 strokes on ice. The homogenates were centrifuged at 800 g for 10 min to remove unbroken cells, large debris, nuclei and intact chloroplasts. The resultant supernatant was subjected to sequential centrifugation at 3000 g for 15 min in a Beckman centrifuge J26 with a JS-5.3 rotor, and then 6000 g for 30 min (mitochondrial fraction), 100,000 g for 60 min (microsome fraction) in a Beckman L-90K centrifuge with a SW 32i rotor. Chloroplast thylakoid membranes were isolated as Gu's method described (Gu et al., 2021). Lipid bodies isolation followed by Liu's method (Liu et al., 2016).

Membrane fractions were solubilized in an extraction buffer (60mM DTT, 60mM
Na2CO3, 2% (w/v) SDS and 12% (w/v) sucrose) by vortex for 20 min at 4°C. Protein concentration was determined using the BCA protein assay, as directed by the manufacturer (Thermo Scientific, USA).

Expression analysis by RT-qPCR
1.5 mL cell culture were harvested by centrifugation at 1,500 g for 5 min and washed twice with the 1×PBS buffer. The cell pellets were stored at -80°C prior to use. Total RNA of each sample was isolated by TransZol up Plus RNA Kit (Transgene, China).
The synthesis of single stranded cDNA was carried out using TransScript All-in-One

Western blotting analysis
Sample preparation for SDS-PAGE were processed through Li's method (Li et al., 2020) and whole protein were separated on 12% (w/v) SDS-PAGE, and transferred to nitrocellulose filter (NC) membrane at constant 25 V, for 20 min by using semi-dry transfer system (Bio-Rad Trans-Blot Turbo Transfer System, USA). The membranes were blocked with 10 ml of 5% (w/v) nonfat milk.
For immunofluorescence detection of Bip, Aox, D1, CrGPATer, CrGPATcp and αtubulin, the membrane was incubated overnight with rabbit primary antibody (diluted at 1: 2000 with TBST containing 2% (w/v) nonfat milk), after washed by TBST buffer three times, membrane incubated with the secondary antibody which were anti-rabbit IgG conjugated with HRP diluted by 1: 1000. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection kit (Bio-Rad Clarity Western ECL Substrate, USA).
The subcellular compartment marker antibodies against Bip, Aox and D1were ordered from Agrisera (Sweden). The tubulin was used as internal reference, which was detected by using the tubulin alpha chain antibody (Agrisera, Sweden). The CrGPATer and CrGPATcp polyclonal antibody was generated by using heterologous expressed recombinant protein in E.coli as antigen (ABclonal, China).

Data processing
Two-tailed Student's t-test was used to compare the differences between control groups (wide type and one empty vector transformant) and experimental groups (target gene transformants). If the test gives P value ≤0.05, the differences between two samples were interpreted as being significant.