Overexpression of acetyl-CoA synthetase (ACS) enhances the biosynthesis of neutral lipids and starch in the green microalga Chlamydomonas reinhardtii

Abstract

Ge netic en gi neer ing can be the so lu tion to achieve the eco nom i cally fea si ble pro duc tion of mi croal gal based bio fu els and other bulk ma te ri als. A good num ber of mi croal gal species can grow mixotroph i cally us ing ac etate as car bon source. More over, ex per i men tal ev i dence sug gests that the biosyn the sis of acetyl-CoA could be a lim it ing step in the com plex mul ti fac tor-de pen dent biosyn the sis of acyl glyc erides and point to acetyl-CoA syn thetase (ACS) as a key en zyme in the process. In or der to test this hy poth e sis we have en gi neered the model chloro phyte Chlamy domonas rein hardtii to over ex press the en doge nous chloro plas tic acetyl-CoA syn thetase, ACS2. Ex pres sion of the ACS2 en cod ing gene un der the con trol of the strong con sti tu tive RbcS2 pro moter in ni tro gen-re plete cul tures re sulted in a 2-fold in crease in starch con tent and 60% higher acyl-CoA pool com pared to the parental line. Un der ni tro gen de pri va tion, the Cr-acs2 trans for mant shows 6-fold higher lev els of ACS2 tran script and a 2.4-fold higher ac cu mu la tion of tri a cyl glyc erol (TAG) than the un trans formed con trol. Analy sis of lipid species and fatty acid pro files in the Cr-acs2 trans for mant re vealed a higher con tent than the parental strain in the ma jor gly col ipids and sug gests that the en hanced syn the sis of tri a cyl glyc erol in the trans for mant is not achieved at ex pense of mem brane lipids, but is due to an in crease in the car bon flux to wards the syn the sis of acetyl-CoA in the chloro plast. This data demon strates the po ten tial of en gi neer ing the chloro plas tic ACS to in crease the car bon flux to wards the syn the sis of fatty acids as an al ter na tive strat egy to en hance the biosyn the sis of lipids in mi croal gae.

Multiomics analysis reveals a distinct mechanism of oleaginousness in the emerging model alga Chromochloris zofingiensis

“…Generating more acetyl‐CoA represents a feasible ‘pushing’ strategy to promote fatty acid synthesis and the subsequent TAG assembly (Yan et al ., ; Avidan et al ., ; Rengel et al ., ). In C. zofingiensis , the major sources of acetyl‐CoA are catalyzed by chloroplastic PDHC and acetyl‐CoA synthetase (ACS), which have the potential to be engineered for promoting fatty acid synthesis.…”

Photosynthetic Metabolism and Nitrogen Reshuffling Are Regulated by Reversible Cysteine Thiol Oxidation Following Nitrogen Deprivation in Chlamydomonas

“…However, this study is the first site-specific determination of differential oxidation on acetyl-CoA synthetase and the functional significance of these cysteine residues is currently unknown. Previous studies have connected acetyl-CoA synthetase with increased TAG production following nitrogen limitation due to increased acetyl-CoA accumulation and upregulation of ACS2 , the gene encoding chloroplastic acetyl-CoA synthetase [ 85 , 86 , 87 ]. It is therefore possible that the reduction of cysteines is used to enhance the flux of acetyl-CoA production as the chloroplast remodels toward increasing neutral lipid production.…”

Characterization of Fatty Acid EXporters involved in fatty acid transport for oil accumulation in the green alga Chlamydomonas reinhardtii

“…In the last few decades, microalgae have been regarded as a powerful raw material for the production of biofuel, the most promising renewable energy source. Microalgae can photoautotrophically grow and produce bulk chemicals, such as triacylglycerol (TAG) and starch, which can be transformed into biodiesel and bioethanol, respectively [1]. However, the lipid content of microalgae needs to be increased for commercial-scale production of biodiesel from microalgae [2, 3].…”