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Carotenoids are a wide group of lipophylic isoprenoids synthesized by all photosynthetic organisms and also by some non-photosynthetic bacteria and fungi. Animals, which cannot synthesize carotenoids de novo, must include them in their diet to fulfil essential provitamin, antioxidant, or colouring requirements. Carotenoids are indispensable in light harvesting and energy transfer during photosynthesis and in the protection of the photosynthetic apparatus against photooxidative damage. In this review, we outline the factors inducing carotenoid accumulation in microalgae, the knowledge acquired on the metabolic pathways responsible for their biosynthesis, and the recent achievements in the genetic engineering of this pathway. Despite the considerable progress achieved in understanding and engineering algal carotenogenesis, many aspects remain to be elucidated. The increasing number of sequenced microalgal genomes and the data generated by high-throughput technologies will enable a better understanding of carotenoid biosynthesis in microalgae. Moreover, the growing number of industrial microalgal species genetically modified will allow the production of novel strains with enhanced carotenoid contents.

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Chlorella sorokiniana has been selected for lutein production, after a screening of thirteen species of microalgae, since it showed both a high content in this carotenoid and a high growth rate. The effects of several nutritional and environmental factors on cell growth and lutein accumulation have been studied. Maximal specific growth rate and lutein content were attained at 690 μmol photons m−2 s−1, 28 °C, 2 mM NaCl, 40 mM nitrate and under mixotrophic conditions. In general, optimal conditions for the growth of this strain also lead to maximal lutein productivity. High lutein yielding mutants of C. sorokiniana have been obtained by random mutagenesis, using N-methyl-N′-nitro-nitrosoguanidine (MNNG) as a mutagen and selecting mutants by their resistance to the inhibitors of the carotenogenic pathway nicotine and norflurazon. Among the mutants resistant to the herbicides, those exhibiting both high content in lutein and high growth rate were chosen. Several mutants exhibited higher contents in this carotenoid than the wild type, showing, in addition, either a similar or higher growth rate than the latter strain. The mutant MR-16 exhibited a 2.0-fold higher volumetric lutein content than that of the wild type, attaining values of 42.0 mg L−1 and mutants DMR-5 and DMR-8 attained a lutein cellular content of 7.0 mg g−1 dry weight. The high lutein yield exhibited by C. sorokiniana makes this microalga an excellent candidate for the production of this commercially interesting pigment.

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.

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