Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

Abstract

Summary The green alga Chlamydomonas reinhardtii does not synthesize high‐value ketocarotenoids like canthaxanthin and astaxanthin; however, a β‐carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C‐terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic redesign of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to reddish‐brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio‐accessibility of these pigments were much higher in cell wall‐deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor‐made pigments from this host.

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

“…The gradual distribution of expression levels observed across this intron set hinders generalizations of the degree of IME that can be expected from different intron sequences. The first intron (i1) of RBCS2 has been reported across multiple studies to be a reliable and strong IME effecting intron for C. reinhardtii nuclear transgene expression [31][32][33][39][40][41][42][43]. In this study, higher IME was observed from the introns natively located in closer proximity to the respective promoter, however, with some exceptions (ACTIN, LHCBM1, RPS8, LHCBM3, and RPL20, S1 Fig).…”

“…frequently used for biotechnological studies aimed at metabolic engineering and recombinant protein expression in C. reinhardtii [31,[39][40][41][42][43][44]79]. Here, we identified several intron sequences that induce strong IME comparable or even higher than RBCS2i1 (Fig 1).…”

“…Recently, we developed a sequence optimization strategy which couples effective codon optimization with the systematic insertion of the RBCS2 introns into coding sequences [31]. This transgene design strategy successfully enables reliable nuclear transgene expression in C. reinhardtii and has now been used to demonstrate several examples of concerted metabolic engineering of this host [39][40][41][42][43][44]. Despite the broad applicability of the RBCS2i1, the mechanisms of its IME have not been thoroughly investigated and it was unknown if other introns from the algal genome could have similar behaviors.…”

Metabolic engineering of ketocarotenoids biosynthetic pathway in Chlamydomonas reinhardtii strain CC-4102

“…The authors attributed this finding to the enzyme's inaccessibility to its substrate ß-carotene. In a more recent publication, Perozeni et al 20 also attempted to overexpress CrBKT in Chlamydomonas npq2 mutant, but their study differs from ours. They directed CrBKT to the thylakoid membrane via fusion of the psaD chloroplast transit peptide (cTP 53 ) to its N-terminus.…”

“…As a result, both astaxanthin and canthaxanthin are detected in their psaD-CrBKT transformants. In our study, we intentionally did not include cTP because there is evidence of an intrinsic cTP in CrBKT sequence, both from in silico sequence analysis with PredAlgo 54 as well as fluorescence translocation assay 20 . The lack of astaxanthin leads to the assumption that CrBKT was expressed and imported into chloroplast but not into thylakoid membrane, thus leaving it unable to metabolize zeaxanthin.…”