Expression of bkt and bch genes from Haematococcus pluvialis in transgenic Chlamydomonas

Abstract

β-carotene ketolase and β-carotene hydroxylase encoded by bkt and bch, respectively, are key enzymes required for astaxanthin biosynthesis in Haematococcu pluvialis 34-1n. Two expression vectors containing cDNA sequences of bkt and bch were constructed and co-transformed into cell-wall-deficient Chlamydomonas reinhardtii CC-849. Transgenic algae were screened on TAP agar plates containing 10 μg mL 1 Zeomycin. PCR-Southern analysis showed that bkt and bch were integrated into the genomes of C. reinhardtii. Transcripts of bkt and bch were further confirmed by RT-PCR-Southern analysis. Compared with the wild type, transgenic algae produced 29.04% and 30.27% more carotenoids and xanthophylls, respectively. Moreover, the transgenic algae could accumulate 34% more astaxanthin than wild type. These results indicate that foreign bkt and bch genes were successfully translated into β-carotene ketolase and β-carotene hydroxylase, which were responsible for catalyzing the biosynthesis of astaxanthin in transgenic algae.Chlamydomonas, β-carotene ketolase, β-carotene hydroxylase, carotenoids, xanthophylls, astaxanthin Citation:Zheng KJ, Wang CG, Xiao M, Chen J, Li JC, Hu ZL. Expression of bkt and bch genes from Haematococcus pluvialis in transgenic Chlamydomonas.

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

“…Re-insertion of only CrBKT into the nuclear genome of C. reinhardtii allowed the synthesis of high amounts of astaxanthin and other ketocarotenoids that normally do not accumulate in this organism (Figure 2). In contrast to previous work (Zheng et al, 2014), the overexpression of CrBKT alone was sufficient to induce ketocarotenoid formation in C. reinhardtii: these findings indicate that the native CrCHYB is highly functional and able to participate in ketocarotenoid biosynthesis. When CrBKT was overexpressed in the alga, astaxanthin became the major carotenoid (up to 50% of total carotenoids) with ketocarotenoids corresponding tõ 70% of total carotenoids (Table 1).…”

“…In order to develop a sustainable alternative to traditional astaxanthin production, we sought to engineer the common freshwater microalga Chlamydomonas reinhardtii to constitutively produce astaxanthin and canthaxanthin. In contrast to previous attempts by others (Leon et al, 2007;Tan et al, 2007;Zheng et al, 2014), our approach is based on the synthetic redesign and revival of an endogenous yet inactive (pseudogene) b-carotene ketolase sequence present in the nuclear genome of C. reinhardtii. Strains resulting from the application of this strategy generated astaxanthin, exhibited reddish-brown phenotypes and reached productivities comparable to H. lacustris cultivation without many of its natural process constraints.…”

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

“…In order to develop a sustainable alternative to traditional astaxanthin production, we sought to engineer the common freshwater microalga Chlamydomonas reinhardtii to constitutively produce astaxanthin and canthaxanthin. In contrast to previous attempts by others (Leon et al, 2007;Tan et al, 2007;Zheng et al, 2014), our approach is based on the synthetic redesign and revival of an endogenous yet inactive (pseudogene) beta-ketolase sequence present in the nuclear genome of C. reinhardtii. Strains resulting from the application of this strategy generated astaxanthin, exhibited reddish-brown phenotypes, and reached productivities comparable to H. lacustris cultivation without many of its natural process constraints.…”

Construction of astaxanthin metabolic pathway in the green microalga Dunaliella viridis

“…Therefore, expression of the bkt gene is preferred for astaxanthin production in most transgenic systems [5]. Furthermore, CRTR-B is also required to obtain higher productivity of astaxanthin [6][7][8].…”