BIOSYNTHESIS OF POLY-3-HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGACHLAMYDOMONAS REINHARDTII

Abstract

A cell-wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC-849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris-acetate-phosphate media containing 10 lg AE mL )1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly-3-hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol AE min )1 AE mg protein )1 to 126 nmol AE min )1 AE mg protein )1 . The amount of PHB in double transgenic algae was determined by gas chromatography-mass spectrometry (GC-MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.

PHA Bioplastics, Biochemicals, and Energy from Crops

“…High levels (10.6% DW) have, however, been produced in the cytosol of the microalgae Phaeodactylum tricornutu, a diatom capable of depositing lipids in its cytosol, which could be indicative of a large acetyl-CoA pool (Hempel et al, 2011). In contrast, engineering of Chlamydomonas reinhardtii for cytosolic PHB production yielded only 6 9 10 À4 % DW PHB (Chaogang et al, 2010).…”

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

“…This versatile freshwater species is currently the most genetically tractable microalga with established molecular tools for engineering both the nuclear and chloroplast genomes (Scaife et al 2015;Crozet et al 2018). As such, C. reinhardtii has been proposed as a cell factory for a wide range of products including pharmaceutical proteins (Dyo and Purton 2018;Yan et al 2016), RNA-based vaccines for aquaculture (Somchai et al 2016;Charoonnart et al 2019), dietary enzymes for livestock (Erpel et al 2016;Manuell et al 2007), novel isoprenoids as bioactives (Lauersen 2019), biopolymers (Chaogang et al 2010) and specialist biofuels such as biohydrogen and bisabolene (Torzillo et al 2015;Wichmann et al 2018). All of these engineered strains could be 'retrofitted' using our ptxD cassette to allow selective growth on phosphite media, thereby avoiding the production costs associated with sterilising the media and protecting the algal crop from spoilage.…”

Selenoprotein-Transgenic Chlamydomonas reinhardtii

“…In order to achieve high transformation efficiency, three Sep15 expression plasmids were Not I-digested into linear plasmids before they were transformed into CC-849 cells by the glass-bead method [13]. Briefly, CC-849 cells in the mid-log phase were harvested by centrifugation at 5000 rpm for 5 min, suspended with fresh TAP, and adjusted to 2 × 10 8 cells/mL.…”

“…This expression system has the advantages of non-toxic, low cost and short growth cycle compared with other systems. Several proteins have been already expressed successfully in C. reinhardtii through either nuclear transformation or chloroplast transformation [12,13], which provide references for our present study. Hereby three forms of human Sep15 gene fragments were constructed and expressed in C. reinhardtii .…”