Developing metabolic engineering tools for enhanced synthesis of high-value products in Chlamydomonas reinhardtii

This work aimed to improve the biotechnological potential of Chlamydomonas reinhardtii by increasing its capacity for lutein production using forward and reverse genetic engineering approaches, and by developing genetic tools to enhance and expand the metabolic engineering toolkit available for this model green microalga.
A post-translational regulator of a key enzyme in the carotenoid biosynthetic pathway in higher plants was identified; its overexpression has previously led to the increased accumulation of carotenoids in several plant species. Here, a C. reinhardtii homologue of the regulator was identified, cloned, and overexpressed from the C. reinhardtii nuclear genome for the first time, which resulted in a 2.0-fold increase in lutein production compared to the wild-type strain.
A semi high-throughput screening platform to isolate C. reinhardtii chemical mutants exhibiting increased carotenoid biosynthesis was developed, generating 5 strains that produce significantly higher total carotenoids than the wild-type, the highest of which (EMS-Mut-5) synthesised 5.4-fold more lutein than the parental strain. EMS-Mut-5 was characterised using a label-free quantitative proteomics workflow, which highlighted potential metabolic engineering targets for further enhancement of lutein production.
Lastly, novel genetic devices for metabolic engineering were developed to facilitate transgene expression from the C. reinhardtii nuclear genome. Promoter sequences of highly expressed genes were computationally analysed to find DNA sequences that contribute to their high expression. 14 DNA motifs identified in this study were cloned into fluorescent protein reporter vectors for in vivo analysis, and their expression activity measured by flow cytometry. Ten out of 14 DNA motifs tested displayed significantly higher fluorescent protein expression compared to a minimal core promoter, and promoter 12 (pCRE-12) presented instances of higher expression than the current strongest C. reinhardtii promoter (Hsp70A-RbcS2). The outcome is a new suite of synthetic promoters that can drive a dynamic range of recombinant protein expression levels.