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Investigation of heterologous expression of the non-ribosomal peptide blue pigment synthase and its activator from the nuclear genome of the model microalga Chlamydomonas reinhardtiiShlbi, Manar 31 March 2022 (has links)
The non-ribosomal peptide synthase (NRPS) blue pigment synthase (BpsA)
has been shown in several heterologous hosts to mediate the production of the blue pigment indigoidine from two molecules of L-glutamine. Activation of BpsA is mediated by transfer of a coenzyme A (CoA) by a 4′-phosphopantetheinyl transferase (4′-PPTase). In this thesis, I explored heterologous co-expression of BpsA and the Pseudomonas aeruginosa 4′-PPTase (PaPcpS) and their co- localization to either cytoplasm or chloroplast stroma of the green model microalga Chlamydomonas reinhardtii. The alga represents a potentially sustainable production host for indigoidine, as it is able to grow using CO2 as a sole carbon source and (sun)light for its energy. Both heterologous proteins (BpsA and PaPcpS) could be expressed as full-length fusion proteins with either the mVenus yellow fluorescent reporter or spectinomycin resistance (aadA) selection marker in both subcellular localisations. Dual transformants were identified and subjected to multiple growth conditions to determine whether indigoidine was produced. Under no condition tested was indigoidine detected, indicating that either activation of BpsA or the catalysis of L-glutamine to indigoidine was not occurring in alga. Future work will be required to determine whether it is possible to activate the BpsA in C. reinhardtii. However, this represents the first documented example of expression of a heterologous NRPS in a eukaryotic alga and may serve as foundational work for other target NRPS expression projects.
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