Improving the genetic tractability of the green alga Chlamydomonas reinhardtii.

摘要

Green algae present a unique platform for bioengineering and biomanufacturing; they grow rapidly, photosynthetically, and inexpensively and thus are suitable for large-scale cultivation, yet they are sophisticated eukaryotic cells with vast potential for introducing complex products, traits, or pathways. A long and growing list of publications has established that algae are capable of producing large, intractable proteins that exceed the folding capacity of prokaryotic systems. More recently, studies involving metabolic engineering and systematic manipulation of the photosynthetic machinery have demonstrated that these algae are amenable to customization of complex endogenous processes such as photosynthetic anabolism and lipid metabolism.;While algae's successes and potential for industrial and therapeutic applications are covered extensively in this dissertation - including chapters on recombinant therapeutics, bioenergy applications, and oral vaccine development - these must be viewed in the context of the work that remains. Despite its moniker "the green yeast", even the model green alga Chlamydomonas reinhardtii falls short of other model organisms with regard to genetic tractability due to a relative lack of genetic engineering tools. The latter half of the dissertation addresses several of these shortfalls using innovative strategies inspired by synthetic biology approaches and high-throughput technologies.;In the chloroplast, gene targeting is routine but expression is regulated in translation. A better understanding of gene regulatory elements within transcripts was achieved at the intersection of a novel oligonucleotide library synthesis platform, high-efficiency seamless cloning, and next-generation sequencing technology. In the nuclear genome, a number of problems - including lack of facile reporters, robust promoters, and strong transgene expression cassettes - were addressed using optimized versions of endogenous genes, concomitantly alleviating concerns with genetically modified organism (GMO) regulations. Furthermore, the first reliable and reproducible strategy to measure incremental improvements in the gene targeting efficiency within the algal nuclear genome has been developed. This system is uniquely able to capture and characterize aberrant events at the recombination site, a phenomenon that had been predicted previously but proved difficult to elucidate unequivocally. Taken together, the advances described in this dissertation have significantly advanced the genetic malleability of the model alga C. reinhardtii, with potential application to additional vital algal species.