Enabling eukaryotic algal engineering by optimized transgene design strategies

The ability of microalgae to grow as primary producers from light and carbon dioxide has led to incredible interest in the use of these organisms for a range of applications from sustainable generation of biofuels to consumer products. Although microalgae are natural sources of many interesting compounds such as pigments, lipids, and polysaccharides, expanding the range of products via genetic engineering can offer new incentives for development of photosynthesis-driven bio-processes. Green microalgae represent a large group of photosynthetic microorganisms which are amenable to scalable cultivation and have been models for fundamental research studies for decades. However, genetic engineering in these hosts has been hampered by issues of effective foreign transgene expression from the nuclear genome. We have developed a novel transgene design strategy which employs both codon optimization and a technique of synthetic intron spreading. When coupled to optimized expression vectors and reporters, this strategy has allowed high-level transgene expression of a range of gene targets from the nuclear genome of the green microalga Chlamydomonas reinhardtii. The expression of transgenes designed in this way benefits from the added recruitment of transcriptional machinery, mediated by inherent enhancer elements within the introns, and increased mRNA stability by minimizing exon lengths, presumably matching host genomic organization. The technique enabled modification of the native terpenoid metabolism to increase carbon flux through the 2-c-methyl-d-erythritol 4-phosphate (MEP) precursor pathway. Additionally, we show that the expression of sesqui- and di-terpene synthases from plants is possible in C. reinhardtii and these can convert native precursor metabolites into non-native terpenoid molecules in the algal host. This presentation will overview our novel transgene design strategy and expression vector toolkit, the implementation of these for photosynthetically driven biotechnological concepts in microalgae for a range of applications, and our current understanding of algal terpenoid metabolism in this alga. 

Relevant publications from the presenter:

1. Baier T., Wichmann J., Kruse O, Lauersen K.J. 2018. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Research. 10.1093/nar/gky532 

2. Wichmann J., Baier T., Wentnagel E., Lauersen K. J., Kruse O. 2018. Tailored carbon

partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metabolic Engineering 45: 211-222.
 

3. Lauersen K. J., Baier T., Wichmann J., Wördenweber R., Hübner W., Huser T., Kruse O. 2016. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metabolic Engineering 38: 331-343. 

4. Lauersen K. J., Kruse O., Mussgnug J. H. 2015. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology & Biotechnology 99:3491–3503.

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