Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii

Baier T, Jacobebbinghaus N, Einhaus A, Lauersen KJ, Kruse O. Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii. PLOS Genetics. 2020;16(7): e1008944.Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression

Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit

Lauersen KJ, Kruse O, Mussgnug JH. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 2015;99(8):3491-3503

Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii

Baier T, Wichmann J, Kruse O, Lauersen KJ. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Research. 2018;46(13):6909-6919.Among green freshwater microalgae, Chlamydomonas reinhardtii has the most comprehensive and developed molecular toolkit, however, advanced genetic and metabolic engineering driven from the nuclear genome is generally hindered by inherently low transgene expression levels. Progressive strain development and synthetic promoters have improved the capacity of transgene expression; however, the responsible regulatory mechanisms are still not fully understood. Here, we elucidate the sequence specific dynamics of native regulatory element insertion into nuclear transgenes. Systematic insertions of the first intron of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 2 (rbcS2i1) throughout codon-optimized coding sequences (CDS) generates optimized algal transgenes which express reliably in C. reinhardtii. The optimal rbcS2i1 insertion site for efficient splicing was systematically determined and improved gene expression rates were shown using a codon-optimized sesquiterpene synthase CDS. Sequential insertions of rbcS2i1 were found to have a step-wise additive effect on all levels of transgene expression, which is likely correlated to a synergy of transcriptional machinery recruitment and mimicking the short average exon lengths natively found in the C. reinhardtii genome. We further demonstrate the value of this optimization with five representative transgene examples and provide guidelines for the design of any desired sequence with this strategy

Surveying a Diverse Pool of Microalgae as a Bioresource for Future Biotechnological Applications

Jakob G, Wolf J, Bui TVL, et al. Surveying a Diverse Pool of Microalgae as a Bioresource for Future Biotechnological Applications. Journal of Petroleum & Environmental Biotechnology. 2013;4(05):153.Resource limitation is an escalating concern given human expansion and development. Algae are increasingly recognised as a promising bioresource and the range of cultivated species and their products is expanding. Compared to terrestrial crops, microalgae are very biodiverse and offer considerable versatility for a range of biotechnological applications including the production of animal feeds, fuels, high value products and waste-water treatment. Despite their versatility and capacity for high biomass productivity on non-arable land, attempts to harness microalgae for commercial benefit have been limited. This is in large part due to capital costs and energy inputs remaining high, the necessity of identifying ‘suitable’ land with proximal resource and infrastructure availability and the need for process and strain optimisation. Microalgae represent a relatively unexplored bioresource both for native and engineered strains. Success in this area requires (1) appropriate methods to source and isolate microalgae strains, (2) efficient maintenance of motherstocks, (3) rapid strain characterisation and correct matching of strains to applications, (4) ensuring productive and stable cultivation at scale, and (5) ongoing strain development (breeding, adaptation and engineering). This article illustrates a survey and isolation of over 150 local microalgae strains as a bioresource for ongoing strain development and biotechnological applications

Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii

Wichmann J, Baier T, Wentnagel E, Lauersen KJ, Kruse O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metabolic Engineering. 2018;45:211-222

A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass

Klassen V, Blifernez-Klassen O, Hoekzema Y, Mussgnug JH, Kruse O. A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass. Journal of Biotechnology. 2015;215:44-51