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Overcoming limitations of engineering eukaryotic algae

July 15, 2018
AlgaeIndustryMagazine.com

Have some of the final engineering limitations of microalgae been overcome? Can microalgae be hosts for genetic engineering as powerful as bacteria and yeast? A promising new technique for gene design seems to overcome expression limitations of nuclear transgene expression for practical eukaryotic algal engineering1.

Researchers from the Center for Biotechnology (CeBiTec) at Bielefeld University in Germany have just published a report in Nucleic Acids Research which outlines a strategy of synthetic transgene design that seems to overcome expression limitations from the nuclear genome of the green alga Chlamydomonas reinhardtii1.

Cultivation technologies for the generation of algal biomass are constantly improving and the adoption of algal production processes are growing around the world. In addition to the valuable algal biomass, it has been a long-term goal of algal biotechnologists to create genetically engineered algae with improved traits and producing high value bio-products from sun-light energy and carbon dioxide.

Green algae like C. reinhardtii are among some of the fastest growing microalgae. Transformation of the nuclear genome in this alga was shown to be possible already in 19892. Although it is generally possible to express small transgenes from the nuclear genome, antibiotic selection markers and small fluorescent reporters, engineering robust transgene over-expression has not been as successful and mature engineering concepts have not become common-place for this or other green algae.

The research team, from left to right: Thomas Baier, Julian Wichmann, Prof. Olaf Kruse, Dr. Kyle J. Lauersen

Very strong promoters can reliably express cDNA or codon optimized gene fragments in bacteria, yeasts, and plants for high level engineering concepts. In eukaryotic algae, however, viral promoters that are commonly used in plant biotechnology do not drive robust transgene expression. Algae should have as much, if not more, metabolic capacities than traditional fermentation hosts (bacteria and yeasts), which are commonly used for bio-engineering of a range of products: recombinant proteins, novel metabolites like pigments, and medicines. However, their poor transgene expression rates have limited their accelerated development as hosts for biotechnological production processes.

The team in Bielefeld started working on this problem in late 2014, driven by a desire to use the algal cells as light-driven green cell factories for the production of non-native products.

The key solution for efficient nuclear transgene expression came from the alga itself. C. reinhardtii, like many fast-growing green algae, has a very high intron density: 92% of all genes contain introns with an average of ~6.3 introns per gene3. Its nuclear genome also has a high GC content, ~64%, and a narrow codon bias3. It has been known for 20 years already that insertion of the first intron of the small subunit 2 of Rubisco from this algal into promoters and antibiotic selection markers can boost their expression in C. reinhardtii4.

The group from Bielefeld combined state-of-the-art codon optimization of transgenes with spreading of this intron throughout larger codon-optimized transgenes. The group found that this additional optimization step enabled reliable gene overexpression from the nuclear genome and systematically analyzed the influence of intron addition both in terms of insertion sequence (splice site) and the distance from one intron to the next (exon-length). From their investigations, they were able to create a rule-set of intron spreading that could be applied to any codon optimized transgene and demonstrated the reliable use of this strategy on a number of target recombinant proteins expressed from the nuclear genome of C. reinhardtii.

This is a significant milestone for algal engineering, because mediocre expression of transgenes in green algae has limited overall engineering concepts from this host. Many target transgenes require high-level over-expression to have a metabolic effect and expression from the nuclear genome holds the potentials of targeting recombinant products to any sub-cellular compartment while also enabling all post-translational modifications of target proteins. The team previously managed to use this strategy to express two different terpene synthases in C. reinhardtii leading to production of one perfume component and one potential biofuel5,6. Here they characterized the systematic application of this gene design strategy and offer a guide for others who want to do the same.

The team at Bielefeld thinks that their results will serve as inspiration for other algal biotechnologists to enhance engineering capacities of green algae already used for outdoor cultivation concepts, such as Chlorella and Scenedesmus strains. The results presented by the group on terpenoid engineering in Chlamydomonas already show that green algae have an impressive capacity as biotechnological light-driven hosts for sustainable bioproduction processes and perhaps can inspire the next wave of biotechnological applications of these photosynthetic hosts.

Algal cultivation technologies continue to improve while engineering eukaryotic algae is clearly becoming more reliable and sophisticated. Perhaps it is time to take another look at the use of green algae as light-driven green cell bio-factories for biotechnological production concepts.

References: Key article:

  1. Baier, T., Wichmann, J., Kruse, O. & Lauersen, K. J. Intron-containing algal transgenes mediate efficient recombinant gene expression in the green microalga Chlamydomonas reinhardtii. Nucleic Acids Res. In Press, (2018).

Others

  1. Kindle, K. L., Schnell, R. A., Fernández, E. & Lefebvre, P. A. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J. Cell Biol. 109, 2589–2601 (1989).
  2. Merchant, S. S. et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science (80-. ). 318, 245–250 (2007).
  3. Lumbreras, V., Stevens R., D., Purton, S., Stevens, D. R. & Purton, S. Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J. 14, 441–447 (1998).
  4. Lauersen, K. J. et al. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metab. Eng. 38, 331–343 (2016).
  5. Wichmann, J., Baier, T., Wentnagel, E., Lauersen, K. J. & Kruse, O. Tailored carbon partitioning for phototrophic production of (E)-α-bisabolene from the green microalga Chlamydomonas reinhardtii. Metab. Eng. 45, 211–222 (2018).

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