Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology.

Diatoms are photosynthetic microeukaryotes that dominate phytoplankton populations and have increasing applicability in biotechnology. Uncovering their complex biology and elevating strains to commercial standards depends heavily on robust genetic engineering tools. However, engineering microalgal genomes predominantly relies on random integration of transgenes into nuclear DNA, often resulting in detrimental "position-effects" such as transgene silencing, integration into transcriptionally-inactive regions, and endogenous sequence disruption. With the recent development of extrachromosomal transgene expression via independent episomes, it is timely to investigate both strategies at the phenotypic and genomic level. Here, we engineered the model diatom Phaeodactylum tricornutum to produce the high-value heterologous monoterpenoid geraniol, which, besides applications as fragrance and insect repellent, is a key intermediate of high-value pharmaceuticals. Using high-throughput phenotyping we confirmed the suitability of episomes for synthetic biology applications and identified superior geraniol-yielding strains following random integration. We used third generation long-read sequencing technology to generate a complete analysis of all transgene integration events including their genomic locations and arrangements associated with high-performing strains at a genome-wide scale with subchromosomal detail, never before reported in any microalga. This revealed very large, highly concatenated insertion islands, offering profound implications on diatom functional genetics and next generation genome editing technologies, and is key for developing more precise genome engineering approaches in diatoms, including possible genomic safe harbour locations to support high transgene expression for targeted integration approaches. Furthermore, we have demonstrated that exogenous DNA is not integrated inadvertently into the nuclear genome of extrachromosomal-expression clones, an important characterisation of this novel engineering approach that paves the road to synthetic biology applications.

George J ，Kahlke T ，Abbriano RM ，Kuzhiumparambil U ，Ralph PJ ，Fabris M ... -  《Frontiers in Bioengineering and Biotechnology》

Extrachromosomal Genetic Engineering of the Marine Diatom Phaeodactylum tricornutum Enables the Heterologous Production of Monoterpenoids.

Fabris M ，George J ，Kuzhiumparambil U ，Lawson CA ，Jaramillo-Madrid AC ，Abbriano RM ，Vickers CE ，Ralph P ... -  《ACS Synthetic Biology》

Towards developing algal synthetic biology.

The genetic, physiological and metabolic diversity of microalgae has driven fundamental research into photosynthesis, flagella structure and function, and eukaryotic evolution. Within the last 10 years these organisms have also been investigated as potential biotechnology platforms, for example to produce high value compounds such as long chain polyunsaturated fatty acids, pigments and antioxidants, and for biodiesel precursors, in particular triacylglycerols (TAGs). Transformation protocols, molecular tools and genome sequences are available for a number of model species including the green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum tricornutum, although for both species there are bottlenecks to be overcome to allow rapid and predictable genetic manipulation. One approach to do this would be to apply the principles of synthetic biology to microalgae, namely the cycle of Design-Build-Test, which requires more robust, predictable and high throughput methods. In this mini-review we highlight recent progress in the areas of improving transgene expression, genome editing, identification and design of standard genetic elements (parts), and the use of microfluidics to increase throughput. We suggest that combining these approaches will provide the means to establish algal synthetic biology, and that application of standard parts and workflows will avoid parallel development and capitalize on lessons learned from other systems.

Scaife MA ，Smith AG 《-》

Identification of Loci Enabling Stable and High-Level Heterologous Gene Expression.

Efficient and reliable genome engineering technologies have yet to be developed for diatoms. The delivery of DNA in diatoms results in the random integration of multiple copies, quite often leading to heterogeneous gene activity, as well as host instability. Transgenic diatoms are generally selected on the basis of transgene expression or high enzyme activity, without consideration of the copy number or the integration locus. Here, we propose an integrated pipeline for the diatom, Phaeodactylum tricornutum, that accurately quantifies transgene activity using a β-glucuronidase assay and the number of transgene copies integrated into the genome through Droplet Digital PCR (ddPCR). An exhaustive and systematic analysis performed on 93 strains indicated that 42% of them exhibited high β-glucuronidase activity. Though most were attributed to high transgene copy numbers, we succeeded in isolating single-copy clones, as well as sequencing the integration loci. In addition to demonstrating the impact of the genomic integration site on gene activity, this study identifies integration sites for stable transgene expression in Phaeodactylum tricornutum.

Defrel G ，Marsaud N ，Rifa E ，Martins F ，Daboussi F ... -  《-》

Recent Progress on Systems and Synthetic Biology of Diatoms for Improving Algal Productivity.

Microalgae have drawn much attention for their potential applications as a sustainable source for developing bioactive compounds, functional foods, feeds, and biofuels. Diatoms, as one major group of microalgae with high yields and strong adaptability to the environment, have shown advantages in developing photosynthetic cell factories to produce value-added compounds, including heterologous bioactive products. However, the commercialization of diatoms has encountered several obstacles that limit the potential mass production, such as the limitation of algal productivity and low photosynthetic efficiency. In recent years, systems and synthetic biology have dramatically improved the efficiency of diatom cell factories. In this review, we discussed first the genome sequencing and genome-scale metabolic models (GEMs) of diatoms. Then, approaches to optimizing photosynthetic efficiency are introduced with a focus on the enhancement of biomass productivity in diatoms. We also reviewed genome engineering technologies, including CRISPR (clustered regularly interspaced short palindromic repeats) gene-editing to produce bioactive compounds in diatoms. Finally, we summarized the recent progress on the diatom cell factory for producing heterologous compounds through genome engineering to introduce foreign genes into host diatoms. This review also pinpointed the bottlenecks in algal engineering development and provided critical insights into the future direction of algal production.

Chen J ，Huang Y ，Shu Y ，Hu X ，Wu D ，Jiang H ，Wang K ，Liu W ，Fu W ... -  《-》