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 ... -  《Frontiers in Bioengineering and Biotechnology》

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》

No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum.

Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom. Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein (eGFP). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system. Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.

Diaz-Garza AM ，Merindol N ，Dos Santos KCG ，Lavoie-Marchand F ，Ingalls B ，Desgagné-Penix I ... -  《Microbial Cell Factories》

Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylum tricornutum.

Xie WH ，Zhu CC ，Zhang NS ，Li DW ，Yang WD ，Liu JS ，Sathishkumar R ，Li HY ... -  《-》

Genome Annotation of a Model Diatom Phaeodactylum tricornutum Using an Integrated Proteogenomic Pipeline.

Diatoms comprise a diverse and ecologically important group of eukaryotic phytoplankton that significantly contributes to marine primary production and global carbon cycling. Phaeodactylum tricornutum is commonly used as a model organism for studying diatom biology. Although its genome was sequenced in 2008, a high-quality genome annotation is still not available for this diatom. Here we report the development of an integrated proteogenomic pipeline and its application for improved annotation of P. tricornutum genome using mass spectrometry (MS)-based proteomics data. Our proteogenomic analysis unambiguously identified approximately 8300 genes and revealed 606 novel proteins, 506 revised genes, 94 splice variants, 58 single amino acid variants, and a holistic view of post-translational modifications in P. tricornutum. We experimentally confirmed a subset of novel events and obtained MS evidence for more than 200 micropeptides in P. tricornutum. These findings expand the genomic landscape of P. tricornutum and provide a rich resource for the study of diatom biology. The proteogenomic pipeline we developed in this study is applicable to any sequenced eukaryote and thus represents a significant contribution to the toolset for eukaryotic proteogenomic analysis. The pipeline and its source code are freely available at https://sourceforge.net/projects/gapeproteogenomic.

Yang M ，Lin X ，Liu X ，Zhang J ，Ge F ... -  《-》