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

Jestin George,Unnikrishnan Kuzhiumparambil,Tim Kahlke,Raffaela M Abbriano,Peter J Ralph,Michele Fabris

doi:10.3389/fbioe.2020.00513

Abstract

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.

Highlights

Diatoms are a diverse group of unicellular Stramenopile microalgae that have received substantial attention for their ecological importance (Armbrust, 2009) and biotechnological potential (Huang and Daboussi, 2017)
We demonstrated that extrachromosomal expression (EE) can be used to efficiently express the fusion protein CrGES-mVenus in P. tricornutum cytosol to produce up to 0.21 μg/107 cells (0.309 mg/L) geraniol following bacterial conjugation (Fabris et al, 2020)
Transformant RICE_GmV-89 even showed three populations (Figure 1E). These results demonstrate that individual cells within a clonal transformant randomly integrated chromosomal expression (RICE) cell line, generally assumed to have identical phenotypes, can be highly heterogeneous with regard to transgene expression, but that this heterogeneity can be distributed into unique, discrete populations

Summary

Introduction

Diatoms are a diverse group of unicellular Stramenopile microalgae that have received substantial attention for their ecological importance (Armbrust, 2009) and biotechnological potential (Huang and Daboussi, 2017). Extrachromosomal transformation is anticipated to become increasingly widely-used in diatom genetic engineering (Huang and Daboussi, 2017) These nextgeneration engineering strategies are central to diatom genetics and synthetic biology primarily because they allow multigene stacking approaches (Goyal et al, 2009; Ainley et al, 2013). The recently developed Universal Loop (uLoop) assembly kit provides a collection of useful parts for modular DNA assembly and high-throughput testing as well as more complex gene-stacking designs for P. tricornutum and other diatoms (Pollak et al, 2019). These resources offer unparalleled potential of diatoms such as P. tricornutum compared to other model algal chassis

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