Elsevier

Algal Research

Volume 53, March 2021, 102159
Algal Research

Characterisation of novel regulatory sequences compatible with modular assembly in the diatom Phaeodactylum tricornutum

https://doi.org/10.1016/j.algal.2020.102159Get rights and content

Highlights

  • Transcriptomics-guided selection of putative promoters/terminators in P. tricornutum.

  • Episomal expression of mVenus demonstrates promoters are suitable for genetic engineering.

  • Promoters drive extended expression during dark periods and nitrogen starvation.

  • Novel genetic parts are compatible with standardized TypeIIS cloning systems.

  • Clone-to-clone variation depends on promoter strength.

Abstract

Phaeodactylum tricornutum is a model pennate diatom for molecular studies due to its sequenced genome, genetic tractability, and capacity to produce a myriad of compounds of biotechnological interest. While tools for genetic engineering and heterologous gene expression have been developed for this species, the available set of characterised promoters remains limited. A diverse and well-understood set of genetic regulatory elements is necessary to enable fine tuning of gene expression for metabolic engineering and synthetic biology approaches. In this study, we investigated the expression profile of four endogenous constitutive promoter and terminator pairs Nub (Phatr3_J14260), SVP (Phatr3_J16798), 45582 (Phatr3_J45582), and Pbt (Phatr3_J54958) selected from a multi-experiment, integrated transcriptomics database under a variety of growth conditions, including various light regimes and nitrogen (N) limitation. Expression of the fluorescent reporter mVenus was driven from non-integrative episomes and monitored via flow cytometry. Gene expression was validated on a transcript and protein level using RT-qPCR and western blot, respectively. We found that all four promoter and terminator pairs exhibited stable expression throughout exponential growth, with distinct expression patterns and reduced light dependency compared to the widely-used FcpB promoter. Furthermore, the activity of these promoters was less impacted by N depletion than for FcpB. Our results indicate these novel promoters and terminators are promising tools for fine tuning single or multiple gene expression in P. tricornutum.

Introduction

The pennate diatom Phaeodactylum tricornutum has been developed as a model laboratory species for functional molecular studies in microalgae due to fast growth [1], scalability, and robustness. In addition, P. tricornutum may be suitable as a potential future platform for the sustainable production of biomolecules [2], due to its photosynthetic capabilities and ability to grow in seawater. Therefore, it has been increasingly explored through metabolic engineering studies for a wide range of products [3], including recombinant therapeutic proteins, polyhydroxybutyrate (PHB), and terpenoids [[4], [5], [6], [7], [8]]. Advantages of P. tricornutum include its sequenced genome and the availability of transcriptomic and proteomic datasets [9]. The molecular toolbox for P. tricornutum is advanced and includes transformation methods like electroporation, biolistic bombardment, and bacterial conjugation [[10], [11], [12], [13]], as well as genome editing strategies like CRISPR/Cas9 and TALEN [14,15]. Furthermore, the development of an episomal expression (EE) system that allows for extrachromosomal replication of expression vectors differentiates P. tricornutum from other microalgae [12,16]. Unlike random integrated chromosomal expression (RICE), EE of transgenes is not influenced by position effects and results in more genetically similar transformants in P. tricornutum [17]. Indeed, compared to RICE, EE of the fluorescent protein mVenus was lower but more stable throughout a library of P. tricornutum transformants, indicating a more controllable, consistent, and reproducible system for genetic studies [17]. This, in combination with the availability of selection markers [10], reporter genes [18], and a variety of promoters [19], facilitates stable expression of transgenes in P. tricornutum [20].

While several factors can directly influence gene expression levels, promoter and terminator choice allow the most flexible and efficient adjustment of transgene expression [[21], [22], [23]]. While the promoter sequence affects expression via initiation of transcription, the terminator sequence releases the RNA polymerase and is involved in post-transcriptional regulation, influencing the stability and half-life of mRNA [24]. To our knowledge, only one study to date has determined the influence of terminator sequences on transgene expression in diatoms [25]. Given that well-characterised and effective transcriptional regulators (including both terminators and promoters) are a critical part of the genetic toolbox needed to enable gene stacking and fine tuning of heterologous gene expression [25], the limited empirical data available on terminator choice in diatoms currently impedes more sophisticated genetic engineering approaches.

Previously developed inducible promoter systems in diatoms depend on sensitivity to nutrient concentrations like nitrate or phosphate [[26], [27], [28]], allowing expression to be induced under specific media conditions. However, nutrient limitation may lead to confounding factors such as undesired or competitive synthesis of lipids [29], as well as unanticipated metabolic responses to the starvation treatment. Fine-tuning of complex pathways using constitutive promoters, which continuously drive expression, can be an alternative approach to maintaining a balance between metabolic requirements and carbon flux towards the desired product [30,31]. In some cases, moderate rather than maximum target gene expression can lead to the most efficient phenotypes that express appropriate levels of the intended bioproduct; this is because the diversion of energy and carbon away from necessary cell maintenance and repair to synthesise the targeted product can result in a high metabolic burden and may lead to an erosion of cell viability [32,33].

Knowledge of the genetic structure and core elements of promoters and terminators in diatoms is still limited, unlike in widely-used host organisms such as bacteria and yeasts [30], and only a handful of promoters have been validated and described. One of the most frequently used constitutive promoters in diatoms is the fucoxanthin chlorophyll a/c binding protein B (FcpB) promoter, which is part of the highly expressed light-harvesting complex [10]. However, FcpB shows high variability in expression during light and dark cycles [34]. Other characterised endogenous constitutive promoters include those of genes encoding the elongation factor 2 (EF2) [35], purine permease, actin/actin like protein (Act2) [36], glutamine synthethase (GLNA) [37] and the histone H4 [38]. Heterologous promoters, such as the constitutive diatom infecting virus promoter CIP1 [39] have also been characterised, however results concerning CIP1's efficiency have been contradictory [37,39].

A broad range of promoters are needed to facilitate complex pathway expression, as multiple uses of the same promoter can result in transcriptional silencing [40]. EE can accelerate the characterisation process because it allows investigation of the true nature of promoters and their terminators in a reproducible and unbiased manner without a wide range of expression due to RICE [17].

In this study, we characterise novel endogenous constitutive promoters in combination with their corresponding terminators in P. tricornutum. In a comprehensive approach to promoter selection, we leveraged a consolidated transcriptomic resource consisting of the publically available transcriptomes from P. tricornutum [41]. The integration of transcriptomic data across multiple experiments enables comparative evaluation of gene expression across a broad range of culturing conditions and physiological states of P. tricornutum, and is a unique approach to identify gene candidates with consistently high expression in diatoms. We employed the newly developed modular uLoop system [42] to generate a series of EE constructs for a more reliable evaluation of the activity and behavior of selected promoter-terminator pairs, and describe 4 pairs that efficiently drive gene expression of mVenus under different growth conditions. These genetic elements contribute to a growing molecular toolbox for P. tricornutum, and are available as modular parts compatible with standardized Type IIS cloning strategies.

Section snippets

Cell culture

The diatom P. tricornutum (strain CCAP1055/1) was supplied by the Bigelow National Center of Marine Algae and Microbiota collection (https://ncma.bigelow.org). Cells were maintained in f/2 medium made from artificial sea water base [43,44] under continuous light at 21 °C and light intensity of 200 μmol photons m−1 s−1 in a fully controlled incubator (Kühner, Switzerland). Culture vessels were agitated at 95 rpm to keep cells suspended.

Screened transformants were maintained on half strength f/2

Integrated transcriptomic datasets are a resource for constitutive promoter identification

Since constitutive promoters drive gene expression over a range of different growth conditions, transcriptomic analysis is particularly useful in identifying new stable and robust promoter targets from genes based on expression pattern. We analysed expression data from 416 remapped and normalised P. tricornutum RNA-seq samples, the collation of which is available online (https://alganaut.uts.edu.au) (accessed January 2019) [41]. Promoters for genes whose transcript levels were most strongly and

Conclusion

Due to increasing knowledge of their genetic regulation and metabolic capabilities, eukaryotic microalgae such as P. tricornutum have been pushed into the spotlight of metabolic engineering and synthetic biology [2,71]. However, constraints such as limited numbers of experimentally characterised promoter and terminator sequences are roadblocks to unlocking P. tricornutum's full potential as an efficient cell factory.

In this study we used an RNAseq-informed approach for the identification of

CRediT authorship contribution statement

M.W.: Investigation, Formal Analysis, Writing – original draft, Visualisation; R.M.A. and M.D.: Conceptualisation, Supervision; J.A.: Software; R.M.A, M.P., L.B., and A.C.J.M.: provided experimental support; All authors: Writing – review and editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by an International Research Scholarship by the University of Technology Sydney to MW and operational funding by the UTS:C3 and the Faculty of Science. We thank Kun Xiao for technical assistance with flow cytometry; Dr. Nahshon Siboni for assistance with RT-qPCR; Dr. Chris Dupont and Dr. Bernardo Pollak (J. Craig Venter Institute) for kindly providing the Pt_FcpB promoter and terminator as part of uLoop cloning system. The authors acknowledge the use of the equipment

Statement of informed consent

No conflicts, informed consent or human or animal rights are applicable to this study.

References (71)

  • E. Calo et al.

    Modification of enhancer chromatin: what, how, and why?

    Mol. Cell

    (2013)
  • M. Bulger et al.

    Functional and mechanistic diversity of distal transcription enhancers

    Cell

    (2011)
  • Y. Liu et al.

    On the dependency of cellular protein levels on mRNA abundance

    Cell

    (2016)
  • M. Ramarajan et al.

    Novel endogenous promoters for genetic engineering of the marine microalga Nannochloropsis gaditana CCMP526

    Algal Res.

    (2019)
  • G. Markou et al.

    Microalgae for high-value compounds and biofuels production: a review with focus on cultivation under stress conditions

    Biotechnol. Adv.

    (2013)
  • S. Qin et al.

    Advances in genetic engineering of marine algae

    Biotechnol. Adv.

    (2012)
  • M. Branco-Vieira et al.

    Potential of Phaeodactylum tricornutum for biodiesel production under natural conditions in Chile

    Energies

    (2018)
  • Butler, T.; Kapoore, R.V.; Vaidyanathan, S. Phaeodactylum tricornutum: a diatom cell factory. Trends Biotechnol. 2020,...
  • F. Hempel et al.

    Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum

    PLoS One

    (2011)
  • F. Hempel et al.

    Microalgae as bioreactors for bioplastic production

    Microb. Cell Factories

    (2011)
  • M. Fabris et al.

    Extrachromosomal genetic engineering of the marine diatom Phaeodactylum tricornutum enables the heterologous production of monoterpenoids

    ACS Synth. Biol.

    (2020)
  • S. D’Adamo et al.

    Engineering the unicellular alga Phaeodactylum tricornutum for high-value plant triterpenoid production

    Plant Biotechnol. J.

    (2019)
  • C. Bowler et al.

    The Phaeodactylum genome reveals the evolutionary history of diatom genomes

    Nature

    (2008)
  • K.E. Apt et al.

    Stable nuclear transformation of the diatom Phaeodactylum tricornutum

    Mol. Gen. Genet.

    (1996)
  • A. Falciatore et al.

    Transformation of nonselectable reporter genes in marine diatoms

    Mar. Biotechnol.

    (1999)
  • B.J. Karas et al.

    Designer diatom episomes delivered by bacterial conjugation

    Nat. Commun.

    (2015)
  • F. Daboussi et al.

    Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

    Nat. Commun.

    (2014)
  • D. Stukenberg et al.

    Optimizing CRISPR/Cas9 for the diatom Phaeodactylum tricornutum

    Front. Plant Sci.

    (2018)
  • R.E. Diner et al.

    Refinement of the diatom episome maintenance sequence and improvement of conjugation-based DNA delivery methods

    Front. Bioeng. Biotechnol.

    (2016)
  • J. George et al.

    Metabolic engineering strategies in diatoms reveal unique phenotypes and genetic configurations with implications for algal genetics and synthetic biology

    Front. Bioeng. Biotechnol.

    (2020)
  • L.A. Zaslavskaia et al.

    Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

    J. Phycol.

    (2000)
  • W. Huang et al.

    Genetic and metabolic engineering in diatoms

    Philos. Trans. R. Soc. B Biol. Sci.

    (2017)
  • L. Doron et al.

    Transgene expression in microalgae—from tools to applications

    Front. Plant Sci.

    (2016)
  • D. Feike et al.

    Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots

    Plant Biotechnol. J.

    (2019)
  • H.E. Mischo et al.

    Disengaging polymerase: terminating RNA polymerase II transcription in budding yeast

    Biochim. Biophys. Acta BBA - Gene Regul. Mech.

    (1829)
  • Cited by (11)

    • Production of recombinant and therapeutic proteins in microalgae

      2022, Current Opinion in Biotechnology
      Citation Excerpt :

      Currently, most species of algae only have a handful of well-characterized promoters and terminators available for use. To expand this, an RNAseq-driven approach to designing promoters and terminators for P. tricornutum was successfully applied for expression of mVenus [22]. This study also demonstrated the importance of testing promoters under a variety of conditions as most were found to be sensitive to light intensity and/or nitrogen depletion.

    • Characterization of Chaetoceros lorenzianus-infecting DNA virus-derived promoters of genes from open reading frames of unknown function in Phaeodactylum tricornutum

      2022, Marine Genomics
      Citation Excerpt :

      We can access public databases of genome sequences of both centric diatoms and pennate diatoms (Falciatore et al., 2020). Gene delivery methods and molecular tools, including selectable markers, reporter genes, promoters, and terminators for diatom transformation, have been developed and can be applied for genetic engineering of diatoms (Huang and Daboussi, 2017; Kadono et al., 2020; Velmurugan and Deka, 2018; Windhagauer et al., 2021). More recent studies have led to the development of genome editing techniques (Kroth et al., 2018).

    View all citing articles on Scopus
    View full text