Characterisation of novel regulatory sequences compatible with modular assembly in the diatom Phaeodactylum 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)
- et al.
Development of endogenous promoters that drive high-level expression of introduced genes in the model diatom Phaeodactylum tricornutum
Mar. Genomics
(2018) - et al.
Phytosterol biosynthesis and production by diatoms (Bacillariophyceae)
Phytochemistry
(2019) - et al.
High-efficiency nuclear transformation of the diatom Phaeodactylum tricornutum by electroporation
Mar. Genomics
(2014) - et al.
Chapter 18 - production of biopharmaceuticals in microalgae
- et al.
Manipulating gene expression for the metabolic engineering of plants
Metab. Eng.
(2002) - et al.
Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum
Algal Res.
(2017) - et al.
Effect of nitrogen concentration on growth, lipid production and fatty acid profiles of the marine diatom Phaeodactylum tricornutum
Agric. Nat. Resour.
(2017) - et al.
Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum
Gene
(2007) - et al.
Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum
Algal Res.
(2015) - et al.
Evaluation of potential reference genes for reverse transcription-qPCR studies of physiological responses in Drosophila melanogaster
J. Insect Physiol.
(2011)
Modification of enhancer chromatin: what, how, and why?
Mol. Cell
Functional and mechanistic diversity of distal transcription enhancers
Cell
On the dependency of cellular protein levels on mRNA abundance
Cell
Novel endogenous promoters for genetic engineering of the marine microalga Nannochloropsis gaditana CCMP526
Algal Res.
Microalgae for high-value compounds and biofuels production: a review with focus on cultivation under stress conditions
Biotechnol. Adv.
Advances in genetic engineering of marine algae
Biotechnol. Adv.
Potential of Phaeodactylum tricornutum for biodiesel production under natural conditions in Chile
Energies
Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum
PLoS One
Microalgae as bioreactors for bioplastic production
Microb. Cell Factories
Extrachromosomal genetic engineering of the marine diatom Phaeodactylum tricornutum enables the heterologous production of monoterpenoids
ACS Synth. Biol.
Engineering the unicellular alga Phaeodactylum tricornutum for high-value plant triterpenoid production
Plant Biotechnol. J.
The Phaeodactylum genome reveals the evolutionary history of diatom genomes
Nature
Stable nuclear transformation of the diatom Phaeodactylum tricornutum
Mol. Gen. Genet.
Transformation of nonselectable reporter genes in marine diatoms
Mar. Biotechnol.
Designer diatom episomes delivered by bacterial conjugation
Nat. Commun.
Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology
Nat. Commun.
Optimizing CRISPR/Cas9 for the diatom Phaeodactylum tricornutum
Front. Plant Sci.
Refinement of the diatom episome maintenance sequence and improvement of conjugation-based DNA delivery methods
Front. Bioeng. Biotechnol.
Metabolic engineering strategies in diatoms reveal unique phenotypes and genetic configurations with implications for algal genetics and synthetic biology
Front. Bioeng. Biotechnol.
Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes
J. Phycol.
Genetic and metabolic engineering in diatoms
Philos. Trans. R. Soc. B Biol. Sci.
Transgene expression in microalgae—from tools to applications
Front. Plant Sci.
Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots
Plant Biotechnol. J.
Disengaging polymerase: terminating RNA polymerase II transcription in budding yeast
Biochim. Biophys. Acta BBA - Gene Regul. Mech.
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