N-terminal sequences affect expression of triterpene biosynthesis enzymes in Chlamydomonas chloroplasts

Hsu, Shih-Chi; Browne, Daniel R.; Tatli, Mehmet; Devarenne, Timothy P.; Stern, David B.

doi:10.1016/j.algal.2019.101662

Cited by 5 publications

(4 citation statements)

References 61 publications

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“…The addition of C-terminal tags to a geraniol synthase-FPPS(F96W-N127W) fusion protein also increased protein expression and improved geraniol production in yeast 52 . In a Chlamydomonas reinhardtii study, levels of FPPS and SQS-like enzymes were elevated by modification of the N-end terminal residues, which suggested decreased protein degradation by the N-end rule 53 .…”

Section: Resultsmentioning

confidence: 96%

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Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

Cheah

Stark

Plan

et al. 2022

Preprint

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The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), which corresponds to increases of a similar magnitude in terpene synthase expression. Therefore, increased in vivo enzyme levels, resulting from improved expression and/or stability, is likely to be a major driver of catalytic enhancement from enzyme fusion.

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Section: Resultsmentioning

confidence: 96%

“…In a Chlamydomonas reinhardtii study, levels of FPPS and SQS-like enzymes were elevated by modification of the N-end terminal residues, which suggested decreased protein degradation by the Nend rule 53 .…”

Section: Stabilisation Of Diverse Terpene Synthases By Translational ...mentioning

confidence: 97%

Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

Cheah

Stark

Plan

et al. 2022

Preprint

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“…It was previously unknown if, and to which extent, FPP would be freely available in the chloroplast. In one previous investigation [ 17 ], it was attempted with no success to use FPP as a substrate in the chloroplast of C. reinhardtii . In our previous work [ 7 ], a patchoulol synthase was targeted to the algal chloroplast and mitochondria without generating the sesquiterpenoid patchoulol.…”

Section: Discussionmentioning

confidence: 99%

Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-α-bisabolene production

et al. 2022

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Background Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts. Results In this study, we investigated the accessibility of different isoprenoid precursor pools for C15 sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase (AgBS) as a reporter. The abundance of the C15 sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of AgBS with different FPP synthases (FPPSs), indicating limited C5 precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when AgBS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO2 as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations. Conclusions Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.

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“…However, the use of the same cis element in more than one transcription unit needs to be avoided to prevent unwanted recombination events between direct repeats leading to transgene instability (Gimpel et al, 2016;Larrea-Alvarez and Purton, 2020). An alternative strategy to creating separate transcriptional units is to express multiple transgenes as a single synthetic operon (Bock, 2013;Macedo-Osorio et al, 2018;Hsu et al, 2019). Many endogenous genes in the chloroplast are co-expressed from a single promoter as polycistronic transcripts separated by "intercistronic expression elements" (IEEs).…”

Section: Multigenic Expressionmentioning

confidence: 99%

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

et al. 2021

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Sustainable and economically viable support for an ever-increasing global population requires a paradigm shift in agricultural productivity, including the application of biotechnology to generate future crop plants. Current genetic engineering approaches aimed at enhancing the photosynthetic efficiency or composition of the harvested tissues involve relatively simple manipulations of endogenous metabolism. However, radical rewiring of central metabolism using new-to-nature pathways, so-called “synthetic metabolism”, may be needed to really bring about significant step changes. In many cases, this will require re-programming the metabolism of the chloroplast, or other plastids in non-green tissues, through a combination of chloroplast and nuclear engineering. However, current technologies for sophisticated chloroplast engineering (“transplastomics”) of plants are limited to just a handful of species. Moreover, the testing of metabolic rewiring in the chloroplast of plant models is often impractical given their obligate phototrophy, the extended time needed to create stable non-chimeric transplastomic lines, and the technical challenges associated with regeneration of whole plants. In contrast, the unicellular green alga, Chlamydomonas reinhardtii is a facultative heterotroph that allows for extensive modification of chloroplast function, including non-photosynthetic designs. Moreover, chloroplast engineering in C. reinhardtii is facile, with the ability to generate novel lines in a matter of weeks, and a well-defined molecular toolbox allows for rapid iterations of the “Design-Build-Test-Learn” (DBTL) cycle of modern synthetic biology approaches. The recent development of combinatorial DNA assembly pipelines for designing and building transgene clusters, simple methods for marker-free delivery of these clusters into the chloroplast genome, and the pre-existing wealth of knowledge regarding chloroplast gene expression and regulation in C. reinhardtii further adds to the versatility of transplastomics using this organism. Herein, we review the inherent advantages of the algal chloroplast as a simple and tractable testbed for metabolic engineering designs, which could then be implemented in higher plants.

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N-terminal sequences affect expression of triterpene biosynthesis enzymes in Chlamydomonas chloroplasts

Cited by 5 publications

References 61 publications

Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

Translational fusion of terpene synthases enhances metabolic flux by increasing protein stability

Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-α-bisabolene production

The Algal Chloroplast as a Testbed for Synthetic Biology Designs Aimed at Radically Rewiring Plant Metabolism

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