Synthetic Biology in Cyanobacteria and Applications for Biotechnology

Hudson, Elton P.

doi:10.1002/9783527824908.ch5

Cited by 7 publications

(5 citation statements)

References 182 publications

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“…The gram-negative bacteria exhibit a large ecological variety as well as a broad morphological diversity ( Bennett and Bogorad, 1973 ; Rippka et al., 1979 ; Schirrmeister et al., 2013 ). Their physiological diversity makes them promising biological chassis for the synthesis of a variety of natural products, including bioactive metabolites like cytotoxins and potential pharmaceutical lead compounds, food supplements, animal feed, pigments, as well as biofuels ( Pulz and Gross, 2004 ; Hays and Ducat, 2015 ; Jain et al., 2017 ; Hudson et al., 2021 ; Barone et al., 2023 ). Their ability to convert sunlight and atmospheric CO 2 directly into valuable organic compounds could make the chemical and pharmaceutical industry more sustainable and therefore mitigate climate change if high production yields are achieved ( Choi et al, 2020 ; Posten and Schaub, 2009 ; Oliver and Atsumi, 2015 ; Oliver et al., 2016 ).…”

Section: Introductionmentioning

confidence: 99%

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803

Germann,

Nakielski,

Dietsch

et al. 2023

Front. Plant Sci.

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Cyanobacteria are a promising platform for the production of the triterpene squalene (C30), a precursor for all plant and animal sterols, and a highly attractive intermediate towards triterpenoids, a large group of secondary plant metabolites. Synechocystis sp. PCC 6803 natively produces squalene from CO2 through the MEP pathway. Based on the predictions of a constraint-based metabolic model, we took a systematic overexpression approach to quantify native Synechocystis gene’s impact on squalene production in a squalene-hopene cyclase gene knock-out strain (Δshc). Our in silico analysis revealed an increased flux through the Calvin-Benson-Bassham cycle in the Δshc mutant compared to the wildtype, including the pentose phosphate pathway, as well as lower glycolysis, while the tricarboxylic acid cycle predicted to be downregulated. Further, all enzymes of the MEP pathway and terpenoid synthesis, as well as enzymes from the central carbon metabolism, Gap2, Tpi and PyrK, were predicted to positively contribute to squalene production upon their overexpression. Each identified target gene was integrated into the genome of Synechocystis Δshc under the control of the rhamnose-inducible promoter Prha. Squalene production was increased in an inducer concentration dependent manner through the overexpression of most predicted genes, which are genes of the MEP pathway, ispH, ispE, and idi, leading to the greatest improvements. Moreover, we were able to overexpress the native squalene synthase gene (sqs) in Synechocystis Δshc, which reached the highest production titer of 13.72 mg l-1 reported for squalene in Synechocystis sp. PCC 6803 so far, thereby providing a promising and sustainable platform for triterpene production.

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Section: Introductionmentioning

confidence: 99%

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803

Germann,

Nakielski,

Dietsch

et al. 2023

Front. Plant Sci.

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“…Cyanobacteria are photosynthetic prokaryotes that have gained interest as cell factories for sustainable production of various compounds. − However, in order for large-scale processes to become economically feasible, further study and significant engineering of these cyanobacterial hosts is required. While the toolset to engineer cyanobacteria is steadily growing, − there is still need for reliable tools that allow for precise, markerless, rapid, and multiplexed editing in these polyploid organisms that are commonly time-consuming to engineer.…”

Section: Introductionmentioning

confidence: 99%

Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803

et al. 2022

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Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here, we describe a riboswitch-inducible CRISPR/Cas9 system, contained on a single replicative vector, for the model cyanobacterium Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector into Synechocystis . Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits performed better, reaching, e.g., 100% for insertion of a FLAG-tag onto rbcL . Importantly, the single-vector CRISPR/Cas9 system mediated multiplexed editing of up to three targets in parallel in Synechocystis . All single-target and several double-target mutants were also fully segregated after the first round of induction. Lastly, a vector curing system based on the nickel-inducible expression of the toxic mazF (from Escherichia coli ) was added to the CRISPR/Cas9 vector. This inducible system allowed for curing of the vector in 25–75% of screened colonies, enabling edited mutants to become markerless.

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“…[1][2][3] However, in order for such large-scale processes to become economically feasible, further study and significant engineering of these cyanobacterial hosts is required. While the toolset to engineer cyanobacteria is steadily growing, [4][5][6] there is still need for reliable tools that allow for precise, markerless, rapid, and multiplexed editing in these polyploid organisms 7 that are commonly time-consuming to engineer.…”

Section: Introductionmentioning

confidence: 99%

Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

Cengic

Cañadas

Minton

et al. 2022

Preprint

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Establishing various synthetic biology tools is crucial for the development of cyanobacteria for biotechnology use, especially tools that allow for precise and markerless genome editing in a time-efficient manner. Here we describe a riboswitch-inducible CRISPR/Cas9 system, contained on one single replicative vector, for the model cyanobacteria Synechocystis sp. PCC 6803. A theophylline-responsive riboswitch allowed tight control of Cas9 expression, which enabled reliable transformation of the CRISPR/Cas9 vector into Synechocystis. Induction of the CRISPR/Cas9 mediated various types of genomic edits, specifically deletions and insertions of varying size. The editing efficiency varied depending on the target and intended edit; smaller edits overall performed better, reaching e.g. 100% for insertion of a FLAG-tag onto rbcL. Importantly, the single-vector CRISPR/Cas9 system described herein was also shown to mediate multiplexed editing of up to three targets in parallel in Synechocystis. All single-target and several double-target mutants were also fully segregated after the first round of induction, adding to the usefulness of this system. Further, a vector curing system that is separately induced by nickel and contained on the CRISPR/Cas9 vector itself, improved curing efficiencies by roughly 4-fold, enabling the final mutants to become truly markerless.

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Synthetic Biology in Cyanobacteria and Applications for Biotechnology

Cited by 7 publications

References 182 publications

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803

A systematic overexpression approach reveals native targets to increase squalene production in Synechocystis sp. PCC 6803

Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803

Inducible CRISPR/Cas9 allows for multiplexed and rapidly segregated single target genome editing in Synechocystis sp. PCC 6803

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