Genome Engineering in Cyanobacteria: Where We Are and Where We Need To Go

Ramey, Christopher Josh; Barón‐Solá, Ángel; Aucoin, Hanna R.; Boyle, Nanette R.

doi:10.1021/acssynbio.5b00043

Cited by 57 publications

(42 citation statements)

References 128 publications

(155 reference statements)

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“…Genetic control systems, which can boost production through precise regulation of heterologous gene expression, are limited in cyanobacteria (Berla et al, 2013;Huang et al, 2010;Ramey et al, 2015;Zess et al, 2016). Inducible promoters provide a defined transcriptional response upon sensing specific inputs.…”

Section: Introductionmentioning

confidence: 99%

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Immethun

DeLorenzo

Focht

et al. 2017

Biotech & Bioengineering

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Many under-developed organisms possess important traits that can boost the effectiveness and sustainability of microbial biotechnology. Photoautotrophic cyanobacteria can utilize the energy captured from light to fix carbon dioxide for their metabolic needs while living in environments not suited for growing crops. Various value-added compounds have been produced by cyanobacteria in the laboratory; yet, the products' titers and yields are often not industrially relevant and lag behind what have been accomplished in heterotrophic microbes. Genetic tools for biological process control are needed to take advantage of cyanobacteria's beneficial qualities, as tool development also lags behind what has been created in common heterotrophic hosts. To address this problem, we developed a suite of sensors that regulate transcription in the model cyanobacterium Synechocystis sp. PCC 6803 in response to metabolically relevant signals, including light and the cell's nitrogen status, and a family of sensors that respond to the inexpensive chemical, l-arabinose. Increasing the number of available tools enables more complex and precise control of gene expression. Expanding the synthetic biology toolbox for this cyanobacterium also improves our ability to utilize this important under-developed organism in biotechnology. Biotechnol. Bioeng. 2017;114: 1561-1569. © 2017 Wiley Periodicals, Inc.

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Section: Introductionmentioning

confidence: 99%

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Immethun

DeLorenzo

Focht

et al. 2017

Biotech & Bioengineering

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“…However, generation of a single gene knockout mutant may take >3 weeks using conventional methods [6] due to its long doubling time and oligoploidy nature [1]. Sometimes deletion of certain genes essential for metabolic balances is not feasible or easily achieved as the deletion might be lethal to the cells.…”

Section: Introductionmentioning

confidence: 99%

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

et al. 2016

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BackgroundCyanobacterium Synechococcus elongatus PCC 7942 holds promise for biochemical conversion, but gene deletion in PCC 7942 is time-consuming and may be lethal to cells. CRISPR interference (CRISPRi) is an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single guide RNA (sgRNA) to repress sequence-specific genes without the need of gene knockout, and is repurposed to rewire metabolic networks in various procaryotic cells.ResultsTo employ CRISPRi for the manipulation of gene network in PCC 7942, we integrated the cassettes expressing enhanced yellow fluorescent protein (EYFP), dCas9 and sgRNA targeting different regions on eyfp into the PCC 7942 chromosome. Co-expression of dCas9 and sgRNA conferred effective and stable suppression of EYFP production at efficiencies exceeding 99%, without impairing cell growth. We next integrated the dCas9 and sgRNA targeting endogenous genes essential for glycogen accumulation (glgc) and succinate conversion to fumarate (sdhA and sdhB). Transcription levels of glgc, sdhA and sdhB were effectively suppressed with efficiencies depending on the sgRNA binding site. Targeted suppression of glgc reduced the expression to 6.2%, attenuated the glycogen accumulation to 4.8% and significantly enhanced the succinate titer. Targeting sdhA or sdhB also effectively downregulated the gene expression and enhanced the succinate titer ≈12.5-fold to ≈0.58–0.63 mg/L.ConclusionsThese data demonstrated that CRISPRi-mediated gene suppression allowed for re-directing the cellular carbon flow, thus paving a new avenue to rationally fine-tune the metabolic pathways in PCC 7942 for the production of biotechnological products.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0595-3) contains supplementary material, which is available to authorized users.

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“…Rabinovitich-Deere et al, (2013) 205 Reviews metabolic engineering and synthetic biology approaches to produce biofuels. Ramey et al, (2015) 206 Reviews synthetic biology tools for cyanobacterial genome engineering efforts. Rosgaard et al, (2012) 32 Provides a summary of engineering of carbon fixation in cyanobacteria.…”

Section: Cyanobacteria and Algae As Sources Of Biofuelmentioning

confidence: 99%

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review

Johnson

Gibbons

et al. 2016

Biotechnology Progress

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The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO , H O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357-1371, 2016.

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Genome Engineering in Cyanobacteria: Where We Are and Where We Need To Go

Cited by 57 publications

References 128 publications

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803

CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review

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