CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production

Kim, Seong Keun; Han, Gui Hwan; Seong, Wonjae; Kim, Haseong; Kim, Seon-Won; Lee, Dae-Hee

doi:10.1016/j.ymben.2016.08.006

Cited by 133 publications

(103 citation statements)

References 67 publications

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“…4d). This highlights the future potential of dCas9 as a simple tool for balancing between production of interest and growth, as also reported in E. coli [41]. Also, though S. cerevisiae is neither a natural producer of carotenoids, nor a preferred microbial cell factory for TAG biosynthesis, the renowned orthogonality of CRISPR/dCas9 should be amenable for implementation in natural carotenoid and high-TAG producing hosts like Xanthophyllomyces dendrorhous and Yarrowia lipolytica , respectively.…”

Section: Discussionmentioning

confidence: 84%

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

et al. 2017

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BackgroundTranscriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. In order to allow flexible and timely control over gene expression without the interference of native gene expression machinery, a large number of studies have focused on developing synthetic biology tools for orthogonal control of transcription. Most recently, the nuclease-deficient Cas9 (dCas9) has emerged as a flexible tool for controlling activation and repression of target genes, by the simple RNA-guided positioning of dCas9 in the vicinity of the target gene transcription start site.ResultsIn this study we compared two different systems of dCas9-mediated transcriptional reprogramming, and applied them to genes controlling two biosynthetic pathways for biobased production of isoprenoids and triacylglycerols (TAGs) in baker’s yeast Saccharomyces cerevisiae. By testing 101 guide-RNA (gRNA) structures on a total of 14 different yeast promoters, we identified the best-performing combinations based on reporter assays. Though a larger number of gRNA-promoter combinations do not perturb gene expression, some gRNAs support expression perturbations up to ~threefold. The best-performing gRNAs were used for single and multiplex reprogramming strategies for redirecting flux related to isoprenoid production and optimization of TAG profiles. From these studies, we identified both constitutive and inducible multiplex reprogramming strategies enabling significant changes in isoprenoid production and increases in TAG.ConclusionTaken together, we show similar performance for a constitutive and an inducible dCas9 approach, and identify multiplex gRNA designs that can significantly perturb isoprenoid production and TAG profiles in yeast without editing the genomic context of the target genes. We also identify a large number of gRNA positions in 14 native yeast target pomoters that do not affect expression, suggesting the need for further optimization of gRNA design tools and dCas9 engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0664-2) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 84%

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

et al. 2017

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“…In particular, a number of recent studies have exploited CRISPRi to regulate the metabolic pathways in E. coli for enhanced production of various biotechnological products including poly(3-hydroxybutyrate- co -4-hydroxybutyrate) [23], terpenoid [8], pinosylvin [46], flavonoid [47] and mevalonate [48]. Escherichia coli is a popular host as it has been extensively studied, grows fast, possesses a single chromosome and plenty of genetic engineering toolkits have been developed for E. coli engineering.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, in many cases intermediate levels of enzyme expression may result in better product titer [7]. Therefore, tunable and balanced gene expression is desirable for high productivity, product titer, and conversion yield, and controllable gene repression/knockdown may be preferable than gene deletion for certain biotechnological applications and synthetic biological manipulations [8]. …”

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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“…The 3-hydoxy-3-methylglutaryl reductase (HMG1) gene was repressed while the geranyl diphosphate synthase (GPS1) gene was overexpressed (-2.616 and 1.642 fold changes, respectively; Table 1, Fig 4). A recent work altering the expression of all the mevalonate pathway genes in the context of operon using CRISPRi system led to the striking of a balance between terpenoid production and cell growth [88]. Furthermore, non-volatile isoprenoids, such as carotenoids, and phenylpropanoids play a recognized antioxidant function in plant response to different environmental constrains, including drought stress [89].…”

Section: Resultsmentioning

confidence: 99%

Different adaptation strategies of two citrus scion/rootstock combinations in response to drought stress

et al. 2017

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Scion/rootstock interaction is important for plant development and for breeding programs. In this context, polyploid rootstocks presented several advantages, mainly in relation to biotic and abiotic stresses. Here we analyzed the response to drought of two different scion/rootstock combinations presenting different polyploidy: the diploid (2x) and autotetraploid (4x) Rangpur lime (Citrus limonia, Osbeck) rootstocks grafted with 2x Valencia Delta sweet orange (Citrus sinensis) scions, named V/2xRL and V/4xRL, respectively. Based on previous gene expression data, we developed an interactomic approach to identify proteins involved in V/2xRL and V/4xRL response to drought. A main interactomic network containing 3,830 nodes and 97,652 edges was built from V/2xRL and V/4xRL data. Exclusive proteins of the V/2xRL and V/4xRL networks (2,056 and 1,001, respectively), as well as common to both networks (773) were identified. Functional clusters were obtained and two models of drought stress response for the V/2xRL and V/4xRL genotypes were designed. Even if the V/2xRL plant implement some tolerance mechanisms, the global plant response to drought was rapid and quickly exhaustive resulting in a general tendency to dehydration avoidance, which presented some advantage in short and strong drought stress conditions, but which, in long terms, does not allow the plant survival. At the contrary, the V/4xRL plants presented a response which strong impacts on development but that present some advantages in case of prolonged drought. Finally, some specific proteins, which presented high centrality on interactomic analysis were identified as good candidates for subsequent functional analysis of citrus genes related to drought response, as well as be good markers of one or another physiological mechanism implemented by the plants.

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CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production

Cited by 133 publications

References 67 publications

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

Transcriptional reprogramming in yeast using dCas9 and combinatorial gRNA strategies

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

Different adaptation strategies of two citrus scion/rootstock combinations in response to drought stress

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