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Abstract
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.
Highlights
Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues
We found that positioning of scaffold RNA (scRNA) in regions with predicted low nucleosome occupancy for pHMG1 correlated with higher transcriptional impact in accordance with current literature [23, 34, 35]
With the results presented in our study using inducible CRISPR/deficient Cas9 (dCas9) and the scRNA-mediated combinatorial reprogramming for scaffolding synthetic transcription machineries, metabolic engineers have more tools to allow for inducible and multiplex control of expression of essential target genes, which simultaneously can sustain growth
Summary
Transcriptional reprogramming is a fundamental process of living cells in order to adapt to environmental and endogenous cues. Several studies have reported the overexpression and downregulation of key MVA pathway genes, including the ones encoding farnesyl pyrophosphate (FPP) synthase (ERG20), squalene synthase (ERG9), and the HMG-CoA reductase (HMG1), in order to increase production of value-added isoprenoids from simple sugars, while simultaneously maintaining ergosterol levels to support growth [9,10,11]. In another example, triacylglycerols (TAGs) are key molecules for cell functioning as essential energy storage compounds, and potential industrial feedstocks for the production of food ingredients, oleochemicals and biodiesel [12, 13]. This calls for the development and application of new molecular tools to enable testing and identification of optimal expression levels of several genes simultaneously
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