Journal ArticleDOI
Multiplexed CRISPR Activation of Cryptic Sugar Metabolism Enables Yarrowia Lipolytica Growth on Cellobiose.
TLDR
This work expands the synthetic biology toolbox for metabolic engineering in Y. lipolytica and demonstrates how the programmability of the CRISPR-Cas9 system can enable facile investigation of transcriptionally silent regions of the genome.Abstract:
The yeast Yarrowia lipolytica has been widely studied for its ability to synthesize and accumulate intracellular lipids to high levels. Recent studies have identified native genes that enable growth on biomass-derived sugars, but these genes are not sufficiently expressed to facilitate robust metabolism. In this work, a CRISPR-dCas9 activation (CRISPRa) system in Y. lipolytica is developed and is used it to activate native β-glucosidase expression to support growth on cellobiose. A series of different transcriptional activators are compared for their effectiveness in Y. lipolytica, with the synthetic tripartite activator VPR yielding the highest activation. A VPR-dCas9 fusion is then targeted to various locations in a synthetic promoter driving hrGFP expression, and activation is achieved. Subsequently, the CRISPRa system is used to activate transcription of two different native β-glucosidase genes, facilitating enhanced growth on cellobiose as the sole carbon source. This work expands the synthetic biology toolbox for metabolic engineering in Y. lipolytica and demonstrates how the programmability of the CRISPR-Cas9 system can enable facile investigation of transcriptionally silent regions of the genome.read more
Citations
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Journal ArticleDOI
Multiplexed CRISPR technologies for gene editing and transcriptional regulation.
TL;DR: This review discusses multiplexed CRISPR technologies and describes methods for the assembly, expression and processing of synthetic guide RNA arrays in vivo, and offers a glimpse of emerging challenges and emphasize experimental considerations for future studies.
Journal ArticleDOI
Metabolic engineering in the host Yarrowia lipolytica
A. M. Abdel-Mawgoud,Kelly A. Markham,Claire M. Palmer,Nian Liu,Gregory Stephanopoulos,Hal S. Alper +5 more
TL;DR: A case is established for Y. lipolytica as a premier metabolic engineering host based on innate metabolic capacity, emerging synthetic tools, and engineering examples.
Journal ArticleDOI
Synthetic biology tools for engineering Yarrowia lipolytica.
TL;DR: Recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica are presented, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools.
Journal ArticleDOI
CRISPR-mediated genome editing in non-conventional yeasts for biotechnological applications.
TL;DR: The current advances and barriers of CRISPR–Cas9 for genome editing in non-conventional yeasts are reviewed and the possible solutions in enhancing its efficiency for precise genetic engineering are proposed.
Journal ArticleDOI
Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts.
TL;DR: In this article, the authors provide an insight into the recent advances and challenges of systems and synthetic biology as well as metabolic engineering endeavours towards the commercial usage of non-conventional yeasts.
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