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      YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica

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          Abstract

          Effective metabolic engineering of microorganisms relies on balanced expression of both heterologous and endogenous genes to channel metabolic flux towards products of interest while achieving reasonable biomass buildup. To facilitate combinatorial pathway engineering and facile genetic operation, we engineered a set of modular cloning vectors compatible with BioBrick standards, called YaliBricks, to allow for rapid assembly of multigene pathways with customized genetic control elements (promoters, intronic sequences and terminators) in the oleaginous yeast Yarrowia lipolytica. We established a sensitive luciferase reporter and characterized a set of 12 native promoters to expand the oleaginous yeast genetic toolbox for transcriptional fine-tuning. We harnessed the intron alternative splicing mechanism and explored three unique gene configurations that allow us to encode genetic structural variations into metabolic function. We elucidated the role of how these genetic structural variations affect gene expression. To demonstrate the simplicity and effectiveness of streamlined genetic operations, we assembled the 12 kb five-gene violacein biosynthetic pathway in one week. We also expanded this set of vectors to accommodate self-cleavage ribozymes and efficiently deliver guide RNA (gRNA) for targeted genome-editing with a codon-optimized CRISPR-Cas9 nuclease. Taken together, the tools built in this study provide a standard procedure to streamline and accelerate metabolic pathway engineering and genetic circuits construction in Yarrowia lipolytica.

          Highlights

          • •

            Twelve modular cloning vectors YaliBricks developed for Y. lipolytica.

          • •

            Luciferase reporter established and 12 native promoters characterized.

          • •

            Intron alternative splicing harnessed to construct 3 gene configurations.

          • •

            A 12-kb five-gene violacein biosynthetic pathway assembled in one week.

          • •

            CRISPR-Cas9 genome-editing feature was incorporated into YaliBricks.

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          Most cited references62

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          CRISPR/Cas9 in Genome Editing and Beyond

          Haifeng Wang, Marie La Russa, Lei S Qi (2016)
          The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.
          Article 0 comments Cited 393 times – based on 0 reviews     Review now
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            Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing.

            Yangbin Gao, Yunde Zhao (2014)
            CRISPR/Cas9 uses a guide RNA (gRNA) molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated. © 2013 Institute of Botany, Chinese Academy of Sciences.
            Article 0 comments Cited 220 times – based on 0 reviews     Review now
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              Transcriptional interference--a crash course.

              Keith E. Shearwin, Benjamin P Callen, J. Barry Egan (2005)
              The term "transcriptional interference" (TI) is widely used but poorly defined in the literature. There are a variety of methods by which one can interfere with the process or the product of transcription but the term TI usually refers to the direct negative impact of one transcriptional activity on a second transcriptional activity in cis. Two recent studies, one examining Saccharomyces cerevisiae and the other Escherichia coli, clearly show TI at one promoter caused by the arrival of a transcribing complex initiating at a distant promoter. TI is potentially widespread throughout biology; therefore, it is timely to assess exactly its nature, significance and operative mechanisms. In this article, we will address the following questions: what is TI, how important and widespread is it, how does it work and where should we focus our future research efforts?
              Article 0 comments Cited 217 times – based on 0 reviews     Review now
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                Author and article information

                Contributors
                Peng Xu
                Journal
                Journal ID (nlm-ta): Metab Eng Commun
                Journal ID (iso-abbrev): Metab Eng Commun
                Title: Metabolic Engineering Communications
                Publisher: Elsevier
                ISSN (Electronic): 2214-0301
                Publication date PMC-release: 01 October 2017
                Publication date Collection: December 2017
                Publication date (Electronic): 01 October 2017
                Volume: 5
                Pages: 68-77
                Affiliations
                [a ]Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States
                [b ]Department of Food Science and Engineering, Jinan University, 601 West Huangpu Road, Guangzhou 510632, China
                Author notes
                [* ]Corresponding author. pengxu@ 123456umbc.edu
                Article
                Publisher ID: S2214-0301(17)30023-8
                DOI: 10.1016/j.meteno.2017.09.001
                PMC ID: 5699529
                PubMed ID: 29188186
                SO-VID: b1caf4bd-4892-4962-8ad6-b49d328ee0d3
                Copyright © © 2017 The Authors
                License:

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                Date received : 25 August 2017
                Date revision received : 22 September 2017
                Date accepted : 25 September 2017
                Categories
                Subject: Article

                Keywords: gene assembly,intron alternative splicing,luciferase reporter,promoter activity,yarrowia lipolytica,gene configuration,genome-editing
                Data availability:
                Keywords: gene assembly, intron alternative splicing, luciferase reporter, promoter activity, yarrowia lipolytica, gene configuration, genome-editing

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