Research Article pubs.acs.org/synthbio
Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with Automatically Simulated Executable Documentation Filipa Pereira,† Flávio Azevedo,† Nadia Skorupa Parachin,‡,§ Bar̈ bel Hahn-Hag̈ erdal,‡ Marie F. Gorwa-Grauslund,‡ and Björn Johansson*,† †
ACS Synth. Biol. 2016.5:386-394. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/21/18. For personal use only.
CBMACentre of Molecular and Environmental Biology, Department of Biology, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal ‡ Division of Applied Microbiology, Department of Chemistry, Lund University, SE-22100 Lund, Sweden S Supporting Information *
ABSTRACT: We have developed the Yeast Pathway Kit (YPK) for rational and random metabolic pathway assembly in Saccharomyces cerevisiae using reusable and redistributable genetic elements. Genetic elements are cloned in a suicide vector in a rapid process that omits PCR product purification. Single-gene expression cassettes are assembled in vivo using genetic elements that are both promoters and terminators (TP). Cassettes sharing genetic elements are assembled by recombination into multigene pathways. A wide selection of prefabricated TP elements makes assembly both rapid and inexpensive. An innovative software tool automatically produces detailed self-contained executable documentation in the form of pydna code in the narrative Jupyter notebook format to facilitate planning and sharing YPK projects. A D-xylose catabolic pathway was created using YPK with four or eight genes that resulted in one of the highest growth rates reported on D-xylose (0.18 h−1) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improve the yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool. KEYWORDS: metabolic engineering, Saccharomyces cerevisiae, D-xylose, synthetic biology, bioinformatics
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etabolic engineering of Saccharomyces cerevisiae has been applied for the production of a wide range of fuels and chemicals (for review, see refs 1 and 2). Engineering of production strains usually requires the expression of a considerable number of genes because it is rare for a single enzyme to exert considerable control over a trait, such as flux along a metabolic pathway. The need to express multiple genes has led to the application of techniques that allow the simultaneous assembly of multiple promoters, genes, and terminators into metabolic pathways. Among the available techniques are the Gibson assembly protocol,3 which is a general technique for enzymatic assembly in vitro, and many variations of in vivo assembly by homologous recombination between flanking sequences added by PCR.4,5 Gene copy number,6 promoter strength,7 and positional effects of individual genes in a pathway8 may affect efficiency, but rationally designing and testing all permutations can be infeasible for longer pathways. Alternatively, a pool of randomly assembled pathways can be created from which the best performing pathways can be selected based on a screening strategy. Protocols for random pathway assembly engineering based on in vivo homologous recombination,9,10 Gibson assembly methods,11 or both12 have also been described. Common for most protocols is that genetic parts such as promoters and terminators are not easily shared and reused © 2016 American Chemical Society
because they are usually PCR products from chromosomal DNA or other sources and, as such, cannot be propagated. Most assembly protocols are “all or nothing” in the sense that multiple genes and regulatory sequences are joined in one reaction. Strategy or implementation errors such as a faulty PCR primer will yield little information to pinpoint the error as no pathway will be created. Furthermore, published pathway assembly protocols are designed for either rational or random assembly, but to do both requires reamplifying the genetic parts with new PCR primers. In this work, we have developed an alternative pathway assembly approach, called the Yeast Pathway Kit (YPK). YPK differs from previous methods in that promoters, genes, and terminators are cloned in one of three closely spaced blunt restriction sites in pYPKa, a highly efficient Escherichia coli positive selection vector designed for the rapid cloning of unpurified PCR products. Episomal yeast single-gene expression vectors are constructed from these basic elements by in vivo gap repair in S. cerevisiae three at a time in a promoter− gene−terminator configuration. This assembly is directed by Received: November 25, 2015 Published: February 25, 2016 386
DOI: 10.1021/acssynbio.5b00250 ACS Synth. Biol. 2016, 5, 386−394
ACS Synthetic Biology
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Research Article
RESULTS AND DISCUSSION YPK Strategy. The YPK metabolic pathway assembly strategy takes advantage of the observation that natural intergenic sequences from genes expressed in tandem are both terminators of the upstream gene and promoters for the downstream gene. These intergenic sequences (designated terminator−promoters or TPs) are used both for transcription regulation and for aiding the assembly of multiple gene metabolic pathways from single-gene expression cassettes. TPs from the intergenic sequences upstream of the genes TEF1 (579), TDH3 (698), PGI1 (1302), FBA1 (630), PDC1 (955), RPS19b (626), RPS19a (544), TPI1 (583), and ENO2 (520) were PCR-amplified from S. cerevisiae chromosomal DNA (sizes given in parentheses). These TPs have been used for the expression of heterologous proteins in S. cerevisiae, except for ribosomal protein promoters RPS19b and RPS19a. The TPs were first cloned into the ZraI or EcoRV site of the pYPKa suicide vector (Figure 1A). Genes to be expressed were cloned
short overlaps of plasmid backbone sequences between the three cloning sites. The single-gene expression vectors are subsequently joined into pathways by homologous recombination between promoters and terminators of each cassette. This can be done by the pairwise use of the same sequence as a promoter or terminator in the two vectors. The YPK promoters and terminators are intergenic sequences from tandemly expressed S. cerevisiae genes that naturally serve both as terminators and promoters (terminator−promoters or TPs) of the two adjacent genes. This second stage of the assembly is directed by the relatively long TPs of each cassette (500−1300 bp). YPK provides reusable genetic elements at several levels in the assembly process as there is an E. coli vector for each genetic element that is easily verified, stored, propagated, and distributed. The single-gene yeast expression vectors constitute a second level of reusable genetic elements as well as a way to study and verify each gene expression cassette separately. Assembly and verification of pathways can be performed using only two specific primers per gene together with a set of eight short (
