Combinatorial assembly of large biochemical pathways into yeast chromosomes for improved production of value-added compounds.

Saccharomyces cerevisiae as a eukaryotic organism is particularly suitable as microbial cell factory because it has interesting features such as membrane environments for supporting membrane-associated enzymes and its capability for post-translational modifications of enzymes from plants. However, S. cerevisiae does not readily express polycistronic transcriptional units, which represents a significant challenge for constructing large biochemical pathways in budding yeast. In the present study, we developed a novel approach for rapid construction of large biochemical pathways into yeast chromosomes. Our approach takes advantage of antibiotic selection for combinatorial assembly of large pathways into the δ-sites of retrotransposon elements of yeast chromosomes. As proof-of-principle, a five-gene isobutanol pathway and an eight-gene mevalonate pathway were successfully assembled into yeast chromosomes in one-step fashion. To our knowledge, this is the first report to exploit δ-integration coupled with antibiotic selection for rapid assembly of large biochemical pathways in budding yeast. We envision our new approach could serve as a generalized technique for large pathway construction in yeast-a method that would be of significant interest to the synthetic biology community.

[1]  Thomas H Segall-Shapiro,et al.  Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome , 2010, Science.

[2]  Christian Weber,et al.  Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae , 2012, Biotechnology for Biofuels.

[3]  G. Stephanopoulos,et al.  Tuning genetic control through promoter engineering. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  L. Grivell,et al.  Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein. , 1984, The EMBO journal.

[5]  Huimin Zhao,et al.  Iowa State University From the SelectedWorks of Zengyi Shao 2012 Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae , 2017 .

[6]  R. Müller,et al.  Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. , 1994, Nucleic acids research.

[7]  K. Oldenburg,et al.  Recombination-mediated PCR-directed plasmid construction in vivo in yeast. , 1997, Nucleic acids research.

[8]  D. Tipper,et al.  Use of β‐lactamase as a secreted reporter of promoter function in yeast , 1994 .

[9]  Yajun Yan,et al.  Conversion of proteins into biofuels by engineering nitrogen flux , 2011, Nature Biotechnology.

[10]  G. Fink,et al.  Ty elements transpose through an RNA intermediate , 1985, Cell.

[11]  Zengyi Shao,et al.  DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways , 2008, Nucleic acids research.

[12]  Irina Borodina,et al.  Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism , 2011, Biotechnology for biofuels.

[13]  J. Liao,et al.  Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels , 2008, Nature.

[14]  Yong-Su Jin,et al.  Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes , 2012, Bioprocess and Biosystems Engineering.

[15]  Jun Ishii,et al.  Genetic engineering to enhance the Ehrlich pathway and alter carbon flux for increased isobutanol production from glucose by Saccharomyces cerevisiae. , 2012, Journal of biotechnology.

[16]  Keith E. J. Tyo,et al.  Isoprenoid Pathway Optimization for Taxol Precursor Overproduction in Escherichia coli , 2010, Science.

[17]  Akihiko Kondo,et al.  Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains , 2010, Microbial cell factories.

[18]  Timothy S. Ham,et al.  Production of the antimalarial drug precursor artemisinic acid in engineered yeast , 2006, Nature.

[19]  H. Feldmann,et al.  Nucleotide sequence and characteristics of a Ty element from yeast. , 1985, Nucleic acids research.

[20]  Jay D Keasling,et al.  Redirection of flux through the FPP branch‐point in Saccharomyces cerevisiae by down‐regulating squalene synthase , 2008, Biotechnology and bioengineering.

[21]  Jay D Keasling,et al.  Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. , 2009, Metabolic engineering.

[22]  Yousef Haj-Ahmad,et al.  Counter-selection facilitated plasmid construction by homologous recombination in Saccharomyces cerevisiae. , 2003, BioTechniques.

[23]  Jay D. Keasling,et al.  Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin , 2012, Proceedings of the National Academy of Sciences.

[24]  G. Butler,et al.  GAL1-GAL10 divergent promoter region of Saccharomyces cerevisiae contains negative control elements in addition to functionally separate and possibly overlapping upstream activating sequences. , 1987, Genes & development.

[25]  Keith E. J. Tyo,et al.  Stabilized gene duplication enables long-term selection-free heterologous pathway expression , 2009, Nature Biotechnology.

[26]  F. Karst,et al.  The Saccharomyces cerevisiae mevalonate diphosphate decarboxylase is essential for viability, and a single Leu-to-Pro mutation in a conserved sequence leads to thermosensitivity , 1997, Journal of bacteriology.

[27]  James C. Liao,et al.  3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation , 2010, Applied Microbiology and Biotechnology.

[28]  M. Eckart,et al.  Quality and authenticity of heterologous proteins synthesized in yeast. , 1996, Current opinion in biotechnology.

[29]  Verena Siewers,et al.  Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae , 2009, Yeast.

[30]  G. Stephanopoulos,et al.  Compartmentalization of metabolic pathways in yeast mitochondria improves production of branched chain alcohols , 2013, Nature Biotechnology.

[31]  J. Keasling,et al.  High-level semi-synthetic production of the potent antimalarial artemisinin , 2013, Nature.

[32]  J. Hegemann,et al.  A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast. , 2002, Nucleic acids research.

[33]  Huimin Zhao,et al.  Rapid characterization and engineering of natural product biosynthetic pathways via DNA assembler. , 2011, Molecular bioSystems.

[34]  D. Thiele,et al.  A widespread transposable element masks expression of a yeast copper transport gene. , 1996, Genes & development.

[35]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[36]  B. Barrell,et al.  Life with 6000 Genes , 1996, Science.

[37]  Ming Jiang,et al.  Downstream reactions and engineering in the microbially reconstituted pathway for Taxol , 2012, Applied Microbiology and Biotechnology.

[38]  D. Pompon,et al.  Yeast expression of animal and plant P450s in optimized redox environments. , 1996, Methods in enzymology.

[39]  Gregory Stephanopoulos,et al.  Engineering of Promoter Replacement Cassettes for Fine-Tuning of Gene Expression in Saccharomyces cerevisiae , 2006, Applied and Environmental Microbiology.