Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system☆
Introduction
Fuel ethanol production from biomass at the industrial level using Saccharomyces cerevisiae shows great promise for satisfying future energy demands, but the limited range of materials that can be fermented remains an obstacle to cost-effective bioethanol production (Farrell et al., 2006, Saha, 2003). Although several genetically engineered strains of S. cerevisiae have been developed that will ferment xylose to ethanol (Karhumaa et al., 2007, Sedlak and Ho, 2004, Wisselink et al., 2007), further optimization is needed. It will require the simultaneous expression, at sufficiently high level, of all the enzymes and proteins needed to allow industrial yeast strains to grow efficiently on pentose as well as hexose sugars anaerobically. In addition, for cost-effective industrial ethanol production from biomass it will be necessary to express the enzymes required to saccharify the lignocellulosic feedstocks that are the source of hexose and pentose sugars. Genes considered necessary for complete fermentation of xylose and arabinose, the two major pentose sugar constituents of lignocellulosic biomass, include those encoding xylose isomerase (XI), xylulokinase (XKS), arabinose A, arabinose B, and arabinose D (Karhumaa et al., 2007, Wisselink et al., 2007), which may be obtained from a microorganism naturally capable of fermenting these sugars. Saccharification of lignocellulosic feedstocks also requires utilization of hydrolytic enzymes including cellulases and hemicellulases after initial chemical pre-treatment (Rudolf et al., 2007, Saha et al., 2005). The cost-effectiveness of the fuel ethanol fermentation process could be further enhanced by obtaining high-value co-products and by-products from the process, such as monomers for polymer production and commercially important proteins and peptides. Genes for these proteins and peptides can be mutagenized, placed in an expression system capable of producing high levels of functional proteins or peptides, and screened in high throughput to optimize desired characteristics. An integrated automated platform is available to carry out all the processes for gene assembly, plasmid library production, expression of proteins, enzymes, and value-added proteins or peptides, and growth testing of the transformed yeast strains (Hughes et al., 2005, Hughes et al., 2006, Hughes et al., 2007). In addition, a system to express numerous genes simultaneously is essential. A yeast plasmid system was designed to accomplish this using three small ubiquitin-like modifier (SUMO) yeast expression vectors (Butt et al., 2005, Malakhov et al., 2004). We integrated this three-plasmid vector system into the automated protocols on the plasmid-based robotic workcell to engineer and screen improved ethanologenic yeast strains expressing (1) xylose isomerase, (2) a library of mutagenized peptides that are putative bioinsecticides, and (3) an enzyme of the pentose phosphate pathway to enhance xylose metabolism. This set of plasmids used on the automated platform offers the possibility of expressing pentose-utilization enzymes and commercially important peptides in yeast, and screening the resulting yeast strains in high throughput for those that grow rapidly anaerobically and produce ethanol at sufficiently high levels for industrial application.
Section snippets
Construction of pSUMOduo three-plasmid vector system designed for use on an automated workcell
For the novel yeast episomal pSUMOduo high-level expression vector set, the protease-cleavable yeast small ubiquitin-related modifier (SUMO) tag (Smt3) was placed in front of an Invitrogen Gateway cassette, producing an AMP-selectable destination vector for recombination of library inserts. The SUMO insert open reading frames (ORF) were expressed behind an ADH promoter and also a T7 modified promoter for in vitro transcription/translation. The vectors contain the yeast high-copy 2 μm origin of
Yeast strain produced using three-plasmid yeast expression technology
The pSUMOduo high-copy expression vector set, containing the protease-cleavable yeast SUMO tag (Smt3) behind an ADH promoter and also a T7 in vitro modified promoter, consists of three vectors, pSUMOduo/URA, pSUMOduo/TRP, and pSUMOduo/LEU, each one having a different yeast selectable marker. Three plasmids were constructed and placed into INVSc1 yeast, a fast growing diploid strain that carries the mutations MATa his3D1 leu2 trp1-289 ura3-52 (Kuyper et al., 2003, Kuyper et al., 2004, and 2005)
Three-plasmid yeast expression strategy
The small ubiquitin-like modifier (SUMO) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Attachment of SUMO (also called Smt3) to the N-terminus of proteins has been found to enhance their expression (Malakhov et al., 2004). The structure of SUMO in aqueous solution consists of two α-helices and one β-sheet with 1 parallel and 3 antiparallel β-strands. Helix α1 (Leu45–Gln56) is strongly amphipathic with hydrophobic residues
Acknowledgments
We thank Karen Hughes for critical reading and formatting of the paper. We also thank Jennifer Steele for help with the sequencing work. The assistance of Watson Chau in performing the molecular biology procedures is greatly appreciated. This research was supported in part by SBIR Phase I CSREES Grant Award: 2006-33610-16796.
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Partially supported by SBIR Phase I CSREES Grant Award: 2006-33610-16796.
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