Ronald E. Hector

Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.

Enhancing alfalfa conversion efficiencies for sugar recovery and ethanol production by altering lignin composition.

Alfalfa (Medicago sativa L.) biomass was evaluated for biochemical conversion into ethanol using dilute-acid and ammonia pretreatments. The two alfalfa lines compared were a reduced S-lignin transgenic cultivar generated through down regulation of the caffeic acid O-methyltransferase gene and a wild-type control. Both were harvested at two maturities. All the samples had similar carbohydrate contents including a mean composition of 316 g glucan and 497 g total neutral carbohydrates per kg dry biomass, which corresponds to a theoretic ethanol yield of 382 l/ton. Ethanol yields for alfalfa stems pretreated with dilute-acid were significantly impacted by harvest maturity and lignin composition, whereas when pretreated with dilute-ammonia, yield was solely affected by lignin composition. Use of a recombinant xylose-fermenting Saccharomyces strain, for converting the ammonia pretreated alfalfa samples, further increased ethanol yields. Ethanol yields for the xylose-fermenting yeast were 232-278 l/ton and were significantly enhanced for the reduced S lignin cultivars.

Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24

BackgroundSaccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation.ResultsFour XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain’s ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 μmole/min/mg protein and a Km of 34 mM.ConclusionThis study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

High titer ethanol production from SPORL-pretreated lodgepole pine by simultaneous enzymatic saccharification and combined fermentation.

Lodgepole wood chips were pretreated by sulfite pretreatment to overcome recalcitrance of lignocelluloses (SPORL) at 25% solids loading and 180 °C for 20 min with sulfuric acid and sodium bisulfite charges of 2.2 and 8 wt/wt% on an oven-dry wood basis, respectively. The pretreated wood chips were disk-milled with pretreatment spent liquor and water, and the solid fraction was separated from the liquor stream. The liquor was neutralized and concentrated through vacuum evaporation. Quasi-simultaneous enzymatic saccharification of the cellulosic solids and combined fermentation with the concentrated liquor was conducted at up to 20% total solids loading. Fed-batching of the solids facilitated liquefaction and saccharification, as well as managing instantaneous inhibitor concentrations. At a commercial cellulase (CTec2) loading of only 9 FPU or 0.06 mL/g untreated wood, a maximum ethanol titer of 47.4 g/L was achieved, resulting in a calculated yield of 285 L/tonne of wood using Saccharomyces cerevisiae YRH400 at 35 °C and pH 5.5.

Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a different selectable marker (URA, TRP, or LEU), and the system provides high expression levels of three different proteins simultaneously. This system was integrated into the protocols on a fully automated plasmid-based robotic platform to screen engineered strains of S. cerevisiae for improved growth on xylose. First, a novel PCR assembly strategy was used to clone a xylose isomerase (XI) gene into the URA-selectable SUMO vector and the plasmid was placed into the S. cerevisiae INVSc1 strain to give the strain designated INVSc1-XI. Second, amino acid scanning mutagenesis was used to generate a library of mutagenized genes encoding the bioinsecticidal peptide lycotoxin-1 (Lyt-1) and the library was cloned into the TRP-selectable SUMO vector and placed into INVSc1-XI to give the strain designated INVSc1-XI-Lyt-1. Third, the Yersinia pestis xylulokinase gene was cloned into the LEU-selectable SUMO vector and placed into the INVSc1-XI-Lyt-1 yeast. Yeast strains expressing XI and xylulokinase with or without Lyt-1 showed improved growth on xylose compared to INVSc1-XI yeast.

Saccharomyces cerevisiae engineered for xylose metabolism requires gluconeogenesis and the oxidative branch of the pentose phosphate pathway for aerobic xylose assimilation.

Saccharomyces strains engineered to ferment xylose using Scheffersomyces stipitis xylose reductase (XR) and xylitol dehydrogenase (XDH) genes appear to be limited by metabolic imbalances, due to differing cofactor specificities of XR and XDH. The S. stipitis XR, which uses both NADH and NADPH, is hypothesized to reduce the cofactor imbalance, allowing xylose fermentation in this yeast. However, unadapted S. cerevisiae strains expressing this XR grow poorly on xylose, suggesting that metabolism is still imbalanced, even under aerobic conditions. In this study, we investigated the possible reasons for this imbalance by deleting genes required for NADPH production and gluconeogenesis in S. cerevisiae. S. cerevisiae cells expressing the XR–XDH, but not a xylose isomerase, pathway required the oxidative branch of the pentose phosphate pathway (PPP) and gluconeogenic production of glucose‐6‐P for xylose assimilation. The requirement for generating glucose‐6‐P from xylose was also shown for Kluyveromyces lactis. When grown in xylose medium, both K. lactis and S. stipitis showed increases in enzyme activity required for producing glucose‐6‐P. Thus, natural xylose‐assimilating yeast respond to xylose, in part, by upregulating enzymes required for recycling xylose back to glucose‐6‐P for the production of NADPH via the oxidative branch of the PPP. Finally, we show that induction of these enzymes correlated with increased tolerance to the NADPH‐depleting compound diamide and the fermentation inhibitors furfural and hydroxymethyl furfural; S. cerevisiae was not able to increase enzyme activity for glucose‐6‐P production when grown in xylose medium and was more sensitive to these inhibitors in xylose medium compared to glucose. Published in 2011 by John Wiley & Sons, Ltd.

Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost‐effectiveness of the process. Spider venom is one of many potential sources of novel insect‐specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin‐1 from the wolf spider were produced in high throughput using an automated integrated plasmid‐based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin‐1 open reading frames (ORF) in a novel small ubiquitin‐like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so‐called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin α‐helix to interact with insect cell membranes or to increase its pore‐forming ability, leading to cell lysis. The toxin peptides have potential as value‐added coproducts to increase the cost‐effectiveness of fuel ethanol bioproduction. Copyright

Automated Yeast Mating Protocol Using Open Reading Frames from Saccharomyces cerevisiae Genome to Improve Yeast Strains for Cellulosic Ethanol Production

Engineering the industrial ethanologen Saccharomyces cerevisiae to use pentose sugars from lignocellulosic biomass is critical for commercializing cellulosic fuel ethanol production. Approaches to engineer pentose-fermenting yeasts have required expression of additional genes. We implemented a high-throughput strategy to improve anaerobic growth on xylose and rate of ethanol production by evaluating overexpression of each native S. cerevisiae gene from a collection of haploid PJ69–4 MATa strains expressing the gene open reading frames (ORFs) mated to a haploid PJ69–4 MATalpha strain expressing the Piromyces sp.E2 xylose isomerase (XI) gene. The resulting 6113 diploid strains containing the XI gene and a different yeast gene ORF were screened for growth on xylose in anaerobic plate cultures using an integrated robotic workcell. Nine unique strains were isolated; two were found to no longer grow on glucose; seven were further evaluated for fermentation of alkaline peroxide pretreated enzymatically saccharified wheat straw hydrolysate. All successfully used glucose and xylose, consuming most of the glucose and a small amount of the xylose. Transforming the strains with an additional vector expressing xylulokinase gene did not improve anaerobic growth on xylose but improved glucose use and ethanol production on the hydrolysate, with three strains giving maximum ethanol production ≥ 14.0 g L −1 .

The Saccharomyces cerevisiae YMR315W gene encodes an NADP(H)-specific oxidoreductase regulated by the transcription factor Stb5p in response to NADPH limitation

Engineered xylose-metabolizing Saccharomyces cerevisiae cells grown on xylose show increased expression of YMR315W at both the mRNA and protein levels. Additionally, the YMR315W promoter contains a putative binding site for the transcription factor Stb5p, which has been shown to regulate genes involved in NADPH production such as ZWF1, GND1 and GND2. We hypothesized that Ymr315wp, a conserved protein of unknown function, is an additional source of NADPH in wild-type cells. In this study, we purified histidine-tagged enzyme and determined that Ymr315wp is an NADP(H)-specific oxidoreductase. We also showed that YMR315W transcription is regulated by Stb5p in response to diamide induced NADPH depletion. Overexpression of Ymr315wp in BY4727 cells resulted in elevated NADPH levels and increased resistance to diamide. However, the presence of Ymr315wp in cells lacking the oxidative branch of the pentose phosphate pathway resulted in decreased NADPH levels and increased diamide sensitivity. These results suggest that in BY4727 cells Ymr315wp contributes to NADPH production as an alternative source of NADPH.

Cost-Effective High-Throughput Fully Automated Construction of a Multiplex Library of Mutagenized Open Reading Frames for an Insecticidal Peptide Using a Plasmid-Based Functional Proteomic Robotic Workcell with Improved Vacuum System

Robotic platforms are essential for production of large numbers of expression-ready plasmid sets to develop optimized clones and improved microbial strains for crucial bioenergy applications and simultaneous high-value peptide expression. Here we demonstrate a plasmid-based integrated robotic workcell, modified with a motorized vacuum filtration system, for performing fully automated molecular biology protocols, including assembly of mutagenized gene sequences, purification of PCR amplicons, ligation of PCR products into vectors, transformation of competent Escherichia coli, plating of recovered transformants, plasmid preparation, cloning, and expression of optimized genes. A library of genes encoding variants of wolf spider lycotoxin-1, a candidate bioinsecticide, was produced using PCR mutagenesis in an amino acid scanning strategy to generate a complete set of mutations across the lycotoxin-1 gene. The improved vacuum filtration system permits automated purification of PCR products. Methods for recovery and growth of bacteria containing plasmids with PCR inserts allow individual colony formation on a novel solid medium in a deepwell plate. Inserts are cloned into a bacterial vector to verify expression. These protocols form the core of a fully automated molecular biology platform that reduces the cost and time required to perform all operations. (JALA 2007;12:202–12)