Performance of xylose-fermenting yeasts in oat and soybean hulls hydrolysate and improvement of ethanol production using immobilized cell systems.

We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg-1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g-1, and productivities of 0.39 and 0.29 g (L h)-1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g-1 and productivities from 0.14 to 0.23 g (L h)-1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates.

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes of fungal oxido-reductive pathway needed for xylose fermentation integrated into its genome (YRH1415) was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than YRH1415 strain and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting from the hydrolysis of lignocellulosic biomass (switchgrass). The rate of xylose consumption did not appear to be affected by the ploidy of strains or the presence of two copies of the xylose fermentation genes but by heterozygosity of alleles for xylose metabolism in YRH1415. Furthermore, inhibitor tolerance was influenced by the heterozygous genome of the industrial strain, which also showed a marked influenced on tolerance to increasing concentrations of toxic compounds, such as furfural. In this work, selection of haploid derivatives was found to be a useful strategy to develop efficient xylose-fermenting industrial yeast strains.

Triacetic acid lactone production in industrial Saccharomyces yeast strains.

Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into 13 industrial yeast strains of varied genetic background. TAL production varied 63-fold between strains when compared in batch culture with glucose. Ethanol, acetate, and glycerol were also tested as potential carbon sources. Batch cultures with ethanol medium produced the highest titers. Therefore, fed-batch cultivation with ethanol feed was assayed for TAL production in bioreactors, producing our highest TAL titer, 5.2 g/L. Higher feed rates resulted in a loss of TAL and subsequent production of additional TAL side products. Finally, TAL efflux was measured and TAL is actively exported from S. cerevisiae cells. Percent yield for all strains was low, indicating that further metabolic engineering of the strains is required.

Plant cell walls to ethanol.

Conversion of plant cell walls to ethanol constitutes second generation bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation and separation. Ultimately, it is desirable to combine as many of the biochemical steps as possible in a single organism to achieve CBP (consolidated bioprocessing). A commercially ready CBP organism is currently unreported. Production of second generation bioethanol is hindered by economics, particularly in the cost of pretreatment (including waste management and solvent recovery), the cost of saccharification enzymes (particularly exocellulases and endocellulases displaying kcat ~1 s-1 on crystalline cellulose), and the inefficiency of co-fermentation of 5- and 6-carbon monosaccharides (owing in part to redox cofactor imbalances in Saccharomyces cerevisiae).

Metabolic engineering of a stable haploid strain derived from lignocellulosic inhibitor tolerant Saccharomyces cerevisiae natural isolate YB-2625.

BACKGROUND: Significant genetic diversity exists across Saccharomyces strains. Natural isolates and domesticated brewery and industrial strains are typically more robust than laboratory strains when challenged with inhibitory lignocellulosic hydrolysates. These strains also contain genes that are not present in lab strains and likely contribute to their superior inhibitor tolerance. However, many of these strains have poor sporulation efficiencies and low spore viability making subsequent gene analysis, further metabolic engineering, and genomic analyses of the strains challenging. This work aimed to develop an inhibitor tolerant haploid with stable mating type from S. cerevisiae YB-2625, which was originally isolated from bagasse. RESULTS: Haploid spores isolated from four tetrads from strain YB-2625 were tested for tolerance to furfural and HMF. Due to natural mutations present in the HO-endonuclease, all haploid strains maintained a stable mating type. One of the haploids, YRH1946, did not flocculate and showed enhanced tolerance to furfural and HMF. The tolerant haploid strain was further engineered for xylose fermentation by integration of the genes for xylose metabolism at two separate genomic locations (ho∆ and pho13∆). In fermentations supplemented with inhibitors from acid hydrolyzed corn stover, the engineered haploid strain derived from YB-2625 was able to ferment all of the glucose and 19% of the xylose, whereas the engineered lab strains performed poorly in fermentations. CONCLUSIONS: Understanding the molecular mechanisms of inhibitor tolerance will aid in developing strains with improved growth and fermentation performance using biomass-derived sugars. The inhibitor tolerant, xylose fermenting, haploid strain described in this work has potential to serve as a platform strain for identifying pathways required for inhibitor tolerance, and for metabolic engineering to produce fuels and chemicals from undiluted lignocellulosic hydrolysates.

Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon.

Expression of a new fluorescent reporter protein called mNeonGreen, that is not based on the jellyfish green fluorescent protein (GFP) sequence, shows increased brightness and folding speed compared to enhanced GFP. However, in vivo brightness of mNeonGreen and its yeast-optimized variant ymNeonGreen in S. cerevisiae is lower than expected, limiting the use of this high quantum yield, fast-folding reporter in budding yeast. This study shows that secondary RNA structure near the start codon in the ymNeonGreen ORF inhibits expression in S. cerevisiae. Removing secondary structure, without altering the ymNeonGreen protein sequence, led to a 2 and 4-fold increase in fluorescence when expressed in S. cerevisiae and E. coli, respectively. In S. cerevisiae, increased fluorescence was seen with strong and weak promoters and led to higher transcript levels suggesting greater transcript stability and improved expression in the absence of stable secondary RNA structure near the start codon.

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.

Expression and characterization of fifteen Rhizopus oryzae 99-880 polygalacturonase enzymes in Pichia pastoris.

Polygalacturonase (PG) enzymes hydrolyze the long polygalacturonic acid chains found in the smooth regions of pectin. Interest in this enzyme class continues due to their ability to macerate tissues of economically important crops and their use in a number of industrial processes. Rhizopus oryzae has a large PG gene family with 15 of 18 genes encoding unique active enzymes. The PG enzymes, 12 endo-PG and 3 exo-galacturonases, were expressed in Pichia pastoris and purified enabling biochemical characterization to gain insight into the maintenance of this large gene family within the Rhizopus genome. The 15 PG enzymes have a pH optima ranging from 4.0 to 5.0. Temperature optima of the 15 PG enzymes vary from 30 to 40 °C. While the pH and temperature optima do little to separate the enzymes, the specific activity of the enzymes is highly variable ranging from over 200 to less than 1 µmol/min/mg. A general pattern related to the groupings found in the phylogentic tree was visible with the group containing the exo-PG enzymes demonstrating the lowest specific activity. Finally, the progress curves of the PG enzymes, contained within the phylogenetic group that includes the exo-PG enzymes, acting on trigalacturonic acid lend additional support to the idea that the ancestral form of PG in Rhizopus is endolytic and exolytic function evolved later.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from Selenomonas ruminantium.

Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pK(a) 5.0) and catalytic acid (E186, pK(a) 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the catalytically-inactive, dianionic enzyme (D14(-)E186(-)). Enzyme (E) and aminoalcohols (A) form E-A with the affinity progression: triethanolamine>diethanolamine>ethanolamine. E186A mutation raises the K(i)(triethanolamine) 1000-fold. By occupying subsite -1 with aminoalcohols, affinity of monosaccharide inhibitors (I) for subsite +1 is demonstrated. The single access route for ligands into the active site dictates ordered formation of E-A followed by E-A-I. E-A-I forms with the affinity progression: ethanolamine>diethanolamine>triethanolamine. The latter affinity progression is seen in formation of E-A-substrate complexes with substrate 4-nitrophenyl-beta-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on the strength of E-A-4NPX. E-A-substrate complexes form weakly with substrate 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA), and inhibition patterns appear competitive. Biphasic inhibition by triethanolamine reveals minor (<0.03%) contamination of E186A preparations (including a His-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts when used unwarily as buffer components.

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

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.

Development and characterization of vectors for tunable expression of both xylose-regulated and constitutive gene expression in Saccharomyces yeasts.

Synthetic hybrid promoters for xylose-regulated gene expression in the yeast Saccharomyces cerevisiae have recently been developed. However, the narrow range of expression level from these new hybrid promoters limits their utility for pathway optimization in engineered strains. To expand the range of xylose-regulated gene expression, a series of expression vectors was created using a xylose derepressible promoter (PXYL) and varied termination regions from several S. cerevisiae genes. The new set of vectors showed a 26-fold range of gene expression under inducing conditions and a 13-fold average induction due to xylose. In the presence of the XylR repressor, gene expression was very sensitive to xylose concentration and full induction was observed with 0.10 g/L xylose. In the absence of XylR, gene expression from the vector set did not require xylose and was constitutive over a similar 26-fold range of expression. These results show that the vectors are extremely versatile for constitutive expression as well as for fine-tuning both the timing of gene expression and expression level using xylose as an inexpensive inducer.

Identification, biochemical characterization, and evolution of the Rhizopus oryzae 99-880 polygalacturonase gene family.

A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with two genes being identical and only one with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level. The cDNA of the different genes was isolated directly or recombinantly and used to express the encoded proteins in Pichia pastoris. Recombinant protein expression demonstrated that 15 of the 17 genes encode active enzymes with twelve genes encoding for endo-polygalacturonase enzymes and three genes encoding for exo-polygalacturonase enzymes. Phylogenetic analysis indicates that the genes form a distinct monophyletic group among fungal polygalacturonase enzymes. Finally, our results also suggest that the ancestral form of polygalacturonase in fungi is endolytic and exolytic function evolved later, at least two independent times.

Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism.

Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid (FA) and coenzyme A intermediates. Two P. putida transposon mutants were isolated that had impaired growth on furfural and FA, and DNA flanking the transposon insertion site was cloned from both mutants. The transposons disrupted psfB, a LysR-family regulatory gene in mutant PSF2 and psfF, a GcvR-type regulatory gene in PSF9. Disruption of two genes adjacent to psfB demonstrated that both are important for growth on FA, and ORFs in the proximity of psfB and psfF were transcriptionally activated during growth of P. putida on FA. Transcript levels increased in response to FA by 10-fold (a putative permease gene) to >1000-fold (a putative decarboxylase gene). The LysR-family gene appears to act positively, and the GcvR-family gene negatively, in regulating expression of neighboring genes in response to FA.

Molecular cloning and expression of three polygalacturonase cDNAs from the tarnished plant bug, Lygus lineolaris.

Three unique cDNAs encoding putative polygalacturonase enzymes were isolated from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae). The three nucleotide sequences were dissimilar to one another, but the deduced amino acid sequences were similar to each other and to other polygalacturonases from insects, fungi, plants, and bacteria. Four conserved segments characteristic of polygalacturonases were present, but with some notable semiconservative substitutions. Two of four expected disulfide bridge-forming cysteine pairs were present. All three inferred protein translations included predicted signal sequences of 17 to 20 amino acids. Amplification of genomic DNA identified an intron in one of the genes, Llpg1, in the 5' untranslated region. Semiquantitative RT-PCR revealed expression in all stages of the insect except the eggs. Expression in adults, male and female, was highly variable, indicating a family of highly inducible and diverse enzymes adapted to the generalist polyphagous nature of this important pest.

A Synthetic Hybrid Promoter for Xylose-Regulated Control of Gene Expression in Saccharomyces Yeasts.

Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DNA-binding repressor (XylR) from Caulobacter crescentus was used to block transcription from synthetic promoters based on the constitutive Ashbya gossypii TEF promoter. The new hybrid promoters were repressed in the absence of xylose and showed up to a 25-fold increase in the presence of xylose. Activation of the promoter was highly sensitive to xylose with activity seen at concentrations below 2 µM xylose. These new xylose-inducible promoters allow improved control of gene expression for engineered strains of Saccharomyces yeasts.

High-throughput screening of cellulase F mutants from multiplexed plasmid sets using an automated plate assay on a functional proteomic robotic workcell.

BACKGROUND: The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins. RESULTS: We used a functional proteomic assay in a multiplexed setting on an integrated plasmid-based robotic workcell for high-throughput screening of mutants of cellulase F, an endoglucanase from the anaerobic fungus Orpinomyces PC-2. This allowed us to identify plasmids containing optimized clones expressing mutants with improved activity at lower pH. A plasmid library of mutagenized clones of the celF gene with targeted variations in the last four codons was constructed by site-directed PCR mutagenesis and transformed into Escherichia coli. A robotic picker integrated into the workcell was used to inoculate medium in a 96-well deep well plate, combining the transformants into a multiplexed set in each well, and the plate was incubated on the workcell. Plasmids were prepared from the multiplexed culture on the liquid handler component of the workcell and used for in vitro transcription/translation. The multiplexed expressed recombinant proteins were screened for improved activity and stability in an azo-carboxymethylcellulose plate assay. The multiplexed wells containing mutants with improved activity were identified and linked back to the corresponding multiplexed cultures stored in glycerol. Spread plates were prepared from the glycerol stocks and the workcell was used to pick single colonies from the spread plates, prepare plasmid, produce recombinant protein, and assay for activity. The screening assay and subsequent deconvolution of the multiplexed wells resulted in identification of improved CelF mutants and corresponding optimized clones in expression-ready plasmids. CONCLUSION: The multiplex method using an integrated automated platform for high-throughput screening in a functional proteomic assay allows rapid identification of plasmids containing optimized clones ready for use in subsequent applications including transformations to produce improved strains or cell lines.

Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756.

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°C and 75°C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.

Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations.

Switchgrass (Panicum virgatum, L.) is a potential renewable source of carbohydrates for use in microbial conversion to biofuels. Xylan comprises approximately 30% of the switchgrass cell wall. To understand the limitations of commercial enzyme mixtures, alkali-extracted, isolated switchgrass xylan was hydrolyzed by the action of two commercial enzyme cocktails, in the presence and absence of an additional α-arabinofuranosidase enzyme. The two most abundant enzymatic digestion products from each commercial enzyme treatment were separated and characterized by LC-MS(n), linkage analysis, and NMR. The most abundant oligosaccharide from each commercial cocktail was susceptible to hydrolysis when supplemented with a GH62 α-arabinofuranosidase enzyme; further characterization confirmed the presence of (1→3)-α-arabinose linkages. These results demonstrate the lack of the required selectivity for arabinose-containing substrates in the commercial enzyme preparations tested. One product from each condition remained intact and was found to contain (1→2)-ß-xylose-(1→3)-α-arabinose side chains; this linkage acts as a source of oligosaccharide recalcitrance.