Genome-wide screening of the genes required for tolerance to vanillin,which is a potential inhibitor of bioethanol fermentation,in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Lignocellulosic materials are abundant and among the most important potential sources for bioethanol production. Although the pretreatment of lignocellulose is necessary for efficient saccharification and fermentation, numerous by-products, including furan derivatives, weak acids, and phenolic compounds, are generated in the pretreatment step. Many of these components inhibit the growth and fermentation of yeast. In particular, vanillin is one of the most effective inhibitors in lignocellulose hydrolysates because it inhibits fermentation at very low concentrations. To identify the genes required for tolerance to vanillin, we screened a set of diploid yeast deletion mutants, which are powerful tools for clarifying the function of particular genes.     

Molecular mechanisms of yeast tolerance and in situ detoxification of lignocellulose hydrolysates

Pretreatment of lignocellulose biomass for biofuel production generates inhibitory compounds that interfere with microbial growth and subsequent fermentation. Remediation of the inhibitors by current physical, chemical, and biological abatement means is economically impractical, and overcoming the inhibitory effects of lignocellulose hydrolysate poses a significant technical challenge for lower-cost cellulosic ethanol production. Development of tolerant ethanologenic yeast strains has demonstrated the potential of in situ detoxification for numerous aldehyde inhibitors derived from lignocellulose biomass pretreatment and conversion. In the last decade, significant progress has been made in understanding mechanisms of yeast tolerance for tolerant strain development. Enriched genetic backgrounds, enhanced expression, interplays, and global integration of many key genes enable yeast tolerance. Reprogrammed pathways support yeast functions to withstand the inhibitor stress, detoxify the toxic compounds, maintain energy and redox balance, and complete active metabolism for ethanol fermentation. Complex gene interactions and regulatory networks as well as co-regulation are well recognized as involved in yeast adaptation and tolerance. This review presents our current knowledge on mechanisms of the inhibitor detoxification based on molecular studies and genomic-based approaches. Our improved understanding of yeast tolerance and in situ detoxification provide insight into phenotype-genotype relationships, dissection of tolerance mechanisms, and strategies for more tolerant strain development for biofuels applications.     

Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors

Recent results show that treatments with reducing agents, including the sulfur oxyanions dithionite and hydrogen sulfite, efficiently improve the fermentability of inhibitory lignocellulose hydrolysates, and that the treatments are effective when the reducing agents are added in situ into the fermentation vessel at low temperature. In the present investigation, dithionite was added to medium with model inhibitors (coniferyl aldehyde, furfural, 5-hydroxymethylfurfural, or acetic acid) and the effects on the fermentability with yeast were studied. Addition of 10 mM dithionite to medium containing 2.5 mM coniferyl aldehyde resulted in a nine-fold increase in the glucose consumption rate and a three-fold increase in the ethanol yield. To investigate the mechanism behind the positive effects of adding sulfur oxyanions, mixtures containing 2.5 mM of a model inhibitor (an aromatic compound, a furan aldehyde, or an aliphatic acid) and 15 mM dithionite or hydrogen sulfite were analyzed using mass spectrometry (MS). The results of the analyses, which were performed by using UHPLC-ESI-TOF-MS and UHPLC-LTQ/Orbitrap-MS/MS, indicate that the positive effects of sulfur oxyanions are primarily due to their capability to react with and sulfonate inhibitory aromatic compounds and furan aldehydes at low temperature and slightly acidic pH (such as 25°C and pH 5.5).     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Improving the fermentation performance of saccharomyces cerevisiae by laccase during ethanol production from steam‐exploded wheat straw at high‐substrate loadings

Operating the saccharification and fermentation processes at high‐substrate loadings is a key factor for making ethanol production from lignocellulosic biomass economically viable. However, increasing the substrate loading presents some disadvantages, including a higher concentration of inhibitors (furan derivatives, weak acids, and phenolic compounds) in the media, which negatively affect the fermentation performance. One strategy to eliminate soluble inhibitors is filtering and washing the pretreated material. In this study, it was observed that even if the material was previously washed, inhibitory compounds were released during the enzymatic hydrolysis step. Laccase enzymatic treatment was evaluated as a method to reduce these inhibitory effects. The laccase efficiency was analyzed in a presaccharification and simultaneous saccharification and fermentation process at high‐substrate loadings. Water‐insoluble solids fraction from steam‐exploded wheat straw was used as substrate and Saccharomyces cerevisiae as fermenting microorganism. Laccase supplementation reduced strongly the phenolic content in the media, without affecting weak acids and furan derivatives. This strategy resulted in an improved yeast performance during simultaneous saccharification and fermentation process, increasing significantly ethanol productivity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Development of a Saccharomyces cerevisiae Strain with Enhanced Resistance to Phenolic Fermentation Inhibitors in Lignocellulose Hydrolysates by Heterologous Expression of Laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.     

Directing cell catalysis of glucose to 2-keto-d-gluconic acid using Gluconobacter oxydans NL71

2-Keto-d-gluconic acid (2-KGA) is a high-value-added product from lignocellulosic straw-based biorefining process obtained via cell catalysis using Gluconobacter oxydans (G. oxydans); nonetheless, the economical production has some limitations because of the formation of undesirable mixture products of keto-gluconic acid from glucose. In this study, it was demonstrated that pulse addition of hydrogen peroxide (H₂O₂) to synthetic media was beneficial to improve the conversion rate of glucose to 2-KGA by 1.47-fold in G. oxydans. Moreover, some lignocellulosic degradation compounds served as substitute and played the role of H₂O₂ on corn stover hydrolysate for the production of 2-KGA. By conducting qRT-PCR analysis, eight dehydrogenase genes were identified in G. oxydans, which were responsible for regulating 2-KGA production. Moreover, most of the identified genes were found to produce membrane-bound glucose dehydrogenase and gluconate 2-dehydrogenase, and were up-regulated by adding H₂O₂ or degradation compounds. These up-regulated genes could be responsible for directing cell catalysis of glucose to 2-KGA in G. oxydans.     

Production of free gluconic acid by cells ofGluconobacter oxydans

Summary Microbial oxidation of glucose to free gluconic acid by growingG.oxydans batch cultures was investigated. Kinetic data for the discussed process were obtained and attention was paid mainly to the influence of the initial glucose concentration and of the conversion degree on the course of the process. It was determined that relatively high maximum specific growth rates of about 0,39 h⁻¹ and gluconic acid volumetric productivities up to 53 mmol/h could be reached usingG.oxydans NBIMCC 1043 in runs without pH control. A maximum conversion degree of 90,4% was achieved.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Catalytic and biocatalytic oxidation of glucose to gluconic acid in a modified three-phase reactor

A comparative study of catalytic and biocatalytic glucose oxidation was carried out. Gluconobacter oxydans NBIMCC 1043 strain was used for biocatalytic glucose conversion. In the case of cell recycle coupled with cross-flow microfiltration the productivity and biomass concentration reached 40% and 3 g l^–1 respectively, in comparison to those of batch fermentation (21% and 2.3 g l^–1, respectively).     

Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase

To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.     

Biotransformation of glucose to 5-keto-<Emphasis Type="SmallCaps">d</Emphasis>-gluconic acid by recombinant<Emphasis Type="Italic"> Gluconobacter oxydans</Emphasis> DSM 2343

For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a precursor of the industrially important l-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP⁺, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.     

A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO₂, 0.05% pCO₂) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.     

High cell density fermentation of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> DSM 2003 for glycolic acid production

Gluconobacter oxydans has a lower biomass yield. Uniform design (UD) was applied to determine the optimum composition of the critical media and their mutual interactions for increased biomass yield of Gluconobacter oxydans DSM 2003 in shake flasks. Fed-batch fermentation process for biomass was optimized in a 3.7-l fermentor. By undertaking a preliminary and improved fed-batch fermentation-process strategy, a cell density of 6.0 g/l (DCW) was achieved in 22 h and 14.1 g/l (DCW) in 35 h, which is the highest cell density of G. oxydans produced thus far in a 3.7-l bioreactor. The biomass production was increased by 135% compared with that using the original cultivation strategy. Bioconversion of ethylene glycol to glycolic acid was catalyzed by the resting cells of G. oxydans DSM 2003, and conversion rate reached 86.7% in 48 h. In summary, the approach including high-density fermentation of G. oxydans DSM 2003 and bioconversion process was established and proved to be an effective method for glycolic acid production.     

Oxidative d-xylose metabolism of Gluconobacter oxydans

Summary Gluconobacter oxydans subsp. suboxydans ATCC 621 oxidizes d-xylose to xylonic acid very efficiently, although it cannot grow on xylose as sole carbon source. The oxidation of xylose was found to be catalyzed by a membrane-bound xylose dehydrogenase. The xylono--lactone formed in the oxidation reaction is subsequently hydrolyzed to xylonic acid by a -lactonase. The complete oxidation pathway of d-xylose in G. oxydans is evidently located in the periplasmic space.     

Comparison of Pseudomonas fragi and Gluconobacter oxydans for production of xylonic acid from hemicellulose hydrolyzates

Summary Xylonic acid was produced efficiently from pure xylose by Pseudomonas fragi ATCC 4973 and Gluconobacter oxydans subsp. suboxydans ATCC 621. The yield from 10% xylose was in both cases over 95% of the theoretical. However, the sensitivities of the strains towards the major inhibitors found in hemicellulose hydrolyzates, ie. acetic acid, furfural and two lignin-derived compounds, varied. G. oxydans tolerated all these inhibitors better than P. fragi. In tests using steamed hemicellulose hydrolyzate, G. oxydans was able to utilize the substrate only at dilute xylose concentrations. After ether extraction or mixed bed resin pretreatment, the fermentability of the hydrolyzate was increased significantly.     

Comparison of the resistance of industrial and laboratory strains of Saccharomyces and Zygosaccharomyces to lignocellulose-derived fermentation inhibitors

Low-molecular weight aliphatic acids, furaldehydes and a broad range of different aromatic compounds are known to inhibit the fermentation of lignocellulose hydrolysates by yeasts. In this work, a cocktail of different lignocellulose-derived inhibitors was used to compare the inhibitor resistance of eleven different industrial and laboratory Saccharomyces cerevisiae strains and two Zygosaccharomyces strains. The inhibitor cocktail was composed of two aliphatic acids, formic and acetic acid, two furaldehydes, furfural and 5-hydroxymethylfurfural (HMF), and two aromatic compounds, cinnamic acid and coniferyl aldehyde. Fermentations were performed under oxygen-limited conditions and with different levels (100, 75, 50, 25 and 0%) of the inhibitor cocktail present. The ethanol yield on initial glucose, the volumetric and specific ethanol productivity, the biomass yield and the glucose consumption rates were used as criteria for the performance of the strains. The results revealed major differences in inhibitor resistance between yeast strains within the same species. The ethanol yield of the S. cerevisiae strain that was least affected decreased only with 10% at an inhibitor cocktail concentration of 100%, while the decrease in ethanol yield for the most sensitive S. cerevisiae strain was more than 50% already at an inhibitor cocktail concentration of 25%. Ethanol formation was generally less affected than growth and ethanol yield less than ethanol productivity. The two most resistant strains were an S. cerevisiae strain isolated from a spent sulphite liquor plant and one of the laboratory S. cerevisiae strains. Additional fermentations with either HMF or coniferyl aldehyde revealed that the degree of resistance of different yeast strains was highly dependent on the inhibitor used. A mutant strain of S. cerevisiae displaying enhanced resistance against coniferyl aldehyde compared with the parental strains was identified.