Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate.

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two ß-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both ß-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.

Waste and by-products as sources of lycopene, phytoene, and phytofluene - Integrative review with bibliometric analysis.

Food loss and waste are severe social, economic, and environmental issues. An example is the incorrect handling of waste or by-products used to obtain bioactive compounds, such as carotenoids. This review aimed to present a comprehensive overview of research on lycopene, phytoene, and phytofluene obtained from waste and by-products. In this study, an integrative literature approach was coupled with bibliometric analysis to provide a broad perspective of the topic. PRISMA guidelines were used to search studies in the Web of Science database systematically. Articles were included if (1) employed waste or by-products to obtain lycopene, phytoene, and phytofluene or (2) performed applications of the carotenoids previously extracted from waste sources. Two hundred and four articles were included in the study, and the prevalent theme was research on the recovery of lycopene from tomato processing. However, the scarcity of studies on colorless carotenoids (phytoene and phytofluene) was evidenced, although these are generally associated with lycopene. Different technologies were used to extract lycopene from plant matrices, with a clear current trend toward choosing environmentally friendly alternatives. Microbial production of carotenoids from various wastes is a highly competitive alternative to conventional processes. The results described here can guide future forays into the subject, especially regarding research on phytoene and phytofluene, potential and untapped sources of carotenoids from waste and by-products, and in choosing more efficient, safe, and environmentally sustainable extraction protocols.

Oxidative cleavage of polysaccharides by a termite-derived superoxide dismutase boosts the degradation of biomass by glycoside hydrolases.

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite Coptotermes gestroi, a Cu/Zn superoxide dismutase (CgSOD-1), plays a previously unknown role in plant biomass degradation. We show that CgSOD-1 transcripts and peptides are up-regulated in response to an increased level of lignocellulose recalcitrance and that CgSOD-1 localizes in the lumen of the fore- and midguts of C. gestroi together with termite main cellulase, CgEG-1-GH9. CgSOD-1 boosts the saccharification of polysaccharides by CgEG-1-GH9. We show that the boosting effect of CgSOD-1 involves an oxidative mechanism of action in which CgSOD-1 generates reactive oxygen species that subsequently cleave the polysaccharide. SOD-type enzymes constitute a new addition to the growing family of oxidases, ones which are up-regulated when exposed to recalcitrant polysaccharides, and that are used by Nature for biomass degradation.

Xylo-oligosaccharides, fermentable sugars, and bioenergy production from sugarcane straw using steam explosion pretreatment at pilot-scale.

This study investigated the production of xylo-oligosaccharides (XOS) from sugarcane straw (SCS) using steam explosion (SE) pretreatment at pilot-scale, as well as co-production of fermentable sugars and lignin-rich residues for bioethanol and bioenergy, respectively. SE conditions 200 °C; 15 bar; 10 min led to 1) soluble XOS yields of up to 35 % (w/w) of initial xylan with âˆ¼50 % of the recovered XOS corresponding to xylobiose and xylotriose, considered the most valuable sugars for prebiotic applications; 2) fermentable glucose yields from the enzymatic hydrolysis of SE-pretreated SCS of up to âˆ¼78 %; 3) increase in the energy content of saccharified SCS residues (16 %) compared to the untreated material. From an integrated biorefinery perspective, it demonstrated the potential use of SCS for the production of value-added XOS ingredients as well as liquid and solid biofuel products.

Enzymatic removal of inhibitory compounds from lignocellulosic hydrolysates for biomass to bioproducts applications.

The physicochemical pretreatment is an important step to reduce biomass recalcitrance and facilitate further processing of plant lignocellulose into bioproducts. This process results in soluble and insoluble biomass fractions, and both may contain by-products that inhibit enzymatic biocatalysts and microbial fermentation. These fermentation inhibitory compounds (ICs) are produced during the degradation of lignin and sugars, resulting in phenolic and furanic compounds, and carboxylic acids. Therefore, detoxification steps may be required to improve lignocellulose conversion by microoganisms. Several physical and chemical methods, such as neutralization, use of activated charcoal and organic solvents, have been developed and recommended for removal of ICs. However, biological processes, especially enzyme-based, have been shown to efficiently remove ICs with the advantage of minimizing environmental issues since they are biogenic catalysts and used in low quantities. This review focuses on describing several enzymatic approaches to promote detoxification of lignocellulosic hydrolysates and improve the performance of microbial fermentation for the generation of bioproducts. Novel strategies using classical carbohydrate active enzymes (CAZymes), such as laccases (AA1) and peroxidases (AA2), as well as more advanced strategies using prooxidant, antioxidant and detoxification enzymes (dubbed as PADs), i.e. superoxide dismutases, are discussed as perspectives in the field.

Screening of potential endoglucanases, hydrolysis conditions and different sugarcane straws pretreatments for cello-oligosaccharides production.

Cello-oligosaccharides (COS) are oligomers with 2 to 6 ß-1,4-linked glucose units, with potential applications in the food/feed and bioenergy industrial sectors. In this study, the combination of five heterologous expressed endoglucanases varying the temperature and pH conditions were evaluated by design of experiments for COS production. Afterwards, the best combination was tested to produce COS from different pretreated sugarcane straws: ionic liquid, diluted acid, hydrothermal and steam-explosion. The results showed that steam explosion pretreated sugarcane straw treated with CtCel9R enzyme at 50 °C and pH 5.0 yielded 13.4 mg COS g biomass-1, 5-18-fold higher compared to the other pretreated straws. Under the conditions evaluated, the removal of hemicellulose and decrease in the cellulose crystallinity can benefits the enzymatic hydrolysis. This is the first study that combined the evaluation of different enzymes, conditions, and sugarcane straw pretreatments to optimize COS production in a single step without glucose formation.

An integrated approach to obtain xylo-oligosaccharides from sugarcane straw: From lab to pilot scale.

Sugarcane straw (SS) is a widely available agricultural processing feedstock with the potential to produce 2nd generation bioethanol and bioproducts, in addition to the more conventional use for heat and/or electrical power generation. In this study, we investigated the operational parameters to maximize the production of xylo-oligosaccharides (XOS) using mild deacetylation, followed by hydrothermal pretreatment. From the laboratory to the pilot-scale, the optimized two-stage pretreatment promoted 81.5% and 70.5% hemicellulose solubilization and led to XOS yields up to 9.8% and 9.1% (w/w of initial straw), respectively. Moreover, different fungal xylanases were also tested to hydrolyze XOS into xylobiose (X2) and xylotriose (X3). GH10 from Aspergillus nidulans performed better than GH11 xylanases and the ratio of the desired products (X2 + X3) increased to 72% due to minimal monomeric sugar formation. Furthermore, a cellulose-rich fraction was obtained, which can be used in other high value-added applications, such as for the production of cello-oligomers.

Designing a cocktail containing redox enzymes to improve hemicellulosic hydrolysate fermentability by microorganisms.

Bioproducts production using monomeric sugars derived from lignocellulosic biomass presents several challenges, such as to require a physicochemical pretreatment to improve its conversion yields. Hydrothermal lignocellulose pretreatment has several advantages and results in solid and liquid streams. The former is called hemicellulosic hydrolysate (HH), which contains inhibitory phenolic compounds and sugar degradation products that hinder microbial fermentation products from pentose sugars. Here, we developed and applied a novel enzyme process to detoxify HH. Initially, the design of experiments with different redox activities enzymes was carried out. The enzyme mixture containing the peroxidase (from Armoracia rusticana) together with superoxide dismutase (from Coptotermes gestroi) are the most effective to detoxify HH derived from sugarcane bagasse. Butanol fermentation by the bacteria Clostridium saccharoperbutylacetonicum and ethanol production by the yeast Scheffersomyces stipitis increased by 24.0× and 2.4×, respectively, relative to the untreated hemicellulosic hydrolysates. Detoxified HH was analyzed by chromatographic and spectrometric methods elucidating the mechanisms of phenolic compound modifications by enzymatic treatment. The enzyme mixture degraded and reduced the hydroxyphenyl- and feruloyl-derived units and polymerized the lignin fragments. This strategy uses biocatalysts under environmentally friendly conditions and could be applied in the fuel, food, and chemical industries.

Rheological and antioxidant properties of chitosan/gelatin-based materials functionalized by pomegranate peel extract.

Biopolymer-based materials are potential candidates for food coatings application. In this study, pomegranate (Punica granatum L.) peel extract (PPE) at different concentrations was incorporated to chitosan/gelatin gels and the rheological, antioxidant and structural properties were evaluated. Due to its high phenolic content, PPE enhanced the antioxidant capacity of chitosan/gelatin mixtures. PPE addition extended linear viscoelastic range and enabled the samples to easily flow under the applied shear rate. Rheological properties indicated that both viscosity and activation energy of materials containing natural compounds are highly dependent on temperature. Scanning electron microscopy (SEM) images revealed the influence of PPE concentration in the scaffolds pores size. Findings of this study proved that PPE was capable to improve the functional characteristics of chitosan/gelatin-based materials enhancing the desired properties for their potential application as food coatings.

Novel redox-active enzymes for ligninolytic applications revealed from multiomics analyses of Peniophora sp. CBMAI 1063, a laccase hyper-producer strain.

The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.

Lignolytic-consortium omics analyses reveal novel genomes and pathways involved in lignin modification and valorization.

BACKGROUND: Lignin is a heterogeneous polymer representing a renewable source of aromatic and phenolic bio-derived products for the chemical industry. However, the inherent structural complexity and recalcitrance of lignin makes its conversion into valuable chemicals a challenge. Natural microbial communities produce biocatalysts derived from a large number of microorganisms, including those considered unculturable, which operate synergistically to perform a variety of bioconversion processes. Thus, metagenomic approaches are a powerful tool to reveal novel optimized metabolic pathways for lignin conversion and valorization. RESULTS: The lignin-degrading consortium (LigMet) was obtained from a sugarcane plantation soil sample. The LigMet taxonomical analyses (based on 16S rRNA) indicated prevalence of Proteobacteria, Actinobacteria and Firmicutes members, including the Alcaligenaceae and Micrococcaceae families, which were enriched in the LigMet compared to sugarcane soil. Analysis of global DNA sequencing revealed around 240,000 gene models, and 65 draft bacterial genomes were predicted. Along with depicting several peroxidases, dye-decolorizing peroxidases, laccases, carbohydrate esterases, and lignocellulosic auxiliary (redox) activities, the major pathways related to aromatic degradation were identified, including benzoate (or methylbenzoate) degradation to catechol (or methylcatechol), catechol ortho-cleavage, catechol meta-cleavage, and phthalate degradation. A novel Paenarthrobacter strain harboring eight gene clusters related to aromatic degradation was isolated from LigMet and was able to grow on lignin as major carbon source. Furthermore, a recombinant pathway for vanillin production was designed based on novel gene sequences coding for a feruloyl-CoA synthetase and an enoyl-CoA hydratase/aldolase retrieved from the metagenomic data set. CONCLUSION: The enrichment protocol described in the present study was successful for a microbial consortium establishment towards the lignin and aromatic metabolism, providing pathways and enzyme sets for synthetic biology engineering approaches. This work represents a pioneering study on lignin conversion and valorization strategies based on metagenomics, revealing several novel lignin conversion enzymes, aromatic-degrading bacterial genomes, and a novel bacterial strain of potential biotechnological interest. The validation of a biosynthetic route for vanillin synthesis confirmed the applicability of the targeted metagenome discovery approach for lignin valorization strategies.

Laccase-derived lignin compounds boost cellulose oxidative enzymes AA9.

BACKGROUND: The discovery of lignin as activator for the redox enzyme lytic polysaccharide monooxygenases (LPMOs) for the oxidation of cell-wall polysaccharides opens a new scenario for investigation of the interplay between different lignocellulose-degrading enzymes. The lignin-active enzymes in one hand, and the carbohydrate active in the other, are linked through a variety of electrons carrier molecules either derived from lignin or enzymatically transferred. Likewise, in nature, many lignocellulose-degrading organisms are expressing those enzymes simultaneously, and we wanted to test if a major commercial available lignin oxidase enzyme, i.e., laccase could benefit and synergize the activity of the LPMOs by depolymerizing the insoluble lignin. RESULTS: In this work, two fungal laccases together with a mediator (ABTS) were used to isolate low-molecular-weight lignin from lignocellulosic biomass. The isolated lignins were used as electron donors for activation of LPMOs. A direct correlation between the low-molecular-weight lignin isolated with laccases and an increased activity of a cellulolytic cocktail containing LPMO was found when pure cellulose was hydrolyzed. We then tried to implement existing commercial cellulases cocktail with laccase enzymes, but under the conditions tested, the co-incubation of laccases with LPMOs showed a substrate competition towards oxygen inhibiting the LPMO. In addition, we found that laccase treatment may cause other modifications to pure cellulose, rendering the material more recalcitrant for enzymatic saccharification. CONCLUSIONS: Laccase-mediated system was able to depolymerize lignin from pre-treated and native sugarcane bagasse and wheat straw, and the released phenolic molecules were able to donate electrons to LPMO enzymes boosting the overall enzymatic hydrolysis of cellulose. Likewise, other poly-phenol oxidase, we might have just started showing possible pros or cons in applying several oxidase enzymes for a simultaneous degradation of cellulose and lignin, and we found that the competition towards oxygen and their different consumption rates must be taken into account for any possible co-application.

Expanding the Knowledge on Lignocellulolytic and Redox Enzymes of Worker and Soldier Castes from the Lower Termite Coptotermes gestroi.

Termites are considered one of the most efficient decomposers of lignocelluloses on Earth due to their ability to produce, along with its microbial symbionts, a repertoire of carbohydrate-active enzymes (CAZymes). Recently, a set of Pro-oxidant, Antioxidant, and Detoxification enzymes (PAD) were also correlated with the metabolism of carbohydrates and lignin in termites. The lower termite Coptotermes gestroi is considered the main urban pest in Brazil, causing damage to wood constructions. Recently, analysis of the enzymatic repertoire of C. gestroi unveiled the presence of different CAZymes. Because the gene profile of CAZy/PAD enzymes endogenously synthesized by C. gestroi and also by their symbiotic protists remains unclear, the aim of this study was to explore the eukaryotic repertoire of these enzymes in worker and soldier castes of C. gestroi. Our findings showed that worker and soldier castes present similar repertoires of CAZy/PAD enzymes, and also confirmed that endo-glucanases (GH9) and beta-glucosidases (GH1) were the most important glycoside hydrolase families related to lignocellulose degradation in both castes. Classical cellulases such as exo-glucanases (GH7) and endo-glucanases (GH5 and GH45), as well as classical xylanases (GH10 and GH11), were found in both castes only taxonomically related to protists, highlighting the importance of symbiosis in C. gestroi. Moreover, our analysis revealed the presence of Auxiliary Activity enzyme families (AAs), which could be related to lignin modifications in termite digestomes. In conclusion, this report expanded the knowledge on genes and proteins related to CAZy/PAD enzymes from worker and soldier castes of lower termites, revealing new potential enzyme candidates for second-generation biofuel processes.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.