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Biomass has been profiled as a potential source of a portfolio of value-added products and energy vectors with applications in different industrial sectors. The Territory of the Southern Coast of Bahia (TSC) in Brazil has a high potential to provide agricultural and forest biomass to small and medium-scale biorefineries. The Territory of the Southern Coast of Bahia (TSC) in Brazil has a high potential to provide agricultural and forest biomass for these bioindustries. This work aims to quantify the biomass of the main agricultural crops of the TSC over the period of 1999 to 2019. For this purpose, the biomass classification in the Municipal Agricultural Production (PAM) of the Brazilian Institute of Geography and Statistics (IBGE) was used, according to its applications in pharmaceutical industries and other industries using the Classification and Regression Tree (CART) algorithm. Moreover, the identification of the main forest species of the Atlantic Forest that can supply bio-based products was included. Thus, a time series analysis was performed. Biomass classification was performed with the inverted tree using the Gini Index. The results indicate that banana production (1.3 Mt) exceeded, over a period of 20 years, cocoa production (1.1 Mt). This occurs despite the higher occupying area of cocoa (5.8 million hectares) compared with that of bananas. Coffee biomass has been profiled as a raw material to be upgraded in various industries, while bananas are oriented toward supplying inputs for the pharmaceutical industry. Forest species also have the potential to be used in the pharmaceutical industry. In conclusion, small and medium-scale biorefineries can help to increase the use of agricultural and forest biomass as a way of providing inputs for several industries and guaranteeing positive social impacts for the population.

Commercial cultivation of sugarcane is usually carried out by planting culm segments (sett) carrying buds in their internodes. However, this is an inefficient practice due to high sprouting irregularity. In this work, we inspect the first stages of the physiological preparation of the culm for sprouting, trying to identify compounds that actively participate in this process. We compared, during the first 48 h, the metabolic profile of sugarcane against energy cane, a cultivar known to have higher sprouting speed and consistency. In fact, during this short period it was possible to observe that energy cane already had a higher physiological activity than sugarcane, with significant changes in the catabolism of amino acids, increased levels of reducing sugars, lipids and metabolic activity in the phenylpropanoid pathway. On the other hand, sugarcane samples had just begun their activity during this same period, with an increase in the level of glutamate as the most significant change, which may be linked to the strategy of these cultivars to develop their roots before leaves, opposite of what is seen for energy cane. These results contribute to the development of strategies for increasing the efficiency of sprouting in sugarcane.

Agave plants are well-known for their drought resilience and commercial applications. Among them, Agave sisalana (sisal) is the species most used to produce hard fibers, and it is of great importance for semiarid regions. Agaves also show potential as bioenergy feedstocks, as they can accumulate large amounts of biomass and fermentable sugar. This study aimed to reconstruct the A. sisalana interactome, and identify key genes and modules involved in multiple plant tissues (root, stem, and leaf) through RNA-Seq analysis. We integrated A. sisalana transcriptome sequences and gene expression generated from stem, leaf, and root tissues to build global and conditional co-expression networks across the entire transcriptome. By combining the co-expression network, module classification, and function enrichment tools, we identified 20 functional modules related to at least one A. sisalana tissue, covering functions such as photosynthesis, leaf formation, auxin-activated signaling pathway, floral organ abscission, response to farnesol, brassinosteroid mediated signaling pathway, and light-harvesting. The final interactome of A. sisalana contains 2,582 nodes and 15,083 edges. In the reconstructed interactome, we identified submodules related to plant processes to validate the reconstruction. In addition, we identified 6 hub genes that were searched for in the co-expression modules. The intersection of hub genes identified by both the protein-protein interaction networks (PPI networks) and co-expression analyses using gene significance and module membership revealed six potential candidate genes for key genes. In conclusion, we identified six potential key genes for specific studies in Agave transcriptome atlas studies, biological processes related to plant survival in unfavorable environments, and provide strategies for breeding programs.

Aspergillus welwitschiae causes bole rot disease in sisal (Agave sisalana and related species) which affects the production of natural fibers in Brazil, the main worldwide producer of sisal fibers. This fungus is a saprotroph with a broad host range. Previous research established A. welwitschiae as the only causative agent of bole rot in the field, but little is known about the evolution of this species and its strains. In this work, we performed a comparative genomics analysis of 40 Aspergillus strains. We show the conflicting molecular identity of this species, with one sisal-infecting strain sharing its last common ancestor with Aspergillus niger, having diverged only 833 thousand years ago. Furthermore, our analysis of positive selection reveals sites under selection in genes coding for siderophore transporters, Sodium‑calcium exchangers, and Phosphatidylethanolamine-binding proteins (PEBPs). Herein, we discuss the possible impacts of these gene functions on the pathogenicity in sisal.

A plethora of studies have focused on improvements of xylitol production. The challenges of establishing a biotechnological route for the industrial production of this sugar have been explored using different microorganisms and renewable feedstock. Nevertheless, sugarcane biomass has been neglected as the pentose source for xylitol production using Saccharomyces cerevisiae. Therefore, here we investigate the use of an industrial S. cerevisiae strain for xylitol production in batch fermentation of non-detoxified sugarcane straw hydrolysate, envisioning the diversification of the current infrastructure used for second-generation bioethanol production from the same lignocellulosic material. In order to optimize the xylose conversion in a non-fed cultivation system, guidelines in cell inoculum and medium supplementation are suggested, as well as the first attempt to use electro-fermentation for this purpose. Accordingly, our results show that the increase in initial cell density and hydrolysate supplementation allows a xylitol production of 19.24 ± 0.68 g/L, representing 0,132 g/L.h productivity.

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.

Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae's metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4

BACKGROUNDSugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate.RESULTSPotential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5’-NGG 20 nt sequences, and guidelines for the fast and cost-effective CRISPR-editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer influence of the genetic background. Near-theoretical yields were achieved for all strains, however the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane bagasse hydrolysate with remaining solid particles represented a challenge for xylose metabolization and 3.65 ± 0.16 g/L xylitol titre was achieved by FMYX. Finally, quantification of NADPH - cofactor implied in XR activity - revealed that FMYX has 33% more available cofactors than CENPKX.CONCLUSIONSAlthough widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains’ performance. We also pioneer the use of sugarcane bagasse as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor production of reduced sugars.

Made available in DSpace on 2022-04-28T19:44:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-15 Agaves have been used for centuries as a feedstock in dryland areas for fibers, food, and beverages, and have enormous potential for biofuel production. Brazil is the world's largest producer of Agave fiber (sisal). However, since the development of synthetic fibers, the national investment in Agave research has decreased drastically, leading to the cessation of the country's breeding programs. What is left of the Brazilian elite cultivars were planted at a germplasm bank in the middle of the semiarid. Surprisingly, after 7 years of abandonment, the plants were still healthy and did not show any clear signs of stress. Here, we aimed to investigate how these plants managed to cope with this environment and the molecular basis of their biomass traits. We assembled the transcriptomic atlas of Agave sisalana, Agave fourcroydes, and Agave hybrid 11648 ((A. amaniensis x A. angustifolia) x A. amaniensis). We observed that the cultivars activated a highly overlapping set of stress-response genes, which were the most expressed transcripts. Also, raffinose was detected at high concentrations, possibly acting as an osmolyte, though differences at its biosynthesis have been found depending on cultivar. Finally, we observed differences in recalcitrance that could be attributed to lignin composition and its biosynthetic pathway. Our data contribute new insights that can help molecular breeders to correspond to emerging expectations for Agave as biorenewables feedstocks for dryland areas. Laboratório de Genômica e BioEnergia Departamento de Genética Evolução Microbiologia e Imunologia UNICAMP Departamento de Bioprocessos e Biotecnologia Faculdade de Ciências Agronômicas UNESP Departamento de Botânica Instituto de Biociências USP Laboratório de Anatomia Vegetal Departamento de Biologia Vegetal UNICAMP Empresa Brasileira de Pesquisa Agropecuária - Algodão EMBRAPA High-Throughput Sequencing Facility School of Medicine UNC Centro para Computação em Engenharia e Ciências UNICAMP Departamento de Bioprocessos e Biotecnologia Faculdade de Ciências Agronômicas UNESP

The conversion of lignocellulosic polymers into monomeric sugars demands a plethora of enzymatic activities generally not produced by a single microorganism and induced by the carbon source. In this vein, this work investigates the synergy between the enzymes secreted by the cellulolytic model fungi Trichoderma reesei RUT-C30 (TR) and Penicillium oxalicum (PO) to deconstruct sugarcane straw (SCS) and energy cane bagasse (ECB). TR and PO secrete a similar profile of cellulose-active enzymes resulting in a comparable conversion of SCS and ECB into glucose. The synergy between the enzymes produced by both fungi to break down the cellulose fraction becomes evident by the improvement of glucose titers from ~35-54% and from ~10-17% in SCS and ECB conditions, respectively, reached with the mixture of the secretomes of both fungi. The effect of a hemicellulase-enriched secretome produced by TR is particularly seen in SCS where the xylose yield reached ~15% compared to 5% by PO, remaining unaltered following the mixture of secretomes. However, the secretion of enzymes active in the decorations of the main chain polymers possibly aid PO to access the hemicellulose fraction of ECB reaching xylose yields similar to TR in this condition.

BACKGROUNDSaccharomyces cerevisiae is largely applied in many biotechnological processes, from traditional food and beverage industries to modern biofuel and biochemicals factories. During the fermentation process, yeast cells are usually challenged in different harsh conditions, which often impact productivity. Regarding bioethanol production, cell exposure to acidic environments is related to productivity loss on both first and second generation ethanol. In this scenario, indigenous strains traditionally used in fermentation stand out as a source of complex genetic architecture, mainly due to their highly robust background - including low pH tolerance. RESULTSIn this work, we pioneer the use of QTL mapping to uncover the genetic basis that endow industrial strain Pedra-2 (PE-2) with outstanding acid resistance. First, we developed a fluorescence-based high-throughput approach to collect a large number of haploid cells using flow cytometry. Then, we were able to apply a bulk segregant analysis to solve the genetic basis of low pH resistance in PE-2, which uncovered a region in chromosome XIII as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed allele GAS1, encoding a β-1,3-glucanosyltransferase, as the major contributor to this phenotype. The GAS1 sequence alignment of 48 S. cerevisiae strains pointed out a non-synonymous mutation (T211A) prevalence in wild type isolates, which is absent in laboratory strains. We further showcase that GAS1 allele swap between PE-2 and a low pH-susceptible strain can improve cell viability on the latter of up to 12% after a sulfuric acid wash process.CONCLUSIONThis work revealed GAS1 as the major causative gene associated with low pH resistance in PE-2, harboring a non-synonymous mutation persistent in industrial strains. We also showcase how GAS1PE-2 can improve acid resistance of a susceptible strain, suggesting that these findings can be a powerful foundation for the development of more robust and acid-tolerant strains for the industrial production of economically-relevant goods. Our results collectively show the importance of tailored industrial isolated strains in the discovery of the genetic architecture of relevant traits and its implications over productivity.

Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.

Xylose assimilation and fermentation are important traits for second generation ethanol production. However, some genomic features associated with this pentose sugar’s metabolism remain unknown in yeasts. Comparative genomics studies have led to important insights in this field, but we are still far from completely understanding endogenous yeasts’ xylose metabolism. In this work, we carried out a deep evolutionary analysis suited for comparative genomics of xylose-consuming yeasts, searching for of positive selection on genes associated with glucose and xylose metabolism in the xylose-fermenters’ clade. Our investigation detected positive selection fingerprints at this clade not only among sequences of important genes for xylose metabolism, such as xylose reductase and xylitol dehydrogenase, but also in genes expected to undergo neutral evolution, such as the glycolytic gene phosphoglycerate mutase. In addition, we present expansion, positive selection marks, and convergence as evidence supporting the hypothesis that natural selection is shaping the evolution of the little studied methylglyoxal reductases. We propose a metabolic model suggesting that selected codons among these proteins caused a putative change in cofactor preference from NADPH to NADH that alleviates cellular redox imbalance. These findings provide a wider look into pentose metabolism of yeasts and add this previously overlooked piece into the intricate puzzle of oxidative imbalance. Although being extensively discussed in evolutionary works the awareness of selection patterns is recent in biotechnology researches, rendering insights to surpass the reached status quo in many of its subareas.

The fungus Moniliophthora perniciosa is the causal agent of witches' broom disease (WBD), one of the most devastating diseases of cacao, the chocolate tree. Many strategies to control WBD have been tested so far, including the use of agrochemicals such as the strobilurins. Strobilurins are fungicides of the QoI family, and they are used in the control of a wide array of fungal diseases in many different crops, including cereals, field crops, fruits, tree nuts, and vegetables. These drugs act by specifically inhibiting fungal respiration at the Qo site of complex III, which is a component of the main mitochondrial respiratory chain. However, M. perniciosa is resistant to this family of chemicals. It has been postulated that this resistant phenotype is, at least in part, a result of the strong ability of this fungus to counteract the oxidative stress generated by the impairment of the main mitochondrial respiratory chain, through the activation of an alternative oxidase (Mp-AOX). To test this hypothesis, we expressed functional mitochondria-localized Mp-AOX in the model yeast Saccharomyces cerevisiae. We demonstrated that heterologous expression of Mp-AOX strongly inhibits hydrogen peroxide production by mitochondria. It also diminishes the total cell amount of oxidized glutathione (GSSG), resulting in a fifty-fold higher GSH/GSSG ratio in cells expressing Mp-AOX than in wild type cells. In addition, Mp-AOX activity decreases yeast growth rate and leads to low biomass production. Therefore, we propose the use of this heterologous expression system to direct the development of new inhibitors of fungal AOX by comparing the differences in optical density of Mp-AOX-expressing cells in the presence and absence of potential AOX inhibitors. Together, our results confirm the antioxidant role of Mp-AOX and provide an in vivo platform to be used in the screening of new fungicides based on Mp-AOX inhibition.

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.

Brazil was the first country to use bioethanol in large volumes, still in the 70′s, to cope with the oil crisis. The development of vehicles powered by ethanol included the biofuel definitively in the Brazilian energy matrix, ensuring its demand. To increase the ethanol production, a new cane variety has been developed, the energy cane, which can potentially triple the productivity of biomass per hectare and reduce the production costs. Since most of the sugar produced by energy cane is insoluble, second-generation mills have been installed in Brazil, and nowadays the technology is in a consolidation phase. To increase the stability of the biofuels market and to attend the Nationally Determined Contribution at COP21, Brazil has created the RenovaBio, a program by which the biofuels producers receive financial titles, the CBIOs, in the proportion of the biofuel production volume and efficiency, and under the condition of respecting the environmental legislation. Fuels distributors will have an obligation to buy CBIOs to compensate emissions beyond their mandates, but CBIOs will also be available to any interested investor, with significant potential for a strong appreciation. As a result, the expectation is to have a substantial increase in sustainable biofuels production, mainly ethanol, which could become a world commodity to supply international markets in the bioeconomy world.

The Polyploid Gene Assembler (PGA), developed and tested in this study, represents a new strategy to perform gene-space assembly from complex genomes using low coverage DNA sequencing. The pipeline integrates reference-assisted loci and de novo assembly strategies to construct high-quality sequences focused on gene content. Pipeline validation was conducted with wheat (Triticum aestivum), a hexaploid species, using barley (Hordeum vulgare) as reference, that resulted in the identification of more than 90% of genes and several new genes. Moreover, PGA was used to assemble gene content in Saccharum spontaneum species, a parental lineage for hybrid sugarcane cultivars. Saccharum spontaneum gene sequence obtained was used to reference-guided transcriptome analysis of six different tissues. A total of 39,234 genes were identified, 60.4% clustered into known grass gene families. Thirty-seven gene families were expanded when compared with other grasses, three of them highlighted by the number of gene copies potentially involved in initial development and stress response. In addition, 3,108 promoters (many showing tissue specificity) were identified in this work. In summary, PGA can reconstruct high-quality gene sequences from polyploid genomes, as shown for wheat and S. spontaneum species, and it is more efficient than conventional genome assemblers using low coverage DNA sequencing.

The need to replace fossil fuels has motivated research to develop alternative technologies for renewable energy production. For second generation ethanol, this includes the high-throughput screening and selection of yeast strains resistant to inhibitory byproducts of biomass pretreatment. In this work, we evaluated the use of static growth in 96-well microplates as a fermentative assay for Saccharomyces cerevisiae high-throughput screening, together with robust OD measurement correlations that overcome the low absorbance limit found in conventional plate spectrophotometers. This resulted in a method of yeast cultivation in microplates that requires no agitation and results in accurate kinetic parameters that can be used for the screening of S. cerevisiae. In order to automate calculation of these physiological parameters, we created OCHT®, an open-source high-throughput phenotype profiling software for processing, analyzing and visualizing S. cerevisiae growth data. Together, the cultivation standardization and data analysis toolbox allowed the evaluation and comparison of four S. cerevisiae strains in media containing aldehydes growth inhibitors, typically found in hydrolysates produced from sugarcane biomass. We identified FMY001 (SA1-derived) as a tolerant strain at high concentrations of HMF, furfural and vanillin. The individual analysis of segregants derived from FMY001 demonstrated their potential application in emerging biotechnological processes and gave us preliminary insights about the genetic mechanisms related to its high HMF concentration tolerance.

In this work, we evaluated the fermentative performance and metabolism modifications of a second generation (2G) industrial yeast by comparing an industrial condition during laboratory and industrial scale fermentations. Fermentations were done using industrial lignocellulosic hydrolysate and a synthetic medium containing inhibitors and analyses were carried out through transcriptomics and proteomics of these experimental conditions. We found that fermentation profiles were very similar, but there was an increase in xylose consumption rate during fermentations using synthetic medium when compared to lignocellulosic hydrolysate, likely due to the presence of unknown growth inhibitors contained in the hydrolysate. We also evaluated the bacterial community composition of the industrial fermentation setting and found that the presence of homofermentative and heterofermentative bacteria did not significantly change the performance of yeast fermentation. In parallel, temporal differentially expressed genes (tDEG) showed differences in gene expression profiles between compared conditions, including heat shocks and the presence of up-regulated genes from the TCA cycle during anaerobic xylose fermentation. Thus, we indicate HMF as a possible electron acceptor in this rapid respiratory process performed by yeast, in addition to demonstrating the importance of culture medium for the performance of yeast within industrial fermentation processes, highlighting the uniquenesses according to scales.

Background Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches’ broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. Results Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host–pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. Conclusions Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

Moniliophthora perniciosaandMoniliophthora roreriare hemibiotrophic fungi that harbor a large number of Pathogenesis-Related 1 genes, many of which are induced in the biotrophic interaction withTheobroma cacao.Here, we provide evidence that the evolution of PR-1 inMoniliophthorawas adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Phylogenetic analysis revealed conserved PR-1 genes, shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity inMoniliophthora, as well as in recent specialization cases within both species. PR-1 families inMoniliophthorawith higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arm race. Furthermore, we show that the highly diversifiedMpPR-1genes are not induced by two phytoalexins, suggesting detoxification might not be their main function as proposed before.

Viral infections pose intense burdens to healthcare systems and global economies. The correct diagnosis of viral diseases represents a crucial step towards effective treatments and control. Biosensors have been successfully implemented as accessible and accurate detection tests for some of the most important viruses. While most biosensors are based on physical or chemical interactions of cell-free components, the complexity of living microorganisms holds a poorly explored potential for viral detection in the face of the advances of synthetic biology. Indeed, cell-based biosensors have been praised for their versatility and economic attractiveness, however, yeast platforms for viral disease diagnostics are still limited to indirect antibody recognition. Here we propose a novel strategy for viral detection in Saccharomyces cerevisiae, which combines the transductive properties of G Protein-Coupled Receptors (GPCRs) with the Yeast Surface Display (YSD) of specific enzymes enrolled in the viral recognition process. The GPCR/YSD complex might allow for active virus detection through a modulated signal activated by a GPCR agonist, whose concentration correlates to the viral titer. Additionally, we explore this methodology in a case study for the detection of highly pathogenic coronaviruses that share the same cell receptor upon infection (i.e. the Angiotensin-Converting Enzyme 2, ACE2), as a conceptual example of the potential of the GPCR/YSD strategy for the diagnosis of COVID-19.

Ethanol production has key differences between the two largest producing countries of this biofuel, Brazil and the USA, such as feedstock source, sugar concentration and ethanol titers in industrial fermentation. Therefore, it is highly probable that these specificities have led to genome adaptation of the Saccharomyces cerevisiae strains employed in each process to tolerate different environments. In order to identify particular adaptations, in this work, we have compared the genomes of industrial yeast strains widely used to produce ethanol from sugarcane, corn and sweet sorghum, and also two laboratory strains as reference. The genes were predicted and then 4524 single-copy orthologous were selected to build the phylogenetic tree. We found that the geographic location and industrial process were shown as the main evolutionary drivers: for sugarcane fermentation, positive selection was identified for metal homeostasis and stress response genes, whereas genes involved in membrane modeling have been connected with corn fermentation. In addition, the corn specialized strain Ethanol Red showed an increased number of copies of MAL31, a gene encoding a maltose transporter. In summary, our work can help to guide new strain chassis selection for engineering strategies, to produce more robust strains for biofuel production and other industrial applications.

Made available in DSpace on 2020-12-10T20:10:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-14 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Serrapilheira Institute Background The need to restructure the world's energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineeredSaccharomyces cerevisiaestrains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeastS. cerevisiaeis not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes. Results Assessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeastCandida sojaeas a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters. Conclusions Considering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast. Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Biorenewable Natl Lab LNBR, BR-13083100 Campinas, SP, Brazil Univ Campinas UNICAMP, Inst Biol, Genet & Mol Biol Grad Program, Campinas, Brazil Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Biosci Natl Lab LNBio, BR-13083970 Campinas, SP, Brazil Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Synchrotron Light Lab LNLS, BR-13083970 Campinas, SP, Brazil Sao Paulo State Univ, UNESP, Dept Phys, Inst Biosci Humanities & Exact Sci, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Sao Paulo State Univ, UNESP, Dept Phys, Inst Biosci Humanities & Exact Sci, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil FAPESP: 2017/08519-6 FAPESP: 2017/05078-9 FAPESP: 2018/00888-5 CNPq: 430291/2018-3 CAPES: 001 Serrapilheira Institute: Serra1708-16205

When comparing gene expression data of different tissues it is often interesting to identify tissue-specific genes or transcripts. Even though there are several metrics to measure tissue-specificity, a user-friendly tool that facilitates this analysis is not available yet. We present tspex, a software that allows easy computation of a comprehensive set of different tissue-specificity metrics from gene expression data. tspex can be used through a web interface, command-line or the Python API. Its package version also provides visualization functions that facilitate inspection of results. The documentation and the source code of tspex are available at https://apcamargo.github.io/tspex/ and the web application can be accessed at https://tspex.lge.ibi.unicamp.br/

Agaves, which have been grown commercially for fiber or alcoholic beverages, are emerging as a candidate crop for biochemicals and biofuels production in semiarid regions because of their high productivity in low rainfall areas, drought tolerance, and low lignin content.In this work, we present the transcriptomic atlas of Agave sisalana, Agave fourcroydes, and agave hybrid 11648 (A. amaniensis x A. angustifolia) under prolonged drought in field conditions. Leaf, stem, and root tissues were sequenced, and gene expression profiles were correlated with biomass composition, enzymatic hydrolysis of cell wall carbohydrates, histochemical analysis, and non-structural carbohydrates content.Differences in biomass accessibility were attributed to either lignin content or lignin composition, possibly through modification of s/g ratio promoted by changes in Caffeic Acid 3-O-Methyltransferase (COMT) transcript abundance. Unlike most plants, the most highly expressed transcripts do not encode photosynthetic proteins, but rather involved in stress response. Although the three cultivars presented quantitative differences in global gene expression, they activated a highly overlapping set of genes. The main molecular strategies employed by agave to cope with high-temperature and drought seem to consist in overexpressing HSP and LEA, as well as promoting raffinose accumulation as an osmolyte.In conclusion, our data provide vital new genetic information for the study of Agave species and provide new insights into cell wall architecture, recalcitrance, and resistance to abiotic stresses for these species.

Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches’ broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry., Barsottini et al. characterize the alternative oxidase (MpAOX), which is crucial for the survival of Moniliophthora perniciosa, a fungal pathogen of cocoa. They identify key residues for the interactions between this enzyme and its inhibitors, guiding the future development of AOX-targeting antifungal agents that would benefit the chocolate industry.