Targeted Redesign and Optimization of Culture Media for Ethylene Glycol Biosynthesis in Komagataella phaffii Thályta Fraga Pacheco, João Ricardo Moreira de Almeida Fermentation, 2025 Tailoring culture media and supplementation strategies to the specific requirements of a target product is essential for enhancing microbial production efficiency. This work addresses an unexplored aspect of K. phaffii cultivation: optimizing culture media for metabolite production from xylose, diverging from the conventional focus on recombinant protein expression and the use of glycerol or methanol as primary substrates. Ethylene glycol biosynthesis in an engineered K. phaffii strain was improved by evaluating media and nutrient supplementation. Among the seven evaluated formulations, FM22 and d’Anjou were the most effective, with inositol and thiamine dichloride playing key roles in enhancing production. Salt concentrations in both media were optimized using Central Composite Design (CCD), reducing complexity while increasing yields. Ethylene glycol production increased by 54% in FM22 and 21% in d’Anjou, accompanied by a threefold and 26% reduction in the total salt content, respectively. The vitamin solution was streamlined from seven to two components, each at half the standard concentration. Trace element solutions were reduced to 25% of the original volume without compromising productivity. These findings underscore the dual benefit of culture medium optimization: improved ethylene glycol yields and simplified formulations, establishing a foundation for the development of more efficient and cost-effective bioprocesses using K. phaffii.
Metabolic Engineering of Komagataella phaffii and Process Optimization for Biosynthesis of 1,2,4-Butanetriol From Xylose Débora Trichez, Thályta Pacheco, Clara Vida G. C. Carneiro, Jessica C. Bergmann, João Ricardo M. de Almeida Biotechnology Journal, 2025 1,2,4‐butanetriol (BTO) is a four‐carbon polyol used as a precursor for synthesizing pharmaceuticals, polymers, and energetic plasticizers. The present study demonstrates the microbial production of BTO from xylose by engineered Komagataella phaffii yeast strains for the first time. The pathway was established through the overexpression of the enzymes xylose dehydrogenase (XylB), xylonate dehydratase (XylD), and 2‐ketoacid decarboxylase (KDC). Two xylonate dehydratase genes, xylD‐CC from Caulobacter crescentus and xylD‐HL from Halomonas lutea , were evaluated in the constructions, both enabling BTO production. Furthermore, to improve BTO production, a central composite design analysis (CCD) was employed, identifying the best cultivation conditions to improve yeast performance. Under these optimized conditions, the engineered K. phaffii strain produced 1.3 g/L of BTO, achieving a 147% increase compared to the initial setup. Although further genetic engineering efforts are required to enhance BTO production, this study provides insights into potential improvement targets and highlights K. phaffii as a promising platform for the bio‐based synthesis of chemical compounds like BTO.
Improvement of Laccase Activity in Co-Culture of Panus lecomtei and Sporidiobolus pararoseus and Its Application as an Enzymatic Additive in Biomass Hydrolysis and Dye Decolorization Rubén Darío Romero Peláez, Luana Assis Serra, Daiana Wischral, Joice Raísa Barbosa Cunha, Thais Demarchi Mendes, Thályta Fraga Pacheco, Felix Gonçalves de Siqueira, João Ricardo Moreira de Almeida Fermentation, 2023 This work investigates the effects of the co-culture between the filamentous fungus Panus lecomtei and the yeast Sporidiobolus pararoseus in the production of laccases. The variations of time interval and inoculum volume of S. pararoseus in co-cultures with P. lecomtei stimulated laccase production, reaching its highest activity at nearly 2960.7 ± 244 U/mL with a maximum time point of 120 h and 2.0% (v/v), respectively. Further application in the pretreated sugarcane bagasse hydrolysis was performed, using P. lecomtei and S. pararoseus extract added to an enzyme mixture from the co-culture of P. lecomtei and Trichoderma reesei that positively favored the hydrolysis efficiency by 66.87%. Furthermore, the addition of P. lecomtei and S. pararoseus extract increased the degradation of industrial anthraquinone Remazol Brilliant Blue R by 78.98%. As a result, the extract derived from the co-culture of P. lecomtei and S. pararoseus rich in laccases presents potential in biotechnological applications, being suitable in the hydrolysis of lignocellulosic biomass and the degradation of unwanted dyes released in the environment.
Evaluation of different strains of Saccharomyces cerevisiae for ethanol production from high-amylopectin BRS AG rice (Oryza sativa L.) Isabela C. Almeida, Thályta F. Pacheco, Fabricio Machado, Sílvia B. Gonçalves Scientific Reports, 2022 Ethanol is the main biofuel produced by fermentation route and the search for new feedstocks to produce fuel ethanol is still a great challenge. This work aims to compare the ethanol production from a new irrigated rice cultivar BRS AG to the conventional cultivar BRS PAMPA applied in Brazil. Six different commercial strains ofSaccharomyces cerevisiae(BG-1, CAT-1, FT-858, JP-1, PE-2, and SA-1) were applied in fermentation reactions. Fermentations performed with BRS PAMPA rice revealed that the highest yields were achieved with strain SA-1, corresponding to 93.0% of the theoretical maximum and final ethanol concentration of 58.92 g L−1, and with CAT-1, a yield of 92.7% and final ethanol concentration of 58.93 g L−1. For the fermentations with BRS AG rice, the highest yields were obtained with strain FT-858, exhibiting a 89.6% yield and final ethanol concentration of 62.45 g L−1, and with CAT-1, 87.9% yield and final ethanol concentration of 61.25 g L−1were achieved. The most appropriate microorganism for ethanol production using BRS PAMPA rice and BRS AG rice was CAT-1. Comparatively, the ethanol yield and productivity using BRS AG were higher than those observed for BRS PAMPA for all strains, except for PE-2 and SA-1 that led to very similar results. The experimental results showed that the giant rice BRS AG is an excellent feedstock for fuel ethanol production in lowland fields.
Advances in Komagataella phaffii Engineering for the Production of Renewable Chemicals and Proteins Clara Vida Galrão Corrêa Carneiro, Luana Assis Serra, Thályta Fraga Pacheco, Letícia Maria Mallmann Ferreira, Lívia Teixeira Duarte Brandão, Mariana Nogueira de Moura Freitas, Débora Trichez, João Ricardo Moreira de Almeida Fermentation, 2022 The need for a more sustainable society has prompted the development of bio-based processes to produce fuels, chemicals, and materials in substitution for fossil-based ones. In this context, microorganisms have been employed to convert renewable carbon sources into various products. The methylotrophic yeast Komagataella phaffii has been extensively used in the production of heterologous proteins. More recently, it has been explored as a host organism to produce various chemicals through new metabolic engineering and synthetic biology tools. This review first summarizes Komagataella taxonomy and diversity and then highlights the recent approaches in cell engineering to produce renewable chemicals and proteins. Finally, strategies to optimize and develop new fermentative processes using K. phaffii as a cell factory are presented and discussed. The yeast K. phaffii shows an outstanding performance for renewable chemicals and protein production due to its ability to metabolize different carbon sources and the availability of engineering tools. Indeed, it has been employed in producing alcohols, carboxylic acids, proteins, and other compounds using different carbon sources, including glycerol, glucose, xylose, methanol, and even CO2.
Production of Enzymatic Extract with High Cellulolytic and Oxidative Activities by Co-Culture of Trichoderma reesei and Panus lecomtei Rubén Darío Romero Peláez, Daiana Wischral, Joice Raísa Barbosa Cunha, Thais Demarchi Mendes, Thályta Fraga Pacheco, Félix Gonçalves de Siqueira, João Ricardo Moreira de Almeida Fermentation, 2022 This work aimed to produce enzymatic fungi extracts with hydrolytic and oxidative activities to hydrolyze lignocellulosic biomasses efficiently. For this, the fungi Trichoderma reesei and Panus lecomtei were co-cultured using the vegetable biomasses oil palm decanter cake, wheat bran, and cottonseed cake as substrates in submerged fermentation. T. reesei and P. lecomtei showed partially compatible positive interaction on plates. The co-cultures respond positively to variations of temperature and inoculum interval, generating extracts responsible for higher hydrolysis yield when grown at 25 °C, and P. lecomtei is inoculated 24 h after T. reesei. The enzymatic extract production of co-cultures was also improved by modifying the components of the initial media and evaluating enzymatic activities, hydrolysis of sugarcane bagasse pretreated by autohydrolysis and ethanol production as a response. Five culture media were evaluated with variations in the composition of nutritional elements, minerals and substrates. The best extract showed a maximum cellulose hydrolysis efficiency of 68.7% compared with 44.8% of the initial medium. The ethanolic fermentation of hydrolysates obtained by co-culture extracts showed higher ethanol yields than monocultures. This work demonstrates the use of fungi co-cultures to produce enzymatic extracts composed of cellulolytic, hemicellulolytic, and ligninolytic enzymes complexes, which allow hydrolyzing pretreated lignocellulosic biomass with high efficiency, generating hydrolysates that are easier fermented by yeast.
Colonization of oil palm empty fruit bunches by basidiomycetes from the Brazilian cerrado: Enzyme production Elias Alves da Silva, Thais Demarchi Mendes, Thályta Fraga Pacheco, Daiana Wischral, Dulce Cristine dos Santos, Simone Mendonça, Marli Camassola, Félix Gonçalves de Siqueira, Manoel Teixeira Souza Energy Science and Engineering, 2022 The use of residual lignocellulosic biomass appears as an opportunity to obtain high added value products like enzymes. The present work goal was to select basidiomycetes capable of metabolizing empty fruit bunches (EB) or sludge decanter (SD) from the oil palm industry to produce enzymatic cocktails. First, eight macro‐basidiomycetes strains from the Brazilian cerrado were selected based on genomic DNA analyses out of an initial group of 30. The selected basidiomycetes and two ascomycetes (commercial mushrooms) were cultured to obtain ligninolytic and cellulolytic enzymes, respectively. All the extracts produced had proteins, and enzymatic profiles evaluated. The enzymatic hydrolysis of pretreated EB used cocktails of basidiomycetes extracts combined with commercial cellulases or ascomycetes extracts. The best combination of enzymatic extracts for hydrolyses of hydrothermally pretreated EB was the cocktail with Trichoderma reesei, Aspergillus aculeatus, and Pleurotus sp. extracts, reaching 19.7% of glucose yield and 22.8% of xylose yield. These results show that enzymes and sugars are products derivable from residues produced in large quantities by the oil palm industry. This study corroborates the hypothesis that basidiomycetes from the Brazilian cerrado present an enormous biotechnological potential, still unexplored.
Silencing of a BAHD acyltransferase in sugarcane increases biomass digestibility Wagner Rodrigo de Souza, Thályta Fraga Pacheco, Karoline Estefani Duarte, Bruno Leite Sampaio, Patrícia Abrão de Oliveira Molinari, Polyana Kelly Martins, Thaís Ribeiro Santiago, Eduardo Fernandes Formighieri, Felipe Vinecky, Ana Paula Ribeiro, Bárbara Andrade Dias Brito da Cunha, Adilson Kenji Kobayashi, Rowan Andrew Craig Mitchell, Dasciana de Sousa Rodrigues Gambetta, Hugo Bruno Correa Molinari Biotechnology for Biofuels, 2019
Biotechnological application of non-conventional yeasts for xylose valorization Jessica C. Bergmann, Débora Trichez, Wilson Galvão de Morais Junior, Talita Gabriela Salles Ramos, Thályta Fraga Pacheco, Clara Vida G. C. Carneiro, Victor Mendes Honorato, Luana Assis Serra, João Ricardo M. Almeida Non Conventional Yeasts from Basic Research to Application, 2019
Suppression of a single BAHD gene in Setaria viridis causes large, stable decreases in cell wall feruloylation and increases biomass digestibility Wagner R. de Souza, Polyana K. Martins, Jackie Freeman, Till K. Pellny, Louise V. Michaelson, Bruno L. Sampaio, Felipe Vinecky, Ana P. Ribeiro, Barbara A. D. B. da Cunha, Adilson K. Kobayashi, Patricia A. de Oliveira, Raquel B. Campanha, Thályta F. Pacheco, Danielly C. I. Martarello, Rogério Marchiosi, Osvaldo Ferrarese‐Filho, Wanderley D. dos Santos, Robson Tramontina, Fabio M. Squina, Danilo C. Centeno, Marília Gaspar, Marcia R. Braga, Marco A. S. Tiné, John Ralph, Rowan A. C. Mitchell, Hugo B. C. Molinari New Phytologist, 2018
