Intensification of 2G bioethanol process: yeast development to overcome challenges derived from lignocellulosic processing

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017ortuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI"01"0145"FEDER "006684), BioTecNorte operation (NORTE"01"0145"FEDER" 000004) and MultiBiorefinery project (POCI"01"0145"FEDER"016403info:eu-repo/semantics/publishedVersio

Strategic yeast engineering for sustainable biorefineries

The astonishing growth of the world population, the global climate changes, and the depletion of fossil fuels are strong drivers for the development of a resource-efficient and sustainable economy. In this regard, the biotechnological manufacturing of lignocellulosic-based biofuels/biochemicals is pivotal. In these processes, fermentation is a core operation and the microorganism used, generally, Saccharomyces cerevisiae, plays a central role. Designing a tailor-made yeast considering the strain background and the particular raw material and process is of major importance[1] and, in this context, industrial S. cerevisiae, presenting robustness traits[2] is a promising platform. Following a process-oriented yeast design, the successful engineering of xylose metabolism[3], inhibitors tolerance[4,5] or cell-surface display of hydrolytic enzymes[6], resulting in ethanol-producing competitive processes have been attained[7,8]. Nevertheless, integration with the production of high-value low-volume biochemicals can further improve economic feasibility[9]. The design of yeast to produce value-added compounds like xylitol[10] or arabitol, resveratrol[11] or to be used as whole-cell biocatalysts for bioconversion of highly versatile compounds present in lignocellulose like valuable furan-derivatives could also be demonstrated. Overall, our results show that a rational yeast design based on the strain metabolic background under a process integration framework is a potential strategy for biorefineries sustainable development.Portuguese Foundation for Science and Technology under the scope of the strategic funding of UIDB/04469/2020 unit and BIOVINO (0688_BIOVINO_6_E).info:eu-repo/semantics/publishedVersio

Optimizing CRISPR/Cas9 for high-expression genome loci in industrial yeast strains

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] The yeast Saccharomyces cerevisiae is one of the key cell factories for the production of bio-based chemicals, from fuels and bulk chemicals to active pharmaceuticals. Generally recognized as safe (GRAS) by the U. S. Food and Drug Administration and with a broad array of tools available at the molecular level, S. cerevisiae has been successfully manipulated for a wide range of applications. For large-scale fermentations, particularly in biorefineries, yeast cells must perform under harsh conditions, such as fluctuating pH and temperature, high osmotic pressure and presence of inhibitors derived from biomass hydrolysis. In this context, robust and stress-tolerant yeast chassis are required to attain high titers and product yields [1]. Industrial environments have been identified as a bioresource of yeast strains with higher robustness and fermentation performance and distinct strains have been isolated. [...]info:eu-repo/semantics/publishedVersio

Genetic engineering approaches for enhanced lignocellulosic-based bioprocesses

Book of Abstracts of CEB Annual Meeting 2017Lignocellulosic biomass is the most abundant, low-cost, bio-renewable resource. It has a recognised potential as a sustainable platform for the production of biofuels and other bio-chemicals. To improve the accessibility of the cellulose component from complex lignocellulosic structures to the enzymes, a pretreatment step is necessary. Enzymatic saccharification of resulting whole slurry is highly desirable as it avoids the solid-liquid separation step, the need for detoxification and related waste disposal problem, and increases final sugar concentration. However, lignin residues and other inhibitory compounds resulting from pretreatment negatively affect the digestibility of the whole slurry and compromise fermentation efficiency. To tackle these pitfalls, genetic engineering strategies have been developed and integrated in the process to improve both stages. For improving the fermentation efficiency, our approach has been to intensify the process by using high solid loadings and both pentose and hexoses fractions, enriching sugar concentration available for fermentation. To work under such demanding conditions robust yeast strains are crucial. We have selected natural robust yeast isolates and identified key genes necessary for yeast growth and maximal fermentation rate in hydrolysates. Selected robust yeast chassis have been metabolic engineered for cofermentation of glucose and xylose from hemicellulose fraction using a novel metabolic assembly tool and key tolerance genes expression has been simultaneously evaluated for the valorization of biomass of different origins. Results obtained pointed to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process [1]. The flexibility of the metabolic assembly tool developed and the selected robust yeast backgrounds envisioned the developing of effective yeast platforms for biomass processing into different products. For improving the saccharification of whole slurry, our strategy has been to use the efficient recombinant protein production system from Escherichia coli to produce hydrolysis enhancers, namely a family 3 carbohydrate-binding module (CBM3). The purified CBM3 was used as an additive in the enzymatic hydrolysis of the whole slurry from hydrothermally-pretreated Eucalyptus globulus wood among other biotechnological applications [2]. The results obtained show an increase in glucose yield when CBM3 was added, compensating the negative effect of inhibitors on the enzymatic efficiency of whole slurry saccharification. Thus, CBM3 is a valid additive for enhanced lignocellulosics saccharification and a valuable alternative to costly additives (e.g. BSA) as it can be affordably obtained from heterologous bacterium or integrated in the developed yeast platforms, thus contributing to more cost-efficient and environmental-friendly biomass conversion bioprocesses.info:eu-repo/semantics/publishedVersio

New promotors for recombinant gene expression control in Ashbya gossypii identified through analysis of transcriptomic data

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New promotors for recombinant gene expression control in Ashbya gossypii identified through analysis of transcriptomic data

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Development of molecular and enzymatic kits for the detection of total coliforms and Escherichia coli in water samples

The drinking water is one of the main sources of infectious diseases. It is of major importance to keep a good water quality monitoring. The need for more rapid, sensitive and specific tests is essential; not only for water industry, but for a better public safety. Therefore, detection of microbial indicators of potential pathogens in water is the solution to the prevention and recognition of problems related to human health and safety. The main purpose of this work is to develop commercial kits for the detection of the extensively used as indicator organisms: Escherichia coli and total coliforms. An enzymatic method of detection of these microorganisms is being developed based on the enzymes β-Dglucuronidase and β-D-galactosidase, respectively. The results are visible in 18 h for 1 CFU. In order to achieve a higher level of sensitivity and specificity, molecular detection using the Polymerase Chain Reaction (PCR) technique is being investigated. Three primers were selected for identification of total coliforms, E. coli and E. coli with other enteric pathogens. At this moment, we achieved a sensitivity level of 676 CFU in 8 h, which is already a good achievement but there is still more research to be done in order to accomplish the 1 CFU detection

Transformation of a flocculating saccharomyces cerevisiae using lithium acetate and pYAC4

A flocculating yeast Saccharomyces cerevisiae ura3 was transformed by the method based on treatment of intact cells with lithium acetate plus single-stranded carrier DNA using the shuttle vector pYAC4. The transformation efficiency was above 10³ transformants per microgram of plasmid DNA which is similar to other described yeast transformation systems. Under selective pressure, the transformed cells were stable and maintained the flocculation ability. Thus, this simple transformation system can be used for gene expression studies in flocculating yeasts, overcoming disadvantages of conventional methods such as the spheroplast one.Fundação para a Ciência e Tecnologia (FCT) - PRAXIS XXI/BD/11306/9

Eucalyptus wood and cheese whey valorization for biofuels production

In this work, two raw materials (Eucalyptus wood and cheese whey) were used for ethanol production. Eucalyptus wood was hydrothermally pretreated at 233 ºC in order to increase the enzymatic saccharification of cellulose. Pretreated Eucalyptus wood mixed or not with cheese whey were used as substrates for ethanol production by simultaneous saccharification and fermentation (SSF) using two Saccharomyces cerevisiae strains (industrial Ethanol Red® and laboratory CEN.PK1137D). The use of cheese whey mixed with Eucalyptus wood increased 1.3 and 1.5-fold the ethanol concentration in comparison with Eucalyptus without cheese whey using S. cerevisiae Ethanol Red® and CEN.PK113 7D strains, respectively. Higher ethanol concentration was obtained by Ethanol Red® than ethanol produced by CEN.PK113-7D with cheese whey supplementation (93 g/L and 65 g/L corresponding to 94 % and 66 % of ethanol yield, respectively). Results obtained in this work showed an interesting strategy for the valorization of two raw materials in order to produce high concentrations of ethanol.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI -01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01- 0145-FEDER -000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio