Biotechnological production of xylitol: engineering industrial Saccharomyces cerevisiae for valorization of lignocellulosic biomass

The use of renewable biomass, such as lignocellulosic materials, for the production of biofuels and chemicals within a biorefinery scheme contributes to achieve a sustainable development. Xylitol has been identified as one of the top 12 value added chemicals to be obtained from biomass, and can be produced from hemicellulose-derived xylose through biotechnological processes [1]. In this work, xylitol was produced from xylose (using glucose as co-substrate for co-factor regeneration) in batch fermentations by the industrial Saccharomyces cerevisiae PE-2 strain (over)expressing (1) a wild type xylose reductase from Pichia spititis (XR); (2) a NADH-preferable xylose reductase mutant (mut-XR) from Pichia spititis and (3) the endogenous GRE3 gene which encodes for an unspecific aldose reductase (AR). Maximum yield (0.98 g g-1) was obtained by the strain overexpressing the GRE3 gene. Moreover, the recombinant strain PE-2-GRE3 showed significantly higher xylitol productivity than the laboratory strain, CENPK.113-5D overexpressing the same gene. This strain (PE-2-GRE3) was selected for bioconversion of 160 g L-1 of xylose in a fed-batch fermentation, which resulted in 149 g L-1 of xylitol concentration with a productivity of 1.2 g L-1 h-1. These results open new perspectives and opportunities for the valorisation of hemicellulosic hydrolysates through the production of xylitol within a biorefinery concept.info:eu-repo/semantics/publishedVersio

Multi-feedstock biorefinery concept: Valorization of winery wastes by engineered yeast

The wine industry produces significant amounts of by-products and residues that are not properly managed, posing an environmental problem. Grape must surplus, vine shoots, and wine lees have the potential to be used as renewable resources for the production of energy and chemicals. Metabolic engineering efforts have established Saccharomyces cerevisiae as an efficient microbial cell factory for biorefineries. Current biorefineries designed for producing multiple products often rely on just one feedstock, but the bioeconomy would clearly benefit if these biorefineries could efficiently convert multiple feedstocks. Moreover, to reduce the environmental impact of fossil fuel consumption and maximize production economics, a biorefinery should be capable to supplement the manufacture of biofuel with the production of high-value products. This study proposes an integrated approach for the valorization of diverse wastes resulting from winemaking processes through the biosynthesis of xylitol and ethanol. Using genetically modified S. cerevisiae strains, the xylose-rich hemicellulosic fraction of hydrothermally pretreated vine shoots was converted into xylitol, and the cellulosic fraction was used to produce bioethanol. In addition, grape must, enriched in sugars, was efficiently used as a low-cost source for yeast propagation. The production of xylitol was optimized, in a Simultaneous Saccharification and Fermentation process configuration, by adjusting the inoculum size and enzyme loading. Furthermore, a yeast strain displaying cellulases in the cell surface was applied for the production of bioethanol from the glucan-rich cellulosic. With the addition of grape must and/or wine lees, high ethanol concentrations were reached, which are crucial for the economic feasibility of distillation. This integrated multi-feedstock valorization provides a synergistic alternative for converting a range of winery wastes and by-products into biofuel and an added-value chemical while decreasing waste released to the environment.info:eu-repo/semantics/publishedVersio

Integrated approach for selecting efficient Saccharomyces cerevisiae for industrial lignocellulosic fermentations: importance of yeast chassis linked to process conditions

In this work, four robust yeast chassis isolated from industrial environments were engineered with the same xylose metabolic pathway. The recombinant strains were physiologically characterized in synthetic xylose and xylose-glucose medium, on non-detoxified hemicellulosic hydrolysates of fast-growing hardwoods (Eucalyptus and Paulownia) and agricultural residues (corn cob and wheat straw) and on Eucalyptus hydrolysate at different temperatures. Results show that the co-consumption of xylose-glucose was dependent on the yeast background. Moreover, heterogeneous results were obtained among different hydrolysates and temperatures for each individual strain pointing to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process.The authors acknowledge the financial support from the Strategic Project of UID/BIO/04469/2013 CEB Unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte

Xylose fermentation efficiency of industrial Saccharomyces cerevisiae yeast with separate or combined xylose reductase/xylitol dehydrogenase and xylose isomerase pathways

Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains.This study was supported by the Portuguese Foundation for Science and Technology (FCT, Portugal) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to Joana T. Cunha), the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte, the MultiBiorefinery project (POCI-01–0145FEDER-016403) and the Biomass and Bioenergy Research Infrastructure (PINFRA/22059/2016).info:eu-repo/semantics/publishedVersio

Contribution of PRS3, RPB4 and ZWF1 to the resistance of industrial Saccharomyces cerevisiae CCUG53310 and PE-2 strains to lignocellulosic hydrolysate-derived inhibitors

PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes.Fundação para a Ciência e a Tecnologia, Portugal, through: Grant SFRH/BDP/63831/2009 to Grant SFRH/BPD/77995/2011; Strategic Project PEst-OE/EQB/LA0023/2013; and Project BioInd NORTE-07-0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER

Towards a cost-effective bioethanol process: yeast development to overcome challenges derived from lignocellulosic processing

The use of renewable biomass to supply the increasing energetic needs and to partially replace fossil fuels is nowadays recognized as a suitable and desirable alternative to attain a sustainable growth based on a bioeconomy. In spite of the intensive research on lignocellulose-to-ethanol production processes, second generation (2G) bioethanol is still not cost competitive and specific challenges remain. These processes are as a whole substantially more complex than initially thought as several types of biomass may be used as substrate, each with specific challenges. A range of biomass pre-treatments may be applied for biomass fractionation, each with its own specificities and leading to different inhibitor profiles. Finally, different hydrolysis/fermentation schemes may be used. The optimization of these processes has to consider the integration of all the stages of the process and should be done together. One of the key aspects for the development of cost-effective lignocellulose-to-bioethanol processes is the engineering of the yeast strain. There is still a lack of robust and sugars-fast fermentation yeast strains for 2G bioethanol. Recently, we have screened and selected naturally robust yeast strains from industrial environments[1] and engineering some of the more promising strains with the xylose metabolic pathway[2] and inhibitor tolerance genes[3]. We will present the results of the simultaneous engineering of xylose metabolization pathway together with inhibitor tolerance in diverse robust background strains and its performance in different hydrolysates obtained from distinct types of biomass. The heterogeneous outcome of the genetic engineering in different hydrolysates show that tolerance and xylose engineering must be customized to the strain background and hydrolysate used in the process. The results obtained highlight that yeast development must not only be integrated in the process but it must also be tailor-made for each specific process. [1]Pereira et al.(2014)Bioresource Technology 161:192-199. [2]Romani et al.(2015)Bioresource Technology 179:150-158. [3]Cunha et al.(2015)Bioresource Technology 191:7-16

Nanocellulose production: exploring the enzymatic route and residues of pulp and paper industry

Increasing environmental and sustainability concerns, caused by current population growth, has promoted a raising utilization of renewable bio-resources for the production of materials and energy. Recently, nanocellulose (NC) has been receiving great attention due to its many attractive features such as non-toxic nature, biocompatibility, and biodegradability, associated with its mechanical properties and those related to its nanoscale, emerging as a promising material in many sectors, namely packaging, regenerative medicine, and electronics, among others. Nanofibers and nanocrystals, derived from cellulose sources, have been mainly produced by mechanical and chemical treatments; however, the use of cellulases to obtain NC attracted much attention due to their environmentally friendly character. This review presents an overview of general concepts in NC production. Especial emphasis is given to enzymatic hydrolysis processes using cellulases and the utilization of pulp and paper industry residues. Integrated process for the production of NC and other high-value products through enzymatic hydrolysis is also approached. Major challenges found in this context are discussed along with its properties, potential application, and future perspectives of the use of enzymatic hydrolysis as a pretreatment in the scale-up of NC production.This work was carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)- LISBOA-01-0145-FEDER-022059, supported by Operational Programme for Competitiveness andInternationalization (PORTUGAL2020), by Lisbon Portugal Regional Operational Programme (Lisboa 2020)and by North Portugal Regional Operational Program (Norte 2020) under the Portugal 2020 PartnershipAgreement, through the European Regional Development Fund (ERDF) and was supported by the PortugueseFoundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 and through Project EcoTech (POCI-01-0145-FEDER-032206/FAPESP 2018/07522-6) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope ofNorte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Enhancing the enzymatic saccharification of whole slurry from autohydrolyzed Eucalyptus globulus wood by supplementation with a recombinant carbohydrate-binding module

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017Background: Lignocellulosic biomass has a recognised potential as a sustainable platform for the production of biofuels and other biochemicals. However, lignin residues and other inhibitory compounds resulting from lignocellulosics pretreatment affect the digestibility of resulting whole slurries. The addition of synergistic proteins that can cooperate with cellulases is an emerging strategy for enhanced lignocellulosics hydrolysis. Carbohydrate-binding modules (CBMs) have been shown to improve the enzymatic hydrolysis of pure cellulose models but their effect on the enzymatic hydrolysis of lignocellulosic materials has not yet been evaluated. Thus, in this work, the potential synergistic effect of a family 3 CBM on the enzymatic saccharification of a pretreated lignocellulosic biomass was studied for the first time. [...]C. Oliveira and D. Gomes acknowledge support from Fundação para a Ciência e a Tecnologia (FCT), Portugal (grants SFRH/BPD/110640/2015 and SFRH/BD/88623/2012, respectively). This study was supported by the FCT under the scope of the GlycoCBMs Project PTDC/AGR-FOR/3090/2012–FCOMP-01-0124-FEDER-027948 and the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-010145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

False negative results from using common PCR reagents

Background\ud The sensitivity of the PCR reaction makes it ideal for use when identifying potentially novel viral infections in human disease. Unfortunately, this same sensitivity also leaves this popular technique open to potential contamination with previously amplified PCR products, or "carry-over" contamination. PCR product carry-over contamination can be prevented with uracil-DNA-glycosylase (UNG), and it is for this reason that it is commonly included in many commercial PCR master-mixes. While testing the sensitivity of PCR assays to detect murine DNA contamination in human tissue samples, we inadvertently discovered that the use of this common PCR reagent may lead to the production of false-negative PCR results.\ud \ud Findings\ud We show here that contamination with minute quantities of UNG-digested PCR product or any negative control PCR reactions containing primer-dimers regardless of UNG presence can completely block amplification from as much as 60 ng of legitimate target DNA.\ud \ud Conclusions\ud These findings could potentially explain discrepant results from laboratories attempting to amplify MLV-related viruses including XMRV from human samples, as none of the published reports used internal-tube controls for amplification. The potential for false negative results needs to be considered and carefully controlled in PCR experiments, especially when the target copy number may be low - just as the potential for false positive results already is

Efficacy and patient satisfaction with autoadjusting CPAP with variable expiratory pressure vs standard CPAP: a two-night randomized crossover trial

Expiratory pressure relief (C-Flex) technology monitors the patient’s airflow during expiration and reduces the pressure in response to the patient. Increased comfort levels associated with C-Flex therapy have potential to improve patient adherence to therapy. The purpose of this study was to assess the combination of autoadjusting CPAP (APAP) and C-Flex in terms of (1) treatment efficacy, and (2) patient preference when compared to standard CPAP. Fifteen patients who had previously undergone formal CPAP titration polysomnography were treated with either one night of the APAP with C-Flex or one night of conventional CPAP, in a crossover trial. Patient satisfaction levels were recorded using visual analog scales (VAS) on the morning after the study. Mean patient age was 50 ± 12 years, body mass index (BMI) was 36 ± 6 kg/m(2), baseline AHI was 53 ± 31 events/h, and CPAP Pressure was 11 ± 2 cm/H(2)O. APAP with C-Flex was as effective as CPAP, with no differences detected in sleep latency (17 ± 5 vs 12.3 ± 3 min, p = 0.4), or respiratory indices (AHI of 4.2 ± 2 vs 2.4 ± 0.7 events/h, p = 0.1). VAS scores (scale 0–10) indicated a trend towards increased patient satisfaction while using APAP with C-Flex (7.9 vs 7.2, p = 0.07). 10 patients expressed a preference for APAP with C-Flex (VAS, 0 to10) over standard CPAP (total positive score of 68, mean score of 4.8 ± 4.3). One patient expressed no preference. Four patients expressed a preference for CPAP (total positive score of 13, mean score of 0.9 ± 1.9) (APAP with C-Flex vs standard CPAP, p < 0.01 paired t test). APAP with C-Flex eliminates sleep disordered breathing as effectively as standard CPAP. Patients indicated a preference for APAP with C-Flex suggesting a possible advantage in terms of patient adherence for this mode of treatment