RESUMO
Seven Kluyveromyces marxianus isolates from the elaboration process of pulque and henequen mezcal were characterized. The isolates were identified based on the sequences of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer (ITS-5.8S) region. Genetic differences were found between pulque and henequen mezcal isolates and within henequen mezcal isolates, as shown by different branching patterns in the ITS-5.8S phylogenetic tree and (GTG)5 microsatellite profiles, suggesting that the substrate and process selective conditions may give rise to different K. marxianus populations. All the isolates fermented and assimilated inulin and lactose and some henequen isolates could also assimilate xylose and cellobiose. Henequen isolates were more thermotolerant than pulque ones, which, in contrast, presented more tolerance to the cell wall-disturbing agent calcofluor white (CFW), suggesting that they had different cell wall structures. Additionally, depending on their origin, the isolates presented different maximum specific growth rate (µmax) patterns at different temperatures. Concerning tolerance to stress factors relevant for lignocellulosic hydrolysates fermentation, their tolerance limits were lower at 42 than 30 °C, except for glucose and furfural. Pulque isolates were less tolerant to ethanol, NaCl, and Cd. Finally, all the isolates could produce ethanol by simultaneous saccharification and fermentation (SSF) of a corncob hydrolysate under laboratory conditions at 42 °C.
RESUMO
The production of biofuels, such as bioethanol from lignocellulosic biomass, is an important task within the sustainable energy concept. Understanding the metabolism of ethanologenic microorganisms for the consumption of sugar mixtures contained in lignocellulosic hydrolysates could allow the improvement of the fermentation process. In this study, the ethanologenic strain Escherichia coli MS04 was used to ferment hydrolysates from five different lignocellulosic agroindustrial wastes, which contained different glucose and xylose concentrations. The volumetric rates of glucose and xylose consumption and ethanol production depend on the initial concentration of glucose and xylose, concentrations of inhibitors, and the positive effect of acetate in the fermentation to ethanol. Ethanol yields above 80% and productivities up to 1.85 gEtOH/Lh were obtained. Furthermore, in all evaluations, a simultaneous co-consumption of glucose and xylose was observed. The effect of deleting the xyIR regulator was studied, concluding that it plays an important role in the metabolism of monosaccharides and in xylose consumption. Moreover, the importance of acetate was confirmed for the ethanologenic strain, showing the positive effect of acetate on the co-consumption rates of glucose and xylose in cultivation media and hydrolysates containing sugar mixtures.
Assuntos
Repressão Catabólica , Escherichia coli , Fermentação , Escherichia coli/metabolismo , Xilose/metabolismo , Glucose/metabolismo , Açúcares/metabolismo , Etanol/metabolismoRESUMO
The yeast Saccharomyces cerevisiae is an excellent candidate for establishing cell factories to convert lignocellulosic biomass into chemicals and fuels. To enable this technology, yeast robustness must be improved to withstand the fermentation inhibitors (e.g., weak organic acids, phenols, and furan aldehydes) resulting from biomass pretreatment and hydrolysis. Here, we discuss how evolution experiments performed in the lab, a method commonly known as adaptive laboratory evolution (ALE), may contribute to lifting yeast tolerance against the inhibitors of lignocellulosic hydrolysates (LCHs). The key is that, through the combination of whole-genome sequencing and reverse engineering, ALE provides a robust platform for discovering and testing adaptive alleles, allowing to explore the genetic underpinnings of yeast responses to LCHs. We review the insights gained from past evolution experiments with S. cerevisiae in LCH inhibitors and propose experimental designs to optimise the discovery of genetic variants adaptive to biomass toxicity. The knowledge gathered through ALE projects is envisaged as a roadmap to engineer superior yeast strains for biomass-based bioprocesses.
Assuntos
Etanol , Saccharomyces cerevisiae , Fermentação , Hidrólise , Lignina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Kluyveromyces marxianus is an ascomycetous yeast which has shown promising results in cellulosic ethanol and renewable chemicals production. It can survive on a variety of carbon sources under industrially favorable conditions due to its fast growth rate, thermotolerance, and acid tolerance. K. marxianus, is generally regarded as a safe (GRAS) microorganism, is widely recognized as a powerhouse for the production of heterologous proteins and is accepted by the US Food and Drug Administration (USFDA) for its pharmaceutical and food applications. Since lignocellulosic hydrolysates are comprised of diverse monomeric sugars, oligosaccharides and potential metabolism inhibiting compounds, this microorganism can play a pivotal role as it can grow on lignocellulosic hydrolysates coping with vegetal cell wall derived inhibitors. Furthermore, advancements in synthetic biology, for example CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, will enable development of an engineered yeast for the production of biochemicals and biopharmaceuticals having a myriad of industrial applications. Genetic engineering companies such as Cargill, Ginkgo Bioworks, DuPont, Global Yeast, Genomatica, and several others are actively working to develop designer yeasts. Given the important traits and properties of K. marxianus, these companies may find it to be a suitable biocatalyst for renewable chemicals and fuel production on the large scale. This paper reviews the recent progress made with K. marxianus biotechnology for sustainable production of ethanol, and other products utilizing lignocellulosic sugars.