Engineering the pentose phosphate pathway of Saccharomyces cerevisiae for production of ethanol and xylitol

The baker s yeast Saccharomyces cerevisiae has a long tradition in alcohol production from D-glucose of e.g. starch. However, without genetic modifications it is unable to utilise the 5-carbon sugars D-xylose and L arabinose present in plant biomass. In this study, one key metabolic step of the catabolic D-xylose pathway in recombinant D-xylose-utilising S. cerevisiae strains was studied. This step, carried out by xylulokinase (XK), was shown to be rate-limiting, because overexpression of the xylulokinase-encoding gene XKS1 increased both the specific ethanol production rate and the yield from D xylose. In addition, less of the unwanted side product xylitol was produced. Recombinant D-xylose-utilizing S. cerevisiae strains have been constructed by expressing the genes coding for the first two enzymes of the pathway, D-xylose reductase (XR) and xylitol dehydrogenase (XDH) from the D-xylose-utilising yeast Pichia stipitis. In this study, the ability of endogenous genes of S. cerevisiae to enable D-xylose utilisation was evaluated. Overexpression of the GRE3 gene coding for an unspecific aldose reductase and the ScXYL2 gene coding for a xylitol dehydrogenase homologue enabled growth on D-xylose in aerobic conditions. However, the strain with GRE3 and ScXYL2 had a lower growth rate and accumulated more xylitol compared to the strain with the corresponding enzymes from P. stipitis. Use of the strictly NADPH-dependent Gre3p instead of the P. stipitis XR able to utilise both NADH and NADPH leads to a more severe redox imbalance. In a S. cerevisiae strain not engineered for D-xylose utilisation the presence of D-xylose increased xylitol dehydrogenase activity and the expression of the genes SOR1 or SOR2 coding for sorbitol dehydrogenase. Thus, D-xylose utilisation by S. cerevisiae with activities encoded by ScXYL2 or possibly SOR1 or SOR2, and GRE3 is feasible, but requires efficient redox balance engineering. Compared to D-xylose, D-glucose is a cheap and readily available substrate and thus an attractive alternative for xylitol manufacture. In this study, the pentose phosphate pathway (PPP) of S. cerevisiae was engineered for production of xylitol from D-glucose. Xylitol was formed from D-xylulose 5-phosphate in strains lacking transketolase activity and expressing the gene coding for XDH from P. stipitis. In addition to xylitol, ribitol, D-ribose and D-ribulose were also formed. Deletion of the xylulokinase-encoding gene increased xylitol production, whereas the expression of DOG1 coding for sugar phosphate phosphatase increased ribitol, D-ribose and D-ribulose production. Strains lacking phosphoglucose isomerase (Pgi1p) activity were shown to produce 5 carbon compounds through PPP when DOG1 was overexpressed. Expression of genes encoding glyceraldehyde 3-phosphate dehydrogenase of Bacillus subtilis, GapB, or NAD-dependent glutamate dehydrogenase Gdh2p of S. cerevisiae, altered the cellular redox balance and enhanced growth of pgi1 strains on D glucose, but co-expression with DOG1 reduced growth on higher D-glucose concentrations. Strains lacking both transketolase and phosphoglucose isomerase activities tolerated only low D-glucose concentrations, but the yield of 5-carbon sugars and sugar alcohols on D-glucose was about 50% (w/w).Polttoaine-etanolin valmistaminen uusiutuvasta raaka-aineesta, kuten kasvimateriaalista, on tärkeää fossiilisten raaka-aineiden rajallisuuden ja ilmastonmuutoksen takia. Saccharomyces cerevisiae -leivinhiiva pystyy tuottamaan tehokkaasti etanolia glukoosista, mutta se ei pysty käyttämään kasvimateriaalin viisihiilisiä sokereita, ksyloosia ja arabinoosia, ilman geneettistä muokkausta. Tutkimuksessa havaittiin, että yksi ksyloosi-sokerin käyttöä rajoittava tekijä on ksylulokinaasi-entsyymi. Lisäämällä tämän entsyymin määrää solussa sekä etanolin tuottonopeus että saanto ksyloosista kasvoivat. Lisäksi ksylitoli-sivutuotteen määrä pieneni. Ksyloosia käyttäviin leivinhiivakantoihin on tuotu tämän sokerin käytön mahdollistavat entsyymit ksyloosi reduktaasi (XR) ja ksylitoli dehydrogenaasi (XDH) ksyloosia luontaisesti käyttävästä Pichia stipitis -hiivasta. Tutkimuksessa osoitettiin, että myös leivinhiivasta itsestään löytyvät geenit GRE3 ja ScXYL2, joiden koodaamilla entsyymeillä on vastaavasti XR ja XDH aktiivisuutta, mahdollistavat kasvun ksyloosilla. GRE3 ja ScXYL2 geenien avulla rakennettu leivinhiivakanta ei kuitenkaan ollut yhtä tehokas kuin vastaava P. stipitis -hiivan geenejä ilmentävä kanta, johtuen Gre3- ja P. stipitis -hiivan XR-entsyymien erilaisista kofaktori vaatimuksista. Jotta leivinhiivan omia entsyymejä voitaisiin hyödyntää kantarakennuksessa, olisi ensin löydettävä tehokkaita keinoja ksyloosi-reitin hapetus-pelkistys tasapainon parantamiseen. Ksyloosi-sokeria on perinteisesti käytetty ksylitolin valmistamiseen. Ennen kemiallista prosessia ksyloosi pitää kuitenkin eristää puumateriaalista. Elintarviketeollisuudessa laajalti käytetty glukoosi-sokeri olisi halpa ja helposti saatavilla oleva vaihtoehto ksylitolin raaka-aineeksi. Tutkimuksessa osoitettiin, että leivinhiiva voidaan muokata tuottamaan ksylitolia, ribitolia ja riboosia glukoosista poistamalla joko sen transketolaasi- tai fosfoglukoosi-isomeraasi-aktiivisuutta koodaavat geenit tai molemmat. Näiden viisihiilisten sokerien ja sokerialkoholien määrää ja suhteita voitiin lisäksi muuttaa tuottamalla Dog1-sokerifosfataasi aktiivisuutta, poistamalla ksylulokinaasi aktiivisuus ja erilaisilla hapetus-pelkistystasapainoon vaikuttavilla entsyymeillä

Narratives of thriving : Black lesbian and queer women negotiating racism, sexism, and heterosexism

This qualitative exploratory study explores the narratives that Black lesbian and queer women age 21 to 35 tell about their lived experience by addressing racism, sexism, and heterosexism and how Black lesbian and queer women live and negotiate in the world. In exploring these narratives, the research focused on the following questions: What are the ways in which Black Lesbian and Queer Women create their own story as they negotiate at the margins of society? How do Black lesbian women create meaning out of their experiences in the face of racism, sexism, and heterosexism? The study found that these 12 self-identified Black lesbian and queer Women were proactive and intentional about creating public and private spaces where they and other Black lesbian queer women could feel safe, comfortable, and free in being their full complex selves. The major findings included each participant exercised resistance strategies to maintain the integrity and expression of their identities, including engaging in practices of renaming to allow space for an intersectional and complex understanding of their identities. They were proactive in finding and building homeplaces to help them manage their complex and individually unique experiences of racism, sexism, and heterosexism. Their narratives revealed important themes regarding coming out and negotiating their identities within their family, faith communities, work, and other social groups. This study revealed Black lesbian and queer women are not simply surviving they are thriving in their communities and in their lives. I conclude with a recommendation that clinicians develop a sense of how their own identities interact and intersect within systems of oppression, and of how Black lesbian and queer women might be impacted by racism, sexism, heterosexism, and other oppressions

Identification and Characterization of a Novel Diterpene Gene Cluster in Aspergillus nidulans

Fungal secondary metabolites are a rich source of medically useful compounds due to their pharmaceutical and toxic properties. Sequencing of fungal genomes has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in normal laboratory conditions. Therefore, to discover new metabolites, it is important to find ways to induce the expression of genes in these otherwise silent biosynthetic clusters. We discovered a novel secondary metabolite in Aspergillus nidulans by predicting a biosynthetic gene cluster with genomic mining. A Zn(II)2Cys6–type transcription factor, PbcR, was identified, and its role as a pathway-specific activator for the predicted gene cluster was demonstrated. Overexpression of pbcR upregulated the transcription of seven genes in the identified cluster and led to the production of a diterpene compound, which was characterized with GC/MS as ent-pimara-8(14),15-diene. A change in morphology was also observed in the strains overexpressing pbcR. The activation of a cryptic gene cluster by overexpression of its putative Zn(II)2Cys6–type transcription factor led to discovery of a novel secondary metabolite in Aspergillus nidulans. Quantitative real-time PCR and DNA array analysis allowed us to predict the borders of the biosynthetic gene cluster. Furthermore, we identified a novel fungal pimaradiene cyclase gene as well as genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase and a geranylgeranyl pyrophosphate (GGPP) synthase. None of these genes have been previously implicated in the biosynthesis of terpenes in Aspergillus nidulans. These results identify the first Aspergillus nidulans diterpene gene cluster and suggest a biosynthetic pathway for ent-pimara-8(14),15-diene

Low oxygen levels as a trigger for enhancement of respiratory metabolism in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The industrially important yeast <it>Saccharomyces cerevisiae </it>is able to grow both in the presence and absence of oxygen. However, the regulation of its metabolism in conditions of intermediate oxygen availability is not well characterised. We assessed the effect of oxygen provision on the transcriptome and proteome of <it>S. cerevisiae </it>in glucose-limited chemostat cultivations in anaerobic and aerobic conditions, and with three intermediate (0.5, 1.0 and 2.8% oxygen) levels of oxygen in the feed gas.</p> <p>Results</p> <p>The main differences in the transcriptome were observed in the comparison of fully aerobic, intermediate oxygen and anaerobic conditions, while the transcriptome was generally unchanged in conditions receiving different intermediate levels (0.5, 1.0 or 2.8% O₂) of oxygen in the feed gas. Comparison of the transcriptome and proteome data suggested post-transcriptional regulation was important, especially in 0.5% oxygen. In the conditions of intermediate oxygen, the genes encoding enzymes of the respiratory pathway were more highly expressed than in either aerobic or anaerobic conditions. A similar trend was also seen in the proteome and in enzyme activities of the TCA cycle. Further, genes encoding proteins of the mitochondrial translation machinery were present at higher levels in all oxygen-limited and anaerobic conditions, compared to fully aerobic conditions.</p> <p>Conclusion</p> <p>Global upregulation of genes encoding components of the respiratory pathway under conditions of intermediate oxygen suggested a regulatory mechanism to control these genes as a response to the need of more efficient energy production. Further, cells grown in three different intermediate oxygen levels were highly similar at the level of transcription, while they differed at the proteome level, suggesting post-transcriptional mechanisms leading to distinct physiological modes of respiro-fermentative metabolism.</p

Oxygen dependence of metabolic fluxes and energy generation of Saccharomyces cerevisiae CEN.PK113-1A

<p>Abstract</p> <p>Background</p> <p>The yeast <it>Saccharomyces cerevisiae </it>is able to adjust to external oxygen availability by utilizing both respirative and fermentative metabolic modes. Adjusting the metabolic mode involves alteration of the intracellular metabolic fluxes that are determined by the cell's multilevel regulatory network. Oxygen is a major determinant of the physiology of <it>S. cerevisiae </it>but understanding of the oxygen dependence of intracellular flux distributions is still scarce.</p> <p>Results</p> <p>Metabolic flux distributions of <it>S. cerevisiae </it>CEN.PK113-1A growing in glucose-limited chemostat cultures at a dilution rate of 0.1 h^-1with 20.9%, 2.8%, 1.0%, 0.5% or 0.0% O₂in the inlet gas were quantified by ¹³C-MFA. Metabolic flux ratios from fractional [U-¹³C]glucose labelling experiments were used to solve the underdetermined MFA system of central carbon metabolism of <it>S. cerevisiae</it>.</p> <p>While ethanol production was observed already in 2.8% oxygen, only minor differences in the flux distribution were observed, compared to fully aerobic conditions. However, in 1.0% and 0.5% oxygen the respiratory rate was severely restricted, resulting in progressively reduced fluxes through the TCA cycle and the direction of major fluxes to the fermentative pathway. A redistribution of fluxes was observed in all branching points of central carbon metabolism. Yet only when oxygen provision was reduced to 0.5%, was the biomass yield exceeded by the yields of ethanol and CO₂. Respirative ATP generation provided 59% of the ATP demand in fully aerobic conditions and still a substantial 25% in 0.5% oxygenation. An extensive redistribution of fluxes was observed in anaerobic conditions compared to all the aerobic conditions. Positive correlation between the transcriptional levels of metabolic enzymes and the corresponding fluxes in the different oxygenation conditions was found only in the respirative pathway.</p> <p>Conclusion</p> <p>¹³C-constrained MFA enabled quantitative determination of intracellular fluxes in conditions of different redox challenges without including redox cofactors in metabolite mass balances. A redistribution of fluxes was observed not only for respirative, respiro-fermentative and fermentative metabolisms, but also for cells grown with 2.8%, 1.0% and 0.5% oxygen. Although the cellular metabolism was respiro-fermentative in each of these low oxygen conditions, the actual amount of oxygen available resulted in different contributions through respirative and fermentative pathways.</p