FEMS yeast research最新文献

Successful conversion of cellulosic biomass into biofuels requires organisms capable of efficiently utilizing xylose as well as cellodextrins and glucose. Ogataea (Hansenula) polymorpha is the natural xylose-metabolizing organism and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50°C. Cellobiose-fermenting strains, derivatives of an improved ethanol producer from xylose O. polymorpha BEP/cat8∆, were constructed in this work by the introduction of heterologous genes encoding cellodextrin transporters (CDTs) and intracellular enzymes (β-glucosidase or cellobiose phosphorylase) that hydrolyze cellobiose. For this purpose, the genes gh1-1 of β-glucosidase, CDT-1m and CDT-2m of cellodextrin transporters from Neurospora crassa and the CBP gene coding for cellobiose phosphorylase from Saccharophagus degradans, were successfully expressed in O. polymorpha. Through metabolic engineering and mutagenesis, strains BEP/cat8∆/gh1-1/CDT-1m and BEP/cat8∆/CBP-1/CDT-2mAM were developed, showing improved parameters for high-temperature alcoholic fermentation of cellobiose. The study highlights the need for further optimization to enhance ethanol yields and elucidate cellobiose metabolism intricacies in O. polymorpha yeast. This is the first report of the successful development of stable methylotrophic thermotolerant strains of O. polymorpha capable of coutilizing cellobiose, glucose, and xylose under high-temperature alcoholic fermentation conditions at 45°C.

The use of non-Saccharomyces yeasts in winemaking is gaining traction due to their specific phenotypes of technological interest, including their unique profile of central carbon metabolites and volatile compounds. However, the lack of knowledge about their physiology hinders their industrial exploitation. The intracellular redox status, involving NAD/NADH and NADP/NADPH cofactors, is a key driver of yeast activity during fermentation, notably directing the formation of metabolites that contribute to the wine bouquet. The biosynthesis of these cofactors can be modulated by the availability of their precursors, nicotinic acid and tryptophan, and their ratio by that of thiamine. In this study, a multifactorial experiment was designed to assess the effects of these three nutrients and their interactions on the metabolic response of various wine yeast species. The data indicated that limiting concentrations of nicotinic acid led to a species-dependent decrease in intracellular NAD(H) concentrations, resulting in variations of fermentation performance and production of metabolic sinks. Thiamine limitation did not directly affect redox cofactor concentrations or balance, but influenced redox management and subsequently the production of metabolites. Overall, this study identified nicotinic acid and thiamine as key factors to consider for species-specific modulation of the metabolic footprint of wine yeasts.

Candida tropicalis is a leading cause of nonalbicans candidemia in tropical/subtropical areas and a predominant genotype of azole-resistant C. tropicalis clinical isolates belongs to clade 4. The aim of this study was to reveal markers for rapidly identifying the predominant azole-resistant C. tropicalis genotype. We analysed XYR1, one of the six genes used in the multilocus sequence typing analysis, and SNQ2, an ATP-binding cassette transporter in 281 C. tropicalis, including 120 and 161 from Taiwan and global areas, respectively. Intriguingly, the first 4-mer of codon sequences ATRA of CTRG_05978 (96/119 versus 21/162, P < .001, at phi = 0. 679) and the SNQ2 A2977G resulting in amino acid I993V alternation (105/118 versus 12/163, P < .001, at phi = 0.81) was significantly associated with the clade 4 genotype. The sensitivity and specificity of the clade 4 genotype detection with a combination of SNPs of CTRG_05978 and SNQ2 were 0.812 and 0.994, respectively, at phi = 0.838. Furthermore, we successfully established a TaqMan SNP genotyping assay to identify the clade 4 genotype. Our findings suggest that to improve the management of C. tropicalis infections, rapidly identifying azole-resistant C. tropicalis by detecting SNPs of CTRG_05978 and SNQ2 is promising.

Traditional industrial Saccharomyces cerevisiae could not metabolize xylose due to the lack of a specific enzyme system for the reaction from xylose to xylulose. This study aims to metabolically remould industrial S. cerevisiae for the purpose of utilizing both glucose and xylose with high efficiency. Heterologous gene xylA from Piromyces and homologous genes related to xylose utilization were selected to construct expression cassettes and integrated into genome. The engineered strain was domesticated with industrial material under optimizing conditions subsequently to further improve xylose utilization rates. The resulting S. cerevisiae strain ABX0928-0630 exhibits a rapid growth rate and possesses near 100% xylose utilization efficiency to produce ethanol with industrial material. Pilot-scale fermentation indicated the predominant feature of ABX0928-0630 for industrial application, with ethanol yield of 0.48 g/g sugars after 48 hours and volumetric xylose consumption rate of 0.87 g/l/h during the first 24 hours. Transcriptome analysis during the modification and domestication process revealed a significant increase in the expression level of pathways associated with sugar metabolism and sugar sensing. Meanwhile, genes related to glycerol lipid metabolism exhibited a pattern of initial increase followed by a subsequent decrease, providing a valuable reference for the construction of efficient xylose-fermenting strains.

In this article we explore the intersection of science and art through a collaboration between us scientists and the bioartists Anna Dimitriu and Alex May, focusing on the interface of yeast biotechnology and art. The collaboration, originally initiated in 2018, resulted in three major artworks: CULTURE, depicting the evolution of yeast and human societies; FERMENTING FUTURES, illustrating a synthetic autotrophic yeast and its link to lactic acid production; and WOOD SPIRIT-AMBER ACID, inspired by the VIVALDI project targeting CO2 reduction to methanol. We emphasize the reciprocal nature of the collaboration, detailing the scientific insights gained and the impact of artistic perspectives on us as researchers. We also highlight the historical connection between art and science, particularly in the Renaissance periods, and underscore the educational value of integrating art into science not only to support public engagement and science dissemination, but also to widen our own perceptions in our research.

Controllable regulatory elements, like inducible, titratable promoters, are highly desired in synthetic biology toolboxes. A set of previously developed erythritol-inducible promoters along with an engineered Yarrowia lipolytica host strain were shown to be a very potent expression platform. In this study, we push the previously encountered limits of the synthetic promoters' titratability (by the number of upstream motifs) by using a compatible transcription factor, Euf1, as the promoter titrator. Overexpression of spliced EUF1 turned out to be very efficient in promoting expression from the compatible promoter, however, the erythritol-inducible character of the promoter was then lost. Analysis of the EUF1's splicing pattern suggests that the intron removal is promoted in the presence of erythritol, but is not dependent on it. The 3D structures of spliced versus unspliced Euf1 were modeled, and ligand-binding strength was calculated and compared. Furthermore, the EUF1-dependent expression profile under different chemical stimulants was investigated. Depletion of carbon source was identified as the significant factor upregulating the expression from the Euf1-dependent promoter (2-10-fold). Considering these findings and transcriptomics data, a new mechanism of the Euf1-regulated promoter action is proposed, involving a 'catabolite repression' transcription factor-Adr1, both acting on the same ERY-inducible promoter.

Emerging low-emission production technologies make ethanol an interesting substrate for yeast biotechnology, but information on growth rates and biomass yields of yeasts on ethanol is scarce. Strains of 52 Saccharomycotina yeasts were screened for growth on ethanol. The 21 fastest strains, among which representatives of the Phaffomycetales order were overrepresented, showed specific growth rates in ethanol-grown shake-flask cultures between 0.12 and 0.46 h-1. Seven strains were studied in aerobic, ethanol-limited chemostats (dilution rate 0.10 h-1). Saccharomyces cerevisiae and Kluyveromyces lactis, whose genomes do not encode Complex-I-type NADH dehydrogenases, showed biomass yields of 0.59 and 0.56 gbiomass gethanol-1, respectively. Different biomass yields were observed among species whose genomes do harbour Complex-I-encoding genes: Phaffomyces thermotolerans (0.58 g g-1), Pichia ethanolica (0.59 g g-1), Saturnispora dispora (0.66 g g-1), Ogataea parapolymorpha (0.67 g g-1), and Cyberlindnera jadinii (0.73 g g-1). Cyberlindnera jadinii biomass showed the highest protein content (59 ± 2%) of these yeasts. Its biomass yield corresponded to 88% of the theoretical maximum that is reached when growth is limited by assimilation rather than by energy availability. This study suggests that energy coupling of mitochondrial respiration and its regulation will become key factors for selecting and improving yeast strains for ethanol-based processes.

Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.

Lignocellulose (dry plant biomass) is an abundant cheap inedible residue of agriculture and wood industry with great potential as a feedstock for biotechnological processes. Lignocellulosic substrates can serve as valuable resources in fermentation processes, allowing the production of a wide array of chemicals, fuels, and food additives. The main obstacle for cost-effective conversion of lignocellulosic hydrolysates to target products is poor metabolism of the major pentoses, xylose and L-arabinose, which are the second and third most abundant sugars of lignocellulose after glucose. We study the oversynthesis of riboflavin in the flavinogenic yeast Candida famata and found that all major lignocellulosic sugars, including xylose and L-arabinose, support robust growth and riboflavin synthesis in the available strains of C. famata. To further increase riboflavin production from xylose and lignocellulose hydrolysate, genes XYL1 and XYL2 coding for xylose reductase and xylitol dehydrogenase were overexpressed. The resulting strains exhibited increased riboflavin production in both shake flasks and bioreactors using diluted hydrolysate, reaching 1.5 g L-1.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.