Activation of the yeast Retrograde Response pathway by adaptive laboratory evolution with S-(2-aminoethyl)-L-cysteine reduces ethanol and increases glycerol during winemaking
Ontology highlight
ABSTRACT:
SUBMITTER: Garrigos V
PROVIDER: S-EPMC11337681 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:In yeast, ethanol is produced as a by-product of fermentation through glycolysis. Ethanol also stimulates a developmental foraging response called filamentous growth and is thought to act as a quorum-sensing molecule. Ethanol-inducible filamentous growth was examined in a small collection of wine/European strains, which validated ethanol as an inducer of filamentous growth. Wine strains also showed variability in their filamentation responses, which illustrates the striking phenotypic differences that can occur among individuals. Ethanol-inducible filamentous growth in Σ1278b strains was independent of several of the major filamentation regulatory pathways [including fMAPK, RAS-cAMP, Snf1, Rpd3(L), and Rim101] but required the mitochondrial retrograde (RTG) pathway, an inter-organellar signaling pathway that controls the nuclear response to defects in mitochondrial function. The RTG pathway regulated ethanol-dependent filamentous growth by maintaining flux through the TCA cycle. The ethanol-dependent invasive growth response required the polarisome and transcriptional induction of the cell adhesion molecule Flo11p. Our results validate established stimuli that trigger filamentous growth and show how stimuli can trigger highly specific responses among individuals. Our results also connect an inter-organellar pathway to a quorum sensing response in fungi.
| S-EPMC5372830 | biostudies-literature
Project description:Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate-glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.
| S-EPMC6888959 | biostudies-literature
Project description:Production and balance of glycerol is essential for the survival of yeast cells in certain stressful conditions as hyperosmotic or cold shock that occur during industrial processes as winemaking. These stress responses are well-known in S. cerevisiae, however, little is known in other phylogenetically close related Saccharomyces species associated with natural or fermentation environments such as S. uvarum, S. paradoxus or S. kudriavzevii. In this work we have investigated the expression of four genes (GPD1, GPD2, STL1, and FPS1) crucial in the glycerol pool balance in the four species with a biotechnological potential (S. cerevisiae; S. paradoxus; S. uvarum; and S. kudriavzevii), and the ability of strains to grow under osmotic and cold stresses. The results show different pattern and level of expression among the different species, especially for STL1. We also studied the function of Stl1 glycerol symporter in the survival to osmotic changes and cell growth capacity in winemaking environments. These experiments also revealed a different functionality of the glycerol transporters among the different species studied. All these data point to different strategies to handle glycerol accumulation in response to winemaking stresses as hyperosmotic or cold-hyperosmotic stress in the different species, with variable emphasis in the production, influx, or efflux of glycerol.
| S-EPMC4814467 | biostudies-literature
Project description:Glycerol is a major by-product of industrial ethanol production and its formation consumes up to 4 % of the sugar substrate. This study modified the glycerol decomposition pathway of an industrial strain of Saccharomyces cerevisiae to optimize the consumption of substrate and yield of ethanol. This study is the first to couple glycerol degradation with ethanol formation, to the best of our knowledge. The recombinant strain overexpressing GCY1 and DAK1, encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, in glycerol degradation pathway, exhibited a moderate increase in ethanol yield (2.9 %) and decrease in glycerol yield (24.9 %) compared to the wild type with the initial glucose concentration of 15 % under anaerobic conditions. However, when the mhpF gene, encoding acetylating NAD?-dependent acetaldehyde dehydrogenase from Escherichia coli, was co-expressed in the aforementioned recombinant strain, a further increase in ethanol yield by 5.5 % and decrease in glycerol yield by 48 % were observed for the resultant recombinant strain GDMS1 when acetic acid was added into the medium prior to inoculation compared to the wild type. The process outlined in this study which enhances glycerol consumption and cofactor regulation in an industrial yeast is a promising metabolic engineering strategy to increase ethanol production by reducing the formation of glycerol.
| S-EPMC3769588 | biostudies-literature
Project description:Biotechnology suggests that microorganisms can be used as chemical factories that transform renewable feedstock into value-added chemicals. Conversion of glycerol, using direct transformation or fermentation, into valuable products such as polymers, surfactants, solvents, and chemical intermediates has attained growing interest in recent years due to the dramatic growth of the biodiesel industry. However, the use of cell factories could be limited by low growth and uptake rates under certain environmental conditions, thus understanding microbial nutritional requirements is a critical point to use them as cell factories. Here, we compared E. coli ATCC 8739 transcriptomic responses to glycerol under aerobic conditions in an optimized culture medium (Condition 3) and one evolved strain in glycerol using as a reference a glycerol-based medium (Condition 11). Our analyses revealed 478 and 431 differentially expressed genes (DEGs) with log2 fold change (FC) > |2| and p Adjusted value < 0.05, for the bacteria growing in the optimized culture medium and the evolved strain, respectively. Among the DEGs, glp operon was found to be up-regulated as a response to glycerol uptake. Interestingly, between them, it was found genes that requires the use of phosphorous to ovoid the toxicity during glycerol consumption. Previously, we identified using a computational approach that phosphorous and nitrogen are essential compound that support high glycerol consumption in E. coli.
2019-11-23 | GSE140847 | GEO
Project description:An S288c-derived lab strain of Saccharomyces cerevisiae has lower ethanol resistance than either the vineyard strain M22 or the oak strain YPS163. We performed a 60 minute time-course experiment in the presence of 5% ethanol and compared the transcriptional response to ethanol between strains. We additionally analyzed the time-point of maximal response (30 min.) in wild type cells, YPS163 msn2D, and YPS163 hap1D cells.
2010-12-17 | GSE22904 | GEO
Project description:BackgroundAdaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation.ResultsHere, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmolEtOH gCDW-1 h-1 and a growth rate of 0.15 ± 0.01 h-1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements.ConclusionsThe established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.
| S-EPMC10483779 | biostudies-literature
Project description:Diacetyl contributes to the flavor profile of many fermented products. Its typical buttery flavor is considered as an off flavor in lager-style beers, and its removal has a major impact on time and energy expenditure in breweries. Here, we investigated the possibility of lowering beer diacetyl levels through evolutionary engineering of lager yeast for altered synthesis of α-acetolactate, the precursor of diacetyl. Cells were exposed repeatedly to a sub-lethal level of chlorsulfuron, which inhibits the acetohydroxy acid synthase responsible for α-acetolactate production. Initial screening of 7 adapted isolates showed a lower level of diacetyl during wort fermentation and no apparent negative influence on fermentation rate or alcohol yield. Pilot-scale fermentation was carried out with one isolate and results confirmed the positive effect of chlorsulfuron adaptation. Diacetyl levels were over 60% lower at the end of primary fermentation relative to the non-adapted lager yeast and no significant change in fermentation performance or volatile flavor profile was observed due to the adaptation. Whole-genome sequencing revealed a non-synonymous SNP in the ILV2 gene of the adapted isolate. This mutation is known to confer general tolerance to sulfonylurea compounds, and is the most likely cause of the improved tolerance. Adaptive laboratory evolution appears to be a natural, simple and cost-effective strategy for diacetyl control in brewing.
| S-EPMC6267509 | biostudies-literature
Project description:An S288c-derived lab strain of Saccharomyces cerevisiae has lower ethanol resistance than either the vineyard strain M22 or the oak strain YPS163. We performed a 60 minute time-course experiment in the presence of 5% ethanol and compared the transcriptional response to ethanol between strains. We additionally analyzed the time-point of maximal response (30 min.) in wild type cells, YPS163 msn2D, and YPS163 hap1D cells. The genomic expression response to 5% ethanol stress was measured in 3 different strain backgrounds, and 2 different mutant backgrounds. Basal gene expression was also compared in two non-lab strains to the S288c reference. A single replicate was performed for the time-course experiment. All of the single time-point experiments were performed using biological triplicates.
2010-12-17 | E-GEOD-22904 | biostudies-arrayexpress
Project description:Thermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids, conferring partial AEC resistance to the wild type, were obtained. Two cosmids carried a common genomic region from TTC0795 to TTC0810. This region contains genes encoding an ATP-binding cassette (ABC) transporter consisting of TTC0806/TTC0795, using TTC0807 as the periplasmic substrate-binding protein. Sequencing revealed that AT14 carries mutations in TTC0795 and TTC0969, causing decreases in the thermostability of the products. TTC0969 encodes the nucleotide-binding protein of a different ABC transporter consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0966 as the periplasmic substrate-binding protein. By similar screening for cosmids constructed for the mutant AT10, mutations were found at TTC0807 and TTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substrate-binding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.
| S-EPMC3754599 | biostudies-literature