Mechanisms underlying the acquisition of resistance to octanoic-acid-induced-death following exposure of Saccharomyces cerevisiae to mild stress imposed by octanoic acid or ethanol

2001 ◽  
Vol 175 (4) ◽  
pp. 301-307 ◽  
Author(s):  
M. Guadalupe Cabral ◽  
Cristina A. Viegas ◽  
Isabel Sá-Correia
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Octanoic Acid ◽  
Mild Stress

Regulation of the expression of the H+-ATPase genes PMA1 and PMA2 during growth and effects of octanoic acid in Saccharomyces cerevisiae

1994 ◽  
Vol 1217 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Cristina A. Viegas ◽  
Philip Supply ◽  
Etienne Capieaux ◽  
Luc Van Dyck ◽  
André Goffeau ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Octanoic Acid

scholarly journals Mild Pretreatments to Increase Fructose Consumption in Saccharomyces cerevisiae Wine Yeast Strains

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1129
Author(s):  
Hatice Aybuke Karaoglan ◽  
Filiz Ozcelik ◽  
Alida Musatti ◽  
Manuela Rollini
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Wine Yeast ◽  
Temperature Treatment ◽  
Mean Bias Error ◽  
Bias Error ◽  
Model Parameters ◽  
Suitable Model ◽  
Mild Stress ◽  
Chi Square ◽  
Sigmoidal Model

The present research investigates the effect of different pretreatments on glucose and fructose consumption and ethanol production by four Saccharomyces cerevisiae wine strains, three isolated and identified from different wine regions in Turkey and one reference strain. A mild stress temperature (45 °C, 1 h) and the presence of ethanol (14% v/v) were selected as pretreatments applied to cell cultures prior to the fermentation step in synthetic must. The goodness fit of the mathematical models was estimated: linear, exponential decay function and sigmoidal model were evaluated with the model parameters R2 (regression coefficient), RMSE (root mean square error), MBE (mean bias error) and χ2 (reduced Chi-square). Sigmoidal function was determined as the most suitable model with the highest R2 and lower RMSE values. Temperature pretreatment allowed for an increase in fructose consumption rate by two strains, evidenced by a t90 value 10% lower than the control. One of the indigenous strains showed particular promise for mild temperature treatment (45 °C, 1 h) prior to the fermentation step to reduce residual glucose and fructose in wine. The described procedure may be effective for indigenous yeasts in preventing undesirable sweetness in wines.

scholarly journals Transcriptomic response of Saccharomyces cerevisiae to octanoic acid production

2021 ◽  
Vol 21 (2) ◽  
Author(s):  
Leonie Baumann ◽  
Tyler Doughty ◽  
Verena Siewers ◽  
Jens Nielsen ◽  
Eckhard Boles ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Acid Production ◽  
Octanoic Acid ◽  
Chain Fatty Acid ◽  
Medium Chain Fatty Acid ◽  
Cell Physiology ◽  
Transcriptomic Response ◽  
Promising Alternative ◽  
Medium Chain

ABSTRACT The medium-chain fatty acid octanoic acid is an important platform compound widely used in industry. The microbial production from sugars in Saccharomyces cerevisiae is a promising alternative to current non-sustainable production methods, however, titers need to be further increased. To achieve this, it is essential to have in-depth knowledge about the cell physiology during octanoic acid production. To this end, we collected the first RNA-Seq data of an octanoic acid producer strain at three time points during fermentation. The strain produced higher levels of octanoic acid and increased levels of fatty acids of other chain lengths (C6–C18) but showed decreased growth compared to the reference. Furthermore, we show that the here analyzed transcriptomic response to internally produced octanoic acid is notably distinct from a wild type's response to externally supplied octanoic acid as reported in previous publications. By comparing the transcriptomic response of different sampling times, we identified several genes that we subsequently overexpressed and knocked out, respectively. Hereby we identified RPL40B, to date unknown to play a role in fatty acid biosynthesis or medium-chain fatty acid tolerance. Overexpression of RPL40B led to an increase in octanoic acid titers by 40%.

scholarly journals Development and implementation of strategies to improve octanoic acid production in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
◽  
Leonie Baumann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Octanoic Acid ◽  
Chain Fatty Acid ◽  
Producer Strain ◽  
Medium Chain ◽  
Improved Strain ◽  
Producer Strains ◽  
Chain Fatty Acids

Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative. Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain. The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated. A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts. In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts. ...

scholarly journals Mannoprotein Content and Volatile Molecule Profiles of Trebbiano Wines Obtained by Saccharomyces cerevisiae and Saccharomyces bayanus Strains

Fermentation ◽  
2019 ◽  
Vol 5 (3) ◽  
pp. 66
Author(s):  
Braschi ◽  
Ricci ◽  
Grazia ◽  
Versari ◽  
Patrignani ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethyl Ester ◽  
Octanoic Acid ◽  
Acid Ethyl Ester ◽  
Principal Component ◽  
Decanoic Acid ◽  
Aroma Compound ◽  
Propanoic Acid ◽  
Saccharomyces Bayanus ◽  
Volatile Profiles

: The production of volatile compounds has become one of the major technological features for yeast selection. In fact, although the aromatic profile of the wine is the sum of varietal-, pre-, post-, and fermentative-aroma compound, yeasts affect the quality of the grape from maturation throughout fermentation, metabolizing sugars and other components into alcohols, esters, organic acids, and aldehydes. Among the new technological features, the production of mannoproteins has gained interest. From this perspective, the main aim of this work was to characterize 9 strains of Saccharomyces cerevisiae and 1 of Saccharomyces bayanus for their volatile profiles and the release of mannoproteins. The strains were inoculated in Trebbiano musts and incubated at 15 °C; at the end of fermentation the wines were evaluated by GC/MS/SPME for their volatile profiles and mannoprotein content by enzymatic assay. The strains were inoculated at level ranging between 4.9 and 6.3 log CFU/mL but only the strains L318 and 12233X6167 were able to reach values of 7.5 log CFU/mL. The aromatic profiles resulted in a strain-specific fingerprinting. According to the principal component analysis, the wines produced by the strains L288, L234, and L318 were characterized by the presence of propanoic acid, butanol, octanoic acid, and 3 methyl pentanol while the wine obtained by the strain 12233x35G2 was characterized by the presence of propanoic acid, butanol, octanoic acid and 3 methyl pentanol while the strain 12233x35G2 was characterized by the presence of decanoic acid ethyl ester, heptanoic acid ethyl ester, and acetic acid 2 phenetyl ester. Regarding mannoproteins, the highest concentration was achieved by strain12233x6167 (104 mg/L). The data allowed to select the strains endowed with the best fermentation performances in terms of aroma and mannoproteins release.

scholarly journals Changes in trehalose content of baker's yeast as affected by octanoic acid

1993 ◽  
Vol 50 (3) ◽  
pp. 460-463 ◽  
Author(s):  
L.E. Gutierrez
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Octanoic Acid ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Ethanolic Fermentation ◽  
Trehalose Content ◽  
Trehalose Accumulation

Octanoic acid inhibited ethanolic fermentation by Saccharomyces cerevisiae (bakers yeast) and the trehalose accumulation, however did not affect the endogenous degradation of trehalose. This inhibition may be explained by the binding of octanoic acid to hexokinase or other proteins of plasma membrane because they are not necessary for endogenous fermentation. The degradation of trehalose may be due to an activation of trehalase.

scholarly journals Metabolic engineering of Saccharomyces cerevisiae for production of medium-chain fatty acids and their derivatives

2021 ◽  
Author(s):  
◽  
Florian Wernig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Metabolic Engineering ◽  
Acid Production ◽  
Fatty Acid Synthase ◽  
Octanoic Acid ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Acetyl Coa

The oleochemical and petrochemical industries provide diverse chemicals used in personal care products, food and pharmaceutical industries or as fuels, oils, polymers and others. However, fossil resources are dwindling and concerns about these conventional production methods have risen due to their strong negative impact on the environment and contribution to climate change. Therefore, alternative, sustainable and environmentally friendly production methods for oleochemical compounds such as fatty acids, fatty alcohols, hydroxy fatty acids and dicarboxylic acids are desired. The biotechnological production by engineered microorganism could fulfill these requirements. The concept of metabolic engineering, which is the modification of metabolic pathways of a host organism for increased production of a target compound, is a widely used strategy in biotechnology to generate cell factories or chassis strains for robust, efficient and high production. In this work, the versatile model and industrial yeast Saccharomyces cerevisiae was manipulated by metabolic engineering strategies for increased production of the medium-chain fatty acid octanoic acid and de novo production the derived 8-hydroxyoctanoic acid. Octanoic acid production was enabled by the fatty acid biosynthesis pathway by use of a mutated fatty acid synthase (FASRK) in a wild type FAS deficient strain. The yeast fatty acid synthase (FAS) consists of two polypeptides, α and β, which assemble to a α6β6 complex in a co-translational manner by interaction of the subunits. Because this step might be subject to cellular regulation, the α- and β- subunits of fatty acid synthase were fused to form a single-chain construct (fusFASRK), which displayed superior octanoic acid production compared with split FASRK. Thus, FASRK expression was identified as a limiting step of octanoic acid production. But the strains that produce octanoic acid have a severe growth defect that is undesirable for biotechnological applications and could lead to lower production titers. One reason is the strong inhibitory effect of octanoic acid. Another possibility is that the mutant FAS no longer produces enough essential long-chain fatty acids. To compensate for this, the mutated split and fused FAS variants were co-expressed individually in a strain harboring genomic wild type FAS alleles. In addition, mutant and wild type variants of fused and split FAS were co-expressed together in a FAS deficient strain. However, both cases resulted in decreased octanoic acid titers potentially by physical and/or metabolic crosstalk of the FAS variants. The fatty acid biosynthesis relies on cytosolic acetyl-CoA for initiation and derived malonyl-CoA for elongation and requires NADPH for reductive power. To increase production of octanoic acid, engineering strategies for increased acetyl-CoA and NADHP supply were investigated. First, the flux through the native cytosolic acetyl-CoA and NADPH providing pyruvate dehydrogenase bypass was enhanced by overexpression of the target genes ADH2, ALD6 and ACSL461P from Salmonella enterica in combination or individually. Next, the acety-CoA forming heterologous phosphoketolase/phosphotransacetylase pathway was expressed and NADPH formation was increased by redirecting the flux of glucose-6-phosphate into the NADPH producing oxidative branch of the pentose phosphate pathway. In particular, the flux through glycolysis and pyruvate dehydrogenase bypass was reduced by downregulating the expression of the phosphoglucose isomerase PGI1 and deleting the acetaldehyde dehydrogenase ALD6. Glucose-6-phosphate was guided into the pentose phosphate pathway by overexpressing the glucose-6-phosphate dehydrogenase ZWF1. The first approach did not influence octanoic acid production but the latter increased yields in the glucose consumption phase by 65 %. However, combining the superior fusFASRK with acetyl-CoA and NADPH supply engineering strategies did not result in additive production effects, indicating that other limitations hinder high octanoic acid accumulation. Limitations could be caused in particular by the strong inhibitory effects of octanoic acid or by intrinsic limitations of the FASRK mutant. To enlarge the octanoic acid production platform towards other derived valuable oleochemical compounds the de novo production of 8-hydroxyoctanoic acid was targeted. Since short- and medium-chain fatty acids have a strong inhibitory effect on Saccharomyces cerevisiae, the inhibitory effect of hydroxy fatty acid and dicarboxylic with eight or ten carbon atoms were compared and revealed only little or no growth impairment. Subsequently, the formation of 8-hydroxyoctanoic acid was targeted by a terminal hydroxylation of externally supplied octanoic acid in a bioconversion. For that, three heterologous genes, encoding for cytochromes P450 enzymes and their cognate cytochrome P450 reductases were expressed and 8-hydroxyoctanoic acid production was compared. In addition, the use of different carbon sources was compared. ...

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