scholarly journals Functional expression of the lactate permease Jen1p of Saccharomyces cerevisiae in Pichia pastoris

2003 ◽  
Vol 376 (3) ◽  
pp. 781-787 ◽  
Author(s):  
Isabel SOARES-SILVA ◽  
Dorit SCHULLER ◽  
Raquel P. ANDRADE ◽  
Fátima BALTAZAR ◽  
Fernanda CÁSSIO ◽  
...  

In Saccharomyces cerevisiae the activity for the lactate–proton symporter is dependent on JEN1 gene expression. Pichia pastoris was transformed with an integrative plasmid containing the JEN1 gene. After 24 h of methanol induction, Northern and Western blotting analyses indicated the expression of JEN1 in the transformants. Lactate permease activity was obtained in P. pastoris cells with a Vmax of 2.1 nmol·s−1·mg of dry weight−1. Reconstitution of the lactate permease activity was achieved by fusing plasma membranes of P. pastoris methanol-induced cells with Escherichia coli liposomes containing cytochrome c oxidase, as proton-motive force. These assays in reconstituted heterologous P. pastoris membrane vesicles demonstrate that S. cerevisiae Jen1p is a functional lactate transporter. Moreover, a S. cerevisiae strain deleted in the JEN1 gene was transformed with a centromeric plasmid containing JEN1 under the control of the glyceraldehyde-3-phosphate dehydrogenase constitutive promotor. Constitutive JEN1 expression and lactic acid uptake were observed in cells grown on either glucose and/or acetic acid. The highest Vmax (0.84 nmol·s−1·mg of dry weight−1) was obtained in acetic acid-grown cells. Thus overexpression of the S. cerevisiae JEN1 gene in both S. cerevisiae and P. pastoris cells resulted in increased activity of lactate transport when compared with the data previously reported in lactic acid-grown cells of native S. cerevisiae strains. Jen1p is the only S. cerevisiae secondary porter characterized so far by heterologous expression in P. pastoris at both the cell and the membrane-vesicle levels.

Fermentation ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 59
Author(s):  
Timothy J. Tse ◽  
Daniel J. Wiens ◽  
Jianheng Shen ◽  
Aaron D. Beattie ◽  
Martin J. T. Reaney

As barley and oat production have recently increased in Canada, it has become prudent to investigate these cereal crops as potential feedstocks for alcoholic fermentation. Ethanol and other coproduct yields can vary substantially among fermented feedstocks, which currently consist primarily of wheat and corn. In this study, the liquified mash of milled grains from 28 barley (hulled and hull-less) and 12 oat cultivars were fermented with Saccharomyces cerevisiae to determine concentrations of fermentation products (ethanol, isopropanol, acetic acid, lactic acid, succinic acid, α-glycerylphosphorylcholine (α-GPC), and glycerol). On average, the fermentation of barley produced significantly higher amounts of ethanol, isopropanol, acetic acid, succinic acid, α-GPC, and glycerol than that of oats. The best performing barley cultivars were able to produce up to 78.48 g/L (CDC Clear) ethanol and 1.81 g/L α-GPC (CDC Cowboy). Furthermore, the presence of milled hulls did not impact ethanol yield amongst barley cultivars. Due to its superior ethanol yield compared to oats, barley is a suitable feedstock for ethanol production. In addition, the accumulation of α-GPC could add considerable value to the fermentation of these cereal crops.


2019 ◽  
Vol 20 (7) ◽  
pp. 1659
Author(s):  
Katarzyna Pielech-Przybylska ◽  
Maria Balcerek ◽  
Grzegorz Ciepielowski ◽  
Barbara Pacholczyk-Sienicka ◽  
Łukasz Albrecht ◽  
...  

The qualitative and quantitative composition of volatile compounds in fermented distillery mash determines the quality of the obtained distillate of agricultural origin (i.e., raw spirit) and the effectiveness of further purification steps. Propan-2-ol (syn. isopropyl alcohol), due to its low boiling point, is difficult to remove by rectification. Therefore, its synthesis needs to be limited during fermentation by Saccharomyces cerevisiae yeast, while at the same time controlling the levels of acetaldehyde and acetic acid, which are likewise known to determine the quality of raw spirit. Lactic acid bacteria (LAB) are a common but undesirable contaminant in distillery mashes. They are responsible for the production of undesirable compounds, which can affect synthesis of propan-2-ol. Some bacteria strains are able to synthesize isopropyl alcohol. This study therefore set out to investigate whether LAB with S. cerevisiae yeast are responsible for conversion of acetone to propan-2-ol, as well as the effects of the amount of LAB inoculum and fermentation parameters (pH and temperature) on the content of isopropyl alcohol, acetaldehyde, lactic acid and acetic acid in fermented mashes. The results of NMR and comprehensive two-dimensional gas chromatography coupled with time of flight mass spectrometry (GC × GC-TOF MS) analysis confirmed the ability of the yeast and LAB strains to metabolize acetone via its reduction to isopropyl alcohol. Efficient fermentation of distillery mashes was observed in all tested mashes with an initial LAB count of 3.34–6.34 log cfu/mL, which had no significant effect on the ethanol content. However, changes were observed in the contents of by-products. Lowering the initial pH of the mashes to 4.5, without and with LAB (3.34–4.34 log cfu/mL), resulted in a decrease in propan-2-ol and a concomitant increase in acetaldehyde content, while a higher pH (5.0 and 5.5) increased the content of propan-2-ol and decreased acetaldehyde content. Higher temperature (35 °C) promoted propan-2-ol synthesis and also resulted in increased acetic acid content in the fermented mashes compared to the controls. Moreover, the acetic acid content rose with increases in the initial pH and the initial LAB count.


Author(s):  
Anna Ylinen ◽  
Hannu Maaheimo ◽  
Adina Anghelescu-Hakala ◽  
Merja Penttilä ◽  
Laura Salusjärvi ◽  
...  

Abstract Polyhydroxyalkanoates (PHA) provide biodegradable and bio-based alternatives to conventional plastics. Incorporation of 2-hydroxy acid monomers into polymer, in addition to 3-hydroxy acids, offers possibility to tailor the polymer properties. In this study, poly(D-lactic acid) (PDLA) and copolymer P(LA-3HB) were produced and characterized for the first time in the yeast Saccharomyces cerevisiae. Expression of engineered PHA synthase PhaC1437Ps6–19, propionyl-CoA transferase Pct540Cp, acetyl-CoA acetyltransferase PhaA, and acetoacetyl-CoA reductase PhaB1 resulted in accumulation of 3.6% P(LA-3HB) and expression of engineered enzymes PhaC1Pre and PctMe resulted in accumulation of 0.73% PDLA of the cell dry weight. According to NMR, P(LA-3HB) contained D-Lactic acid repeating sequences. For reference, expression of PhaA, PhaB1, and PHA synthase PhaC1 resulted in accumulation 11% poly(hydroxybutyrate) (PHB) of the cell dry weight. Weight average molecular weights of these polymers were comparable to similar polymers produced by bacterial strains, 24.6 kDa, 6.3 kDa, and 1 130 kDa, for P(LA-3HB), PDLA, and PHB, respectively. The results suggest that yeast, as a robust and acid tolerant industrial production organism, could be suitable for production of 2-hydroxy acid containing PHAs from sugars or from 2-hydroxy acid containing raw materials. Moreover, the wide substrate specificity of PHA synthase enzymes employed increases the possibilities for modifying copolymer properties in yeast in the future.


2002 ◽  
Vol 68 (4) ◽  
pp. 1616-1623 ◽  
Author(s):  
K. C. Thomas ◽  
S. H. Hynes ◽  
W. M. Ingledew

ABSTRACT Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.


2021 ◽  
Vol 12 ◽  
Author(s):  
Siyuan Sun ◽  
Xiuxiu Xu ◽  
Ling Liang ◽  
Xiaoli Wang ◽  
Xue Bai ◽  
...  

Lactic acid, a metabolic by-product of host and intestinal microbiota, has been recovered as an active signal molecule in the immune system. In this study, a lactic acid biosynthesis pathway that directly produces lactic acid from glucose rather than ethanol with high production was reconstructed in Saccharomyces cerevisiae. The engineered S. cerevisiae showed anti-inflammatory activity in dextran sulfate sodium (DSS)-induced mice with improved histological damage, increased mucosal barrier, and decreased intestinal immune response. Lactic acid regulated the macrophage polarization state and inhibited the expression of pro-inflammatory cytokines in vivo and in vitro. Increasing the macrophage monocarboxylic acid transporter-mediated active lactic acid uptake suppressed the excessive activation of the NLRP3 inflammasome and the downstream caspase-1 pathway in macrophages. Moreover, lactic acid promoted histone H3K9 acetylation and histone H3K18 lactylation. Meanwhile, the engineered S. cerevisiae altered the diversity and composition of the intestinal microbiota and changed the abundance of metabolic products in mice with colitis. In conclusion, this study shows that the application of engineered S. cerevisiae attenuated DSS-induced colitis in mice via suppressing macrophage pyroptosis and modulating the intestinal microbiota, which is an effective and safe treatment strategy for ulcerative colitis.


2018 ◽  
Vol 37 (3) ◽  
pp. 302
Author(s):  
Mulono Apriyanto ◽  
Sutardi Sutardi ◽  
Supriyanto Supriyanto ◽  
Eni Harmayani

The aims of the study was to improve quality of cocoa bans by fermentation of sun dried cocoa beans. The fermentation variations were conducted as follows: first, fermentation without the addition of inoculum (control), the second treatment using inoculum of S. cerevisiae (FNCC 3056), L. lactis (FNC 0086) and A. aceti (FNCC 0016), each of 108 cfu/g  given simultaneously at the beginning of fermentation.and the third treatment wassequential administration, i.e: yeast at the initial fermentation, lactic acid bacteria after 24 hours fermentation, and acetic acid bacteria after 48 hr of fermentation third with the same microbial population with the second treatment. The fermentation was conducted for120 hours. The fermentation temperature were controlled during fermentation as follows: 35 °C  for the first 24 hours, 45 °C  for the next second 24- hours, 55 °C the third 24 hours and 35 °C for the last 48 hours of fermentation. The results showed that after the rehydration, pulp composition of dry beans could be used as a substrate for fermentation. During fermentation, dry cocoa beans showed reduction of total sugar content, pH and total polyphenols for all the three treatments. Cut test of dried cocoa beans during the fermentation showed the increasing percentage of brown color of the three treatments. Reducing sugar and fermentation indexes increasedfor all treatments during fermentation. Concentration of ethanol, lactic acid and acetic acid reached highest level at 24, 60, and 108 hours of fermentationfor all treatments.  Highest populations of S. cerevisiae, L. lactis and A. aceti of three treatments obtained at 24, 48 and 72 hours of fermentation. After fermentation and roasting, dry beans produced hydrophobic amino acids as precursors of flavor and volatile compounds.                                               ABSTRAKPenelitian ini bertujuan untuk mengetahui perubahan sifat kimia pada fermentasi biji kakao kering jemur. Biji kakao kering jemur yang diperoleh dari petani memiliki kadar air yang tidak seragam. Guna menimalkan kegagalan fermentasi maka biji kakao kering jemur diperoleh melalui pengeringan biji kakao segar menggunakan kabinet dryer dengan sebelumnya dikondisikan pada suhu seperti pengeringan dengan sinar matahari, dan masing ditentukan kadar gula reduksinya. Percobaan fermentasi biji kakao kering dilakukan fermentasi pada wadah fermentasi dengan jumlah biji 150 g setiap wadah. Sebelum difermentasi terlebih dahulu biji kakao kering jemur direhidrasi agar didapat kadar air mendekati biji segar, kemudian biji kakao kering jemur diinkubasi selama enam hari dan tanpa dibalik selama fermentasi. Setiap perlakuan diulangi tiga kali dan diamati tiap 24 jam sampai 120 jam. Kadar gula reduksi (kontrol 4,49–11,45%, inokulum diawal (IA) 4,69–11,55%, inokulum bertahap (IB) 4,64–11,54%), kadar asam tertitrasi (kontrol 4,48–6,45%, inokulum diawal (IA) 4,64–6,39%, inokulum bertahap (IB)  4,45–6,59%), populasi Saccharomycescerevisiae (kontrol 5,56–7,28 (log CFU/g), inokulum diawal (IA) 6,45–8,75 (logCFU/g), inokulum bertahap (IB) 6.88 – 8.99 (logCFU/g), Lactobacillus lactis (kontrol 6,66–8,15 (log CFU/g), inokulum diawal (IA) 7,65–8,21(log CFU/g), inokulum bertahap (IB) 7,66–8,95 (log CFU/g) dan Acetobacter aceti (kontrol 4,26–6,95% (log CFU/g), inokulum diawal (IA) 4,85–7,40 (log CFU/g), inokulum bertahap (IB) 4,35–7,91 (log CFU/g)) dalam pulp fermentasi diamati selama proses fermentasi. Untuk mengetahui kualitas biji kakao dilakukan pengukuran pH (kontrol 5,67–3,98, inokulum diawal (IA) 5,67–3,55, inokulum bertahap (IB) 5,67–3,50), kadar etanol (kontrol 0,3–0,5%, inokulum diawal (IA) 0,3–0,52%, inokulum bertahap (IB) 0,35–0,53%) dan indeks fermentasi selama fermentasi (kontrol 0,31–0,88, inokulum diawal (IA) 0,32–0,99, inokulum bertahap (IB) 0,33–1,03).Kata kunci: Acetobacter aceti; biji kakao kering jemur; fermentasi; Lactobacillus lactis; Saccharomyces cerevisiae


2021 ◽  
Vol 7 (11) ◽  
pp. 928
Author(s):  
Byeong-Kwan Jang ◽  
Yebin Ju ◽  
Deokyeol Jeong ◽  
Sung-Keun Jung ◽  
Chang-Kil Kim ◽  
...  

Lactic acid is mainly used to produce bio-based, bio-degradable polylactic acid. For industrial production of lactic acid, engineered Saccharomyces cerevisiae can be used. To avoid cellular toxicity caused by lactic acid accumulation, pH-neutralizing agents are used, leading to increased production costs. In this study, lactic acid-producing S. cerevisiae BK01 was developed with improved lactic acid tolerance through adaptive laboratory evolution (ALE) on 8% lactic acid. The genetic basis of BK01 could not be determined, suggesting complex mechanisms associated with lactic acid tolerance. However, BK01 had distinctive metabolomic traits clearly separated from the parental strain, and lactic acid production was improved by 17% (from 102 g/L to 119 g/L). To the best of our knowledge, this is the highest lactic acid titer produced by engineered S. cerevisiae without the use of pH neutralizers. Moreover, cellulosic lactic acid production by BK01 was demonstrated using acetate-rich buckwheat husk hydrolysates. Particularly, BK01 revealed improved tolerance against acetic acid of the hydrolysates, a major fermentation inhibitor of lignocellulosic biomass. In short, ALE with a high concentration of lactic acid improved lactic acid production as well as acetic acid tolerance of BK01, suggesting a potential for economically viable cellulosic lactic acid production.


2000 ◽  
Vol 13 (5) ◽  
pp. 377-384 ◽  
Author(s):  
Birgit Morawski ◽  
Zhanglin Lin ◽  
Pat Cirino ◽  
Hyun Joo ◽  
Geethani Bandara ◽  
...  

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