Improve the production of d-limonene by regulating the mevalonate pathway of Saccharomyces cerevisiae during alcoholic beverage fermentation

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast.

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Transcriptional regulation of a sterol-biosynthetic enzyme by sterol levels in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.3981 ◽

1996 ◽

Vol 16 (8) ◽

pp. 3981-3989 ◽

Cited By ~ 46

Author(s):

D Dimster-Denk ◽

J Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Regulation ◽

Feedback Control ◽

Dna Sequences ◽

Promoter Region ◽

Mevalonate Pathway ◽

Transcriptional Level ◽

Biosynthetic Enzyme ◽

Animal Cells ◽

Regulatory Response

Sterols and all nonsterol isoprenoids are derived from the highly conserved mevalonate pathway. In animal cells, this pathway is regulated in part at the transcriptional level through the action of sterol response element-binding proteins acting at specific DNA sequences near promoters. Here we extend at least part of this regulatory paradigm to the ERG10 gene, which encodes a sterol-biosynthetic enzyme of Saccharomyces cerevisiae. Specifically, the discovery of sterol-mediated feedback control of ERG10 transcription is reported. Deletion analysis of the ERG10 promoter region identified sequences involved in the expression of ERG10. This regulatory axis appeared to involve sterol levels, as a late block in the pathway that depletes sterol, but not nonsterol isoprenoids, was able to elicit the regulatory response.

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YEAST DYNAMICS AND PHYSIOCHEMICAL EVALUATION OF CARROT WINE PRODUCED WITH Saccharomyces cerevisiae

Fungal Territory ◽

10.36547/ft.2020.3.3.27-29 ◽

2020 ◽

Vol 3 (3) ◽

pp. 27-29

Author(s):

TOCHUKWU VINCENT` BALOGU

Keyword(s):

Saccharomyces Cerevisiae ◽

Crude Protein ◽

Alcoholic Beverage ◽

Extraction Process ◽

Proximate Analysis ◽

Nutritional Composition ◽

Alcohol Content ◽

Juice Extraction ◽

Total Carbohydrates ◽

Yeast Dynamics

Yeast dynamics and physiochemical evaluation of carrot wine produced with Saccharomyces cerevisiae were assessed. Fresh ripe and health carrot (6kg) were sequentially processed (washed, preheated, blended and sieved) into juice and fermented for 60 days with Saccharomyces cerevisiae. Airtight glass jars composed of juice (2000g), distilled water (2000mL) and sugar (200g) at controlled temperature (20 -25 0C) was used for fermentation. Wines were clarified (siphoning), aged (45 days) and pasteurized (500C – 600C) to stop fermentation. Proximate analysis, yeast dynamics, physiochemical and wine qualities were assessed. Result showed that juice extraction process reconstitute nutritional composition of carrot, such that moisture, ash and total carbohydrates increased, while others (fat, crude fiber and crude protein) decreased. A trendy progressive yeast dynamic model of Yeast load = -0.195 (Day) 2 + 1.822 (Day) + 4.566 with coefficient (R² = 0.907) was observed. Fermentation significantly decreased pH and increased total acidity. Observed wine qualities include alcoholic content (7.88 - 9.19%v/v), attenuation (121% - 142%) and calories (0%). Clarification and ageing have diminishing effect on alcohol content. Carrot wine was judged as physically appealing moderate alcoholic beverage, with smooth consistent taste (authors' opinion), and could be modeled with yeast dynamics. Thus this wine is recommended to calories sensitive people.

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Production, Partial Purification and Characterization of Two α-Amylase Isoforms from Saccharomyces cerevisiae strain YOP 1/2-2 Isolated from Tchapalo (Côte d’Ivoire)

Journal of Advances in Biology & Biotechnology ◽

10.9734/jabb/2020/v23i930177 ◽

2020 ◽

pp. 17-30

Author(s):

Ouattara Lacinan ◽

Koné Fankroma Martial Thierry ◽

Djoudy Alix Evrard ◽

N’guessan Kouadio Florent ◽

Dabonné Soumaïla

Keyword(s):

Saccharomyces Cerevisiae ◽

Cote D'ivoire ◽

Côte D’Ivoire ◽

Alcoholic Beverage ◽

Agar Plate ◽

Amylolytic Activity ◽

Partial Purification ◽

Complete Medium ◽

Cote D’Ivoire ◽

Côte D'ivoire

Amylases play an important role in biotechnology and find applications in several industrial fields such as pharmaceutical, food, paper, cosmetics and detergents. Thus, it appears necessary to identify new sources of amylase, especially from microbial origin, due to the low energy consumption, cost-effective, high metabolic diversity, rapid cell growth, non-toxic and eco-friendly characteristics. In the present report, we carried out the production and partial purification of α-amylase by Saccharomyces cerevisiae strains isolated from Tchapalo, a traditional alcoholic beverage of Côte d'Ivoire. Five fungal isolates were screened initially for α-amylase production by using method of wells on Yeast Extract Peptone Dextrose Agar medium, a complete medium for yeast growth. One step DEAE-Sepharose Fast Flow was achieved for partial purification of α-amylase obtained. Among yeasts, isolate S. cerevisiae YOP 1/2-2 was able to provoke starch hydrolysis halo of 15.067±0.12 mm on starch agar plate after 48 h of incubation at 30°C. The partial purification of resulting enzyme showed two protein peaks, designated α-amylase 1 (AMY1) and α-amylase 2 (AMY2) with amylolytic activity and specific activities of 1.57-1.58 U/mg protein. Both isoforms (AMY1 and AMY2) were thermostable with optimal activity at 50 and 55°C, respectively, and at pH ranged from 4.5 to 5.3 in 0.1 M sodium acetate buffer. EDTA and Cd2+ strongly inhibited α-amylase activity by 72-75% and 64-65%, respectively, whereas cations Ca2+ and Mn2+ showed 85-99% and 71% increased amylolytic activity, respectively. All these properties show the potential uses of α-amylases from S. cerevisiae in the industrial transformation of starch.

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Biotransformation of soy whey into soy alcoholic beverage by four commercial strains of Saccharomyces cerevisiae

International Journal of Food Microbiology ◽

10.1016/j.ijfoodmicro.2017.09.007 ◽

2017 ◽

Vol 262 ◽

pp. 14-22 ◽

Cited By ~ 23

Author(s):

Jian-Yong Chua ◽

Yuyun Lu ◽

Shao-Quan Liu

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcoholic Beverage

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Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.00277-09 ◽

2009 ◽

Vol 75 (17) ◽

pp. 5536-5543 ◽

Cited By ~ 68

Author(s):

Kenro Tokuhiro ◽

Masayoshi Muramatsu ◽

Chikara Ohto ◽

Toshiya Kawaguchi ◽

Shusei Obata ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Metabolic Flux ◽

Mevalonate Pathway ◽

Fusion Gene ◽

Pharmaceutical Products ◽

Test Tube ◽

Ethanol Solution ◽

Multicopy Integration ◽

Engineered Saccharomyces Cerevisiae ◽

Rate Limiting

ABSTRACT (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

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Microbiological Quality of a Locally Brewed Alcoholic Beverage (Pito) Sold in a Community within the Greater Accra Region, Ghana

10.20944/preprints201701.0003.v1 ◽

2017 ◽

Cited By ~ 1

Author(s):

Andrew A Minamor ◽

Alberta Mensah Larteley ◽

Emmanuel Nii Laryea ◽

Emmanuel Afutu ◽

Patience B Tetteh-Quarcoo

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcoholic Beverage ◽

Microbiological Quality ◽

Enterobacter Aerogenes ◽

Plate Count ◽

African Countries ◽

Standard Plate Count ◽

Shigella Spp ◽

Food And Drink ◽

Bacteria And Fungi

Pito is a traditionally brewed alcoholic beverage in some African countries. It is gaining much prominence and the patronage among the youth. Therefore, samples of the drink were collected every week for six weeks from three different popular brewing sites at Lower Prampram in the Ningo-Prampram District of Accra, Ghana. The samples were processed and examined for bacteria and fungi using the Standard Plate Count (SPC) technique. A total of six different bacteria and a fungus were isolated. The bacteria were Escherichia coli, Klesiella pneumoniae, Shigella spp, Enterobacter aerogenes, Staphylococcus aureus and Pseudomonas aeroginosa, whiles the fungus was Saccharomyces cerevisiae. Total viable counts as well as individual isolates counts in all the pito samples were found to be less than 104 cfu/ml. It is noteworthy that, Saccharomyces cerevisiae, the only fungus isolated is known to be associated with fermentation and the microbes isolated from the pito samples were found to be within the permissible limits. However, these potentially pathogenic microbes, if found in unacceptable limits, from the fermenting samples could merit public health attention. Therefore, periodic screening of pito and their brewers, coupled with education on the maintenance of recommended guidelines concerning food and drink production is encouraged.

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Alpha-Terpineol production from an engineered Saccharomyces cerevisiae cell factory

Microbial Cell Factories ◽

10.1186/s12934-019-1211-0 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 3

Author(s):

Chuanbo Zhang ◽

Man Li ◽

Guang-Rong Zhao ◽

Wenyu Lu

Keyword(s):

Saccharomyces Cerevisiae ◽

Mevalonate Pathway ◽

Cell Factory ◽

Fed Batch ◽

Cell Factories ◽

Fed Batch Fermentation ◽

Pharmaceutical Industries ◽

Engineered Saccharomyces Cerevisiae ◽

Rate Limiting ◽

Production Cell

Abstract Background Alpha-Terpineol (α-Terpineol), a C10 monoterpenoid alcohol, is widely used in the cosmetic and pharmaceutical industries. Construction Saccharomyces cerevisiae cell factories for producing monoterpenes offers a promising means to substitute chemical synthesis or phytoextraction. Results α-Terpineol was produced by expressing the truncated α-Terpineol synthase (tVvTS) from Vitis vinifera in S. cerevisiae. The α-Terpineol titer was increased to 0.83 mg/L with overexpression of the rate-limiting genes tHMG1, IDI1 and ERG20F96W-N127W. A GSGSGSGSGS linker was applied to fuse ERG20F96W-N127W with tVvTS, and expressing the fusion protein increased the α-Terpineol production by 2.87-fold to 2.39 mg/L when compared with the parental strain. In addition, we found that farnesyl diphosphate (FPP) accumulation by down-regulation of ERG9 expression and deletion of LPP1 and DPP1 did not improve α-Terpineol production. Therefore, ERG9 was overexpressed and the α-Terpineol titer was further increased to 3.32 mg/L. The best α-Terpineol producing strain LCB08 was then used for batch and fed-batch fermentation in a 5 L bioreactor, and the production of α-Terpineol was ultimately improved to 21.88 mg/L. Conclusions An efficient α-Terpineol production cell factory was constructed by engineering the S. cerevisiae mevalonate pathway, and the metabolic engineering strategies could also be applied to produce other valuable monoterpene compounds in yeast.

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Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

Molecular Biology of the Cell ◽

10.1091/mbc.5.6.655 ◽

1994 ◽

Vol 5 (6) ◽

pp. 655-665 ◽

Cited By ~ 51

Author(s):

D Dimster-Denk ◽

M K Thorsness ◽

J Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Coenzyme A ◽

Mevalonate Pathway ◽

Feedback Regulation ◽

Eukaryotic Cells ◽

Yeast Gene ◽

Hmg Coa Reductase ◽

Common Precursor ◽

Coa Reductase

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.

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Schizosaccharomyces pombe Can Reduce Acetic Acid Produced by Baijiu Spontaneous Fermentation Microbiota

Microorganisms ◽

10.3390/microorganisms7120606 ◽

2019 ◽

Vol 7 (12) ◽

pp. 606 ◽

Cited By ~ 5

Author(s):

Zhewei Song ◽

Hai Du ◽

Menghui Zhang ◽

Yao Nie ◽

Yan Xu

Keyword(s):

Acetic Acid ◽

Schizosaccharomyces Pombe ◽

Mevalonate Pathway ◽

Current Knowledge ◽

Alcoholic Beverage ◽

High Stress ◽

Alcoholic Beverages ◽

Spontaneous Fermentation ◽

Saccharomyces Yeasts

The spontaneous fermentation of alcoholic beverage is a bioprocess donated by microbiota with complex stress environments. Among various microbes, non-Saccharomyces yeasts have high stress tolerance and significantly affect the taste and quality of products in process. Although many researchers have focused on the influence of acid stress, the mechanism of non-Saccharomyces yeasts to tolerant stress remains unclear in microbiota. To bridge the gap, we constructed in situ and in vitro studies to explore the reduction pathway of acetic acid in non-Saccharomyces yeasts. In this study, we found Schizosaccharomyces pombe has special capacities to resist 10 g/L acetic acid in laboratory cultures and decrease the average concentration of acetic acid from 9.62 to 6.55 g/kg fermented grains in Chinese Maotai-flavor liquor (Baijiu) production. Moreover, Schi. pombe promoted metabolic level of mevalonate pathway (high expressions of gene ACCAT1, HMGCS1, and HMGCR1) to degrade a high concentration of acetic acid. Meanwhile, Schi. pombe also improved the concentration of mevalonic acid that is the precursor of terpenes to enhance the taste and quality of Baijiu. Overall, the synchronicity of reduction and generation in Schi. pombe advances the current knowledge to guide more suitable strategies for mechanism studies of non-Saccharomyces yeasts in fermented industries of alcoholic beverages.

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