CN105255951B

CN105255951B - A method of Alcohol Production efficiency is improved by overexpression HAC1 genes

Info

Publication number: CN105255951B
Application number: CN201510713933.XA
Authority: CN
Inventors: 蒋伶活; 徐国强; 吴瑶; 杜婕
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2015-10-28
Filing date: 2015-10-28
Publication date: 2018-10-16
Anticipated expiration: 2035-10-28
Also published as: CN105255951A

Abstract

The invention discloses a kind of methods improving Alcohol Production efficiency by overexpression HAC1 genes, belong to bioenergy development technique field.The present invention is overexpression HAC1 and the engineered strain fermenting and producing alcohol that obtains in ace2 single-gene deletion mycopremnas, and compared to wild type zero load control strain, ethanol production improves 200.8%, and alcohol improves 138.9% to thalline yield；Compared to ace2 single-gene deletion mycopremnas, ethanol production improves 13.1%；Important information is provided with good industrial application value and foreground, while for the excellent Saccharomyces cerevisiae gene engineering bacteria strain for building High-alcohol-yield.

Description

A method of Alcohol Production efficiency is improved by overexpression HAC1 genes

Technical field

The present invention relates to a kind of method improving Alcohol Production efficiency by overexpression HAC1 genes, especially one kind is answered Alcohol is improved to the method for thalline yield with the saccharomyces cerevisiae of transcription factor single-gene missing and overexpression, belongs to bioenergy Development technique field.

Background technology

Fossil energy (coal, oil, natural gas etc.) is the most important energy on our times, with the economic society in countries in the world The development of meeting, the mankind are increasingly incremental to the demand of the energy, but the reserves of fossil energy are limited；Meanwhile fossil energy is fired Global warming and caused problem caused by burning the greenhouse gases generated, annoying always people.Therefore, it finds and sends out Open up the hot spot that reproducible new energy has become whole world concern.Fuel alcohol has cleaning, safe, environmental-friendly and raw material The advantages that renewable, it is considered to be the ideal substitute of petroleum resources, market potential are huge.Currently, Europe, the U.S. and bar Xi Deng states use relatively broadly.

Production by Microorganism Fermentation fuel alcohol is the hot spot of current research, and saccharomyces cerevisiae is ideal alcoholic fermentation production Bacterial strain has：Ethyl alcohol can be rapidly grown and produced on simple culture medium；To non-life suitable environment (such as hyperosmosis, height Temperature and ethyl alcohol are poisoned) there is stronger adaptability；Genetic modification is easy to operate；Can utilize cheap substrate, fermentation costs compared with Low characteristic.The technology that ethyl alcohol is produced using thick mash fermentation obtained prodigious progress past 10 years, but flourishing with the U.S. etc. Country is compared to prodigious gap, existing main problem is still had：It is hypertonic caused by the high concentration substrate of earlier fermentation System is constrained thoroughly；Murder by poisoning caused by the high concentration ethanol in later stage of fermenting inhibits；In fermentation process caused by uppity high-temperature Inhibiting effect.The presence of these problems becomes the principal element that limitation thick mash fermentation technology production efficiency improves.

Currently, for saccharomyces cerevisiae (Saccharomyces cerevisae) tolerance hyperosmosis, alcohol in high concentration and height The research of temperature has achieved many progress, but correlation tolerance mechanism not yet study it is clear.Therefore, to passing through the transformation of rationality Means are very difficult to the tolerance of adverse circumstance to improve S.cerevisiae.Although traditional strain improvement means (as sieved naturally Choosing, mutation breeding) in terms of improving bacterial strain to stress tolerance achieved certain effect, but there are heavy workload, exist The shortcomings of randomness.In recent years, it is broad scale research saccharomyces cerevisiae base with the fast development of molecular biology and omics technology Cause and the relationship of alcohol fermentation provide condition.

Invention content

The invention solves first technical problem be to provide it is a kind of improve Alcohol Production efficiency method, the method It is the efficiency that overexpression HAC1 genes improve saccharomycetes to make fermentation production alcohol in ace2 gene-deleted strains.

In one embodiment of the invention, the engineering bacteria is saccharomyces cerevisiae engineered yeast.

In one embodiment of the invention, the nucleotide sequence of the ACE2 genes is as shown in SEQ ID NO.1.

In one embodiment of the invention, the nucleotide sequence of the HAC1 genes is as shown in SEQ ID NO.2.

In one embodiment of the invention, the amino acid sequence of the ACE2 gene codes such as SEQ ID NO.3 institutes Show.

In one embodiment of the invention, the method is overexpressed in the saccharomyces cerevisiae that ace2 single-genes lack HAC1 genes.

In one embodiment of the invention, the saccharomyces cerevisiae of the ace2 single-genes missing is public purchased from Invotrogen Department, number Sc04146899_s1.

In one embodiment of the invention, described be overexpressed is by HAC1 gene fragment clones to expression plasmid Obtain recombination high-expression plasmid pHAC181-HAC1 on pHAC181 (Jiang L, 2004), then by recombinant plasmid transformed to ace2 It is expressed in single-gene gene-deleted strain.

In one embodiment of the invention, the expression plasmid pHAC181 be YCplac181 SphI and What 3 histidine tags obtained be inserted into the sites EcoRV, refers to document：Analyses of the effects of Rck2p mutants on Pbs2p^DD-induced toxicity in Saccharomyces cervisiae identify a MAP kinase docking motif,and unexpected functional inactivation due to acidic substitution of T379,Mol Gen Genomics,2004,271:208–219.

In one embodiment of the invention, the fermenting and producing is that fermentation training is forwarded to after activating Yeast engineering bacteria Support base so that the OD of fermentation medium after inoculation seed bacterium solution₆₀₀For 0.4-0.5,7- is cultivated in 28-30 DEG C, 200-220r/min Switch to stationary culture, stationary culture 50-52h after 8 hours.

In one embodiment of the invention, the fermenting and producing is that fermentation training is forwarded to after activating Yeast engineering bacteria Support base so that the OD of fermentation medium after inoculation seed bacterium solution₆₀₀It is 0.5, switchs to after 30 DEG C, 220r/min are cultivated 8 hours quiet Culture is set, continues to cultivate 52h.

In one embodiment of the invention, the fermentation medium contain glucose 100g/L, ammonium sulfate 7.5g/L, Potassium dihydrogen phosphate 3.5g/L, epsom salt 0.75g/L, yeast extract 0.2g/L, histidine 0.02g/L, uracil 0.02g/L, leucine 0.1g/L.

In one embodiment of the invention, the preferred YPD culture mediums of activation.It crosses on YPD solid mediums, 2d are cultivated in 30 DEG C with primary activation strain, then are forwarded in YPD fluid nutrient mediums, 30 DEG C, 220r/min shaking table cultures 23h extremely Saturation.

The invention solves second technical problem be to provide the saccharomyces cerevisiae engineering that a kind of Alcohol Production efficiency improves Bacterium, the saccharomyces cerevisiae engineered yeast have lacked the ace2 genes of encoding amino acid sequence such as SEQ ID NO.3 and have been overexpressed nucleosides The HAC1 genes of acid sequence such as SEQ ID NO.2.

The saccharomyces cerevisiae engineered yeast is the overexpression HAC1 genes in the saccharomyces cerevisiae that ace2 single-genes lack.

Beneficial effects of the present invention：

The work that the present invention is obtained using the overexpression HAC1 genes in the Wine brewing yeast strain that ace2 single-genes lack Journey yeast strain ferments produce alcohol, and compared to wild type zero load control strain, when fermentation proceeds to 52h, ethanol production improves 200.8%, alcohol improves 138.9% to thalline yield；Compared to the bacterial strain that ace2 single-genes missing only occurs, ethyl alcohol production Amount improves 13.1%.The present invention has good industrial application value and foreground, while being the excellent wine of structure High-alcohol-yield Brewer yeast engineering strain provides important information.

Description of the drawings

Fig. 1：Fungal biodiversity compares figure；Wherein ace2+HAC1 represents the overexpression in ace2 single-gene deletion mycopremnas HAC1 and the engineered strain obtained, WT+V are represented containing unloaded wild type control strain；

Fig. 2：Ethanol production compares figure；Wherein ace2+HAC1 represents the overexpression in ace2 single-gene deletion mycopremnas HAC1 and the engineered strain obtained, WT+V are represented containing unloaded wild type control strain；

Fig. 3：Alcohol getting rate compares figure；Wherein ace2+HAC1 represents the overexpression in ace2 single-gene deletion mycopremnas HAC1 and the engineered strain obtained, WT+V are represented containing unloaded wild type control strain.

Specific implementation mode

The method that high performance liquid chromatography measures ethanol content：The measurement of ethanol content uses high performance liquid chromatography in zymotic fluid Instrument (HPLC) detects.Zymotic fluid is through processing and supernatant is after 0.22 μm of filtering with microporous membrane, utilizes RID (differential pulse polarograplls Device) it is detected, liquid-phase chromatography method is as follows：Chromatographic column：Shodex SH1011 sugar column organic acid columns；Column temperature：50℃；Flowing Phase：0.0275% (v/v) dilute sulfuric acid, through 0.22 μm of membrane filtration and degasification；Flow velocity：1mL/min；Detection time：17min；Into Sample amount：20μL.

Computational methods of the ethyl alcohol to thalline yield：Calculation of yield formula is as follows：

Y is alcohol yied in formula, and p is concentration of alcohol, and X is dry cell weight x=0.2Z, and Z is OD values.

The composition of YPD culture mediums：Glucose 2%, yeast extract 1%, peptone 2%, the deionization water capacity, pH naturally, High pressure sterilization (115 DEG C, 20min).

The composition (g/L) of fermentation medium：Glucose 100, ammonium sulfate 7.5, potassium dihydrogen phosphate 3.5, epsom salt 0.75, yeast extract 0.2, histidine 0.02, uracil 0.02, leucine 0.1, deionized water constant volume, pH is naturally, high pressure is gone out Bacterium (115 DEG C, 20min).

Embodiment 1：The structure of Saccharomyces cerevisiae gene engineering bacteria

To be purchased from Invotrogen companies, ace2 single-gene deletion mycopremnas YLR131C that number is Sc04146899_s1 (http://www.lifetechnologies.com/order/genome-database/browse/gene- expression/keyword/YLR 131CICID=search-gex-YLR131C it is) starting strain, overexpression is to pass through Build high-expression plasmid pHAC181-HAC1, specific method be by HAC1 genetic fragments (comprising 865bp promoter sequences and its ORF frames do not include terminator codon, total 1850bp), then this segment is cloned on high-expression plasmid pHAC181, finally DNA sequencing is carried out to correct recon, verification sequence does not mutate, and finally obtains recombination high-expression plasmid pHAC181- HAC1, then plasmid pHAC181-HAC1 is imported by ace2 single-gene gene-deleted strains by Li-acetate method, it is high to obtain HAC1 genes Express bacterial strain.

Embodiment 2：Ethanol in Saccharomyces cerevisiae ferments

According to the preparation method of solid medium, YPD tablets and YPD fluid nutrient mediums are prepared in advance.

Saccharomyces cerevisiae gene engineering bacteria, wild-type strain BG (resistances containing G418 in the flat lining out activation present invention of YPD The BY4743 of gene) (http://clones.lifetechnologies.com/cloneinfo.phpClone=yeast) (ace2 single-gene gene-deleted strains are utilized in BY4743 background bacterium with the Wine brewing yeast strain YLR131C of ace2 single-genes missing G418 resistant genes replace what ACE2 genes obtained).YPD tablets are put into 30 DEG C of constant incubator culture 2d.In super-clean bench work Make, from taking a big single bacterium colony to be inoculated in the YPD fluid nutrient mediums of 30mL in the bacterial strain flat board of each activation, 30 DEG C, 220r/min shaking table cultures 23h is extremely saturated.After cultures 23 hours, 50 μ L are added in EP pipes and are incubated overnight bacterial strain bacterium solution, OD is surveyed in 20 times of dilution₆₀₀Value.According to the OD measured₆₀₀Value calculates institute's seed addition bacterium solution amount so that the hair after inoculation seed bacterium solution The final OD of ferment culture medium₆₀₀Value is 0.5, and the seed bacterium solution amount obtained by calculating is added on super-clean bench and is trained in the fermentation of 100mL It supports in base.

Fermentation medium is put into 30 DEG C, the shaking table culture of 220r/min switchs to stationary culture after 8 hours.

In 32h and 52h fermentation time points, take the bacterium solution of 1000 μ L in the EP pipes of 1.5mL, in the centrifuge of 12000rpm Middle centrifugation 4min shifts the supernatant of 600 μ L into the EP pipes of two groups of 1.5mL respectively after centrifugation is good, one group adds 600 μ L, tri- chloroethenes Acid is stored in 4 DEG C of refrigerator and preserves overnight, passes through liquid phase measurement concentration of alcohol within second day.Another group is not added with trichloroacetic acid, sets It is saved backup in -20 DEG C of refrigerator.

Meanwhile in 32h and 52h fermentation time points, 3 group of 50 μ L zymocyte liquid sample is taken, dilutes 20 in the EP pipes of 1.5mL Times, OD values are measured, data are recorded, for calculating Fungal biodiversity.

The first group of supernatant samples that will be stored in 4 DEG C of refrigerators, 12000rpm centrifugations 2min, takes 600 μ L's at room temperature Supernatant liquid filtering, filtered fluid are transferred in liquid phase sample bottle, measure concentration of alcohol.The sample of survey liquid phase cannot be arranged at once, protected It is stored in -20 DEG C of refrigerator.

By liquid phase sample to be measured, liquid phase machine copies test data, data is made Excel tables, and make OD, second Alcohol yield, alcohol yied figure apply the overexpression HAC1 in the Wine brewing yeast strain of ace2 gene delections as shown in Figs. 1-3 Gene and obtain Engineering Yeast strain fermentation production alcohol, (contain empty plasmid compared to wild type zero load control strain The wild-type strain BG of pHAC181, is indicated with WT+V), when fermentation proceeds to 52h, ethanol production improves 200.8%, alcohol 138.9% is improved to thalline yield；Compared to ace2 single-gene deletion mycopremnas, ethanol production improves 13.1%.In addition, this Invention also found, individually lack the Yeast engineering bacteria of HAC1 genes, and Alcohol Production efficiency is to reduce.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention Enclosing be subject to what claims were defined.

Claims

1. a kind of method improving Alcohol Production efficiency, which is characterized in that the method is to utilize ace2 gene delections and excess Express the Yeast engineering bacteria fermenting and producing alcohol of HAC1 genes；The amino acid sequence of the ace2 gene codes such as SEQ ID Shown in NO.3；The nucleotide sequence of the HAC1 genes is as shown in SEQ ID NO.2.

2. according to any method of claim 1, which is characterized in that the method is the wine brewing lacked in ace2 single-genes HAC1 genes are overexpressed in yeast.

3. according to the method described in claim 1, it is characterized in that, the fermenting and producing is transferred after activating Yeast engineering bacteria To fermentation medium so that the OD of fermentation medium after inoculation seed bacterium solution₆₀₀For 0.4-0.5, in 28-30 DEG C, 200-220r/ Min switchs to stationary culture, stationary culture 50-52h after cultivating 7-8 hours.

4. according to the method described in claim 3, it is characterized in that, the fermenting and producing is transferred after activating Yeast engineering bacteria To fermentation medium so that the OD of fermentation medium after inoculation seed bacterium solution₆₀₀It is 0.5, is cultivated 8 hours in 30 DEG C, 220r/min After switch to stationary culture, continue cultivate 52h.

5. method according to claim 3 or 4, which is characterized in that the fermentation medium contains glucose 100g/L, sulphur Sour ammonium 7.5g/L, potassium dihydrogen phosphate 3.5g/L, epsom salt 0.75g/L, yeast extract 0.2g/L, histidine 0.02g/L, Uracil 0.02g/L, leucine 0.1g/L.

6. a kind of saccharomyces cerevisiae engineered yeast, which is characterized in that the saccharomyces cerevisiae engineered yeast has lacked encoding amino acid sequence such as The ace2 genes of SEQ ID NO.3 and the HAC1 genes for being overexpressed nucleotide sequence such as SEQ ID NO.2.

7. saccharomyces cerevisiae engineered yeast according to claim 6, which is characterized in that the saccharomyces cerevisiae engineered yeast is in ace2 It is overexpressed HAC1 genes in the saccharomyces cerevisiae of single-gene missing.

8. application of the saccharomyces cerevisiae engineered yeast of claim 6 or 7 in terms of Alcohol Production.