CN109504695B

CN109504695B - Multi-center monamphosterium xylose isomerase gene and application thereof

Info

Publication number: CN109504695B
Application number: CN201910001283.4A
Authority: CN
Inventors: 王智; 顿宝庆; 李桂英
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2019-01-02
Filing date: 2019-01-02
Publication date: 2022-08-09
Anticipated expiration: 2039-01-02
Also published as: CN109504695A

Abstract

The invention discloses a novel xylose isomerase gene from multi-center monarda flagellata, and the nucleotide sequence of the xylose isomerase gene is shown as SEQ ID NO. 2. The invention introduces the recombinant plasmid containing the gene into host cells to obtain a novel yeast engineering bacterium. Experiments prove that after the gene is expressed in saccharomyces cerevisiae, the saccharomyces cerevisiae which does not have the capacity of converting xylose into xylulose originally obtains the conversion capacity. The engineering bacteria can prepare ethanol and other fermentation products by fermenting a culture medium containing xylose.

Description

Multi-center monamphosterium xylose isomerase gene and application thereof

The technical field is as follows:

the invention relates to a xylose isomerase gene, in particular to a xylose isomerase gene from multi-center monarda flagellata and a function of converting xylose into xylulose, and also relates to a recombinant plasmid and an engineering strain containing the gene, and application of the engineering strain in preparing ethanol and other fermentation products by fermenting a culture medium containing xylose.

Background art:

the industrial production of ethanol from lignocellulose is an economical and environment-friendly way. Since lignocellulosic feedstocks from plants are renewable resources that are available in large quantities. However, many yeasts capable of producing ethanol by fermentation, such as Saccharomyces cerevisiae, do not have xylose as a carbon source. Therefore, it is desirable to provide a yeast capable of ethanol fermentation using xylose as a carbon source.

The saccharomyces cerevisiae has a complete enzyme system for metabolism of xylulose, if xylose in lignocellulose of plants can be isomerized to generate xylulose, the saccharomyces cerevisiae can metabolize xylulose to generate products such as ethanol, and the xylose can be used as a carbon source for ethanol fermentation. If xylose can be effectively fermented into ethanol, the yield of the fermentation of the lignocellulose ethanol can be increased by 25 percent on the original basis, and the production cost of the cellulosic ethanol can be reduced by about 20 percent. Therefore, the ethanol production by xylose fermentation is one of the key factors for determining the economic feasibility of producing ethanol by lignocellulose.

Xylose isomerase (D-Xylose isomerase, XI, EC 5.3.1.5) required for Xylose isomerization catalyzes the conversion of D-Xylose, a pentose, to D-xylulose, widely present in bacteria, a small fraction of fungi. Saccharomyces cerevisiae has a complete enzyme system for xylulose metabolism, and xylulose enters a pentose phosphate pathway and is fermented to generate ethanol. If a new xylose isomerase capable of being efficiently expressed in the saccharomyces cerevisiae is found, the saccharomyces cerevisiae can isomerize xylose into xylulose, so that the aim of performing ethanol fermentation by using xylose as a carbon source is fulfilled.

To date, only a few xylose isomerase genes from bacteria have been actively expressed in the traditional strain of ethanol production, Saccharomyces cerevisiae, and at ethanol fermentation temperatures around 30 ℃ it is common that the activity is too low to be the rate-limiting step in the xylose metabolic pathway.

Disclosure of Invention

The invention aims to screen a new xylose isomerase gene from fungi, and the gene is introduced into saccharomyces cerevisiae, so that the obtained yeast genetic engineering bacteria can obtain the capability of converting xylose into xylulose.

The technical problems to be solved by the invention are as follows:

screening a new xylose isomerase gene from fungi;

providing a recombinant vector containing the xylose isomerase and the gene thereof;

providing a recombinant strain comprising the xylose isomerase gene;

the obtained recombinant strain is applied to the conversion of xylose to generate xylulose, and further ethanol is generated.

The inventor obtains a new xylose isomerase from a fungus, namely multi-center monarda flagellata, named XI, obtains a xylose isomerase gene xylA for coding the enzyme, and experiments prove that the gene has the function of converting xylose into xylulose, and is suitable for being used in energy, brewing and food industries.

Although the present invention was originally completed by screening the fungus, the present invention resulted in the acquisition of a gene sequence. Others do not rely on this fungus and can obtain the xylose isomerase of the present invention if the sequence is known.

The nucleotide sequence of the xylose isomerase gene is shown as SEQ ID NO. 2. The amino acid sequence of xylose isomerase XI encoded by xylA gene is shown in SEQ ID NO. 1.

The invention inserts the xylose isomerase gene xylA between proper restriction enzyme cutting sites of an expression vector, so that the nucleotide sequence of the xylose isomerase gene xylA is operably connected with an expression regulation sequence to obtain a recombinant plasmid containing the xylose isomerase gene xylA. The expression vector is preferably a saccharomyces cerevisiae multicopy constitutive expression vector pYES-HXT to obtain a recombinant plasmid pYES-HXT-XI.

As a most preferred embodiment of the present invention, the expression vector preferably has restriction sites between the SpeI and XbaI restriction sites on plasmid pYES2, such that the nucleotide sequence is located downstream of and under the control of the HXT1 promoter, resulting in recombinant yeast expression plasmid pYES-HXT-XI.

The recombinant yeast expression plasmid pYES-HXT-XI is converted into a host cell to obtain the recombinant strain capable of efficiently expressing the xylose isomerase.

The host cell is a Saccharomyces cerevisiae cell, a pichia pastoris cell or a polytype Saccharomyces cerevisiae cell, preferably the host cell is a Saccharomyces cerevisiae cell (Saccharomyces cerevisiae), and more preferably, the recombinant yeast expression plasmid is transformed into Saccharomyces cerevisiae CEN.PK 113-5D to obtain a recombinant strain O1 (see example 4 for details).

The invention also provides a recombinant strain containing the xylose isomerase gene, preferably a recombinant strain O1. The recombinant strain O1 can efficiently express the xylose isomerase, and the enzyme can be used for fermenting substances containing xylose, converting xylose into xylulose, and further metabolizing to generate ethanol and other fermentation products.

The invention also provides an application of the xylose isomerase, which comprises the following steps:

1. transforming the host cell with the recombinant plasmid to obtain a recombinant strain O1 (see example 4 for details);

2. and culturing the recombinant strain O1 to express the recombinant xylose isomerase.

Wherein, the xylose isomerase of the invention has the following application range:

1. energy industry:

d-xylose, which is present in large amounts in lignocellulosic hydrolysates, can be converted into D-xylulose, which in turn can be converted into valuable fuels by traditional ethanol producing strains, such as Saccharomyces cerevisiae.

2. Food industry

Xylose isomerase can catalyze the conversion of D-glucose to D-fructose in vitro and is therefore also referred to as "glucose isomerase". The latter activity can be used in industry for producing high fructose corn syrup, and can be used as food additive. The high fructose corn syrup is a product which can completely replace sucrose, and can be widely applied to the food and beverage industries like sucrose.

Detailed Description

The plasmids and strains shown in the following examples are only for further illustrating the present invention and are not intended to limit the essence of the present invention. Indeed, those skilled in the art can obtain various other genetically engineered strains having the ability to convert xylose to xylulose using the genes and methods discovered in the present invention without departing from the spirit and concepts of the present invention.

The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.

Test materials and reagents

1. Bacterial strain and carrier: saccharomyces cerevisiae expression vector pYES2 was purchased from Invitrogen, and Saccharomyces cerevisiae strain T1308-U and E.coli DH 5. alpha. were stored in the laboratory.

2. Enzymes and other biochemical reagents: the endonuclease and ligase were purchased from NEB, and the other reagents were domestic reagents (all of which were purchased from general Biochemical reagent).

3. Culture medium:

escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, l% NaCl, pH7.0)

② Yeast Medium YPD (1% yeast extract, 2% peptone, 2% glucose, plate added with 2% agar)

③ selection Medium SC (0.67% YNB, sterile glucose, corresponding carbon sources, plate addition 2% agar)

Example 1 extraction of multicenter Monocilia genome Total DNA

Firstly, taking about 0.5g of multicenter monarda flagellata strain, and quickly grinding the multicenter monarda flagellata strain into powder in liquid nitrogen;

② adding 4ml of extracting solution, quickly oscillating and uniformly mixing;

③ adding equal volume of 4ml chloroform: isoamyl alcohol (24: 1), vortex for 3-5 min;

④10000rpm，4℃，5min；

using chloroform with volume for supernatant: isoamyl alcohol (24: 1) is extracted once more (10000rpm, 4 ℃ centrifugation for 5 min);

sixthly, adding 2/3 times volume of precooled isopropanol at the temperature of 20 ℃ or 2.5 times volume of absolute ethyl alcohol into the supernatant for precipitation, uniformly mixing, and standing for about 30 min;

seventhly, picking out flocculent precipitates by using a capillary glass rod, repeatedly rinsing the flocculent precipitates by using 75% ethanol for a plurality of times, rinsing the flocculent precipitates by using absolute ethyl alcohol for 1 time, drying the flocculent precipitates by blowing, and suspending the flocculent precipitates in 500 mu l of TE;

adding 1 mul of RNaseA (10mg/ml), and processing at 37 ℃ for 1 h;

ninthly, phenol: chloroform: isoamyl alcohol (25: 24: 1) and chloroform: extracting isoamyl alcohol (24: 1) for 1 time respectively (10000rpm, 4 ℃ and 5min of centrifugation);

collecting supernatant, 1/10V 3M NaAc, 2.5V absolute ethyl alcohol, and precipitating at-70 deg.C for more than 30 min. Centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, rinsing precipitate with 75% ethanol, air drying, and dissolving in 200 μ l of ddH with pH8.0 ₂ In O, storing at-20 ℃ for later use.

EXAMPLE 2 cloning of the multicenter Monoflagellate xylose isomerase Gene xylA

PCR amplification was performed using the total DNA of the multicenter flagellate extracted in example 1 as a template. The product obtained by amplification is sent to Shanghai Biometrics company for sequencing after being recovered, and the obtained sequence is verified to contain the full-length gene sequence of xylose isomerase with the total length of 1284bp and the sequence shown as SEQ ID NO.2 through comparison analysis of the sequencing sequence,

Example 3 construction of xylose isomerase Gene expression vector plasmid

Primers were designed with 5 'and 3' end sequences of the gene encoding multicenter flagellate xylose isomerase, including EcoRI and Xba I sites. The PCR product was digested with EcoRI and Xba I. The final product was cloned into the vector produced by pYES 2. In this vector, the GAL1 promoter on pYES2 was replaced with the HXT1 promoter to ensure constitutive expression of xylose isomerase, thereby eliminating the need for galactose in the medium. The HXT1 promoter was cloned from the Saccharomyces cerevisiae genome. The promoter was cleaved by enzyme to form a Nhe I-EcoRI fragment. The PCR products of both the HXT1 promoter and the gene encoding xylose isomerase were ligated to pYES2 which was cut with EcoRI and XbaI, resulting in recombinant plasmid pYES-HXT-XI containing the xylose isomerase gene.

Preparation of competent cells of Escherichia coli and transformation of plasmids

1. Preparation of competent cells (calcium chloride transformation)

Picking single colony from activated colibacillus (E.coli) DH5 alpha plate, inoculating to test tube containing 5ml LB liquid culture medium, shaking culturing at 37 deg.C for 2.5-3 h to make OD of bacterial liquid ₆₀₀ Values of 0.4 to 0.6 were reached, the cultures were cooled on ice to 0 ℃;

pouring the culture into a sterile centrifugal tube of 1.5 ml;

③ centrifuging at 4 ℃ and 4000rpm for 10 min;

fourthly, abandoning the supernatant and collecting thalli;

using 0.1M pre-cooled 0.5ml CaCl ₂ Resuspending the thallus, centrifuging, and discarding the supernatant;

sixthly, precooling 0.5ml of 0.1M CaCl ₂ Resuspending the thallus, carrying out ice bath for 15min, centrifuging, and discarding the supernatant;

seventhly, 200 mu l of precooled 0.1M CaCl is added ₂ Resuspending the cells, and placing in ice bath.

2. Plasmid transformed competent cells

Adding 0.5 mu l of plasmid into a tube of competent cells, gently rotating to mix the contents uniformly, and placing on ice for 30 min;

placing the centrifugal tube in a water bath at 42 ℃ for 90s without shaking the centrifugal tube;

thirdly, rapidly placing the centrifugal tube on ice, and cooling for 2 min;

adding 800 mul LB liquid culture medium, shaking and culturing at 37 deg.C and 200rpm for 45 min;

fifthly, taking 50 mul of culture solution to be coated on an LB solid plate containing ampicillin (50 mug/ml), culturing for 12 to 16 hours at 37 ℃, and checking a transformed colony;

sixthly, selecting a single colony, inoculating the single colony into a test tube containing 5ml of LB liquid culture medium, performing shaking culture at 37 ℃ to extract a transformant which is verified to be correct by plasmid enzyme digestion, and then sequencing to prove that the transformant contains xylose isomerase genes.

EXAMPLE 4 construction of the engineered Strain

Recombinant plasmid pYES-HXT-XI containing xylose isomerase gene is transferred into Saccharomyces cerevisiae T1308-U without xylose converting capacity into xylulose through electric shock conversion method, and transformant is screened on SC culture medium plate with 2% glucose as carbon source. Untransformed cells were unable to grow on these plates. PCR identification proves that the O1 transformant contains a plasmid with a multicenter monamphostema xylose isomerase gene.

Preparation of saccharomyces cerevisiae competent cells and transformation of plasmids:

1. preparation of Yeast competent cells (electroporation method)

Culturing saccharomyces cerevisiae in a 50ml centrifuge tube containing 5ml YPD, and standing overnight at 30 ℃;

② 0.1-0.5ml of overnight culture is taken, inoculated with 2L of shake flask containing 500ml of fresh culture medium, grown overnight to OD ₆₀₀ ＝1.3-1.5；

Thirdly, centrifuging at 4 ℃ for 5min at 1500g to collect cells, and suspending the cells by using 500ml of precooled sterile water;

fourthly, centrifuging the mixture as above, and suspending the cells by using 250ml of precooled sterilized water;

fifthly, centrifuging as above, and suspending the cells by using 20ml of precooled 1M sorbitol;

sixthly, centrifuging the mixture as above, and suspending the cells by using 1ml of precooled 1M sorbitol until the final volume is about 1.5 ml;

note that: equal amounts of electrocompetent cells of 80. mu.l can be frozen, but the transformation efficiency is much reduced.

2. Plasmid electroporation transformation of competent cells:

mixing 80 μ l of the cells with 5-20 μ g of linearized DNA (dissolved in 5-10 μ l of TE), and transferring into a precooled 0.2cm electric cuvette;

② placing on ice for 5 min;

thirdly, carrying out electric shock according to the saccharomyces cerevisiae parameters recommended by the used device;

fourthly, 1ml of precooled 1M sorbitol is immediately added into the cup, and the content is transferred into a sterilized centrifuge tube;

dividing into 200 and 600 mul equal parts, and coating on an SC plate;

sixthly, the plate is incubated at 30 ℃ until the clone is generated, and PCR identification proves that the transformant O1 contains the single flagellate with multiple centers

Plasmid pYES-HXT-XI for the genes for the sugar isomerases.

Example 5 determination of enzyme Activity of xylose isomerase of engineered Strain

1. Test object

Experimental strains: recombinant strain O1 of Saccharomyces cerevisiae containing vector pYES-HXT-XI (see example 4 for details);

control strain: saccharomyces cerevisiae strain containing pYES-HXT vector.

2. Experimental methods

The activity of xylose isomerase in the control strain and the recombinant strain O1 of Saccharomyces cerevisiae containing vector pYES-HXT-XI were measured separately for growth in equilibrated culture with glucose/xylose mixture as the sole carbon source. The enzyme activity was measured as follows:

supernatant (by OD) after appropriate Saccharomyces cerevisiae cell disruption ₅₉₅ Assay adjusted to total protein amount consistent) 100mM Tris-HCl pH7.0 buffer, 10mM MgCl ₂ 2U SDH (sorbitol dehydrogenase, Roche) and 0.15mM NADH (reduced nicotinamide adenine dinucleotide, Roche) were mixed, the reaction was initiated with 500mM xylose, and the amount of NADH oxidized was measured at 30 ℃ and 340nm (i.e., time scanning at 340 nm). Of Xylose Isomerase (XI)One enzyme activity unit (U) is defined as 1. mu. mol substrate converted per minute.

3. Specific operation process

Picking single colonies respectively, inoculating the single colonies in 20ml of SC culture solution, and shaking at 30 ℃ and 200rpm for 48 hours;

② 1600g centrifugation for 5min, abandoning the supernatant, and washing the precipitate twice with 10mM potassium-phosphate buffer (containing 2mM EDTA) with pH 7.5;

③ resuspending in 100mM potassium-phosphate buffer (2mM MgCl. sub.7.5) ₂ And 1mM

dithiothreitol)；

And fourthly, carrying out ultrasonic crushing, centrifuging at the temperature of 4 ℃ for 20min at 36000g, and using the supernatant for enzyme activity analysis and total protein determination.

4. Measurement results

No xylose isomerase activity was measured for the control strain;

the activity of xylose isomerase enzyme of the recombinant strain O1 of Saccharomyces cerevisiae containing the vector pYES-HXT-XI was determined to be 0.92U/mg total protein.

5. Conclusion

After the xylose isomerase coded by the multi-center monascus xylose isomerase gene is expressed in saccharomyces cerevisiae, the host cell is endowed with the capability of converting xylose into xylulose.

Example 6 determination of xylose utilization by engineered Strain

1. Test object

Experimental strains: recombinant s.cerevisiae strain O1 containing vector pYES-HXT-XI (see example 4 for details);

control strain: saccharomyces cerevisiae strain containing pYES-HXT vector.

2. Experimental methods

Glucose/xylose mixtures (initial concentrations of 15.0g/L and 7.5g/L respectively) are used as a unique carbon source, shaking is carried out at 30 ℃ and 200rpm for 48 hours, and the glucose content and the xylose content of a supernatant are measured by High Performance Liquid Chromatography (HPLC), so that the sugar utilization efficiency is calculated.

3. Results

The test result shows that: after 48h of culture, the glucose in the recombinant strain P culture medium is almost completely utilized, and the utilization rate of xylose reaches 57.29%; glucose in the culture medium of the control strain is almost completely used, and the utilization rate of xylose only reaches 2.17%.

4. Conclusion

The xylose utilization rate of the multicenter monarda flagellata xylose isomerase gene engineering strain is greatly higher than that of a control strain.

Sequence listing

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Claims

1. A recombinant plasmid is obtained by connecting a gene with a nucleotide sequence shown as SEQ ID NO.2 with a saccharomyces cerevisiae multicopy expression vector pYES 2.

2. An engineered yeast strain comprising the recombinant plasmid of claim 1.

3. The engineered yeast strain of claim 2, wherein the host cell is selected from the group consisting of a Saccharomyces cerevisiae cell, a Pichia pastoris cell, and a Torulopsis polymorpha cell.

4. Use of the engineered yeast strain of claim 2 to convert xylose to xylulose.

5. Use according to claim 4 for the production of ethanol and other fermentation products by fermentation of xylose-containing material.

6. A xylose isomerase has an amino acid sequence shown in SEQ ID NO. 1.