Yarrowia lipolytica engineering bacterium for producing triacetic acid lactone by using xylose and application
1. The yarrowia lipolytica genetically engineered bacterium is characterized in that yarrowia lipolytica is used as an original strain of the genetically engineered bacterium, a 2-pyrone synthetase encoding gene gh2ps is introduced, and a xylose reductase encoding gene, a xylitol dehydrogenase encoding gene and a xylulokinase encoding gene are overexpressed to obtain the recombinant yarrowia lipolytica genetically engineered bacterium.
2. The yarrowia lipolytica genetically engineered bacterium of claim 1, wherein the 2-pyrone synthase encoding gene, xylose reductase encoding gene, xylitol dehydrogenase encoding gene, and xylulokinase encoding gene are expressed by plasmid expression or genome integration expression.
3. The yarrowia lipolytica genetically engineered bacterium of claim 2, wherein the expression vector used for said plasmid expression is the PYLXP' plasmid.
4. The yarrowia lipolytica genetically engineered bacterium of claim 1, wherein the 2-pyrone synthase encodes gene gh2ps, and the nucleotide sequence is shown in SEQ ID No.1 of the sequence Listing;
the xylose reductase coding gene is endogenous gene yl.xyl1, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 2;
the xylitol dehydrogenase coding gene is an endogenous gene yl.xyl2, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 3;
xylulokinase coding gene is endogenous gene yl.xyl3, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 4.
5. The genetically engineered yarrowia lipolytica of claim 1, wherein the starting strain is yarrowia lipolytica po1 f.
6. The use of the genetically engineered yarrowia lipolytica of claim 1 for the production of triacetin.
7. The use of claim 6, wherein the yarrowia lipolytica genetically engineered bacterium is used for the production of triacetin as follows:
inoculating the engineering bacteria seed liquid into a xylose-containing fermentation culture medium according to the inoculation amount of 1-5%, fermenting at 25-32 ℃ and 250rpm for 5-7 days.
8. The use of claim 7, wherein the pH of the fermentation system is controlled at 5-7.5 by the addition of PBS at the beginning of the fermentation; adding cerulenin with final concentration of 1-3mg/L after fermenting for about 24 h.
9. The use of claim 7, wherein the fermentation medium of the engineered bacteria obtained by plasmid expression consists of: 35-50g/L of xylose, 1-2g/L of YNB, 0.5-2g/L of ammonium sulfate, 0.4-1g/L of CSM-LEU and the balance of water;
the fermentation medium of the engineering bacteria obtained by adopting genome integration expression comprises the following components: 35-50g/L of xylose, 15-25g/L of Tryptone, 5-15g/L of Yeast Extract and the balance of water, wherein the pH value is 5-7.5;
the fermentation medium of the engineering bacteria obtained by adopting genome integration expression comprises the following components: 25-35mL of corn straw hydrolysate, 15-25g/L of Tryptone, 5-15g/L of Yeast Extract and the balance of water, wherein the pH value is 5-7.5.
10. The use according to claim 9, wherein 1-10g/L glycerol is added to the culture medium.
Background art:
the triacetyl lactone is a bridge compound which is in a joint biotransformation and chemical catalysis, contains a 2-pyrone structure, can be converted into various high-value intermediates or end products such as acetylacetone and gamma-caprolactone through hydrolysis, catalytic hydrogenation and the like, is determined to be a potential biorenewable platform chemical, is a valuable commercial compound synthesis precursor, and has large demand in the traditional fields such as materials, chemical industry and the like. In addition, triacetyl lactone has the characteristics of no toxicity, no harm and low corrosivity, so the triacetyl lactone is widely applied to the life health fields of food, medicine and the like.
At present, the production method of triacetyl lactone mainly comprises a chemical synthesis method and a biological fermentation method, wherein the chemical method mainly depends on petroleum cracking, acetic acid pyrolysis and the like, the method depends on fossil resources, the energy consumption is high, the environmental pollution is large, the production cost is high, and the price of triacetyl lactone rises along with the exhaustion of fossil energy. The biological method mainly comprises a plant extraction method and a microbial fermentation method. The plant extraction method is mainly used for extracting the plant African daisy, has few sources, needs to occupy a large amount of farmland cultivated land for planting, has low extraction yield and complex process, and can not meet the industrial requirement of thousands of tons per year. Therefore, a microbial production method for producing triacetyl lactone by taking natural renewable resources as raw materials, taking genetic engineering as means and taking microbial fermentation as a way becomes a research hotspot at present. The method has the advantages of low raw material price, low production cost, no environmental pollution and few byproducts, and the development and utilization of biomass energy conversion not only accords with the national conditions of China, but also accords with the strategy of sustainable development. Compared with escherichia coli and saccharomyces cerevisiae, yarrowia lipolytica has high tolerance and strong environment adaptability, has high-flux metabolic flows of polyketide synthesis precursors acetyl coenzyme A and malonyl coenzyme A, and is an advantageous host for industrial production of triacetic acid lactone.
Lignocellulose is derived from agricultural and forestry residues and wastes, is a renewable resource with rich content and low price, and a plurality of reports show that the lignocellulose can be biologically converted into valuable products at present. Xylose is derived from lignin degradation, is the main sugar component of lignin, and accounts for 30-40% of the lignocellulose biomass. Efficient and rapid bioconversion of xylose is a prerequisite for the implementation of an economically viable lignocellulosic hydrolysate bioconversion process. At present, many researches show that xylose can be converted into biofuel and chemicals such as xylitol, 2, 3-butanediol, isobutanol polyhydroxybutyrate and the like, and the application prospect is wide.
The yarrowia lipolytica has a complete xylose metabolic pathway, but key genes of the pathway are in silent expression in natural conditions, and the yarrowia lipolytica cannot naturally grow in a culture medium taking xylose as a sole carbon source, mainly because the activities of three key enzymes of the xylose metabolic pathway, namely xylose reductase, xylitol dehydrogenase and xylulokinase are too low. Therefore, genetic engineering means are needed to enhance the activities of three key enzymes, so as to enhance the xylose metabolism capability of the strain. However, a method for constructing a xylose metabolic pathway in yarrowia lipolytica by using a genetic engineering method to heterologously synthesize triacetolactone has not been reported.
The invention content is as follows:
in order to solve the technical problems, the invention constructs a strain of yarrowia lipolytica capable of utilizing xylose by a genetic engineering means, and provides a method for producing triacetic acid lactone by using xylose as a carbon source.
One of the technical schemes provided by the invention is a yarrowia lipolytica gene engineering bacterium, which takes yarrowia lipolytica as an initial strain, introduces a 2-pyrone synthetase encoding gene gh2ps, and overexpresses a xylose reductase encoding gene, a xylitol dehydrogenase encoding gene and a xylulokinase encoding gene to obtain a recombinant yarrowia lipolytica engineering bacterium;
furthermore, the expression modes of the 2-pyrone synthetase encoding gene, the xylose reductase encoding gene, the xylitol dehydrogenase encoding gene and the xylulokinase encoding gene can be plasmid expression and can also be genome integration expression;
preferably, the expression vector adopted by the plasmid expression is a PYLXP' plasmid;
further, the 2-pyrone synthetase encoding gene is a plant source gene, and comprises but is not limited to a 2-pyrone synthetase encoding gene gh2ps derived from Gerbera hybrida, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 1;
further, the xylose reductase coding gene is an endogenous gene yl.xyl1, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 2;
further, the xylitol dehydrogenase encoding gene is an endogenous gene yl.xyl2, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 3;
further, the xylulokinase coding gene is an endogenous gene yl.xyl3, and the nucleotide sequence is shown in a sequence table SEQ ID NO. 4;
further, the starting strain may be yarrowia lipolytica po1g, yarrowia lipolytica po1f, or the like;
preferably, the starting strain is yarrowia lipolytica po1 f.
The second technical proposal provided by the invention is the application of the yarrowia lipolytica gene engineering bacteria in producing triacetic acid lactone;
the third technical scheme provided by the invention is a method for producing triacetic acid lactone by adopting the yarrowia lipolytica genetic engineering bacteria, which comprises the following steps:
inoculating the engineering bacteria seed liquid into a xylose-containing fermentation culture medium according to the inoculation amount of 1-5%, fermenting at 25-32 ℃ and 200-250rpm for 5-7 days;
preferably, PBS is added at the beginning of fermentation to control the pH of the fermentation system to be about 5-7.5;
preferably, adding cerulenin with final concentration of 1-3mg/L after fermentation for about 24 h;
further, the fermentation medium of the engineering bacteria obtained by adopting plasmid expression comprises the following components: 35-50g/L xylose, 1-2g/L LYNB, 0.5-2g/L ammonium sulfate, 0.4-1g/L CSM-LEU, and water in balance;
further, the fermentation medium of the engineering bacteria obtained by adopting genome integration expression comprises the following components: 35-50g/L of xylose, 15-25g/L of Tryptone, 5-15g/L of Yeast Extract and the balance of water, wherein the pH value is about 5-7.5;
further, the fermentation medium of the engineering bacteria obtained by adopting genome integration expression comprises the following components: 25-35mL of corn straw hydrolysate, 15-25g/L of Tryptone, 5-15g/L of Yeast Extract and the balance of water, wherein the pH value is 5-7.5;
preferably, 1-10g/L of glycerol is added to the above medium.
The invention has the advantages that:
the invention develops the yarrowia lipolytica genetic engineering bacteria capable of efficiently metabolizing xylose to produce the triacetic acid lactone (the metabolic pathway for producing the triacetic acid lactone in the yarrowia lipolytica is shown in figure 1), determines the advantageous pathway for heterogeneously producing the triacetic acid lactone, and realizes the effective application of the xylose. The integration mode of the yarrowia lipolytica strain is determined, the yarrowia lipolytica strain for producing the triacetic acid lactone by using xylose is successfully constructed, the triacetic acid lactone can also reach higher yield in a general YPX culture medium, the fermentation process is further optimized, the optimal production process for producing the triacetic acid lactone by using the xylose by using the yarrowia lipolytica is developed, the yield reaches the highest level in the current report shake flask, the yield is between 3g/L and 6g/L, and a foundation is laid for subsequently developing a cheap carbon source to produce a high-value platform compound triacetic acid lactone and providing a chassis strain.
Description of the drawings:
FIG. 1: a metabolic pathway for producing triacetin in yarrowia lipolytica.
FIG. 2: qPCR was performed to determine the expression levels of yl.xyl1, yl.xyl2, and yl.xyl3.
FIG. 3: and (4) fermenting and verifying yarrowia lipolytica transformants.
FIG. 4: yali01 curve fermentation.
FIG. 5: integration strain PCR verification
Because the integrated fragments are too long and are not easy to perform PCR, the integrated fragments are divided into a plurality of fragments with shorter lengths for PCR verification: the A1 fragment comprises leu delta up, ura3 and a part of gh2ps genes; fragment a2 comprises part of the gh2ps gene, part of yl.xyl3; fragment a3 includes the moieties yl.xyl3, yl.xyl2; fragments a4 include the moieties yl.xyl2, yl.xyl1, leu Δ down; the presence of A1-A4 at the same time indicated that the linearized plasmid was intact.
FIG. 6: and 5, Yali02 curve fermentation results.
FIG. 7: after optimizing the fermentation conditions, the Yali02 fermentation curve is obtained.
FIG. 8: yali02 produced TAL fermentation curves using lignocellulose.
FIG. 9: when glycerol is added, Yali02 utilizes lignocellulose to generate TAL fermentation curves.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
The instruments, materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The present invention is further explained below by means of specific embodiments.
TABLE 1 primer sequences to which the invention relates
Primer name
Primer sequence 5 '-3'
gh2ps-F
5’-ccgaccagcactttttgcagtactaaccgcaggggtcatatagcagtgatgatgtgg-3’
gh2ps-R
5’-ggggacaggccatggaactagtcggtaccttagttgccgttggccactgc-3’
yl.xyl1-F
5’-ccgaccagcactttttgcagtactaaccgcagtccttcaagctcgcctccgg-3’
yl.xyl1-R
5’-ggggacaggccatggaactagtcggtaccttaggcgaaaatgggaagg-3’
yl.xyl2-F
5’-ccgaccagcactttttgcagtactaaccgcagtcttctaacccgtcatttg-3’
yl.xyl2-R
5’-ggggacaggccatggaactagtcggtaccctactcctcctcgggaccg-3’
yl.xyl3-F
5’-ccgaccagcactttttgcagtactaaccgcagtatctcggactggatctttcg-3’
yl.xyl3-R
5’-ggggacaggccatggaactagtcggtaccttatttctccaggcaggcg-3’
Example 1 mechanistic study based on Glycerol as a xylose metabolism "response switch
To investigate the regulation mechanism of glycerol in the xylose metabolism of yarrowia lipolytica, yarrowia lipolytica po1f containing empty plasmid PYLXP' was cultured in 250mL shake flasks containing leucine deficient xylose fermentation medium (xylose 40g/L, YNB1.7g/L, ammonium sulfate 1.1g/L, CSM-Leu0.69g/L, and water), 3 ‰ pure glycerol was added at the same time, 30 ℃, 250rmp/min, cells were extracted when the cultures reached logarithmic growth phase, and qPCR was used to determine the expression levels of yarrowia lipolytica intracellular xylose reductase gene yl.xyl1, xylitol dehydrogenase gene yl.xyl2, and xylulokinase gene yl.xyl3 in the blank control group without glycerol addition and the experiment group with glycerol addition.
Beta actin was selected as an internal reference gene, and the relative mRNA expression levels of these three genes were measured. The results of the measurement are shown in fig. 2, which shows that the expression level of yl.xyl1 gene is increased 9.8 times, the expression level of yl.xyl2 gene is increased 7.6 times, and the expression level of yl.xyl3 gene is increased 5.2 times in the experimental group, compared to the blank control group.
Meanwhile, the growth conditions of the thalli in the blank control group without glycerol and the experimental group with glycerol are measured, and the results show that the thalli in the blank group without glycerol does not grow, xylose is not consumed, and the strain OD of the experimental group with glycerol is added60010 to 12 is achieved, and the xylose consumption is 6 to 7 g/L.
The above results demonstrate that glycerol can act as a "response switch" to activate the xylose metabolism pathway of yarrowia lipolytica, increasing the ability of the strain to metabolize xylose.
Example 2: construction of synthetic pathway for producing triacetic acid lactone by using xylose
In order to further enhance the capability of yarrowia lipolytica in metabolizing xylose to produce high-value chemical triacetic acid lactone, optimized 2-pyrone synthetase gene gh2ps, xylose reductase gene yl.xyl1, xylitol dehydrogenase gene yl.xyl2 and xylulokinase gene yl.xyl3 expression cassettes are introduced into yarrowia lipolytica po1f to obtain yarrowia lipolytica recombinant strain Yali01, and the gene sequences are shown in SEQ ID NO. 1-SEQ ID NO. 4.
(1) Plasmid construction
The optimized gh2ps gene was synthesized by the company, and yl.xyl1, yl.xyl2, yl.xyl3 were all from yarrowia lipolytica po1f genome. The optimized nucleotide sequences gh2ps (SEQ ID NO.1), yl.xyl1(SEQ ID NO.2) and yl.xyl2(SEQ ID NO.3) yl.xyl3 genes (SEQ ID NO.4) are obtained by amplifying with gh2ps-F/gh2ps-R, yl.xyl1-F/yl.xyl1-R, yl.xyl2-F/yl.xyl2-R and yl.xyl3-F/yl.xyl3-R as primers, respectively. The PYLXP 'vector backbone carries leucine selection marker and ampicillin resistance gene, and the four gene fragments obtained by amplification are ligated to expression vector PYLXP' to obtain an expression cassette containing four target genes gh2ps, yl.xyl1, yl.xyl2 and yl.xyl3.
(2) Yarrowia lipolytica transformation
The expression cassettes containing four target genes of gh2ps, yl.xyl1, yl.xyl2 and yl.xyl3 are transferred into the host yarrowia lipolytica by using a chemical conversion method of lithium acetate/single-stranded DNA/PEG4000 to obtain the recombinant yarrowia lipolytica strain Yali 01.
1) Preparation of yeast competent cells:
activating the strain. The strain stored at-80 ℃ was streaked on a solid YPD medium and cultured at 30 ℃ for 1 day.
② selecting single yeast colony to streak passage on solid YPD culture medium, and culturing for 1 day at 30 ℃.
③ in a clean bench 90 uL 50% PEG4000, 5 uL 2mol/L lithium acetate, 5 uL salmon sperm (boiling at 95 ℃ for 5min)
Adding into 1.5mL clean sterile centrifuge tube, vortex shaking for 15s, mixing well, and making into 100 μ L mixed system.
Scraping a proper amount of lipolysis yeast colonies on the YPD plate, inoculating the lipolysis yeast colonies into a mixed system, uniformly mixing the lipolysis yeast colonies and the mixed system by vortex oscillation for 15s, and preparing yeast competent cells
2) Yeast transformation
Adding 8 mu L of target plasmid into the prepared yeast competent cells in a super clean bench, and uniformly mixing by vortex oscillation;
secondly, putting the conversion system into a water bath kettle at 30 ℃ for water bath, taking out the conversion system every 10min, shaking for 15s, totally 4 times, and then turning into a water bath kettle at 39 ℃ for water bath for 10 min;
thirdly, adding 92 mu L of sterile water into the transformation system in a super clean bench, shaking for 15s, and uniformly mixing to obtain a 200 mu L system;
and fourthly, coating 200 mu L of the mixed solution on a leucine defective screening plate, and culturing for 3 days at the temperature of 30 ℃.
3) Fermentation verification of yarrowia lipolytica transformants
Several yeast transformants were picked from the transformation plate and inoculated into 3mL leucine deficient seed medium (xylose 40g/L, YNB1.7g/L, ammonium sulfate 5g/L, CSM-Leu0.69g/L, water for the remainder) for 2-3 days until OD reached600The strain was inoculated to a hair containing 10mL of leucine-deficient strain at an inoculum size of 2% at a value of about 12A50 mL shake flask of the fermentation medium (40g/L xylose, 1.7g/L YNB, 1.1g/L ammonium sulfate, 0.69g/L CSM-LEU, 3g/L glycerol, and the balance water) was incubated at 30 ℃ and 250rpm for 5 days. Collecting fermentation liquor, and analyzing biomass and TAL yield. The determination result is shown in FIG. 3, and the recombinant strain 5# (named Yali01) with the highest triacetic acid lactone yield is selected for subsequent fermentation.
Example 3: fermentation production of triacetic acid lactone by yarrowia lipolytica recombinant strain Yali01
(1) Recombinant bacterium culture
The recombinant strain Yali01 obtained in example 2 was inoculated into a test tube containing 3mL of leucine deficient seed medium (20 g/L xylose; YNB1.7g/L ammonium sulfate; 5g/L ammonium sulfate; CSM-Leu0.69g/L, balance water), cultured for 2-3 days in a shaker at 30 ℃ and 250rpm, inoculated into a 250mL Erlenmeyer flask containing 30mL of leucine deficient fermentation medium at an inoculum size of 2%, cultured for 7 days in a shaker under conditions of 30 ℃ and 250rpm, and sampled and analyzed every 24 hours.
The fermentation medium comprises the following components in percentage by weight: 40g/L xylose, 1.7g/L YNB, 1.1g/L ammonium sulfate, 0.69g/LCSM-LEU, 3g/L glycerol and the balance of water.
(2) Fermentation broth detection
Determination of OD of fermentation broth by use of microplate reader or ultraviolet spectrophotometer600The value is obtained. Monitoring cell growth by conversion to Dry Cell Weight (DCW) from a calibration curve; the yield of triacetic acid lactone and the content of residual sugar were determined by high performance liquid chromatography.
(3) Results
A yarrowia lipolytica host which does not express a 2-pyrone synthase gene gh2ps can not synthesize triacetic acid lactone, a 2-pyrone synthase gene gh2ps is introduced, an endogenous xylose reductase gene yl.xylyl 1, a xylitol dehydrogenase gene yl.xylyl 2 and a xylulokinase gene yl.xylyl 3 expression cassette of the yarrowia lipolytica are overexpressed to obtain a recombinant bacterium Yali01, xylose is used as a unique carbon source, 1.2g/L of triacetic acid lactone is obtained by shake flask fermentation, the fermentation result is shown in figure 4, the yarrowia lipolytica realizes the fermentation production of triacetic acid lactone by using xylose as the unique carbon source, and the foundation is laid for the yarrowia lipolytica to produce acetyl coenzyme A derivatives by using xylose.
Example 4: construction of yarrowia lipolytica integration strain for producing triacetolactone from xylose
In order to obtain a yarrowia lipolytica engineering strain capable of efficiently producing triacetic acid lactone by stably utilizing xylose, genes gh2ps, yl.xyl1, yl.xyl2 and yl.xyl3 which are proved to be beneficial to the growth and fermentation of the strain in experiments are integrated into the genome of the yarrowia lipolytica, and a yari 02 integrating the yarrowia lipolytica capable of directly utilizing xylose to produce triacetic acid lactone is constructed.
1) Plasmid construction
The four genes gh2ps, yl.xyl1, yl.xyl2, yl.xyl3 in example 1 were linked to the integration vector p.DELTA.leu 2loxP (the construction method of the plasmid p.DELTA.leu 2loxP is shown in: Coupling the plasmid with negative amplification to the integration plasmid and the plasmid yield. the integration plasmid Engineering 61(2020) 79-88) to obtain the integration plasmid containing gh2ps, yl.xyl1, yl.xyl2, yl.xyl3, the gene sequences are shown in SEQ ID No. 1-SEQ ID No. 4. The integrated plasmid was digested with Avr II and Not I, and the linearized integrated fragment leu. DELTA. up-ura3-gh2 ps-yl.xyl3-yl.xyl2-yl.xyl1-leu. DELTA. down was purified and recovered.
2) Integration by homologous recombination
The integrated fragment leu. DELTA. up-URA3-gh2 ps-yl.xyl3-yl.xyl2-yl.xyl1-leu. DELTA. down was transformed into yarrowia lipolytica po1f and plated on URA3 deficient selection plates, and only strains that were successfully integrated were able to grow on URA3 deficient selection plates. Selecting a plurality of positive colonies growing on the URA3 plate to a YPD plate with rich nutrition, carrying out subculture for 3-5 generations, then selecting a single colony growing on the YPD plate to a URA3 defective xylose screening plate, wherein the single colony growing on the screening plate is a strain which is successfully integrated, thereby achieving the purpose of screening the lipolytic yeast which is successfully integrated. And extracting a yeast genome, verifying whether the gene is successfully integrated by PCR, and obtaining an electrophoresis result as shown in FIG. 5, wherein the successfully integrated strain is named as Yali 02.
Example 5: integrated strain Yali02 for producing triacetic acid lactone by xylose fermentation
The seed broth of the integrated strain Yali02 was inoculated at 2% inoculum size into a 250mL shake flask containing 30mL YPX fermentation medium for curve fermentation at 30 ℃ and 250 rpm. The biomass, the yield of the triacetyl lactone and the xylose content are sampled and analyzed every 24h, the analysis method is the same as that in example 3, the fermentation curve is shown in figure 6, the integrated strain Yali02 can accumulate 1.8g/L of triacetyl lactone when the fermentation conditions are not optimized, and important reference is provided for further reducing the production cost.
The YPX medium comprises the following components: 40g/L of xylose, 20g/L of Tryptone, 10g/L of Yeast Extract, 3g/L of glycerol and the balance of water, the pH value is about 6.0, and the fermentation process is not controlled.
Example 6: optimizing the fermentation medium to improve the triacetic acid lactone yield of the integrated strain Yali02
To further increase the amount of triacetin, PBS was added at a final concentration of 0.2M at the beginning of the fermentation, the pH value was controlled to 6.0 or more using PBS buffer during the fermentation, cerulenin (fatty acid synthase inhibitor, inhibiting synthesis of by-product fatty acid) was added at a final concentration of 1.5mg/L at 24 hours of the fermentation, and a small amount of sodium hydroxide was added when the pH value was decreased to 6.0 or less at 72 hours of the fermentation. The influence of pH value regulation and byproduct inhibition on triacetic acid lactone accumulation is evaluated.
The fermentation method and the culture medium were the same as those in example 5, while the medium without glycerol was used as a control, and samples were taken every 24 hours for analysis, and the analysis method was the same as that in example 3.
When cerulenin and the pH buffer solution are added simultaneously, the TAL yield is improved by 172 percent and reaches 4.9g/L (the fermentation curve is shown in figure 7 a) compared with that when the cerulenin and the pH buffer solution are not added in the example 5, which indicates that the cerulenin and the PBS buffer solution have a great promotion effect on the TAL yield in the fermentation process.
The experimental group with both cerulenin and pH buffer and glycerol added increased TAL yield by 10.9% compared to the control group with cerulenin and pH buffer added alone and no glycerol added (4.42g/L, fig. 7 b).
The formulation of 0.2M PBS (pH6.0) used in the present invention was as follows (100 ml): 0.2M NaH2PO4 87.7ml、0.2MNa2HPO4 12.3ml。
Example 7: integrated strain Yali02 for producing triacetic acid lactone by utilizing fermentation of lignocellulose hydrolysate
The integrated strain Yali02 was inoculated into a 250mL shake flask containing 30mL of a lignocellulose fermentation medium for curve fermentation at 30 ℃ and 250rpm, PBS buffer was added at the beginning of the fermentation to give a final concentration of 0.2mol/L, cerulenin and PBS were added at a final concentration of 1.5mg/L for 24 hours of the fermentation, and a small amount of sodium hydroxide was added if the pH dropped below 6.0 for 72 hours of the fermentation.
Meanwhile, the fermentation conditions of the thalli in the blank control group without glycerol and the experimental group with glycerol are measured, samples are taken every 24 hours to analyze the biomass and the yield of the triacetyl lactone, and the analysis method is the same as that of the example 3. The yield of the control group triacetolactone without glycerol is 2.8g/L as shown in FIG. 8, while the experimental group with glycerol can accumulate 3.1g/L triacetolactone as shown in FIG. 9, which lays a foundation for further developing cheap carbon sources to produce high-value triacetolactone.
The culture medium of the lignocellulose hydrolysate comprises the following components: 30mL of corn straw hydrolysate, 20g/L of Tryptone, 10g/L of Yeast Extract, 3 per mill of glycerol and the balance of water, wherein the pH value is about 7.0;
the preparation method of the corn stalk hydrolysate comprises the following steps:
collecting corn stalks, crushing the corn stalks, screening the crushed corn stalks by a screen of 80 meshes to obtain residue powder, mixing the residue powder with a 2% dilute sulfuric acid solution according to the solid-to-liquid ratio of 1:10, placing the mixture in a high-pressure reaction kettle, hydrolyzing the mixture at the high temperature of 100 ℃ for 100min to obtain a mixture of waste residues and hydrolysate, carrying out vacuum filtration for 2 to 3 times, and removing the waste residues to obtain the corn stalk hydrolysate.
The above is the technical solution disclosed and proposed by the present invention, and it should be noted that the above preferred embodiments should not be considered as limiting the present invention, and the scope of the present invention should be determined by the scope defined by the claims. Many modifications and variations of the present invention may be made by those skilled in the art while following the teachings herein, and such modifications and enhancements are considered to be within the scope of the present invention.
SEQUENCE LISTING
<110> Beijing university of chemical industry
<120> yarrowia lipolytica engineering bacterium for producing triacetic acid lactone by using xylose and application
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1209
<212> DNA
<213> African daisy (Gerbera hybrid)
<400> 1
atggggtcat atagcagtga tgatgtggag gtgatccgcg aggctggccg cgcacaagga 60
ctggctacta tcctggccat tggaaccgcc acaccaccaa attgcgtggc tcaagccgat 120
tacgcagact attatttccg ggtaactaaa tcagagcaca tggtcgatct gaaggagaag 180
tttaaaagga tttgcgaaaa gactgccatt aaaaagcgat atctcgccct cacggaagat 240
tatctccagg aaaacccaac catgtgtgag ttcatggctc cctcattgaa cgctcgccag 300
gatctcgtgg ttaccggggt ccccatgctt ggcaaggagg ccgccgtcaa agccatcgac 360
gaatggggtt tgccaaagtc aaaaattacc catctgattt tctgcactac cgccggggta 420
gacatgcccg gtgcagacta ccagctggtg aagctgctgg gtctttcccc atctgtgaag 480
cgctatatgc tgtaccagca gggctgtgca gctggtggta ctgtgctgcg cctggctaag 540
gacttggcag agaacaataa ggggtcacgg gtgctgatcg tctgctccga gattacagcc 600
atcctgtttc acggaccaaa cgagaatcac ctcgactcac tggtggctca agctctgttc 660
ggcgacggtg ctgccgcgct gatagtgggg tcagggcccc atctggctgt ggaacggccc 720
atctttgaga ttgttagcac agatcagacc atcttgcccg acactgagaa agcgatgaag 780
cttcacttga gggagggggg tctcacgttc cagctccata gggacgtgcc actgatggtt 840
gcaaaaaaca tcgaaaacgc tgccgagaaa gcgctgtctc cattggggat tacagactgg 900
aactctgtgt tttggatggt tcaccctgga gggagagcaa tcctggatca ggtggagcgc 960
aaactgaacc ttaaagagga caaactgaga gccagcagac acgtgctgag cgagtatgga 1020
aacttgattt ctgcttgcgt gctttttatc atcgacgagg tgcgcaagcg ctccatggcc 1080
gaaggtaaga gcactaccgg ggaggggctg gattgtggag tgctctttgg atttggtcca 1140
ggtatgaccg tcgaaactgt tgtactccga tccgttcgag tgaccgctgc agtggccaac 1200
ggcaactaa 1209
<210> 2
<211> 951
<212> DNA
<213> Yarrowia lipolytica (Yarrowia lipolytica) po1f
<400> 2
atgtccttca agctcgcctc cggaaagtcc atgcccaagg tcggattcgg cctgtggaag 60
gtcccccgtg acaagaccgc cgacaccgtc tacggagcca tcaagaacgg ttacagactg 120
tttgacggcg ccttcgacta ccagaacgag cgagaggccg gcgaaggtat ccgacgagcc 180
atcaaggatg gcctggtcaa gcgagaggac atcttcatca ccaccaagtt gtggaacacc 240
ttccactcaa aggagcacgc tctgcagatc gccaaggagc agaacgagtg gtggggactc 300
gactacatcg atctctacct catccacttc cccatcccca tgcagtacat tcccatctcc 360
gagaaggagt gggctggatg gaccaacgcc actgactcgg gtcctaaccc tctggccaag 420
atccctaccc gagagacctg ggaggctctc gaggagctag ttgataccgg aatcgccaag 480
tccattggtg tctccaactt caccgcccag aacatttacg acgtccagac ctacaacaag 540
caccccattt ctgctctgca gattgagcac cacccctacc tggtgcagcc ccagctgacc 600
cagctcgcaa aggacaacaa catccaggtc actgcctact cttctttcgg ccccgcctcc 660
tttgtggaga ttggcatgga ccagaaggtc cctcctcttt tcgagaacga gaccatcacc 720
aagatcgcca aggctcacaa caagaccccc tcccaggttc tgctgcgatg ggctacccag 780
cgaggcattg ccgtcatccc caagtccaac aacgtcgagc gacaaactca gaacctcgaa 840
tctctggact ttgacctgac cgaggccgag attaaggaga tctccaacct caacaagaac 900
ctgcgattca acgatcccgg tgtctacgct aaccttccca ttttcgccta a 951
<210> 3
<211> 1074
<212> DNA
<213> Yarrowia lipolytica (Yarrowia lipolytica) po1f
<400> 3
atgtcttcta acccgtcatt tgttcttcga aagccattgg atctcgtctt tgaggatcgg 60
cccgacccca agatccagga cccccactcc gtcaaggtgg cagtcaaaaa gaccggagtt 120
tgcggctcgg atgtccacta ctatctgcat ggaggaatcg gcgacttcat tgtcaaggct 180
cccatggttc taggccatga aagtgccgga gaggtggttg aggttggtcc tgaagtcaag 240
gacctcaagg tgggagatcg agtggctctc gagcccggag tgccgtctcg attgtcacag 300
gagtacaagg agggacgata caacctgtgt ccttgcatgg tgtttgctgc cacccctccc 360
tacgacggta ctctgtgtcg tcactacatc attcccgagg acttttgtgt caagctgcct 420
gatcatgtgt ctctcgagga gggagctctt gtggagcctc tgtccgtggc tgtccactgc 480
aacaagctgg ccaagaccac tgcccaggac gtggttattg tgtttggagc tggcccagtc 540
ggactgctag ccgtgggagt ggccaatgcc tttggatcat ctaccattgt gtgtgttgat 600
cttgttcccg agaagctgga gctcgccaag aagttcggtg ccactcatac gtttgtaccc 660
actaagggag acagtcccaa cgagtctgct gacaagatcc gagctctgat caagggcgct 720
ggtctctctg actcgcccaa tgtggctttg gagtgcaccg gagctgagcc ttctattcag 780
actgctgttt ctgtgctggc cacttccggt cgacttgtgc aggtcggcat gggcaaggat 840
gacgtcaact tccctatcac caaatgcatt gtaaaggaga ttaccgtgct cggatcgttc 900
cgatactgcc atggtgacta tcccctggct gttcagctgg ttgcttctgg caagattgac 960
gtcaagaagc tggtgaccaa ccggttcacc ttcaaggagg ctgagcaggc gtacaagacg 1020
gcggccgagg gcaaggccat caagatcatc attgacggtc ccgaggagga gtag 1074
<210> 4
<211> 1623
<212> DNA
<213> Yarrowia lipolytica (Yarrowia lipolytica) po1f
<400> 4
atgtatctcg gactggatct ttcgactcaa cagctcaagg gcatcattct ggacacaaaa 60
acgctggaca cggtcacaca agtccatgtg gactttgagg acgacttgcc gcagttcaac 120
accgaaaagg gcgtctttca cagctctaca gtggccggag aaatcaatgc tcctgtggca 180
atgtgggggg cagctgtgga cttgctgata gagcgtctgt caaaggaaat agacctttcc 240
acgatcaagt ttgtgtcggg ctcgtgccag caacacggct ctgtttatct caacagcagc 300
tacaaggagg gcctgggttc tctggacaaa cacaaagact tgtctacagg agtgtcatcc 360
ttactggcgc tcgaagtcag ccccaattgg caggatgcaa gcacggagaa ggagtgtgcg 420
cagtttgagg ctgcagtcgg cggtcccgag cagctggctg agatcactgg ctctcgagca 480
catactcgtt tcaccgggcc ccagattctc aaggtcaagg aacgcaaccc caaggtattc 540
aaggccacgt cacgggtcca gctcatatcc aactttctag catctctgtt tgccggcaag 600
gcgtgcccct ttgatcttgc tgacgcctgt ggaatgaatc tgtgggacat ccagaatggc 660
cagtggtgca agaaactcac agatctcatc accgatgaca cccactcggt cgagtccctc 720
cttggagacg tggaaacaga ccccaaggct ctactgggca aaatctcgcc ctatttcgtc 780
tccaagggct tctctccctc ttgtcaggtg gcacagttca caggcgacaa cccaggcact 840
atgctggctc tccccttaca ggccaatgac gtgattgtgt ctttgggaac atctacgacc 900
gccctcgtcg taacaaacaa gtacatgccc gaccccggat accatgtgtt caaccacccc 960
atggagggat acatgggcat gctgtgctac tgcaacggag gtctagcacg agagaagatc 1020
cgagacgagc ttggaggctg ggacgagttt aatgaggcgg ccgagaccac caacacagtg 1080
tctgctgacg atgtccatgt tggcatctac tttccactac gagaaatcct tcctcgagca 1140
ggtccctttg aacgacgttt catctacaac agacaaagtg aacagcttac agagatggct 1200
tctccagagg actcactggc aaccgaacac aaaccgcagg ctcaaaatct caaggacacg 1260
tggccgccac aaatggacgc cactgccatc attcaaagcc aggccctcag tatcaaaatg 1320
agactccaac gcatgatgca tggcgatatt ggaaaggtgt attttgtggg aggcgcctcg 1380
gtcaacactg ctatctgcag cgtaatgtct gccatcttaa aaccaacaaa gggcgcttgg 1440
agatgtggtc tggaaatggc aaacgcttgt gccattggaa gtgcccatca cgcctggctt 1500
tgcgacccca acaagacagg ccaggtacag gttcacgaag aagaggtcaa atacaagaat 1560
gtggacacag acgtgctact caaggcgttc aagctggccg aaaacgcctg cctggagaaa 1620
taa 1623
