CN103820375A - Engineering strain for biologically producing ferulic acid and establishing method of engineering strain - Google Patents
Engineering strain for biologically producing ferulic acid and establishing method of engineering strain Download PDFInfo
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- CN103820375A CN103820375A CN201310682186.9A CN201310682186A CN103820375A CN 103820375 A CN103820375 A CN 103820375A CN 201310682186 A CN201310682186 A CN 201310682186A CN 103820375 A CN103820375 A CN 103820375A
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Abstract
The invention discloses an establishing method of an engineering strain for biologically producing ferulic acid and application of the engineering strain in production of the ferulic acid. The engineering strain enables a vanillyl alcohol alcohol oxidase gene, a coniferyl alcohol dehydrogenase gene and a coniferyl aldehyde dehydrogenase gene to be connected in series and multiply strongly expressed in escherichia coli by using a T7 promoter, and then eugenol is catalyzed to generate the ferulic acid through enzymatic reaction. Through the bioconversion method of the ferulic acid, poisoning of substrate eugenol to a production strain is avoided; the characteristics of simple process and high conversion rate are achieved.
Description
Technical field
The invention belongs to medical material production technical field, be specifically related to a kind of recombinant bacterial strain and construction process thereof of simultaneously expressing vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and coniferyl aldehyde desaturase, and utilize the bioconversion method of this bacterial strain production forulic acid.
Background technology
The chemical name of forulic acid (Ferulic Acid) is Ferulic acid, is one of derivative of TRANSCINNAMIC ACID.Because forulic acid can be used as the precursor substance of microorganism fermentative production vanillin food grade,1000.000000ine mesh, with and the characteristic such as anti-oxidant, antithrombotic formation of being had, reducing blood-fat, mediator's body immunity function, ferulic acid and its derivatives is widely used in reducing blood-fat, prevent and treat coronary heart disease and cancer, antisepsis and anti-inflammation, anti-mutation, promoting blood circulation and removing obstruction in channels, eliminate chloasma, and heavy etc. the therapeutic treatment of insomnia, soreness of waist leg, healthcare products are produced and foodstuff additive field.
The preparation method of forulic acid mainly directly extracts from plant both at home and abroad at present, and the now industrial thiaminogen that generally adopts alkaline process to degrade in rice bran or wheat bran is prepared forulic acid, but because this method is subject to the restriction that material content is lower, output is limited, and cost is higher.Chemical synthesis, take 4-hydroxyl-3-methoxylbenxaldehyde and propanedioic acid as raw material, forms by condensation reaction.But this method long reaction time, and what produce be trans and mixture cis forulic acid, not easily separated, there is some problems in while chemical synthesis aspect environmental pollution.
Summary of the invention
The object of the present invention is to provide a strain series connection high efficiency to express the recombination bacillus coli engineering strain of vanillic alcohol oxidase gene, coniferyl-alcohol dehydrogenase gene and coniferyl aldehyde dehydrogenase gene.T7 promoter sequence has been integrated in this vanillic alcohol oxidase gene, coniferyl-alcohol dehydrogenase gene and coniferyl aldehyde dehydrogenase gene upstream, in engineering strain, is started and is transcribed and final high efficient expression by t7 rna polymerase.Specifically by adding that before each gene T7 promotor forms expression cassette, and three expression cassettes are together in series, first gene is by a T7 promoter transcription like this, second gene can be by two T7 promoter transcriptions, the 3rd gene can be by three T7 promoter transcriptions, to increase the expression amount of gene and the activity of enzyme, thereby reach the object that significantly improves enzymatic reaction activity.
Another object of the present invention is to utilize the bioconversion method of this project bacterial strain production forulic acid.Because substrate Eugenol has certain bacteriostasis, in biotransformation, can suppress the fermentation of engineering strain and the expression of biochemical enzyme, the present invention adopts two-step approach to complete respectively the fermentation of engineering strain and the bio-transformation of forulic acid.First ferment and obtain vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and the coniferyl aldehyde desaturase of great expression by induction, then react with substrate Eugenol as enzyme liquid by collection thalline, resuspension, to avoid substrate Eugenol to suppress the murder by poisoning of recombinant bacterial strain., final conversion generates product forulic acid.
The technical scheme that the present invention adopts for realizing its object:
A kind of recombinant bacterial strain, express vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and coniferyl aldehyde desaturase simultaneously, wherein, T7 promotor is integrated respectively in vanillic alcohol oxidase gene, coniferyl-alcohol dehydrogenase gene and coniferyl aldehyde dehydrogenase gene upstream, in engineering strain, is started and is transcribed by t7 rna polymerase.
The construction process of above-mentioned recombinant bacterial strain, comprises the steps:
1. build to produce and transform bacterial strain pET-VAB
1) with Nde I and the Bam HI synthetic vanillic alcohol oxidase gene (vaoA of double digestion respectively, 1683bp), coniferyl-alcohol dehydrogenase gene (calA, 768bp) with coniferyl aldehyde dehydrogenase gene (calB, 1446bp), be cloned on the pET24a carrier of cutting with Nde I and Bam HI enzyme, obtain recombinant vectors pET24a-vaoA, pET24a-calA and pET24a-calB.
2) recombinant vectors pET24a-vaoA is cut with Bam HI and Eco RI enzyme, reclaim as carrier, pET24a-calA cuts with Bgl II and Eco RI enzyme, and reclaiming size is 900bp left and right fragment, be connected, transform with above-mentioned carrier, obtain recombinant vectors pET24a-vaoA-calA.
3) recombinant vectors pET24a-vaoA-calA is cut with Bam HI and Eco RI enzyme, reclaim as carrier, pET24a-calB cuts with Bgl II and Eco RI enzyme, reclaiming size is 1.6kb left and right fragment, be connected, transform with above-mentioned carrier, obtain recombinant vectors pET24a-vaoA-calA-calB(pET24a-VAB).
4) recombinant vectors pET24a-VAB is proceeded to BL21(DE3), obtain expection engineering bacteria.
2. shake-flask culture fermentation, adopts intestinal bacteria cultivate under the following conditions and ferment
1) seed culture medium (mass percent) and culture condition: peptone 1%, yeast extract 0.5%, sodium-chlor 1%, surplus is pure water.Cultivate 16 hours for 37 ℃, shaking table revolution is 200rpm/min.
2) fermention medium: peptone 1.2%, yeast extract 2.4%, glycerine 0.4%, potassium primary phosphate 0.23%, dipotassium hydrogen phosphate 1.25%, surplus is pure water.
3) fermentation culture conditions: by fermentation volume 1% amount inoculation, 37 ℃ of cultivations, shaking table revolution is 200rpm/min; Inoculate after 2 hours, be cooled to 25～28 ℃ and add final concentration 0.6mM IPTG, continue to cultivate 8 hours.
3. the bio-transformation of forulic acid
1) after fermentation culture finishes, the centrifugal collection thalline of 4000rpm/min at 4 ℃.
2) 100mL phosphoric acid buffer suspension thalline, proceeds to 250mL and transforms in bottle, adds the Eugenol substrate of 0.06mL simultaneously.
3) biotransformation condition: 25 ℃ of reactions, shaking table revolution is 200rpm/min, the Eugenol of interpolation 0.06mL per hour.
4) transform and finish, detect forulic acid output and purity by high performance liquid chromatography (HPLC).
4. high performance liquid chromatography detects ferulaic acid content
1) get conversion fluid 25 μ L, add 475 μ L methyl alcohol fully to mix, lysate.
2) sample centrifugal 5min of 10000rpm/min under 4 ℃ of low temperature, gets supernatant liquor.
3) supernatant liquor filters by 0.45 μ m filter, gets filtrate to be measured.
4) high performance liquid chromatography chromatographic condition: chromatographic column be Dia-monsil-C18 (250mm × 4.6mm, 5 μ m); Moving phase is the methyl alcohol of 68:32: 0.1% acetum; Flow velocity is 0.6mL/min, 25 ℃ of column temperatures; Detection wavelength is 320nm; Sample size 5 μ L.
5), according to the peak area production standard curve of forulic acid standard substance, finally calculate the output of forulic acid in conversion fluid.
Compared with currently available technology, the present invention transforms the safe biologic of medical intermediate product forulic acid and provides effectively, simple implementation method, has higher industrialization value.Utilize T7 promotor make vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and coniferyl aldehyde desaturase simultaneously intestinal bacteria in series connection high efficiency express, catalysis Eugenol generates forulic acid.The method for transformation of this forulic acid is single stage method response procedures, has avoided substrate Eugenol to producing the murder by poisoning of bacterial strain simultaneously, and has had the advantages that technique is simple, transformation efficiency is high.
The bacterial strain that the present invention builds, heterogenous expression vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and coniferyl aldehyde desaturase.Conversion process is that Eugenol bottoms stream is added in cell suspension, working condition utilization be biological catalyst, production process is without high-temperature high-voltage reaction, without noxious chemical, normal temperature and pressure operation.Continued to flow and added Eugenol by conversion process, forulic acid accumulated concentrations increases.After reaction finishes, substrate conversion efficiency is high, and by product is few, and purifying is simple, and production efficiency is high.
The present invention adopts biological enzyme to transform Eugenol production forulic acid, and transformation efficiency reaches more than 95%, and end product forulic acid concentration reaches 10g/L, has industrialization potentiality.
Accompanying drawing explanation
Fig. 1. the collection of illustrative plates of recombinant plasmid pET24a-VAB;
Fig. 2 .vaoA gene order;
Fig. 3 .calA gene order;
Fig. 4 .calB gene order.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.Experimental technique in embodiment, condition is carried out routinely, with reference to as Pehanorm Brooker etc., experimental technique described in molecular cloning experiment guide (third edition,, Science Press in 2008).
Embodiment 1: the structure of recombinant bacterial strain and evaluation
1. contain the strain construction of recombinant plasmid pET24a-vaoA
1) plasmid construction
Plasmid pET24a (+): Novagen company product, size is 5.31kb, contains kalamycin resistance gene, lac repressor lac I gene, promotor is T7, and has multiple restriction endonuclease sites.
With Nde I and synthetic vanillic alcohol oxidase gene (vaoA, the 1683bp) DNA fragmentation of Bam HI double digestion.With Nde I and Bam HI double digestion carrier pET24a (+).Enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bam HI1 μ L, Nde I1 μ L, 37 ℃ are incubated 3 hours.
The DNA fragmentation glue that enzyme is cut reclaims, and by T4 ligase enzyme connection vaoA and carrier DNA fragment, linked system is as follows: vaoA7.5 μ L, pET24a carrier 1.5 μ L, buffer1 μ L, T4 ligase enzyme 1 μ L, be incubated 16 ℃ and spend the night, connect product and adopt heat shock method to be converted in e. coli host bacteria DH5 α.Be applied to and contain 1% peptone, 0.5% yeast extract paste, on the LB solid medium of 1% sodium-chlor and 1.5% agar powder kantlex.
2) recombinant bacterial strain is identified
LB flat board is cultured to and grows transformant at 37 ℃, picking list bacterium colony, and by 37 ℃ of overnight incubation of LB liquid nutrient medium, centrifugal 1 minute of 12000rpm/min, extracts plasmid, and extracting method operates according to test kit specification sheets.
With Nde I and Bam HI double digestion recombinant vectors, enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bam HI1 μ L, Nde I1 μ L, 37 ℃ are incubated 3 hours.Electrophoresis is determined the DNA fragmentation that contains object vaoA gene.
2. reference method 1 builds respectively pET24a-calA and pET24a-calB recombinant vectors
3. build pET24a-vaoA-calA-calB(pET24a-VAB) recombinant vectors
1) build pET24a-vaoA-calA recombinant vectors
Recombinant vectors pET24a-vaoA is cut with Bam HI and Eco RI enzyme, and enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bam HI1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, and DNA fragmentation reclaims as carrier.
PET24a-calA cuts with Bgl II and Eco RI enzyme, and enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bgl II1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, and reclaiming size is 900bp left and right fragment.Reference method 1 is connected, transforms with above-mentioned carrier, obtains recombinant vectors pET24a-vaoA-calA.
2) build pET24a-vaoA-calA-calB recombinant vectors
Recombinant vectors pET24a-vaoA-calA is cut with Bam HI and Eco RI enzyme, and enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bam HI1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, reclaim as carrier.
PET24a-calB cuts with Bgl II and Eco RI enzyme, enzyme is cut system: DNA43 μ L, buffer R5 μ L, Bgl II1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, and reclaiming size is 1.6kb left and right fragment, reference method 1 is connected, transforms with above-mentioned carrier, obtains recombinant vectors pET24a-vaoA-calA-calB(and sees accompanying drawing 1).
4. build the production bacterial strain containing pET24a-vaoA-calA-calB recombinant vectors
Adopt heat shock method that recombinant vectors is converted in e. coli host bacteria BL21 (DE3).Be applied to and contain 1% peptone, 0.5% yeast extract paste, on the LB solid medium of 1% sodium-chlor and 1.5% agar powder.LB flat board is cultured to and grows transformant at 37 ℃, and picking list bacterium colony obtains expection engineering bacteria.
Embodiment 2: the fermentation of recombinant bacterial strain
Join seed liquor 100mL, seed liquor contains peptone 1%, yeast extract 0.5%, and sodium-chlor 1%, surplus is pure water.Be contained in 250mL triangular flask after sterilizing, the single bacterium colony on inoculation plate culture medium, shaking table revolution is 200rpm/min.Cultivate after 16 hours for 37 ℃, inoculate in the 500mL triangular flask that contains 200mL fermented liquid, fermented liquid contains peptone 1.2%, yeast extract 2.4%, and glycerine 0.4%, potassium primary phosphate 0.23%, dipotassium hydrogen phosphate 1.25%, surplus is pure water.Fermentation culture conditions: by fermentation volume 1% amount inoculation, 37 ℃ of cultivations, shaking table revolution is 200rpm/min, inoculates after latter 1.5 hours, is cooled to 25～28 ℃, adds final concentration 0.6mM IPTG, cultivates 8 hours.
Embodiment 3: catalysis Eugenol generates forulic acid
After fermentation culture finishes, 4000rpm/min4 ℃ of low-temperature centrifugation collected thalline, with 100mL phosphoric acid buffer suspension thalline, proceeding to 250mL transforms in bottle, add 0.06mL Eugenol, conversion condition is 25 ℃ of reactions, and shaking table revolution is 200rpm/min, the Eugenol substrate of portion-wise addition 0.06mL per hour.After 8-10 hour, whether transform completely by high performance liquid chromatography (HPLC) detection forulic acid output and Eugenol.
Embodiment 4: high performance liquid chromatography detects ferulaic acid content
Sample preparation: get conversion fluid 25 μ L, add 475 μ L methyl alcohol fully to mix, the centrifugal 5min of 10000rpm/min, 0.45 μ m filter filters, and getting filtrate, to proceed to sample injection bottle to be measured.
Chromatographic condition: chromatographic column be Dia-monsil-C18 (250mm × 4.6mm, 5 μ m); Moving phase: B methyl alcohol: A0.1% acetum is 68:32; Flow velocity is 0.6ml/min, column temperature 25; ℃ detect wavelength be 320nm; Sample size 5 μ L.
Claims (10)
1. a recombinant bacterial strain, it is characterized in that, series multiplex strongly expressed vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and coniferyl aldehyde desaturase, wherein, before vanillic alcohol oxidase gene, coniferyl-alcohol dehydrogenase gene and coniferyl aldehyde dehydrogenase gene, add respectively that T7 promotor forms expression cassette, and three expression cassettes are together in series,, in engineering strain, start and transcribe by t7 rna polymerase.
2. the construction process of recombinant bacterial strain as claimed in claim 1, is characterized in that, comprises the steps:
(1) with Nde I and Bam HI synthetic vanillic alcohol oxidase gene, coniferyl-alcohol dehydrogenase gene and the coniferyl aldehyde dehydrogenase gene of double digestion respectively, be cloned on the pET24a carrier of cutting with Nde I and Bam HI enzyme, obtain recombinant vectors pET24a-vaoA, pET24a-calA and pET24a-calB;
(2) recombinant vectors pET24a-vaoA is cut with Bam HI and Eco RI enzyme, reclaim as carrier, pET24a-calA cuts with Bgl II and Eco RI enzyme, reclaims, and is connected, transforms with above-mentioned carrier, obtains recombinant vectors pET24a-vaoA-calA;
(3) recombinant vectors pET24a-vaoA-calA is cut with Bam HI and Eco RI enzyme, reclaim as carrier, pET24a-calB cuts with Bgl II and Eco RI enzyme, reclaims, be connected, transform with above-mentioned carrier, obtain recombinant vectors pET24a-vaoA-calA-calB(pET24a-VAB);
(4) recombinant vectors pET24a-VAB is proceeded to BL21(DE3), must expect engineering bacteria BL21-VAB.
3. the construction process of recombinant bacterial strain as claimed in claim 2, is characterized in that, step (1) further comprises the steps:
With Nde I and synthetic vanillic alcohol oxidase gene (vaoA, the 1683bp) DNA fragmentation of Bam HI double digestion; With Nde I and Bam HI double digestion carrier pET24a (+), enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bam HI1 μ L, Nde I1 μ L, 37 ℃ are incubated 3 hours; The DNA fragmentation glue that enzyme is cut reclaims, and by T4 ligase enzyme connection vaoA and carrier DNA fragment, linked system is as follows: vaoA7.5 μ L, pET24a carrier 1.5 μ L, buffer1 μ L, T4 ligase enzyme 1 μ L, be incubated 16 ℃ and spend the night, connect product and adopt heat shock method to be converted in e. coli host bacteria DH5 α; Be applied to and contain 1% peptone, 0.5% yeast extract paste, on the LB solid medium of 1% sodium-chlor and 1.5% agar powder;
LB flat board is cultured to and grows transformant at 37 ℃, picking list bacterium colony, and by 37 ℃ of overnight incubation of LB liquid nutrient medium, centrifugal 1 minute of 12000rpm/min, extracts plasmid, and extracting method operates according to test kit specification sheets;
With Nde I and Bam HI double digestion recombinant vectors, enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bam HI1 μ L, Nde I1 μ L, 37 ℃ are incubated 3 hours;
Electrophoresis is determined the DNA fragmentation that contains object vaoA gene.
4. the construction process of recombinant bacterial strain as described in claim 2 or 3, is characterized in that, in step (2), reclaiming size is 900bp left and right fragment, and/or in step (3), reclaiming size is 1.6kb left and right fragment.
5. the construction process of recombinant bacterial strain as described in claim 2-4, it is characterized in that, step (2) further comprises the steps: recombinant vectors pET24a-vaoA Bam HI and Eco RI enzyme to cut, enzyme is cut system: DNA43 μ L, buffer R5 μ L, Bam HI1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, and DNA fragmentation reclaims as carrier; PET24a-calA cuts with Bgl II and Eco RI enzyme, and enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bgl II1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, and reclaiming size is 900bp left and right fragment.
6. the construction process of recombinant bacterial strain as described in any one in claim 2-4, it is characterized in that, step (3) further comprises the steps: recombinant vectors pET24a-vaoA-calA Bam HI and Eco RI enzyme to cut, enzyme is cut system: DNA43 μ L, buffer R5 μ L, Bam HI1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, reclaim as carrier; PET24a-calB cuts with Bgl II and Eco RI enzyme, and enzyme is cut system: DNA43 μ L, and buffer R5 μ L, Bgl II1 μ L, Eco RI1 μ L, 37 ℃ are incubated 3 hours, and reclaiming size is 1.6kb left and right fragment.
7. the construction process of recombinant bacterial strain as described in any one in claim 2-6, is characterized in that, step (4) further comprises the steps: to adopt heat shock method that recombinant vectors is converted in e. coli host bacteria BL21 (DE3); Be applied to and contain 1% peptone, 0.5% yeast extract paste, on the LB solid medium of 1% sodium-chlor and 1.5% agar powder; LB flat board is cultured to and grows transformant at 37 ℃, and picking list bacterium colony obtains expection engineering bacteria.
8. a biological process method for transformation that adopts the forulic acid engineering strain of bacterial strain described in claim 1, is characterized in that, comprises the steps:
A. induction fermentation obtains vanillic alcohol oxydase, coniferyl-alcohol dehydrogenase and the coniferyl aldehyde desaturase of great expression;
B. collect thalline, resuspension as enzyme liquid, and add gradually Eugenol, transform and generate product forulic acid.
9. the biological process method for transformation of forulic acid engineering strain as claimed in claim 8, is characterized in that, step a further comprises: join seed liquor 100mL, seed liquor contains peptone 1%, yeast extract 0.5%, sodium-chlor 1%, surplus is pure water, and ammoniacal liquor is adjusted pH7.0～7.2; Be contained in 250mL triangular flask after sterilizing, the single bacterium colony on inoculation plate culture medium, shaking table revolution is 200rpm/min; Cultivate after 16 hours for 37 ℃, inoculate in the 500mL triangular flask that contains 200mL fermented liquid, fermented liquid contains peptone 1.2%, yeast extract 2.4%, and glycerine 0.4%, potassium primary phosphate 0.23%, dipotassium hydrogen phosphate 1.25%, surplus is pure water.Fermentation culture conditions: 37 ℃ of cultivations, shaking table revolution is 200rpm/min, inoculates after latter 1.5 hours, is cooled to 25～28 ℃, adds final concentration 0.6mM IPTG, cultivates 8 hours.
10. the biological process method for transformation of forulic acid engineering strain as claimed in claim 8 or 9, it is characterized in that, step b further comprises: after fermentation culture finishes, 4000rpm4 ℃ of low-temperature centrifugation collected thalline, with 100mL phosphoric acid buffer suspension thalline, proceeds to 250mL and transforms in bottle, add 0.06mL Eugenol, conversion condition is 25 ℃ of reactions, and shaking table revolution is 200rpm/min, the Eugenol substrate of portion-wise addition 0.06ml per hour; After 8-10 hour, whether transform completely by high performance liquid chromatography (HPLC) detection forulic acid output and Eugenol.
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Cited By (5)
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| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN105838626A (en) * | 2016-05-06 | 2016-08-10 | 江南大学 | Gibberella fujikuroi and method for converting eugenol and synthesizing coniferyl aldehyde through same |
| CN110734935A (en) * | 2019-11-07 | 2020-01-31 | 西南交通大学 | optimization and improvement of method for synthesizing ferulic acid and methyl ferulate based on enzyme method |
| CN112961875A (en) * | 2021-03-05 | 2021-06-15 | 安徽师范大学 | Construction method of engineering strain for producing tetrahydropyrimidine by biological method |
| CN116590161A (en) * | 2023-05-10 | 2023-08-15 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis with high vanillin yield, construction method and application thereof |
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2013
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928224A (en) * | 2015-05-08 | 2015-09-23 | 安徽师范大学 | Ferulic acid producing engineering strain, construction method and biotransformation method |
| CN104928224B (en) * | 2015-05-08 | 2018-07-24 | 安徽师范大学 | A kind of ferulic acid production engineering bacterial strain, construction method and bioconversion method |
| CN105838626A (en) * | 2016-05-06 | 2016-08-10 | 江南大学 | Gibberella fujikuroi and method for converting eugenol and synthesizing coniferyl aldehyde through same |
| CN105838626B (en) * | 2016-05-06 | 2019-06-07 | 江南大学 | A kind of method of Gibberella fujikuroi and its conversion eugenol synthesis coniferyl aldehyde |
| CN110734935A (en) * | 2019-11-07 | 2020-01-31 | 西南交通大学 | optimization and improvement of method for synthesizing ferulic acid and methyl ferulate based on enzyme method |
| CN112961875A (en) * | 2021-03-05 | 2021-06-15 | 安徽师范大学 | Construction method of engineering strain for producing tetrahydropyrimidine by biological method |
| CN112961875B (en) * | 2021-03-05 | 2022-07-19 | 安徽师范大学 | Construction method of engineering strain for producing tetrahydropyrimidine by biological method |
| CN116590161A (en) * | 2023-05-10 | 2023-08-15 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis with high vanillin yield, construction method and application thereof |
| CN116590161B (en) * | 2023-05-10 | 2024-05-03 | 陕西海斯夫生物工程有限公司 | Recombinant amycolatopsis for producing vanillin, construction method and application thereof |
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