CN113755422B - Recombinant amycolatopsis capable of highly producing vanillin, construction method and application thereof - Google Patents

Abstract

The invention provides a recombinant Amycolatopsis sp with high vanillin yield, which expresses a ferulic acid decarboxylase gene (opt fdc) and a 4-vinylguaiacol oxygenase gene (opt cso 2). The invention also provides a construction method of the recombinant amycolatopsis and application of the recombinant amycolatopsis in vanillin production. Experiments prove that the ferulic acid decarboxylase gene and the 4-vinyl guaiacol oxygenase gene can effectively increase the vanillin output of the amycolatopsis, and the vanillin synthesis path does not need coenzyme A and ATP to be used as coenzymes to participate in the reaction, thereby providing technical support for further improving the vanillin fermentation output of the amycolatopsis in industrial production.

Description

Recombinant amycolatopsis capable of producing vanillin at high yield, and construction method and application thereof

[ technical field ] A

The present invention belongs to the field of gene engineering technology. More specifically, the invention relates to a recombinant amycolatopsis for high yield of vanillin, a construction method of the recombinant amycolatopsis, and application of the recombinant amycolatopsis in vanillin production by taking ferulic acid as a substrate.

[ background of the invention ]

Vanillin is also known as vanillin (3-methoxy-4-hydroxybenzaldehyde), is one of the most widely used flavorings in the world, and is widely applied in the fields of food, medicine, cosmetics, agriculture and the like. Vanillin has the beauty of 'the king of food spice', has the fragrance of vanilla beans and strong milk fragrance, and can play a role in assisting aroma and enhancing flavor in food; in the aspect of medicine, vanillin is an important raw material for synthesizing various medicines; vanillin can be used as flavoring agent in the fields of cosmetics, perfume, etc.; can also be used as a ripener and a yield increasing agent for crops in agricultural production. Due to the wide application field, the vanillin produced at home and abroad can not meet the current market demand.

At present, the production mode of vanillin in the market mainly comprises plant extraction, chemical synthesis and microbial transformation. With the increasing demand of consumers for natural flavors, the production of vanillin by converting natural substrates with microorganisms is gaining attention. At present, the used natural substrate is mainly a renewable resource, such as ferulic acid or eugenol. Among them, ferulic acid is considered to be the first choice precursor for the microbial production of vanillin because of its wide distribution in nature and low toxicity to microorganisms. Research shows that at present, only a plurality of strains of Amycolatopsis and Streptomyces can convert substrate ferulic acid into vanillin with the concentration of more than 10g/L, and the method can be basically applied to industrial production. Ferulic acid metabolism analysis was performed on Amycolatopsis sp.ATCC 39116 (formerly known as Setepticides setonii ATCC 39116) strain by Steinbuchel team in Germany (Fleige C, meyer F, steinbuchel A. Metabolic Engineering of the Actinomycete Amycolatopsis sp.Strain ATCC 39116 methods engineered product of Natural vanillin applied and Environmental microbiology,2016,82 (11): 3410-3419), which is believed to produce vanillin under the sequential action of feruloyl CoA synthetase, enoyl-CoA aldolase, which is further degraded to vanillic acid decarboxylase under the action of vanillic acid dehydrogenase, and vanillic acid is degraded to guaiacol and catechin under the action of vanillic acid demethylase, respectively. By knocking out the vdh gene coding vanillin dehydrogenase and performing over-expression on the gene coding feruloyl-CoA synthetase (fcs) and the gene coding enoyl-CoA aldolase (ech), the vanillin production concentration of the engineering strain reaches 22.3g/L, which is the highest level reported at present. At present, genes for producing vanillin by converting ferulic acid through amycolatopsis bacteria are analyzed, a vanillin synthesis path is very clear, but the fcs gene needs coenzyme A and ATP to participate in reaction as coenzymes, the coenzyme A and the ATP are very expensive, more coenzymes are needed to further improve the yield of vanillin, and the construction of a coenzyme regeneration system in amycolatopsis bacteria is very complex. If the coenzyme-dependent pathway can be replaced by a coenzyme-independent pathway, the biocatalytic process does not require the construction of a coenzyme regeneration system or the addition of a coenzyme.

In 2014, furuya et al (Furuya T, miura M, kino K.A Coenzyme-independant decarbonylase/Oxygenase cassette for the Efficient Synthesis of Vanillin. Chembiochem: A European journal of chemical biology,2014,15 (15): 2248-2254) co-expressed in E.coli a Coenzyme-Independent ferulic acid Decarboxylase (Fdc) derived from Bacillus pumilus ATCC 14884 and a Coenzyme-Independent 4-vinylguaiacol Oxygenase (Cso) derived from inert stalk Bacillus sp.caulicus (Caulobacter segnis ATCC 21756), the strains obtained were capable of converting ferulic acid to vanillin with a vanillin production of 1.2g/L. At present, no coenzyme-independent vanillin synthesis route has been studied in amycolatopsis.

[ summary of the invention ]

The invention aims to overcome the technical scheme in the prior art and improve the vanillin output of the amycolatopsis through a genetic engineering technology.

The invention provides a recombinant Amycolatopsis sp for high yield of vanillin, which expresses a ferulic acid decarboxylase gene (opt fdc) and a 4-vinylguaiacol oxygenase gene (opt sco 2).

In the present invention, the ferulic acid decarboxylase gene (opt fdc) is codon-optimized for fdc gene derived from Bacillus pumilus (Bacillus pumilus ATCC 14884) and its nucleotide sequence is shown in SEQ ID NO.1, and the 4-vinylguaiacol oxygenase gene (opt cso 2) is codon-optimized for cso gene derived from Bacillus inertius (Caulobacter segnis ATCC 21756) and its nucleotide sequence is shown in SEQ ID NO. 2.

The present invention also provides a method for constructing the recombinant Amycolatopsis sp, which comprises the steps of:

(1) Construction of a plasmid containing the Strong promoter permE

Synthesizing a strong promoter permE, wherein the gene sequence of the strong promoter permE is shown in SEQ ID NO.3, digesting the synthesized gene sequence by XbaI/EcoRI, and connecting the digested fragment to the XbaI/EcoRI site of a pKC1139 plasmid to obtain the plasmid pKC1139-permE;

(2) Construction of a plasmid containing the opt fdc and opt cso2 genes

Respectively using fdc-FOR/fdc-REV as primers and Amycolatopsis sp genome as a template, carrying out PCR amplification on an opt fdc gene sequence, and then connecting an opt fdc fragment to the NsiI site of a pKC1139-permE plasmid through an Assembly kit to obtain a plasmid pKC1139-opt fdc;

respectively using cso 2-FOR/cso-REV as a primer and Amycolatopsis sp genome as a template, carrying out PCR amplification on an opt cso2 gene sequence, and then connecting an opt cso2 fragment to the NsiI site of a pKC1139-permE plasmid through an Assembly kit to obtain the construction of the plasmid pKC1139-opt cso 2;

digesting the opt cso2 gene fragment on the plasmid pKC1139-opt cso2 by XbaI/SpeI enzyme, connecting to the SpeI site of the plasmid pKC1139-opt fdc, and obtaining pKC1139-opt fdc-cso plasmid;

(3) Construction of recombinant Amycolatopsis

And (3) transforming the pKC1139-opt fdc-cso plasmid obtained in the step (2) into amycolatopsis by using a conjugation transfer experimental method, and screening a positive mutant strain according to the resistance of the arabidopsis to obtain the recombinant amycolatopsis expressing the opt fdc and opt cso2 genes simultaneously.

In the above-mentioned construction method, the Amycolatopsis of step (3) is Amycolatopsis sp HM-141, which has been deposited at 9 days 7 and 7 in 2021 in the common microbiology center of the institute of microbiology of china institute of china academy of sciences No.1, north chen west way, no.3, north township, beijing, with the deposit number of CGMCC No.22871. The skilled person can also use other amycolatopsis strains as starting strains to construct recombinant amycolatopsis containing the opt fdc and opt cso2 genes.

Amycolatopsis sp (Amycolatopsis sp.) HM-141 is obtained by collecting soil samples from the periphery of fruits such as Sabina, sabayan, weinan, etc., separating by conventional technique, and identifying after mutagenesis. The mutagenesis is to select and breed compound mutagenesis by taking an original strain obtained by separation as an initial strain, and screen out a strain with high vanillin yield by ultraviolet-sodium nitrite compound mutagenesis:

activating a starting strain on a plate culture medium, then selecting a bacterial colony, dissolving the bacterial colony in 10mL of sterile water filled with glass beads, shaking and uniformly mixing to obtain a bacterial suspension, and performing gradient dilution to obtain OD ₆₀₀ Is 0.4-0.6 bacterial suspension. Placing 10mL of the bacterial suspension in a sterile culture dish, irradiating 30cm of the sterile culture dish by a 15W ultraviolet lamp for 30s, then culturing for 1h in a dark place, adding 0.1M sodium nitrite into the culture dish, and culturing for 3min at 30 ℃ and 200rpm.

Immediately after mutagenesis, the reaction was terminated by adding a disodium hydrogen phosphate solution. The bacterial suspension after mutagenesis is diluted in a gradient way and then coated on a plate culture medium, a single colony which grows out is selected to be inoculated into a 96-well plate which is filled with a seed culture medium (the seed culture medium is 25g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of potassium dihydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, the pH is adjusted to be 7.2, the mixture is sterilized at 121 ℃ for 20 min) for culture, the seed liquid is inoculated into a 96-well plate fermentation culture medium (the fermentation culture medium is 30g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of potassium dihydrogen phosphate, 1g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, the initial pH is adjusted to be 7.2, the fermentation culture medium is cultured at 121 ℃ for 20min at 30 ℃,200rpm, 0.5g/L of calcium chloride is added into a shaker, the content of biotin is measured by an enzyme linked immunosorbent assay, 8978 nm of a dinitrobenzoic acid substrate, and the content is measured by an ELISA method.

The yield of vanillin is measured by adopting a 2,4-dinitrophenylhydrazine chromogenic method: in the later stage of fermentation conversion, 10 mu L of bacterial liquid is taken out from the culture solution of a 96-well plate every 4h, and is put into a 24-deep-well plate, 100 mu L of 2,4-dinitrophenylhydrazine solution and 5mL of 0.8mol/L NaOH solution are added and mixed evenly, distilled water is used as a blank control, a starting strain is used as a control group, and then the absorbance is detected. Screening positive mutant strains, performing shake flask re-screening, respectively inoculating the positive mutant strains activated on a plate culture medium to a seed culture medium, culturing at 30 ℃ and 200rpm for 48-72h, then inoculating the positive mutant strains to a fermentation culture medium according to the inoculum size of 5%, and performing transformation at 30 ℃ and 200rpm, wherein the initial concentration of a substrate ferulic acid is 10g/L. The content of vanillin in the final fermentation broth was determined by HPLC. Finally, screening out the bacterial strain with high vanillin yield.

The obtained strain was identified by the 16s rDNA method, and it was confirmed that the 16s rDNA of the selected strain had 99% homology with Amycolatopsis, and therefore, amycolatopsis HM-141 was named Amycolatopsis sp. The biological characteristics are as follows: the colony morphology can be shown in figure 1, and the colony is white or yellowish, opaque, and has a dry surface. Gram staining is positive, and the thallus is filamentous.

Determination of vanillin production by Amycolatopsis sp.HM-141:

one tube of glycerol strain Amycolatopsis sp.HM-141, which was preserved at-80 deg.C, was diluted and spread on a solid plate (solid medium: glucose 4g/L, yeast extract 4g/L, malt extract 10g/L, agar 20g/L, and water as the rest), and the plate was inverted and cultured in an incubator at 30 deg.C for 3-4 days until colonies grew out.

Inoculating a loopful bacteria from an activation plate to a seed culture medium, and culturing at 30 ℃ for 48-72h at the rotation speed of 200rpm.

The seed solution was inoculated into a 5L fermentor (containing 4L of fermentation medium) at an inoculum size of 5%, and fermentation was carried out at 30 ℃ with a stirring speed of 800rpm and an aeration ratio of 1vvm. After 24h of culture, adding 22.5g/L of ferulic acid (total 90g, ferulic acid concentration calculated according to the volume of 4L of fermentation broth, dissolving ferulic acid in 0.5M NaOH solution to make the concentration of ferulic acid 100 g/L), then adjusting pH to 8.2, and continuing fermentation for 48h.

Since 0.9L ferulic acid solution was added to 4L fermentation broth, and sampling of the broth during fermentation resulted in a decrease in broth, at the end of fermentation, the actual broth volume was measured to be 4.5L, and the following data were calculated using 4L broth in order to keep the data consistent. Measuring the concentration of vanillin in the fermentation liquor by HPLC to be 15.13g/L, the concentration of residual ferulic acid is 0.4g/L, and if the residual ferulic acid which does not participate in the conversion is counted, the molar conversion rate is 85%; if residual ferulic acid not involved in the conversion is not taken into account, the molar conversion is 87%. The content of the byproduct vanillic acid is 0.25g/L, and vanillic alcohol is not detected in the fermentation liquor.

According to an alternative embodiment, in the step (3), the amycolatopsis is spread on a GYM solid medium containing 50. Mu.g/mL of amycolatopsis and 25. Mu.g/mL of nalidixic acid solution, and cultured at 30 ℃ for 4 days until a single colony grows, and the obtained single colony is a positive mutant.

Based on this, the invention also provides the application of the recombinant Amycolatopsis sp in the production of vanillin.

As a particularly preferred embodiment, the above engineered strain is inoculated into 50mL of seed medium M1 and cultured at 30 ℃ and 200rpm for 72 hours;

inoculating the seed solution into a 5L fermentation tank containing 4L fermentation medium M2 according to the inoculation amount of 5% by mass ratio, and performing fermentation culture for 24h under the conditions of 30 ℃, stirring rotation speed of 800rpm and aeration ratio of 1 vvm;

then adding 20g/L of the first batch of substrate ferulic acid, adding 12g/L of the second batch of substrate ferulic acid when the concentration of ferulic acid is reduced to 3-4g/L, adjusting the pH to 8.2 and continuing to ferment for 48h;

the formula of the M1 culture medium is as follows: 25g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and the pH is adjusted to be 7.2;

the formula of the M2 culture medium is as follows: 30g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 1g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and the pH is adjusted to be 7.2.

After the fermentation was stopped, the concentration of vanillin in the fermentation broth was 23.5g/L as determined by HPLC.

On the other hand, the invention also provides application of the expression of the ferulic acid decarboxylase gene (opt fdc) and the 4-vinylguaiacol oxygenase gene (opt cso 2) in improving the yield of vanillin produced by Amycolatopsis sp.

The invention verifies that the opt fdc gene and the opt cso2 gene independent of coenzyme A and ATP are simultaneously expressed in the amycolatopsis through a genetic engineering means, the vanillin yield of the amycolatopsis can be effectively improved, the vanillin yield is improved by 55 percent compared with that of the original strain, and a technical support is provided for further improving the vanillin fermentation yield in industrial production.

The Amycolatopsis sp (HM-141) has been preserved in the general microbiological culture Collection center of the institute of microbiology, china academy of sciences, china Committee for culture Collection of microorganisms, china Union of sciences, no.3, on the North Chen West Lu 1, the south facing Yang, beijing, 7.9.2021, with the preservation number of CGMCC No.22871.

[ description of the drawings ]

FIG. 1 is a map of plasmid pKC1139-opt fdc-cso in the present invention

FIG. 2 is a map of pKC1139-ech-fcs plasmid of the present invention

[ detailed description ] embodiments

The invention will be better understood from the following examples.

In the present invention, "%" used for explaining the concentration is mass percent and ": all the terms "are mass ratios.

The strains and plasmids used in the examples are shown in Table 1, and the sequences of the primers synthesized are shown in Table 2.

TABLE 1 strains and plasmids used in the invention

TABLE 2 primers used in the study

The present invention relates to the following media:

the LB culture medium formula is: 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride.

The formula of the GYM culture medium is as follows: glucose 4g/L, yeast extract 4g/L, malt extract 10g/L.

The formula of the GYM solid culture medium is as follows: 4g/L of glucose, 4g/L of yeast extract, 10g/L of malt extract, 2g/L of calcium carbonate and 20g/L of agar powder.

The formula of the M1 culture medium is as follows: 25g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and the pH is adjusted to be 7.2.

The formula of the M2 culture medium is as follows: 30g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 1g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and the pH is adjusted to be 7.2.

The bond transfer experiments used in the following examples included the following steps:

(1) Amycolatopsis sp (or other engineered strain) is activated on a GyM solid medium, cultured at 30 ℃ for 3-4 days until colonies grow, and then inoculated into 50mL of a GyM liquid medium and cultured at 30 ℃ and 200rpm for 2 days.

(2) The constructed plasmid is heat shocked into E.coli ET12567 (pUZ 8002) strain, spread on LB solid plate containing 25 ug/mL chloramphenicol, 25 ug/mL kanamycin and 50 ug/mL adriamycin resistance, cultured at 37 ℃ for 12h until single colony grows out, inoculated into 4mL LB liquid culture medium containing resistance at 37 ℃ overnight at 200rpm, and then inoculated into 20mL LB liquid culture medium at 37 ℃ for 4-5h at 200rpm according to 1% inoculum size ₆₀₀ Is 0.4-0.6.

(3) 2mL of Amycolatopsis sp.bacterial liquid and 1mL of E.coli ET12567 (pUZ 8002) bacterial liquid carrying a target plasmid, 5000g of the obtained product are respectively centrifuged for 1min, and the obtained product is washed twice with non-resistant LB, 100. Mu.L of non-resistant LB medium is added, the Amycolatopsis and the E.coli ET12567 (pUZ 8002) are mixed according to a volume ratio of 7:1, and 30. Mu.L of the mixed bacterial liquid is spotted on the non-resistant GYM solid medium for 30 ℃ and 14h of positive culture.

(4) Scraping the grown bacterial plaque, spreading the bacterial plaque on a GyM solid culture medium containing 50 mu g/mL of adriamycin and 25 mu g/mL of nalidixic acid solution, and culturing at 30 ℃ for 4 days until a single colony is grown, namely the correct bacterial strain.

Example 1: construction of opt fdc and opt cso2 gene expression strain independent of coenzyme A, ATP

The opt fdc gene sequence is shown as SEQ ID NO.1, and the opt cso2 gene sequence is shown as SEQ ID NO. 2. To construct the opt fdc and opt cso2 gene expression strains, the overexpression plasmids pKC1139-opt fdc-cso:

a strong promoter permE used in the amycolatopsis is synthesized, and the gene sequence of the strong promoter permE is shown as SEQ ID NO. 3. The synthetic permE gene sequence was digested with XbaI/EcoRI, and the digested fragment was ligated to XbaI/EcoRI site of pKC1139 plasmid to obtain plasmid pKC1139-permE.

Then, an opt fdc gene sequence is amplified by PCR by respectively using fdc-FOR/fdc-REV as primers and an amycolatopsis genome as a template, and then an opt fdc fragment is connected to an NsiI site of a pKC1139-permE plasmid through an Assembly kit, so that the construction of the plasmid pKC1139-opt fdc is completed. Respectively using cso 2-FOR/cso-REV as primers and amycolatopsis genome as a template, carrying out PCR amplification on an opt cso2 gene sequence, and then connecting an opt cso2 fragment to the NsiI site of a pKC1139-permE plasmid through an Assembly kit to complete the construction of the plasmid pKC1139-opt cso2. The opt cso2 gene fragment on the plasmid pKC1139-opt cso2 is digested by XbaI/SpeI enzyme and is connected to the SpeI site of the plasmid pKC1139-opt fdc to obtain pKC1139-opt fdc-cso plasmid, and the plasmid map of pKC1139-opt fdc-cso is shown in figure 1.

A conjugative transfer experimental method is utilized to transform pKC1139-opt fdc-cso plasmid into amycolatopsis HM-141, and positive mutants are screened according to the resistance of the adriamycin to obtain a genetic engineering strain AMY/pKC1139-opt fdc-cso for gene expression of opt fdc and opt cso2.

Example 2: construction of ech and fcs Gene overexpression Strain depending on coenzyme A, ATP

To construct ech and fcs overexpressing strains, the overexpression plasmid pKC1139-ech-fcs was first constructed. The construction of plasmid pKC1139-ech was completed by PCR amplification of ech gene sequence using ech-FOR/ech-REV as primers and amycolatopsis genome as template, and then joining ech fragment to NsiI site of pKC1139-permE plasmid by Assembly kit.

Then, respectively using fcs-FOR/fcs-REV as primers and amycolatopsis genome as a template, carrying out PCR amplification on the fcs gene sequence, and then connecting the fcs fragment to the NsiI site of the pKC1139-permE plasmid through an Assembly kit to complete the construction of the plasmid pKC 1139-fcs. The fcs gene fragment on the plasmid pKC1139-fcs is digested by XbaI/SpeI enzyme and is connected to the SpeI site of the plasmid pKC1139-ech to obtain pKC1139-ech-fcs plasmid, and the plasmid map of pKC1139-ech-fcs is shown in figure 2.

A conjugative transfer experimental method is utilized to transform pKC1139-ech-fcs plasmids into amycolatopsis HM-141, positive mutants are screened according to the resistance of the adriamycin, and a gene engineering strain AMY/pKC1139-ech-fcs with ech and fcs gene overexpression is obtained.

Example 3: comparison of fermentation of the original Strain (HM-141) with the recombinant Strain of the present invention

Fermentation experiments for the production of vanillin by biotransformation of ferulic acid were carried out using Amycolatopsis HM-141 and the strains AMY/pKC1139-opt fdc-cso and AMY/pKC1139-ech-fcs constructed in examples 1-2, respectively. The experimental method is as follows:

the above strains were inoculated into 50mL of a seed medium M1, respectively, cultured at 30 ℃ and 200rpm for 72 hours, and the seed solution was inoculated in a 5L fermentor (containing 4L of the fermentation medium M2) at an inoculum size of 5%, fermentation-cultured at 30 ℃ with a stirring rate of 800rpm and an aeration ratio of 1vvm.

After culturing a control strain (amycolatopsis similis HM-141) for 24h, adding a substrate ferulic acid of 22.5g/L (total 90g, ferulic acid is dissolved in 0.5M NaOH solution to make the concentration of ferulic acid 100 g/L), then adjusting the pH value to 8.2, continuing to ferment for 48h, and measuring the concentration of vanillin in the fermentation liquor by HPLC after the fermentation is finished.

After the engineering strains of the examples 1 and 2 are cultured for 24 hours, 20g/L of the first substrate ferulic acid is added, when 3-4g/L of ferulic acid still remains, 8g/L or 12g/L (total 112g or 128 g) of the second substrate ferulic acid is added, then the pH is adjusted to 8.2, the fermentation is continued for 48 hours, and the concentration of vanillin in the fermentation liquor is measured by HPLC after the fermentation is finished.

The HPLC method for measuring vanillin in the fermentation liquor comprises the following steps: the fermentation broth was centrifuged and the supernatant was analyzed by HPLC. Analyzing the transformation product by Agilent HPLC1260, wherein the chromatographic column is Estrit Hypersil ODS2,5 μm,4.6mm × 250mm; the detection wavelength is 295nm; the column temperature is 30 ℃; flow rate: 1mL/min; sample introduction amount: 10 mu L of the solution; the mobile phase is acetonitrile and 0.5% phosphoric acid water solution, 0-20min,10-20% acetonitrile, 20-30min and 20-10% acetonitrile.

The fermentation results are shown in table 1 below.

TABLE 1 fermentation test results of Amycolatopsis and its engineering strains

The results show that under the optimal fermentation condition, the recombinant amycolatopsis AMY/pKC1139-opt fdc-cso realizes the conversion of 32g/L ferulic acid into vanillin, the concentration of the obtained vanillin in the fermentation broth reaches 23.5g/L, and the detection shows that the substrate residue in the fermentation broth is very low, which indicates that the substrate is basically and completely converted into the product, and the conversion path does not depend on coenzyme A and ATP, so that the method has higher economic value. Compared with the recombinant strain disclosed by the invention, the concentration of a vanillin product which can be realized by the original strain is 15.13g/L, namely the recombinant strain disclosed by the invention realizes 55% yield improvement by expressing opt fdc and opt cso2 genes. As a contrast, when ech and fcs genes dependent on coenzyme A and ATP are overexpressed in an original strain, the yield of vanillin is improved by 34% compared with the original strain, but is still obviously lower than that of the recombinant strain, and the way of synthesizing vanillin by the strain depends on the participation of the coenzyme A and the ATP.

In conclusion, the recombinant amycolatopsis realizes the obvious improvement of the yield of vanillin by expressing opt fdc and opt cso2 genes, and the synthetic route of the recombinant amycolatopsis does not depend on coenzyme A and ATP and has economic superiority.

Sequence listing

<110> Shaanxi Haas Schff bioengineering GmbH

<120> recombinant amycolatopsis for high yield of vanillin, construction method and application thereof

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cggctggacg agcggaccac cgacatggtg gtgctcgacg ccctgcggct gggcgagggc 1380

cccgtggcca ccgtgaagct gccgctgcgg atgcggatgg cgctgcacgg caactggagc 1440

cgggcggtga gcccgtcctc catcaaggcg gtgtga 1476

<210> 3

<211> 357

<212> DNA

<213> Strong promoter permE (Unknown)

<400> 3

tctagaagcc cgacccgagc acgcgccggc acgcctggtc gatgtcggac cggagttcga 60

ggtacgcggc ttgcaggtcc aggaagggga cgtccatgcg agtgtccgtt cgagtggcgg 120

cttgcgcccg atgctagtcg cggttgatcg gcgatcgcag gtgcacgcgg tcgatcttga 180

cggctggcga gaggtgcggg gaggatctga ccgacgcggt ccacacgtgg caccgcgatg 240

ctgttgtggg cacaatcgtg ccggttggta ggatccaagg aggcaacaag atgcataaaa 300

tctccaaaaa aaaaggctcc aaaaggagcc tttaattgta tcggtactag tgaattc 357

Claims (6)

1. A recombinant Amycolatopsis sp. (Amycolatopsis sp.) for high yield of vanillin, characterized in that the recombinant Amycolatopsis expresses a ferulic acid decarboxylase gene (opt fdc) and a 4-vinylguaiacol oxygenase gene (opt co 2);

the nucleotide sequence of the ferulic acid decarboxylase gene (opt fdc) is shown as SEQ ID NO.1, and the nucleotide sequence of the 4-vinylguaiacol oxygenase gene (opt cso 2) is shown as SEQ ID NO. 2.

2. A method of constructing a recombinant Amycolatopsis sp.according to claim 1, comprising the steps of:

(1) Construction of a plasmid containing the Strong promoter permE

Synthesizing a strong promoter permE, wherein the gene sequence of the strong promoter permE is shown in SEQ ID NO.3, digesting the synthesized gene sequence by XbaI/EcoRI, and connecting the digested fragment to the XbaI/EcoRI site of a pKC1139 plasmid to obtain the plasmid pKC1139-permE;

(2) Construction of a plasmid containing the opt fdc and opt cso2 genes

Respectively using fdc-FOR/fdc-REV as primers and Amycolatopsis sp genome as a template, carrying out PCR amplification on an opt fdc gene sequence, and then connecting an opt fdc fragment to an NsiI site of a pKC1139-permE plasmid through an Assembly kit to obtain a plasmid pKC1139-opt fdc;

respectively using cso 2-FOR/cso-REV as a primer and Amycolatopsis sp genome as a template, carrying out PCR amplification on an opt cso2 gene sequence, and then connecting an opt cso2 fragment to the NsiI site of a pKC1139-permE plasmid through an Assembly kit to obtain the construction of the plasmid pKC1139-opt cso 2;

digesting the opt cso2 gene fragment on the plasmid pKC1139-opt cso2 by XbaI/SpeI enzyme, connecting to the SpeI site of the plasmid pKC1139-opt fdc, and obtaining pKC1139-opt fdc-cso plasmid;

(3) Construction of recombinant Amycolatopsis

And (3) transforming the pKC1139-opt fdc-cso plasmid obtained in the step (2) into amycolatopsis by using a conjugation transfer experimental method, and screening a positive mutant strain according to the resistance of the arabidopsis to obtain the recombinant amycolatopsis expressing the opt fdc and opt cso2 genes simultaneously.

3. The method according to claim 2, wherein the Amycolatopsis species of step (3) is Amycolatopsis sp (HM-141), which is deposited at the general microorganism center of the institute for microorganism culture preservation, china Committee for culture Collection of microorganisms, china institute for Collection of sciences, china Union of West Lu No.3, north Chen West province, tokyo, chao, 7/9 days 2021, and the collection number is CGMCC No.22871.

4. The method according to claim 3, wherein in the step (3), the screening for the resistance to the arabidopsis bacterium comprises the steps of spreading the amycolatopsis onto a GYM solid medium containing 50 μ g/mL of the arabidopsis bacterium and 25 μ g/mL of a nalidixic acid solution, and culturing at 30 ℃ for 4 days until a single colony grows, wherein the single colony is a positive mutant strain.

5. Use of the recombinant Amycolatopsis sp.according to claim 1 for the production of vanillin.

6. The use according to claim 5, characterized in that the engineered strain is inoculated in 50mL of seed medium M1, cultured at 30 ℃ for 72h at 200 rpm;

inoculating the seed solution into a 5L fermentation tank containing 4L fermentation medium M2 according to the inoculation amount of 5% by mass ratio, and performing fermentation culture for 24h under the conditions of 30 ℃, stirring rotation speed of 800rpm and aeration ratio of 1 vvm;

then adding 20g/L of the first batch of substrate ferulic acid, adding 12g/L of the second batch of substrate ferulic acid when the concentration of ferulic acid is reduced to 3-4g/L, adjusting the pH to 8.2 and continuing to ferment for 48h;

the formula of the M1 culture medium is as follows: 25g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and the pH is adjusted to be 7.2;

the formula of the M2 culture medium is as follows: 30g/L of glucose, 10g/L of yeast extract powder, 0.8g/L of sodium chloride, 5g/L of monopotassium phosphate, 1g/L of magnesium sulfate heptahydrate, 0.05g/L of calcium chloride and the balance of water, and the pH is adjusted to be 7.2.

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