CN116478942A - Isoeugenol monooxygenase mutant, engineering bacteria and application - Google Patents
Isoeugenol monooxygenase mutant, engineering bacteria and application Download PDFInfo
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Abstract
The invention discloses an isoeugenol monooxygenase mutant, engineering bacteria and application thereof, and belongs to the technical field of biology. The isoeugenol monooxygenase gene is derived from pseudomonas putida (pseudoomonasputida), the nucleotide sequence of the isoeugenol monooxygenase gene is shown as SEQ ID NO.1, and the amino acid sequence of the isoeugenol monooxygenase gene is shown as SEQ ID NO. 2. The mutant is a mutant of SEQ ID NO:2, and the amino acid sequence 59, 216 and 304 is obtained by single mutation or multi-site mutation. The isoeugenol monooxygenase constructed by the invention and the engineering strain are constructed, the enzyme activity of the screened mutant is improved by 445.7 percent compared with the highest original mutant, the screened mutant is used for enzymatic synthesis of p-hydroxybenzaldehyde, the highest product concentration can reach 1.27g/L, and the highest conversion rate can reach 41.6 percent.
Description
Technical Field
The invention relates to isoeugenol monooxygenase mutant, engineering bacteria and application thereof, belonging to the technical field of biology.
Background
p-Hydroxybenzaldehyde (p-HBA for short) can be used for synthesizing pharmaceutical raw materials such as heart cerebrovascular drug esmolol, oral antibiotic amoxicillin (amoxicillin), antibiotic sulfanilamide synergist trimethoxybenzyl amine pyrimidine and the like, and can also be used for synthesizing a plurality of precious fragrances such as ethyl vanillin, heliotropin, anisaldehyde, raspberry ketone and the like. The method is widely applied to the fields of medicines, pesticides, spices, chemical industry, electroplating, liquid crystal materials and the like, and has wide prospect. At present, p-HBA is mainly synthesized by a chemical method, takes phenol, p-methylphenol, p-hydroxybenzyl alcohol and p-hydroxyphenylacetic acid as raw materials, and has the problems of high cost, serious environmental pollution, poor synthesis selectivity, low yield, immature process and the like.
The p-HBA synthesized by the microbiological method has great development and application prospect and meets the ideal requirement of green chemistry. In 2006, sachan et al used paecilomyces to degrade coumaric acid to synthesize parahydroxybenzaldehyde, parahydroxybenzoic acid and protocatechuic acid, however, the yield was low and the composition was complicated. The enzyme has high activity, high selectivity, high specificity and high efficiency in the processes of biosynthesis and metabolism, and has great application value. Isoeugenol monooxygenase (isoeugenol monooxygenase, iem) is a monooxygenase, and specifically oxidizes the side chain carbon-carbon double bond of isoeugenol to convert into vanillin and acetaldehyde. Directed evolution is an effective tool for improving the activity and function of proteins, and has been successfully modified into directed evolution of various enzyme molecules, which is a tool capable of improving the performance of enzymes, such as substrate specificity, thermal stability, enzyme activity and the like.
Disclosure of Invention
Aiming at the defects of the existing chemical synthesis process of the parahydroxyben-zaldehyde, the invention provides an isoeugenol monooxygenase mutant, recombinant bacteria and whole cells thereof as catalysts for synthesizing the parahydroxyben-zaldehyde.
The invention is realized by the following technical scheme:
the invention provides an isoeugenol monooxygenase mutant, wherein an isoeugenol monooxygenase gene is derived from pseudomonas putida (Pseudomonas putida) IE27, the nucleotide sequence of which is shown as SEQ ID NO.1 and the amino acid sequence of which is shown as SEQ ID NO.2 after codon optimization; the mutant is a mutant of SEQ ID NO:2, wherein the 59 th, 216 th and 304 th sites of the amino acid sequence are subjected to single mutation or multi-site mutation;
the mutant is one of the following, namely, the 59 th isoleucine is mutated into tyrosine (IEM-I59Y), the 216 th phenylalanine is mutated into tryptophan and the 304 th proline is mutated into phenylalanine (IEM-F216W-P304F).
The invention also relates to a coding gene of the isoeugenol monooxygenase mutant.
The invention provides engineering bacteria containing the coding gene.
The invention also provides application of the isoeugenol monooxygenase mutant in catalyzing the synthesis of parahydroxybenzaldehyde from parahydroxystyrene.
Further, the application method is to take bacterial cells obtained by fermenting and centrifuging engineering bacteria containing isoeugenol monooxygenase mutant genes as a whole-cell catalyst, and the catalytic system is as follows: 1-5 g/L of p-hydroxystyrene, 100g/L of dimethyl sulfoxide (DMSO), 1-5 mM of ferrous sulfate, pH of 9.5, reaction volume of 20mL, loading into a 500mL triangular flask, placing into an air shaking table for open reaction, and rotating at a shaking table speed of 200r/min and at a temperature of 30-40 ℃.
Further, in the conversion system, the catalyst is used in an amount of 50 to 100g/L based on the weight of the wet bacteria.
Further, engineering bacteria wet thalli containing isoeugenol monooxygenase mutant genes are prepared according to the following method: inoculating recombinant escherichia coli containing isoeugenol monooxygenase mutant genes into LB slant culture medium, and culturing at 37 ℃ for 12-16 h; inoculating the slant strain to LB liquid seed culture medium, and culturing for 4-8 h at 37 ℃ under shaking at 200 r/min; inoculating the seed solution into a 500mL triangular flask containing 50mL of fermentation medium according to the volume ratio of 1-5%, culturing for 4h at 37 ℃ under shaking at 220r/min, cooling to 20-30 ℃ and adding 1mmol/L isopropyl thio-beta-D-galactoside (IPTG) for continuous induction expression for 16h, centrifuging for 10min at 10000r/min after fermentation, collecting thalli at 4 ℃ and washing thalli twice with sterile physiological saline to obtain the isoeugenol monooxygenase mutant whole-cell catalyst.
Further, the preparation method of the LB slant culture medium comprises the following steps: the yeast extract powder is prepared by mixing 5g/L yeast extract powder, 10g/L, naCl g/L peptone, 100mg/L ampicillin and 20g/L agar, adjusting pH to 6.8-7.0, and sterilizing at 121deg.C under high pressure steam for 20 min;
further, the preparation method of the LB liquid seed culture medium comprises the following steps: contains 5g/L of yeast extract powder, 10g/L, naCl g/L of peptone and 100mg/L of ampicillin, and is prepared by adjusting pH to 7.0-7.2 and sterilizing at 121 ℃ for 20min by high-pressure steam;
further, the preparation method of the fermentation medium comprises the following steps: comprises glycerin 20g/L, peptone 10g/L, yeast extract 5g/L, malt extract 2g/L, mgSO 4 2 g/L、KH 2 PO 4 15 g/L、NH 4 Cl 2g/L and FeSO 4 50 The mixed solution of mg/L is adjusted to pH 6.7-7.0, and the product is obtained by sterilizing the mixed solution by high-pressure steam at 121 ℃ for 20 min.
Compared with the prior art, the invention has the beneficial effects that:
the invention adopts error-prone PCR technology to successfully mutate isoeugenol monooxygenase derived from pseudomonas putida (Pseudomonas putida) IE27 and construct engineering strains, the enzyme activity of the screened mutant is improved by 445.7% compared with the highest value of the original mutant, the mutant is used for enzymatic synthesis of p-hydroxybenzaldehyde, the concentration of the product can reach 1.27g/L, and the molar conversion rate is 41.6%. Meanwhile, the raw materials used in the production method are cheap and easy to obtain, and the production process is simple and easy to implement and has lower production cost. The whole cell conversion system solves the problems of high cost, serious environmental pollution, poor synthesis selectivity and low yield of the p-hydroxybenzaldehyde synthesized by a chemical method, and realizes the green production of the p-hydroxybenzaldehyde with high yield.
Detailed Description
The technical scheme of the present invention is further explained by examples below, but the scope of the present invention is not limited in any way by the examples.
Example 1: expression vector and engineering bacterium construction
Through literature report and gene sequence homology, isoeugenol monooxygenase (GenBank: BAF 62888.1) of Pseudomonas putida IE27 is queried and selected in NCBI database, isoeugenol monooxygenase IEM (the gene sequence is SEQ ID No.1, the amino acid sequence is SEQ ID No. 2) is synthesized through whole genes, primers are designed, PCR amplification is carried out by homologous recombination, and subcloning is carried out to the corresponding site of the vector pET32a, thus obtaining recombinant plasmid pET32a-IEM. Through heat shock transformation of E.coli BL21 (DE 3), recombinant E.coli BL21 (DE 3)/pET 32a-IEM expressing isoeugenol monooxygenase is obtained.
Constructing a random mutation library by using a recombinant vector pET32a-IEM as a template and utilizing an error-prone PCR technology. The forward primer IEM-F was 5'-acaaggccatggctgatatcggatccatggcaacgtttgaccgcaatg-3' (bold italics homology arms) and the reverse primer IEM-R was 5'-tcagtggtggtggtggtggtgctcgagtcagttcttagactgccaac-3' (bold italics homology arms). The reaction system of error-prone PCR is: PCR Grade Water 39. Mu.L, 10X TITANIUM Taq Buffer. Mu.L, mnSO 4 8 mM 1. Mu.L, dGTP 2mM 1. Mu.L, 50X Diversify dNTPMix. Mu.L, primer mix 1. Mu.L, template 1. Mu.L, TITANIUM TaqPolym. Mu.L, and a total of 50. Mu.L system, wherein Primer mix in the reaction system is a mixed solution of 0.5. Mu.L each of the upstream and downstream primers described in example 1. The error-prone PCR reaction conditions were: 94 ℃ for 30s,1 cycle; 94℃30s,68℃1min30s,25 cycles; 1min at 68℃for 1 cycle. The error-prone PCR amplified product was detected with 1% agarose gel and purified with AxyPrep DNA gel recovery kit. The purified fragment and a double-enzyme-cut vector pET32a (BamH I and Xho I) carry out homologous recombination reaction, and the reaction system is as follows: 4. Mu.L of 5 XCE II Buffer, 2. Mu.L of Exase II, with ddH 2 O was made up to 20. Mu.L. Mixing, fusing at 37deg.C for 30min, immediately cooling on ice for 10min, and then transforming Escherichia coli BL21 (DE 3) to become competent. Culturing at 37deg.C and 200r/min for 1 hr for activation, coating the activated recombinant cells on LB plate containing 100mg/L ampicillin resistance, and culturing at 37deg.C under inverted condition overnight to obtain isoflos CaryophylliPhenol monooxygenase mutant expression library.
From the obtained isoeugenol monooxygenase mutant expression library colonies, high-activity mutant strains are screened from the single colonies by using a high-throughput screening method. The specific screening method comprises the following steps: randomly picking 1800 single colonies from a flat plate, inoculating into a 96 deep hole plate, culturing for 6 hours at 37 ℃ under 180r/min with LB liquid culture medium, inoculating into a fermentation culture medium according to 5% inoculum size, culturing for 6 hours at 37 ℃ under 220r/min, adding IPTG to a final concentration of 0.5mM, and cooling to 20 ℃ for induction expression; and (5) centrifugally collecting thalli, and measuring the enzyme activity. Standard enzyme activity detection system: 10g/L of wet cell catalyst, 2g/L of p-hydroxystyrene, 100g/L of dimethyl sulfoxide (DMSO), 1mM of ferrous sulfate, 9.5 of reaction pH and 1mL of total system. Definition of unit enzyme activity: under standard reaction conditions, the amount of enzyme required to produce 1. Mu. Mol of p-hydroxybenzaldehyde per minute is one enzyme activity unit U. Screening to obtain 2 high-activity isoeugenol monooxygenase mutant strains, and sequencing to obtain SEQ ID NO:2, the 59 th isoleucine is mutated into tyrosine (IEM-I59Y), the 216 th phenylalanine is mutated into tryptophan, and the 304 th proline is mutated into phenylalanine (IEM-F216W-P304F), and the corresponding engineering strains are E.coli BL21 (DE 3)/pET 32a-IEM-I59Y and E.coli BL21 (DE 3)/pET 32a-IEM-F216W-P304F respectively. The enzyme activities of the mutants are shown in Table 1.
TABLE 1 isoeugenol monooxygenase mutant enzyme Activity
Example 2: production of isoeugenol monooxygenase mutant by recombinant bacterium fermentation and production of parahydroxybenzaldehyde by enzymatic conversion of parahydroxystyrene
(1) Inoculating each mutant strain of IEM into LB slant culture medium at 37 ℃ for culturing for 12h; inoculating the slant strain to LB liquid seed culture medium, shake culturing at 37deg.C at 200r/min for 6 hr, inoculating seed liquid into 500mL triangular flask containing 50mL fermentation culture medium according to 5% inoculum size, shake culturing at 37deg.C at 220r/min for 4 hr, cooling to 25deg.C, adding 1mmol/L isopropyl thio-beta-D-galactoside, and continuously inducing expression for 16 hr. And after the fermentation is finished, 10000r/min and centrifugation is carried out for 10min at 4 ℃ to collect thalli, and the thalli are washed twice by sterile normal saline to respectively obtain isoeugenol monooxygenase mutant whole cell catalysts.
(2) The isoeugenol monooxygenase mutant is used for producing parahydroxyben-zaldehyde by whole cell catalysis, and the conversion conditions are as follows: the addition amount of the whole cell catalyst is 50g/L, the addition amount of the para-hydroxystyrene is 2g/L, the addition amount of the dimethyl sulfoxide (DMSO) is 100g/L, the pH is 1mM, the reaction volume is 20mL, a 500mL triangular flask is filled in the reaction volume, the reaction is carried out in an air shaking table with an opening, the shaking table rotating speed is 200r/min, and the reaction is carried out for 3 hours at 30 ℃. As a result, as shown in Table 2, E.coli BL21 (DE 3)/pET 32a-IEM-F216W-P304F was found to have a P-hydroxybenzaldehyde concentration of 0.72g/L and a molar conversion of 35.5%, E.coli BL21 (DE 3)/pET 32a-IEM-I59Y was found to have a P-hydroxybenzaldehyde concentration of 0.53g/L and a molar conversion of 26.1%, but still was significantly higher than that of the original strain E.coli BL21 (DE 3)/pET 32a-IEM.
TABLE 2 transformation of IEM mutant strains to para-hydroxystyrene to para-hydroxybenzaldehyde
Example 3: production of isoeugenol monooxygenase mutant (IEM-F216W-P304F) by recombinant bacterium fermentation and production of parahydroxyben-zaldehyde by enzymatic conversion of parahydroxystyrene
(1) The IEM mutant IEM-F216W-P304F is inoculated into LB slant culture medium for culturing for 14h at 37 ℃; inoculating bevel strain to LB liquid seed culture medium, shake culturing at 37deg.C at 200r/min for 5 hr, inoculating seed liquid into 500mL triangular flask containing 50mL fermentation culture medium according to 3% inoculum size by volume ratio, shake culturing at 37deg.C at 220r/min for 4 hr, cooling to 20deg.C, adding 0.5mmol/L isopropyl thio-beta-D-galactoside, and continuing to induce expression for 16 hr. And after the fermentation is finished, 10000r/min and centrifugation is carried out for 10min at 4 ℃ to collect thalli, and the thalli are washed twice by sterile normal saline to respectively obtain isoeugenol monooxygenase mutant whole cell catalysts.
(2) The isoeugenol monooxygenase mutant IEM-F216W-P304F is utilized to catalyze and produce the parahydroxyben-zaldehyde by whole cells, and the conversion conditions are as follows: the addition amount of the whole cell catalyst is 50g/L, the addition amount of the p-hydroxystyrene is 3g/L, the addition amount of the dimethyl sulfoxide (DMSO) is 100g/L, the pH is 1mM, the reaction volume is 20mL, a 500mL triangular flask is filled in the reaction volume, the reaction is carried out in an air shaking table with an opening, the shaking table rotating speed is 200r/min, and the reaction is carried out for 5 hours at 30 ℃. As a result, the concentration of parahydroxyben-zaldehyde was 1.27g/L and the molar conversion was 41.6%.
SEQ ID No.1:
atggcaacctttgatcgtaatgatccgcagctggcaggtaccatgtttccgacccgtattgaagcaaatgtttttgacctgga
aatcgagggtgaaatcccgcgtgcaattaatggtagcttttttcgtaataccccggaaccgcaggttaccacccagccgttt
catacctttattgatggtgatggtctggccagcgcatttcattttgaagatggtcaggttgatttcgtgagccgttgggtttgtac
cccgcgttttgaagcagaacgtagcgcacgtaaaagcctgtttggtatgtatcgtaatccgtttaccgatgacccgagcgtt
gaaggtattgatcgtaccgttgcaaataccagcattattacccatcatggcaaagtgctggcagcaaaagaagatggtctgc
cgtatgaattagatccgcagaccctggaaacccgtggtcgttatgattataaaggtcaggttaccagccatacccataccgc
acatccgaaatttgatccgcagaccggtgaaatgctgctgtttggtagcgcagcaaaaggtgaacgtaccctggatatggc
atattatattgtggaccgctatggcaaagttacccatgaaacctggtttaaacagccgtatggtgcatttatgcatgactttgca
gttacccgtaattggagcatttttccgattatgccggcaaccaatagcctggaacgtctgaaagcaaaacagccgatttatat
gtgggaaccggaacgtggtagctatattggtgttctgccgcgtcgtggtcagggtaaagatattcgttggtttcgtgcaccg
gcactgtgggtttttcatgttgttaatgcatgggaagagggtaaccgcattctgattgatctgatggaaagcgaaattctgccg
tttccgtttccgaatagccagaatctgccgtttgatccgagcaaagcagttccgcgtctgacccgttgggaaattgatctgaa
tagcggtaatgatgagatgaagcgtacccagctgcatgaatattttgccgaaatgccgattatggactttcgctttgcactgc
aggatcaccgttatgcatatatgggtgttgatgacccgcgtcgtccgctggcacatcaacaagcagaaaaaatttttgccta
caacagcctgggcgtgtgggataatcatcgtaaagattatgagctgtggttcaccggtaaaatgagcgcagcacaggaac
cggcatttgttccgcgtagcccggatgcacctgaaggtgatggttatctgctgagcgttgttggtcgtctggatgaagatcgt
agcgatctggttattctggatacccagtgtctggcagcaggtccggttgcaacagttaaactgccgtttcgtctgcgtgcagc
actgcatggttgttggcagagcaaaaattaa
SEQ ID No.2：
MATFDRNDPQLAGTMFPTRIEANVFDLEIEGEIPRAINGSFFRNTPEPQVTTQP
FHTFIDGDGLASAFHFEDGQVDFVSRWVCTPRFEAERSARKSLFGMYRNPFT
DDPSVEGIDRTVANTSIITHHGKVLAAKEDGLPYELDPQTLETRGRYDYKGQ
VTSHTHTAHPKFDPQTGEMLLFGSAAKGERTLDMAYYIVDRYGKVTHETWF
KQPYGAFMHDFAVTRNWSIFPIMPATNSLERLKAKQPIYMWEPERGSYIGVLP
RRGQGKDIRWFRAPALWVFHVVNAWEEGNRILIDLMESEILPFPFPNSQNLPF
DPSKAVPRLTRWEIDLNSGNDEMKRTQLHEYFAEMPIMDFRFALQDHRYAYM
GVDDPRRPLAHQQAEKIFAYNSLGVWDNHRKDYELWFTGKMSAAQEPAFVP
RSPDAPEGDGYLLSVVGRLDEDRSDLVILDTQCLAAGPVATVKLPFRLRAALHGCWQSKN。
Claims (10)
1. The isoeugenol monooxygenase mutant is characterized in that the isoeugenol monooxygenase gene is derived from pseudomonas putida IE27, the nucleotide sequence of the isoeugenol monooxygenase gene after codon optimization is shown as SEQ ID NO.1, the amino acid sequence of the isoeugenol monooxygenase mutant is shown as SEQ ID NO.2, and the isoeugenol monooxygenase mutant is characterized in that the isoeugenol monooxygenase gene is shown as SEQ ID NO:2, namely mutant IEM-I59Y, or mutant IEM-F216W-P304F, or phenylalanine at position 216, tryptophan and proline at position 304.
2. A gene encoding the isoeugenol monooxygenase mutant of claim 1.
3. An engineering bacterium comprising the gene of claim 2.
4. An application of the isoeugenol monooxygenase mutant according to claim 1 in catalyzing the synthesis of parahydroxybenzaldehyde from parahydroxystyrene.
5. The use according to claim 4, wherein the method of application is characterized in that the engineering bacteria according to claim 3 are used as whole cell catalysts by fermenting and centrifuging the obtained bacterial cells, the components and the final concentration in the catalytic system are 1-5 g/L of p-hydroxystyrene, 100g/L of dimethyl sulfoxide and 1-5 mM of ferrous sulfate, the pH value of the catalytic system is 9.5, the volume is 20mL, the catalytic system is filled into a container, and the container is placed in an air shaking table for an open reaction, the rotation speed of the shaking table is 200r/min, and the temperature is 30-40 ℃.
6. The use according to claim 5, wherein the amount of whole cell catalyst in the catalytic system is 50 to 100g/L based on the wet bacterial weight.
7. The use according to claim 6, wherein the whole cell catalyst is prepared as follows: inoculating recombinant escherichia coli containing the isoeugenol monooxygenase mutant gene as claimed in claim 2 into LB slant culture medium, and culturing at 37 ℃ for 12-16 h; inoculating the slant strain to LB liquid seed culture medium, and culturing for 4-8 h at 37 ℃ under shaking at 200 r/min; inoculating the seed solution into 50mL fermentation medium according to the volume ratio of 1-5%, culturing for 4h at 37 ℃ under 220r/min in an oscillating way, cooling to 20-30 ℃ and adding 1mmol/L isopropyl thio-beta-D-galactoside for continuous induction expression for 16h, centrifuging after fermentation, collecting thalli, washing thalli twice with sterile physiological saline, and obtaining the isoeugenol monooxygenase mutant whole-cell catalyst.
8. The use according to claim 7, wherein the preparation method of the LB slant medium comprises: the yeast extract powder is prepared by mixing solution containing 5g/L yeast extract powder, 10g/L peptone, 5g/LNaCl, 100mg/L ampicillin and 20g/L agar, regulating pH to 6.8-7.0, and sterilizing at 121deg.C under high pressure steam for 20 min.
9. The use according to claim 7, wherein the preparation method of the LB liquid seed medium comprises: the yeast extract powder is prepared by regulating pH to 7.0-7.2 of a mixed solution containing 5g/L yeast extract powder, 10g/L peptone, 5g/LNaCl and 100mg/L ampicillin, and sterilizing the mixed solution by high-pressure steam at 121 ℃ for 20 min.
10. The use according to claim 7, characterized in that the preparation of the fermentation medium is carried out by: mixing 20g/L glycerol, 10g/L peptone, 5g/L yeast extract powder, 2g/L malt extract powder, 2g/LMgSO 4 、15g/LKH 2 PO 4 、2g/LNH 4 Cl and 50 mg/LFASO 4 Adjusting the pH to 6.7-7.0, and sterilizing the mixture for 20min at 121 ℃ by high-pressure steam.
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