CN114891820B - Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, construction method and application - Google Patents

Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, construction method and application Download PDF

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CN114891820B
CN114891820B CN202210595170.3A CN202210595170A CN114891820B CN 114891820 B CN114891820 B CN 114891820B CN 202210595170 A CN202210595170 A CN 202210595170A CN 114891820 B CN114891820 B CN 114891820B
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bacillus licheniformis
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kivd
leu
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陈守文
周飞
占杨杨
许海霞
尹昊
蔡冬波
马昕
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Hubei University
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Abstract

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to bacillus licheniformis for efficiently synthesizing hydroxytyrosol, a construction method and application thereof. Firstly, carrying out various transformation on the basis of bacillus licheniformis DW2 to obtain recombinant bacillus licheniformis DH7; selecting 4-hydroxyphenylacetic acid-3-hydroxylase from escherichia coli and ketoacid decarboxylase from lactococcus lactis, transferring the ketoacid decarboxylase into bacillus licheniformis DH7 chassis cells, and realizing the de-novo synthesis of hydroxytyrosol yield; through the combined expression of 4-hydroxyphenylacetic acid-3-hydroxylase HpaBC and a keto acid decarboxylase KivD mutant, the recombinant bacillus licheniformis can efficiently synthesize hydroxytyrosol by utilizing low-cost carbon sources such as glucose and the like, and the yield is as high as 5.55+/-0.37. The recombinant bacillus licheniformis disclosed by the invention can solve the problems of low yield and high price of hydroxytyrosol, and has wide industrial prospect.

Description

Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, construction method and application
Technical Field
The invention belongs to the technical field of microbial genetic engineering, and particularly relates to bacillus licheniformis for efficiently synthesizing hydroxytyrosol, a construction method and application thereof.
Background
Hydroxytyrosol (HT), also known as 3, 4-dihydroxyphenylethanol, is a biologically active polyphenol, one of the strongest natural antioxidants known at present. Hydroxytyrosol can scavenge free radicals, reduce oxidation of low density lipoprotein, and stimulate calcium deposition. It also has anticancer, anti-inflammatory and antimicrobial activities. Therefore, hydroxytyrosol can be used in cosmetics, foods, health products and medical industries.
Currently, HT is manufactured industrially by plant extraction or chemical synthesis. In nature, HT exists in olive plants in the form of oleuropein, and hydroxytyrosol can be extracted from fresh olive leaves and olive processing wastewater. Despite the abundance and low cost of these materials, there are also disadvantages such as low yields, strong acid vapors and long duration of the process. Chemical synthesis utilizes some analogues of HT, such as 3, 4-dihydroxyphenylacetic acid, 3, 4-dihydroxybenzaldehyde, tyrosol and the like as substrates to synthesize hydroxytyrosol, and the methods are not suitable for large-scale industrial production due to expensive substrates, bad conditions, complex steps or low yield.
With advances in metabolic engineering and synthetic biology, de novo production of HT from simple carbon sources has been achieved by either reconstructing the natural pathway or introducing artificial biosynthetic pathways into microorganisms. Most reports at present are that escherichia coli is utilized to synthesize hydroxytyrosol through microbial transformation, which lays an important foundation for producing hydroxytyrosol by a microbial fermentation method. However, the de novo synthesis of HT using a simple carbon source has the problems of long synthetic pathway, heavy metabolic load, low efficiency and low substrate conversion, and further improvement of de novo efficiency is required to achieve economic production of HT.
Disclosure of Invention
In view of the above problems in the prior art, it is an object of the present invention to provide Bacillus licheniformis which synthesizes hydroxytyrosol efficiently. A recombinant bacillus licheniformis is developed, a gene keto acid decarboxylase gene and a hydroxylase gene for synthesizing hydroxytyrosol are optimally expressed in bacillus licheniformis for producing tyrosol, and a bacillus licheniformis strain for synthesizing hydroxytyrosol in a heterologous way is constructed.
It is a further object of the present invention to provide the use of the recombinant Bacillus licheniformis described above for the production of hydroxytyrosol.
In order to solve the problems, the technical scheme of the invention is as follows:
a bacillus licheniformis for efficiently synthesizing hydroxytyrosol comprises the following steps:
sequentially knocking out a pyruvate kinase gene pyk, a tyrosine/phenylalanine aminotransferase gene hisC and an aldehyde dehydrogenase gene dhaS in bacillus licheniformis DW2, obtaining bacillus licheniformis DH4 by using an alcohol dehydrogenase gene adhA, and obtaining tyrA fbr The gene is integrated to the ldh site of bacillus licheniformis DH4 to obtain bacillus licheniformis DH5, and the genes, aroK and aroA promoters in the bacillus licheniformis DH5 are replaced by PbacA to obtain bacillus licheniformis DH7;
the 4-hydroxyphenylacetic acid-3-hydroxylase gene hpaBC and kivD in the genome of the escherichia coli BL21 (DE 3) V461A Or kivD V461I Co-expression is carried out, and then the mixture is transferred into DH7, kivD V461A The sequence is shown as SEQ ID NO.89, kivD V461I The sequence is shown as SEQ ID NO. 90.
Wherein the promoter of hpaBC may be: pbacA, P43 or Pbay;
kivD V461A or kivD V461I The promoters of (a) may be: pbacA, P43 or Pbay.
The protection scope of the invention also comprises: the bacillus licheniformis is used for synthesizing hydroxytyrosol.
Compared with the prior art, the invention has the following advantages:
1. the invention overexpresses the target genes of KivD mutants (V461A and V461I) and HpaBC in Bacillus licheniformis DH 7. Through the effective expression of the enzymes in the recombinant bacillus licheniformis DH7, the strain can synthesize hydroxytyrosol from the head by using glucose as a substrate, and the synthesis cost is low and the operation is simple.
2. The invention utilizes the strong promoters P43, pbacA and Pboay to efficiently drive the specific expression of the exogenous genes in the recombinant bacillus licheniformis DH7, improves the construction and conversion efficiency of the recombinant expression vector, further effectively improves the capability of synthesizing hydroxytyrosol by the bacillus licheniformis, and improves the yield of the hydroxytyrosol.
Drawings
FIG. 1 shows the synthetic route for hydroxytyrosol.
FIG. 2 is a liquid phase diagram of DW2/pHY300 and HT1 hydroxytyrosol.
Detailed Description
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
Example 1:
construction of genetically engineered strain DH 7:
(1) Knock out pyk, hisC and dhaS: the genome of bacillus licheniformis DW2 is used as a template, the primers pyk-AF/AR and pyk-BF/BR are used for respectively amplifying the upstream and downstream homology arms of the pyruvate kinase gene pyk fragment, and the upstream and downstream homology arm fragments are fused to obtain a pyk knockout box; amplifying the skeleton by using a primer (T2-T5-F/R) and taking a plasmid T2 (2) -ori as a template; inserting a pyk knockout box into a plasmid T2, performing colony PCR by using T2-F/T2-R to obtain a positive transformant, successfully constructing pyk knockout plasmid T2-delta-pyk through DNA sequencing, introducing the pyk knockout plasmid into bacillus licheniformis DW2 (or called bacillus licheniformis DW2, CN 112226437A), and obtaining pyk knockout strain DH1 through homologous recombination; the construction method of the knockout vector of the tyrosine/phenylalanine aminotransferase gene hisC and the aldehyde dehydrogenase gene dhaS is the same as pyk, and a pyk strain DH3 in which the hisC and the dhaS genes are knocked out simultaneously is obtained through iterative knockout;
the primer sequence required by pyk knockout is as follows:
pyk-AF：ctgcagcccgggggatccgggatacagctacatccc
pyk-AR：ttaaagtacgcttgcacgcggcccaattgtacaaact
pyk-BF：agtttgtacaattgggccgcgtgcaagcgtactttaa
pyk-BR：gatcttttctacgagctcgcggcagcctgctttttc
T2-T5-F：ggatcccccgggctgcaggaattc
T2-T5-R：gagctcgtagaaaagatcaaagga
T2-F：atgtgataactcggcgta
T2-R：gcaagcagcagattacgc
pyk-YF:ttctcggattgatcatgg
pyk-YR:aacggcttgacgactttc
primer for knocking out hisC
hisC-AF：ctgcagcccgggggatccaacggcgtcgtgttcagc
hisC-AR：gtaaaatgaggtgacagagaggtgatgattcagtca
hisC-BF：tgactgaatcatcacctctctgtcacctcattttac
hisC-BR：gatcttttctacgagctccttggcggtgaaatgaaa
hisC-YF：gtgcggaaagtggctgat
hisC-YR：acatgaacatcgtaaaagc
Primer for knocking out hisC
dhaS-AF：ctgcagcccgggggatccgtgcgggtgtatgttcaa
dhaS-AR：atttcacgtccgagtccctcgggtgagttgtcttgg
dhaS-BF：ccaagacaactcacccgagggactcggacgtgaaat
dhaS-BR：gatcttttctacgagctcaataccaggaaccgacaa
dhaS-YF：accgcagcaggatgttct
dhaS-YR：cgcatttgctaaaccttc
(2) Knocking out an alcohol dehydrogenase gene adhA: the genome of bacillus licheniformis DW2 is used as a template, primers adhA-aF/AR and adhA-BF/BR are used for respectively amplifying upstream and downstream homology arms of the adhA fragment, the upstream and downstream homology arm fragments are fused to obtain an ahdA knockout box, and a primer (T2-T5-F/R) is used for amplifying a framework by using plasmids T2 (2) -ori as the template; inserting the strain into a plasmid T2, performing colony PCR by using T2-F/T2-R to obtain a positive transformant, successfully constructing an ad hA knockout plasmid T2-deltA-AdhA through DNA sequencing, introducing the ad hA knockout plasmid into bacillus licheniformis DH3, and obtaining an adhA knockout strain D H through homologous recombination;
primers used for adhA knockout were as follows
adhA-AF:ctgcagcccgggggatccagcagtgtagcacgataa
adhA-AR:gggaggcggaatctttccaatcatatgtaatacagagag
adhA-BF:ctctctgtattacatatgattggaaagattccgcctccc
adhA-BR:gatcttttctacgagctcccttaatggagggcggtcaa
adhA-YF:tcatgagtcctccgattc
adhA-YR:ttttatacgagcggtgac。
(3)tyrA fbr Integration to the ldh site: designing primers (ldh-AF/AR, ldh-BF/BR) to amplify upstream and downstream homology arms of the ldh according to the sequence of the ldh in the genomic DNA sequence of bacillus licheniformis; designing a primer (Pylb-F/R) amplification promoter Pylb according to the sequence of Pylb in the bacillus subtilis DNA sequence; according to the sequence of tyrA in the DNA sequence of colibacillus and the mutant site sequence of tyrA (SEQ ID NO. 85), designing a primer (tyrA-F/tyrA-R) to amplify tyrA fbr The method comprises the steps of carrying out a first treatment on the surface of the Designing primers (TamyL-F and TamyL-R) to amplify TamyL according to the sequence of TamyL in the Bacillus licheniformis DNA sequence; primer is used to make the ldh upstream and downstream homology arm, pylb promoter and tyrA fbr Fragment fusion construction tyrA fbr An expression cassette; amplifying the skeleton by using a primer (T2-T5-F/R) and taking a plasmid T2 (2) -ori as a template; inserting it into plasmid T2, performing colony PCR by using T2-F/T2-R to obtain positive transformant, and performing DNA sequencing to successfully construct tyrA fbr Integrated expression plasmid T2-ldh-tyrA fbr Introducing into Bacillus licheniformis DH4, and obtaining tyrA by homologous recombination fbr Integrating the expression strain DH5; the prephenate dehydrogenase TyrA fbr The coded amino acid sequence is shown as SEQ ID NO. 86.
tyrA fbr The introduction for integration into the ldh site is as follows:
ldh-AF：ctgcagcccgggggatccccgacctgtgatggagat
ldh-AR：ggagcgcgttcgacgatgcatattgtgcaatacttc
Pylb-F：gaagtattgcacaatatgcatcgtcgaacgcgctcc
Pylb-R：ggtcaattcagcaaccatacaaatctccccctttgt
TyrA-F：acaaagggggagatttgtatggttgctgaattgacc
TyrA-R：tccgtcctctctgctcttaatgaaggtattgggctg
TamyL-F：cagcccaataccttcattaagagcagagaggacgga
TamyL-R：aacagattcccaaacggacgcaataatgccgtcgca
ldh-BF：tgcgacggcattattgcgtccgtttgggaatctgtt
ldh-BR：gatcttttctacgagctctcaagcctcccatctgtg
ldh-YF:catatcagcggaatcatc
ldh-YR:ccgcttaatacaaggaga
(4) aroK and aroA promoters were replaced with PbacA:
amplifying an upstream and downstream 500bp sequence of an aroK promoter by using a primer ParoK-AF/AR, respectively serving as an upper homologous arm and a lower homologous arm, amplifying a PbacA promoter sequence by using a bacillus licheniformis DW2 genome DNA as a template, fusing the upper homologous arm, the promoter and the lower homologous arm, and designing primers (T2-T5-F and T2-T5-R), wherein plasmids T2 (2) -ori are used as template amplification frameworks; the fusion fragment and the linear plasmid fragment are connected by using a one-step cloning kit, and are transformed into E.coli DH5 alpha competent cells, and the recombinant plasmid T is respectively obtained through PCR verification and sequencing analysis 2 (2) -PbacA-aroK. The recombinant plasmid is electrically transferred into bacillus licheniformis DH5 for homologous recombination exchange, and screening and verification are carried out to obtain a promoter replacement aroK promoter strain DH6.
The aroA enhanced expression vector strain is constructed by the same method as aroK promoter replacement strain, and DH7 strain is finally obtained.
The primers used for aroK promoter replacement are as follows:
ParoK-AF：ctgcagcccgggggatccgtaggctcatttgctgat
PbacA(aroK)-AR：aatctcgccgaaatcgcaggctatttccaccagtcgtcaa
PbacA(aroK)-F：ttgacgactggtggaaatagcctgcgatttcggcgagatt
PbacA(aroK)-R：tctcattgcggcattcatataaaaattctcctttttgat
PbacA(aroK)-BF：atcaaaaaggagaatttttatatgaatgccgcaatgaga
ParoK-BR：gatcttttctacgagctcctttcaagttgtggaatg
ParoK-YF：aggcgttcaggcggaatt
ParoK-YR：gacacagcgatagaaaca
the primers used for aroA promoter replacement are as follows:
ParoA-AF：ctgcagcccgggggatccgaagtggacgcacatttc
PbacA(aroA)-AR：tctcgccgaaatcgcagggttatccatcctttcttt
PbacA(aroA)-F：aaagaaaggatggataaccctgcgatttcggcgaga
PbacA(aroA)-R：aagttcagtgttgctcatataaaaattctccttttt
PbacA(aroA)-BF：aaaaaggagaatttttatatgagcaacactgaactt
ParoA-BR：gatcttttctacgagctccaacacgctttaagattt
ParoA-YF：gttgacgcacgcttcgtt
ParoA-YR：attgatgaattccttcag
example 2:
construction of wild-type kivD and hpaBC Co-expression Strain
Step 1: the sequence of primers (P43-HpaBC-F and HpaBC-R) is designed according to the sequence of HpaBC in the genome of escherichia coli BL21 (DE 3), and the sequence of P43 is designed according to the sequence of P43 promoter (shown as SEQ ID NO. 92) in the genome of bacillus subtilis 168;
wherein, the sequence of Amp-P43-F, P43-HpaBC-R, P-HpaBC-F, hpaBC-R is as follows:
Amp-P43-F：acttttcggggaaatgtctgataggtggtatgtttt
P43-HpaBC-R：gaaatcttctggtttcatgtgtacattcctctctta
P43-HpaBC-F：taagagaggaatgtacacatgaaaccagaagatttc
HpaBC-R：gtaaacttggtctgacagttaaatcgcagcttccattt
step 2: the fusion fragment was obtained by ligating together P43 and hpaBC (shown as SEQ ID NO. 91) by overlap extension PCR (the primers used were Amp-P43-F and HpaBC-R);
step 3: primers (T5-amp-F and T5-amp-R) were designed and the template amplified backbone was the plasmid pHY-PbacA-kivD (ZhanY, et al, efficiency systems of 2-phenylethanol from L-phenylalanine by engineered Bacillus lichenif ormis using molasses as carbon source. Appl Microbiol Biotechnol.2020.104 (17): 7507-7520). The kivD sequence is shown in SEQ ID NO. 88.
Wherein the sequences of T5-amp-F and T5-amp-R are:
T5-amp-F：gacatttccccgaaaagtgccac
T5-amp-R：ctgtcagaccaagtttactcatata；
step 4: connecting the gene fragments obtained in the steps (6) and (7) with a linear plasmid fragment by using a one-step cloning kit, transferring the connection product into escherichia coli DH5 alpha by a calcium chloride transformation method, screening by using a culture medium containing tetracyclic resistance at 37 ℃ to obtain a transformant, screening a plasmid of the transformant to obtain a positive transformant by colony PCR verification (the primers used are amp-YF and amp-YR), and carrying out DNA sequencing verification to obtain a correct co-expression vector pHY-PbacA-kivD-P43-hpaBC;
wherein, the sequences of amp-YF and amp-YR are:
amp-YF：ccctgatctcgacttcgt
amp-YR：ttaaggggtctgacgctc
step 5: the pHY-PbacA-kivD-P43-hpaBC plasmid is electrically transferred into DH7, and the strain PCR is verified, so that the recombinant strain HT1 for successfully transforming the recombinant plasmid pHY-PbacA-kivD-P43-hpaBC is successfully obtained.
Example 3:
construction of mutant kivD (V461A or V461I) and hpaBC Co-expression Strain
Step 1: the vector pHY-PbacA-kivD-P43-hpaBC in example 2 was used as a template to amplify the backbones containing the mutations of V461A and V461I using primers V461A-F/R and V461I-F/R, respectively.
V461A-F：gttatacagctgaaagagaaattcatggaccaaatcaaa
V461A-R：ctctttcagctgtataaccatcattattgataataaagc
V461I-F：gttatacaatcgaaagagaaattcatggaccaaatcaaa
V461I-R：ctctttcgattgtataaccatcattattgataataaagc
Step 2: the linear frameworks of V461A and V461I obtained in the step (1) are subjected to carrier self-ligation by using a one-step cloning kit, and the ligation product is transferred into escherichia coli DH5 alpha by a calcium chloride transformation method, and then the cloning kit is used for preparing the vector self-ligation vectorScreening by four-ring resistance-containing culture medium at 37deg.C to obtain transformant, colony PCR verifying plasmid of transformant (primers are pHY-YF and pHY-YR), DNA sequencing to verify mutation site successful mutation, and respectively named pHY-PbacA-kivD for V461A and V461I mutant vectors V461A -P43-hpaBC and pHY-PbacA-kivD V461I -P43-hp aBC。kivD V461A The sequence is shown as SEQ ID NO.89, kivD V461I The sequence is shown as SEQ ID NO. 90.
Step 3: pHY-PbacA-kivD V461A -P43-hpaBC and pHY-PbacA-kivD V461I The P43-hpaBC plasmid was electrotransferred into recombinant strain DH7, respectively, to obtain HT2 and HT3 strains.
Example 4:
construction of recombinant strains expressing mutant kivD (V461A and V461I) and hpaBC in combination with different promoters
Step 1: the plasmid pHY-PbacA-kivD in example 3 V461A -P43-hpaBC and pHY-PbacA-kivD V461I P43-hpaBC as a template, and PbacA (shown as SEQ ID NO. 93) and Pgay (shown as SEQ ID NO. 94) as promoters, respectively, to construct 18 expression vectors with different combinations. To construct pHY-P43-kivD V461I As an example of-P43-hpaBC, the P43 fragment is used as a template, the P43 promoter is amplified with the P43-kivD-F/R primer, and pHY-PbacA-kivD is used V461I Amplification of pHY-P43-kivD with kivD-T5-F/R primer as template V461I -a P43-hpaBC vector backbone; p43 promoter fragment and pHY-P43-kivD Using one-step cloning kit V461I Connecting the linear skeleton of-P43-hpaBC, transferring the connecting product into colibacillus DH5 alpha by a calcium chloride conversion method, screening by a culture medium containing tetracyclic resistance at 37 ℃ to obtain a transformant, and naming the transformant as a co-expression vector pHY-P43-kivD V461I -P43-hpacc. The construction method of the expression vectors of the other 15 different combinations is similar to that of pHY-P43-kivD V461I -P43-hpaBC。
The remaining 15 combined expression vectors are shown in the following table:
recombinant plasmid Recombinant plasmid
pHY-PbacA-kivD V461A -PbacA-hpaBC pHY-PbacA-kivD V461I -PbacA-hpaBC
pHY-PbacA-kivD V461A -Pbay-hpaBC pHY-PbacA-kivD V461I -Pbay-hpaBC
pHY-P43-kivD V461A -P43-hpaBC pHY-P43-kivD V461I -PbacA-hpaBC
pHY-P43-kivD V461A -PbacA-hpaBC pHY-P43-kivD V461I -Pbay-hpaBC
pHY-P43-kivD V461A -Pbay-hpaBC pHY-Pbay-kivD V461I -P43-hpaBC
pHY-Pbay-kivD V461A -P43-hpaBC pHY-Pbay-kivD V461I -PbacA-hpaBC
pHY-Pbay-kivD V461A -PbacA-hpaBC pHY-Pbay-kivD V461I -Pbay-hpaBC
pHY-Pbay-kivD V461A -Pbay-hpaBC
P43-kivD-F：tttttataacaggaattctgataggtggtatgtttt
P43-kivD-R：atctcctactgtatacatgtgtacattcctctctta
PbacA-kivD-F：tttttataacaggaattccctgcgatttcggcgaga
PbacA-kivD-R：atctcctactgtatacatataaaaattctccttttt
Pbay-kivD-F:tttttataacaggaattccctgcgatttcggcgaga
Pbay-kivD-R：atctcctactgtatacatacaaatctccccctttgt
kivD-T5-F：gaattcctgttataaaaaaaggatc
kivD-T5-R:：atgtatacagtaggagattaccta
P43-hpaBC-F:acttttcggggaaatgtctgataggtggtatgtttt
P43-hpaBC-R:gaaatcttctggtttcatgtgtacattcctctctta
PbacA-hpaBC-F:acttttcggggaaatgtccctgcgatttcggcgaga
PbacA-hpaBC-R:gaaatcttctggtttcatataaaaattctccttttt
Pbay-hpaBC-F:acttttcggggaaatgtccctgcgatttcggcgaga
Pbay-hpaBC-R:gaaatcttctggtttcatacaaatctccccctttgt
hpaBC-T5-F：gacatttccccgaaaagtgccac
HpaBC-T5-R:atgaaaccagaagatttccg
Step 2: the constructed 18 recombinant plasmids expressed in different combinations and a control vector pHY300 are electrically transformed into a recombinant strain DH7 to obtain 18 recombinant Bacillus licheniformis HT 2-HT 19 and HT0 control strains.
Example 5:
recombinant Bacillus licheniformis hydroxytyrosol fermentation
1) Seed fermentation: on-plate activation of Bacillus licheniformis DW2/pHY-300, HT0 and different recombinant Bacillus licheniformis HT 1-HT 19, picking was inoculated into 250mL triangular flasks containing 50mL of liquid LB, respectively, and cultured at 37℃at 230rp m for 13h. Then subsequently inoculating the fermentation medium with an inoculum size of 2% (volume ratio); the hydroxytyrosol fermentation medium was cultured at 37℃and 230rpm for 48 hours, and the hydroxytyrosol yield was measured after the fermentation.
The formula of the hydroxytyrosol fermentation medium is 10g/L peptone, 5g/L yeast powder, 10g/LNaCl and 18.6g/LK 2 HPO 4 ，5.2g/LKH 2 PO 4 70g/L glucose, pH 7.0, the balance being water.
2) Pretreatment of fermentation liquor: diluting the fermentation broth with deionized water, centrifuging to remove thallus, and filtering with 0.22 μm water phase filter membrane.
3) The hydroxytyrosol is detected by High Performance Liquid Chromatography (HPLC). The measurement conditions are specifically as follows: using an Agilent 1260 high performance liquid chromatograph, column C18 column (4.6 mm ID x 250mm,5 μm), mobile phase methanol: 0.1% formic acid = 2:8, the flow rate is 0.6mL/min, the column temperature is 30 ℃, the sample injection amount is 10.0 mu L, the detection wavelength is 224nm, and the elution is 25min. And calculating the content of hydroxytyrosol in the fermentation broth according to a standard curve prepared by the hydroxytyrosol standard substance. The results showed that no hydroxytyrosol was detected in the control strain DW2/pHY-300 and DH0 strain, but that hydroxytyrosol was detected in both recombinant strains, H T (pHY-P43-kivD V461A -Pbay-hpaBC) and HT16 (pHY-P43-kivD V461I Pbay-hpaBC) strains up to 5.55.+ -. 0.37 and 5.49.+ -. 0.33g/L.
TABLE 1 production of hydroxytyrosol by different recombinant strains
Figure BDA0003667646590000091
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Figure BDA0003667646590000101
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Sequence listing
<110> university of Hubei
<120> Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, construction method and application
<160> 94
<170> SIPOSequenceListing 1.0
<210> 1
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ctgcagcccg ggggatccgg gatacagcta catccc 36
<210> 2
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
ttaaagtacg cttgcacgcg gcccaattgt acaaact 37
<210> 3
<211> 37
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
agtttgtaca attgggccgc gtgcaagcgt actttaa 37
<210> 4
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gatcttttct acgagctcgc ggcagcctgc tttttc 36
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
ggatcccccg ggctgcagga attc 24
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
gagctcgtag aaaagatcaa agga 24
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgtgataac tcggcgta 18
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gcaagcagca gattacgc 18
<210> 9
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
ttctcggatt gatcatgg 18
<210> 10
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
aacggcttga cgactttc 18
<210> 11
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ctgcagcccg ggggatccaa cggcgtcgtg ttcagc 36
<210> 12
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gtaaaatgag gtgacagaga ggtgatgatt cagtca 36
<210> 13
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
tgactgaatc atcacctctc tgtcacctca ttttac 36
<210> 14
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gatcttttct acgagctcct tggcggtgaa atgaaa 36
<210> 15
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gtgcggaaag tggctgat 18
<210> 16
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
acatgaacat cgtaaaagc 19
<210> 17
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
ctgcagcccg ggggatccgt gcgggtgtat gttcaa 36
<210> 18
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
atttcacgtc cgagtccctc gggtgagttg tcttgg 36
<210> 19
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
ccaagacaac tcacccgagg gactcggacg tgaaat 36
<210> 20
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gatcttttct acgagctcaa taccaggaac cgacaa 36
<210> 21
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
accgcagcag gatgttct 18
<210> 22
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
cgcatttgct aaaccttc 18
<210> 23
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
ctgcagcccg ggggatccag cagtgtagca cgataa 36
<210> 24
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
gggaggcgga atctttccaa tcatatgtaa tacagagag 39
<210> 25
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
ctctctgtat tacatatgat tggaaagatt ccgcctccc 39
<210> 26
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gatcttttct acgagctccc ttaatggagg gcggtcaa 38
<210> 27
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
tcatgagtcc tccgattc 18
<210> 28
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
ttttatacga gcggtgac 18
<210> 29
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
ctgcagcccg ggggatcccc gacctgtgat ggagat 36
<210> 30
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
ggagcgcgtt cgacgatgca tattgtgcaa tacttc 36
<210> 31
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gaagtattgc acaatatgca tcgtcgaacg cgctcc 36
<210> 32
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
ggtcaattca gcaaccatac aaatctcccc ctttgt 36
<210> 33
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
acaaaggggg agatttgtat ggttgctgaa ttgacc 36
<210> 34
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
tccgtcctct ctgctcttaa tgaaggtatt gggctg 36
<210> 35
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
cagcccaata ccttcattaa gagcagagag gacgga 36
<210> 36
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
aacagattcc caaacggacg caataatgcc gtcgca 36
<210> 37
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
tgcgacggca ttattgcgtc cgtttgggaa tctgtt 36
<210> 38
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gatcttttct acgagctctc aagcctccca tctgtg 36
<210> 39
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
catatcagcg gaatcatc 18
<210> 40
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
ccgcttaata caaggaga 18
<210> 41
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
ctgcagcccg ggggatccgt aggctcattt gctgat 36
<210> 42
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
aatctcgccg aaatcgcagg ctatttccac cagtcgtcaa 40
<210> 43
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
ttgacgactg gtggaaatag cctgcgattt cggcgagatt 40
<210> 44
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
tctcattgcg gcattcatat aaaaattctc ctttttgat 39
<210> 45
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
atcaaaaagg agaattttta tatgaatgcc gcaatgaga 39
<210> 46
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gatcttttct acgagctcct ttcaagttgt ggaatg 36
<210> 47
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
aggcgttcag gcggaatt 18
<210> 48
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
gacacagcga tagaaaca 18
<210> 49
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 49
ctgcagcccg ggggatccga agtggacgca catttc 36
<210> 50
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 50
tctcgccgaa atcgcagggt tatccatcct ttcttt 36
<210> 51
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
aaagaaagga tggataaccc tgcgatttcg gcgaga 36
<210> 52
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 52
aagttcagtg ttgctcatat aaaaattctc cttttt 36
<210> 53
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 53
aaaaaggaga atttttatat gagcaacact gaactt 36
<210> 54
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 54
gatcttttct acgagctcca acacgcttta agattt 36
<210> 55
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 55
gttgacgcac gcttcgtt 18
<210> 56
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 56
attgatgaat tccttcag 18
<210> 57
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 57
acttttcggg gaaatgtctg ataggtggta tgtttt 36
<210> 58
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 58
gaaatcttct ggtttcatgt gtacattcct ctctta 36
<210> 59
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 59
taagagagga atgtacacat gaaaccagaa gatttc 36
<210> 60
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 60
gtaaacttgg tctgacagtt aaatcgcagc ttccattt 38
<210> 61
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 61
gacatttccc cgaaaagtgc cac 23
<210> 62
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 62
ctgtcagacc aagtttactc atata 25
<210> 63
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 63
ccctgatctc gacttcgt 18
<210> 64
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 64
ttaaggggtc tgacgctc 18
<210> 65
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 65
gttatacagc tgaaagagaa attcatggac caaatcaaa 39
<210> 66
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 66
ctctttcagc tgtataacca tcattattga taataaagc 39
<210> 67
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 67
gttatacaat cgaaagagaa attcatggac caaatcaaa 39
<210> 68
<211> 39
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 68
ctctttcgat tgtataacca tcattattga taataaagc 39
<210> 69
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 69
tttttataac aggaattctg ataggtggta tgtttt 36
<210> 70
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 70
atctcctact gtatacatgt gtacattcct ctctta 36
<210> 71
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 71
tttttataac aggaattccc tgcgatttcg gcgaga 36
<210> 72
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 72
atctcctact gtatacatat aaaaattctc cttttt 36
<210> 73
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 73
tttttataac aggaattccc tgcgatttcg gcgaga 36
<210> 74
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 74
atctcctact gtatacatac aaatctcccc ctttgt 36
<210> 75
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 75
gaattcctgt tataaaaaaa ggatc 25
<210> 76
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 76
atgtatacag taggagatta ccta 24
<210> 77
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 77
acttttcggg gaaatgtctg ataggtggta tgtttt 36
<210> 78
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 78
gaaatcttct ggtttcatgt gtacattcct ctctta 36
<210> 79
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 79
acttttcggg gaaatgtccc tgcgatttcg gcgaga 36
<210> 80
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 80
gaaatcttct ggtttcatat aaaaattctc cttttt 36
<210> 81
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 81
acttttcggg gaaatgtccc tgcgatttcg gcgaga 36
<210> 82
<211> 36
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 82
gaaatcttct ggtttcatac aaatctcccc ctttgt 36
<210> 83
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 83
gacatttccc cgaaaagtgc cac 23
<210> 84
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 84
atgaaaccag aagatttccg 20
<210> 85
<211> 1122
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 85
atggttgctg aattgaccgc attacgcgat caaattgatg aagtcgataa agcgctgctg 60
aatttattag cgaagcgtct ggaactggtt gctgaagtgg gcgaggtgaa aagccgcttt 120
ggactgccta tttatgttcc ggagcgcgag gcatctatct tggcctcgcg tcgtgcagag 180
gcggaagctc tgggtgtacc gccagatctg attgaggatg ttttgcgtcg ggtgatgcgt 240
gaatcttact ccagtgaaaa cgacaaagga tttaaaacac tttgtccgtc actgcgtccg 300
gtggttatcg tcggcggtgg cggtcagatg ggacgcctgt tcgagaagat gctgaccctc 360
tcgggttatc aggtgcggat tctggagcaa catgactggg atcgagcggc tgatattgtt 420
gccgatgccg gaatggtgat tgttagtgtg ccaatccacg ttactgagca agttattggc 480
aaattaccgc ctttaccgaa agattgtatt ctggtcgatc tggcatcagt gaaaaatggg 540
ccattacagg ccatgctggt ggcgcatgat ggtccggtgc tggggctaca cccgatgttc 600
ggtccggaca gcggtagcct ggcaaagcaa gttgtggtct ggtgtgatgg acgtaaaccg 660
gaagcatacc aatggtttct ggagcaaatt caggtctggg gcgctcggct gcatcgtatt 720
agcgccgtcg agcacgatca gaatatggcg tttattcagg cactgcgcca ctttgctact 780
tttgcttacg ggctgcacct ggcagaagaa aatgttcagc ttgagcaact tctggcgctc 840
tcttcgccga tttaccgcct tgagctggcg atggtcgggc gactgtttgc tcaggatccg 900
cagctttatg ccgacatcat tatgtcgtca gagcgtaatc tggcgttaat caaacgttac 960
tataagcgtt tcggcgaggc gattgagttg ctggagcagg gcgataagca ggcgtttatt 1020
gacagtttcc gcaaggtgga gcactggttc ggcgattacg tacagcgttt tcagagtgaa 1080
agccgcgtgt tattgcgtca ggcgaatgac aatcgccagt aa 1122
<210> 86
<211> 373
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 86
Met Val Ala Glu Leu Thr Ala Leu Arg Asp Gln Ile Asp Glu Val Asp
1 5 10 15
Lys Ala Leu Leu Asn Leu Leu Ala Lys Arg Leu Glu Leu Val Ala Glu
20 25 30
Val Gly Glu Val Lys Ser Arg Phe Gly Leu Pro Ile Tyr Val Pro Glu
35 40 45
Arg Glu Ala Ser Ile Leu Ala Ser Arg Arg Ala Glu Ala Glu Ala Leu
50 55 60
Gly Val Pro Pro Asp Leu Ile Glu Asp Val Leu Arg Arg Val Met Arg
65 70 75 80
Glu Ser Tyr Ser Ser Glu Asn Asp Lys Gly Phe Lys Thr Leu Cys Pro
85 90 95
Ser Leu Arg Pro Val Val Ile Val Gly Gly Gly Gly Gln Met Gly Arg
100 105 110
Leu Phe Glu Lys Met Leu Thr Leu Ser Gly Tyr Gln Val Arg Ile Leu
115 120 125
Glu Gln His Asp Trp Asp Arg Ala Ala Asp Ile Val Ala Asp Ala Gly
130 135 140
Met Val Ile Val Ser Val Pro Ile His Val Thr Glu Gln Val Ile Gly
145 150 155 160
Lys Leu Pro Pro Leu Pro Lys Asp Cys Ile Leu Val Asp Leu Ala Ser
165 170 175
Val Lys Asn Gly Pro Leu Gln Ala Met Leu Val Ala His Asp Gly Pro
180 185 190
Val Leu Gly Leu His Pro Met Phe Gly Pro Asp Ser Gly Ser Leu Ala
195 200 205
Lys Gln Val Val Val Trp Cys Asp Gly Arg Lys Pro Glu Ala Tyr Gln
210 215 220
Trp Phe Leu Glu Gln Ile Gln Val Trp Gly Ala Arg Leu His Arg Ile
225 230 235 240
Ser Ala Val Glu His Asp Gln Asn Met Ala Phe Ile Gln Ala Leu Arg
245 250 255
His Phe Ala Thr Phe Ala Tyr Gly Leu His Leu Ala Glu Glu Asn Val
260 265 270
Gln Leu Glu Gln Leu Leu Ala Leu Ser Ser Pro Ile Tyr Arg Leu Glu
275 280 285
Leu Ala Met Val Gly Arg Leu Phe Ala Gln Asp Pro Gln Leu Tyr Ala
290 295 300
Asp Ile Ile Met Ser Ser Glu Arg Asn Leu Ala Leu Ile Lys Arg Tyr
305 310 315 320
Tyr Lys Arg Phe Gly Glu Ala Ile Glu Leu Leu Glu Gln Gly Asp Lys
325 330 335
Gln Ala Phe Ile Asp Ser Phe Arg Lys Val Glu His Trp Phe Gly Asp
340 345 350
Tyr Val Gln Arg Phe Gln Ser Glu Ser Arg Val Leu Leu Arg Gln Ala
355 360 365
Asn Asp Asn Arg Gln
370
<210> 87
<211> 1647
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 87
atgtatacag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60
tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccgcaaggat 120
atgaaatggg tcggaaatgc taatgaatta aatgcttcat atatggctga tggctatgct 180
cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240
aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300
acatcaaaag ttcaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360
aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420
aatgcaaccg ttgaaattga ccgagtactt tctgcactat taaaagaaag aaaacctgtc 480
tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540
aaaaaagaaa actcaacttc aaatacaagt gaccaagaga tcttgaacaa aattcaagaa 600
agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagttttggc 660
ttagaaaaaa cagtctctca atttatttca aagacaaaac tacctattac gacattaaac 720
tttggaaaaa gttcagttga tgaagctctc ccttcatttt taggaatcta taatggtaaa 780
ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatcct gatgcttgga 840
gttaaactca cagactcttc aacaggagcc ttcactcatc atttaaatga aaataaaatg 900
atttcactga atatagatga aggaaaaata tttaacgaaa gcatccaaaa ttttgatttt 960
gaatccctca tctcctctct cttagaccta agcgaaatag aatacaaagg aaaatatatc 1020
gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080
caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140
ttctttggcg cttcatcaat tttcttaaaa ccaaagagtc attttattgg tcaaccctta 1200
tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260
agcagacacc ttttatttat tggtgatggt tcacttcaac ttacggtgca agaattagga 1320
ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380
gtcgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440
tcaaaattac cagaatcatt tggagcaaca gaagaacgag tagtctcgaa aatcgttaga 1500
actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560
tggattgagt tagttttggc aaaagaagat gcaccaaaag tactgaaaaa aatgggcaaa 1620
ctatttgctg aacaaaataa atcataa 1647
<210> 88
<211> 548
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 88
Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly
1 5 10 15
Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu
20 25 30
Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn
35 40 45
Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys
50 55 60
Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val
65 70 75 80
Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile
85 90 95
Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His
100 105 110
His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu
115 120 125
Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val
130 135 140
Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val
145 150 155 160
Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro
165 170 175
Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln
180 185 190
Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro
195 200 205
Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr
210 215 220
Val Ser Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn
225 230 235 240
Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile
245 250 255
Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser
260 265 270
Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr
275 280 285
Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn
290 295 300
Ile Asp Glu Gly Lys Ile Phe Asn Glu Ser Ile Gln Asn Phe Asp Phe
305 310 315 320
Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys
325 330 335
Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala
340 345 350
Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln
355 360 365
Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala
370 375 380
Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu
385 390 395 400
Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile
405 410 415
Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu
420 425 430
Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn
435 440 445
Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu
450 455 460
Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr
465 470 475 480
Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Glu Arg Val Val Ser
485 490 495
Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala
500 505 510
Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Ala Lys
515 520 525
Glu Asp Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu
530 535 540
Gln Asn Lys Ser
545
<210> 89
<211> 1647
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
atgtatacag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60
tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccgcaaggat 120
atgaaatggg tcggaaatgc taatgaatta aatgcttcat atatggctga tggctatgct 180
cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240
aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300
acatcaaaag ttcaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360
aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420
aatgcaaccg ttgaaattga ccgagtactt tctgcactat taaaagaaag aaaacctgtc 480
tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540
aaaaaagaaa actcaacttc aaatacaagt gaccaagaga tcttgaacaa aattcaagaa 600
agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagttttggc 660
ttagaaaaaa cagtctctca atttatttca aagacaaaac tacctattac gacattaaac 720
tttggaaaaa gttcagttga tgaagctctc ccttcatttt taggaatcta taatggtaaa 780
ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatcct gatgcttgga 840
gttaaactca cagactcttc aacaggagcc ttcactcatc atttaaatga aaataaaatg 900
atttcactga atatagatga aggaaaaata tttaacgaaa gcatccaaaa ttttgatttt 960
gaatccctca tctcctctct cttagaccta agcgaaatag aatacaaagg aaaatatatc 1020
gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080
caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140
ttctttggcg cttcatcaat tttcttaaaa ccaaagagtc attttattgg tcaaccctta 1200
tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260
agcagacacc ttttatttat tggtgatggt tcacttcaac ttacggtgca agaattagga 1320
ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380
attgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440
tcaaaattac cagaatcatt tggagcaaca gaagaacgag tagtctcgaa aatcgttaga 1500
actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560
tggattgagt tagttttggc aaaagaagat gcaccaaaag tactgaaaaa aatgggcaaa 1620
ctatttgctg aacaaaataa atcataa 1647
<210> 90
<211> 1647
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 90
atgtatacag taggagatta cctattagac cgattacacg agttaggaat tgaagaaatt 60
tttggagtcc ctggagacta taacttacaa tttttagatc aaattatttc ccgcaaggat 120
atgaaatggg tcggaaatgc taatgaatta aatgcttcat atatggctga tggctatgct 180
cgtactaaaa aagctgccgc atttcttaca acctttggag taggtgaatt gagtgcagtt 240
aatggattag caggaagtta cgccgaaaat ttaccagtag tagaaatagt gggatcacct 300
acatcaaaag ttcaaaatga aggaaaattt gttcatcata cgctggctga cggtgatttt 360
aaacacttta tgaaaatgca cgaacctgtt acagcagctc gaactttact gacagcagaa 420
aatgcaaccg ttgaaattga ccgagtactt tctgcactat taaaagaaag aaaacctgtc 480
tatatcaact taccagttga tgttgctgct gcaaaagcag agaaaccctc actccctttg 540
aaaaaagaaa actcaacttc aaatacaagt gaccaagaga tcttgaacaa aattcaagaa 600
agcttgaaaa atgccaaaaa accaatcgtg attacaggac atgaaataat tagttttggc 660
ttagaaaaaa cagtctctca atttatttca aagacaaaac tacctattac gacattaaac 720
tttggaaaaa gttcagttga tgaagctctc ccttcatttt taggaatcta taatggtaaa 780
ctctcagagc ctaatcttaa agaattcgtg gaatcagccg acttcatcct gatgcttgga 840
gttaaactca cagactcttc aacaggagcc ttcactcatc atttaaatga aaataaaatg 900
atttcactga atatagatga aggaaaaata tttaacgaaa gcatccaaaa ttttgatttt 960
gaatccctca tctcctctct cttagaccta agcgaaatag aatacaaagg aaaatatatc 1020
gataaaaagc aagaagactt tgttccatca aatgcgcttt tatcacaaga ccgcctatgg 1080
caagcagttg aaaacctaac tcaaagcaat gaaacaatcg ttgctgaaca agggacatca 1140
ttctttggcg cttcatcaat tttcttaaaa ccaaagagtc attttattgg tcaaccctta 1200
tggggatcaa ttggatatac attcccagca gcattaggaa gccaaattgc agataaagaa 1260
agcagacacc ttttatttat tggtgatggt tcacttcaac ttacggtgca agaattagga 1320
ttagcaatca gagaaaaaat taatccaatt tgctttatta tcaataatga tggttataca 1380
attgaaagag aaattcatgg accaaatcaa agctacaatg atattccaat gtggaattac 1440
tcaaaattac cagaatcatt tggagcaaca gaagaacgag tagtctcgaa aatcgttaga 1500
actgaaaatg aatttgtgtc tgtcatgaaa gaagctcaag cagatccaaa tagaatgtac 1560
tggattgagt tagttttggc aaaagaagat gcaccaaaag tactgaaaaa aatgggcaaa 1620
ctatttgctg aacaaaataa atcataa 1647
<210> 91
<211> 2093
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 91
atgaaaccag aagatttccg cgccagtacc caacgtcctt tcaccgggga agagtatctg 60
aaaagcctgc aggatggtcg cgagatctat atctatggcg agcgagtgaa agacgtcacc 120
actcatccgg catttcgtaa tgcggcagcg tctgttgccc agctgtacga cgcactgcac 180
aaaccggaga tgcaggactc tctgtgttgg aacaccgaca ccggcagcgg cggctatacc 240
cataaattct tccgcgtggc gaaaagtgcc gacgacctgc gccagcaacg cgacgccatc 300
gctgagtggt cacgcctgag ctatggctgg atgggccgta ccccagacta caaagccgct 360
ttcggttgcg cactgggcgc gaatccgggc ttttacggtc agttcgagca gaacgcccgt 420
aactggtaca cccgtattca ggaaactggc ctctacttta accacgcgat tgttaaccca 480
ccgatcgatc gtcatttgcc gaccgataaa gtgaaagacg tttacatcaa gctggaaaaa 540
gagactgacg ccgggattat cgtcagcggt gcgaaagtgg ttgccaccaa ctcggcgctg 600
actcactaca acatgattgg cttcggctcg gcacaagtga tgggcgaaaa cccggacttc 660
gcactgatgt tcgttgcgcc aatggatgcc gatggcgtga aattaatctc ccgcgcctct 720
tatgagatgg tcgcgggtgc taccggctcg ccatacgact acccgctctc cagccgcttc 780
gatgagaacg atgcgattct ggtgatggat aacgtgctga ttccatggga aaacgtgctg 840
atctaccgcg attttgatcg ctgccgtcgc tggacgatgg aaggcggttt tgcccgtatg 900
tatccgctgc aagcctgtgt gcgcctggca gtgaaattag acttcattac ggcactgctg 960
aaaaaatcac tcgaatgtac cggcaccctg gagttccgtg gtgtgcaggc cgatctcggt 1020
gaagtggtag cgtggcgcaa caccttctgg gcattgagtg actcgatgtg ttcagaagca 1080
acgccgtggg tcaacggggc ttatttaccg gatcatgccg cactgcaaac ctatcgcgta 1140
ctggcaccaa tggcctacgc gaagatcaaa aacattatcg aacgcaacgt taccagtggc 1200
ctgatctatc tcccttccag tgcccgtgac ctgaataatc cgcagatcga ccagtatctg 1260
gcgaagtatg tgcgcggttc gaacggtatg gatcacgtcc agcgcatcaa gatcctcaaa 1320
ctgatgtggg atgctattgg cagcgaattt ggtggtcgtc acgaactgta tgaaatcaac 1380
tactccggta gccaggatga gattcgcctg cagtgtctgc gccaggcaca aaactccggc 1440
aatatggaca agatgatggc gatggttgat cgctgcctgt cggaatacga ccaggacggc 1500
tggactgtgc cgcacctgca caacaacgac gatatcaaca tgctggataa gctgctgaaa 1560
taacgcagca ggaggttaag atgcaattag atgaacaacg cctgcgcttt cgtgacgcga 1620
tggccagcct gtcggcagcg gtaaatatta tcaccaccga gggcgacgcc ggacaatgcg 1680
ggattacggc aacggccgtc tgctcggtca cggatacacc accgtcgctg atggtgtgca 1740
ttaacgccaa cagtgcgatg aacccggttt ttcagggcaa cggcaagttg tgcgtcaacg 1800
tcctcaacca tgagcaggaa ctgatggcac gccacttcgc gggcatgaca ggcatggcga 1860
tggaagagcg ttttagcctc tcatgctggc aaaaaggtcc gctggcgcag ccggtgctaa 1920
aaggttcgct ggccagtctt gaaggtgaga tccgcgatgt gcaggcaatt ggcacacatc 1980
tggtgtatct ggtggagatt aaaaacatca tcctcagtgc agaaggtcat ggacttatct 2040
actttaaacg ccgtttccat ccggtgatgc tggaaatgga agctgcgatt taa 2093
<210> 92
<211> 300
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 92
tgataggtgg tatgttttcg cttgaacttt taaatacagc cattgaacat acggttgatt 60
taataactga caaacatcac cctcttgcta aagcggccaa ggacgctgcc gccggggctg 120
tttgcgtttt tgccgtgatt tcgtgtatca ttggtttact tatttttttg ccaaagctgt 180
aatggctgaa aattcttaca tttattttac atttttagaa atgggcgtga aaaaaagcgc 240
gcgattatgt aaaatataaa gtgatagcgg taccattata ggtaagagag gaatgtacac 300
<210> 93
<211> 335
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 93
cctgcgattt cggcgagatt caagcccggg tctaatctat ttttccttct tcggacgctt 60
caaaaattac ttttattata atcggaacag tgttttttag atcttttgat ctatttggtg 120
tttatcttgt ctcataaata catgtttaaa caatgtaaaa tataaaatat ccaattcata 180
aaaaattaac cattattaaa caatattcct atggaaaata atgattattt ttgataatct 240
gttttcacaa gacggaggtt caataaaaaa tcggtaaaag agcaactaca gaccaatatt 300
atggtgaata ttttatcaaa aaggagaatt tttat 335
<210> 94
<211> 333
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 94
cctgcgattt cggcgagatt caagcccggg tctaatctat ttttccttct tcggacgctt 60
caaaaattac ttttattata atcggaacag tgttttttag atcttttgat ctatttggtg 120
tttatcttgt ctcataaata catgtttaaa caatgtaaaa tataaaatat ccaattcata 180
aaaaattaac cattattaaa caatattcct atggaaaata atgattattt ttgataatct 240
gttttcacaa gacggaggtt caataaaaaa tcggtaaaag agcaactaca gaccaatatt 300
atggtgaatg aaacaacaaa gggggagatt tgt 333

Claims (2)

1. A preparation method of bacillus licheniformis for synthesizing hydroxytyrosol comprises the following steps:
the pyruvate kinase gene in bacillus licheniformis DW2 is knocked out in sequencepykTyrosine/phenylalanine aminotransferase genehisCAldehyde dehydrogenase genedhaSAnd alcohol dehydrogenase geneadhAObtaining Bacillus licheniformis DH4, andtyrA fbr gene integration into Bacillus licheniformis DH4ldhObtaining Bacillus licheniformis DH5, and adding the Bacillus licheniformis DH5 into the obtained Bacillus licheniformis DH5aroKPromoters and methods of use aroAThe promoters are replaced by PbacA to obtain bacillus licheniformis DH7;
the PbacA is derived from Bacillus licheniformis DW2, thetyrA fbr The amino acid sequence coded by the gene is shown as SEQ ID NO. 86;
coli BL21 (DE 3) genome4-hydroxyphenylacetic acid-3-hydroxylase gene in (B)hpaBCAnd (3) withkivD V461A Or (b)kivD V461I Co-expression is carried out, then the mixture is transferred into DH7,kivD V461A the sequence is shown as SEQ ID NO.89,kivD V461I the sequence is shown as SEQ ID NO.90, whereinhpaBCThe promoters of (a) are: pbacA, P43 or Pbay;kivD V461A or (b)kivD V461I The promoters of (a) are: pbacA, P43 or Pbay.
2. Use of bacillus licheniformis according to claim 1 for the synthesis of hydroxytyrosol.
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