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

Abstract

The invention belongs to the technical field of microbial genetic engineering, and in particular relates to a bacillus licheniformis capable of efficiently synthesizing hydroxytyrosol, a construction method and an application thereof. In the present invention, various transformations are further carried out on the basis of Bacillus licheniformis DW2 to obtain recombinant Bacillus licheniformis DH7; 4-hydroxyphenylacetic acid-3-hydroxylase derived from Escherichia coli and ketoacid decarboxylase from Lactococcus lactis are selected , and transferred it into Bacillus licheniformis DH7 chassis cells to realize the de novo synthesis of hydroxytyrosol production; through the combined expression of 4-hydroxyphenylacetic acid-3-hydroxylase HpaBC and ketoacid decarboxylase KivD mutants, so that all The recombinant Bacillus licheniformis can efficiently synthesize hydroxytyrosol by using cheap carbon sources such as glucose, 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 broad industrial prospects.

Description

A Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, construction method and application thereof

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and particularly relates to a Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, a construction method and application thereof.

Background Art

Hydroxytyrosol (HT), also known as 3,4-dihydroxyphenylethanol, is a bioactive polyphenol and one of the strongest natural antioxidants known. Hydroxytyrosol can scavenge free radicals, reduce the oxidation of low-density lipoproteins, and stimulate calcium deposition. It also has anti-cancer, anti-inflammatory and antimicrobial activities. Therefore, hydroxytyrosol can be used in cosmetics, food, health products and medical industries.

Currently, HT is manufactured industrially through plant extraction or chemical synthesis. In nature, HT exists in the olive plant in the form of oleuropein, and hydroxytyrosol can be extracted from fresh olive leaves and olive processing wastewater. Although these materials are abundant and inexpensive, they also have some disadvantages, such as low yield, strong acidic water vapor and long duration of the method. Chemical synthesis uses some analogues of HT, such as 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxybenzaldehyde and tyrosol as substrates to synthesize hydroxytyrosol. These methods are not suitable for large-scale industrial production due to expensive substrates, harsh conditions, complex steps or low yields.

With the progress of metabolic engineering and synthetic biology, the de novo production of HT from simple carbon sources has been achieved by reconstructing natural pathways or introducing artificial biosynthetic pathways in microorganisms. Currently, most reports use Escherichia coli to synthesize hydroxytyrosol through microbial transformation, which lays an important foundation for the production of hydroxytyrosol by microbial fermentation. However, the de novo synthesis of HT using simple carbon sources has a long synthesis pathway, a heavy metabolic burden, and also has problems such as low efficiency and low substrate conversion rate. It is necessary to further improve the de novo efficiency to achieve economical production of HT.

Summary of the invention

In view of the problems existing in the above-mentioned prior art, one of the purposes of the present invention is to provide a Bacillus licheniformis that can efficiently synthesize hydroxytyrosol. A recombinant Bacillus licheniformis is developed, and the ketoacid decarboxylase gene and the hydroxylase gene for synthesizing hydroxytyrosol are optimized and expressed in the Bacillus licheniformis that produces tyrosol, so as to construct a Bacillus licheniformis strain that can heterologously synthesize hydroxytyrosol.

Another object of the present invention is to provide the use of the recombinant Bacillus licheniformis in the production of hydroxytyrosol.

In order to solve the above problems, the technical solution of the present invention is as follows:

A Bacillus licheniformis for efficiently synthesizing hydroxytyrosol comprises the following steps:

The pyruvate kinase gene pyk, the tyrosine/phenylalanine transaminase gene hisC, the aldehyde dehydrogenase gene dhaS, and the alcohol dehydrogenase gene adhA in Bacillus licheniformis DW2 were knocked out in sequence to obtain Bacillus licheniformis DH4, the tyrA ^fbr gene was integrated into the ldh site of Bacillus licheniformis DH4 to obtain Bacillus licheniformis DH5, and the genes, aroK and aroA promoters in Bacillus licheniformis DH5 were replaced with PbacA to obtain Bacillus licheniformis DH7;

The 4-hydroxyphenylacetic acid-3-hydroxylase gene hpaBC in the Escherichia coli BL21 (DE3) genome was co-expressed with kivD ^V461A or kivD ^V461I , and then transformed into DH7. The sequence of kivD ^V461A is shown in SEQ ID NO.89, and the sequence of kivD ^V461I is shown in SEQ ID NO.90.

The promoter of hpaBC can be: PbacA, P43 or Pbay;

The promoter of kivD ^V461A or kivD ^V461I can be: PbacA, P43 or Pbay.

The protection scope of the present invention also includes: the above-mentioned Bacillus licheniformis is used to synthesize hydroxytyrosol.

Compared with the prior art, the present invention has the following advantages:

1. The present invention overexpresses KivD mutants (V461A and V461I) and HpaBC target genes in Bacillus licheniformis DH7. Through the effective expression of these enzymes in recombinant Bacillus licheniformis DH7, the strain can use glucose as a substrate to synthesize hydroxytyrosol from scratch, with low synthesis cost and simple operation.

2. The present invention utilizes strong promoters P43, PbacA and Pbay to efficiently drive the specific expression of exogenous genes in recombinant Bacillus licheniformis DH7, thereby improving the construction and transformation efficiency of the recombinant expression vector, thereby effectively improving the ability of Bacillus licheniformis to synthesize hydroxytyrosol and increasing the yield of hydroxytyrosol.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the synthetic route of hydroxytyrosol.

FIG. 2 is a liquid phase diagram of DW2/pHY300 and HT1 hydroxytyrosol.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited to these embodiments.

Embodiment 1:

Construction of genetically engineered strain DH7:

(1) Knockout of pyk, hisC and dhaS: Using the genome of Bacillus licheniformis DW2 as a template, the upstream and downstream homology arms of the pyruvate kinase gene pyk fragment were amplified using primers pyk-AF/AR and pyk-BF/BR, respectively, and the upstream and downstream homology arm fragments were fused to obtain the pyk knockout cassette; using primers (T2-T5-F/R) and plasmid T2(2)-ori as a template to amplify the backbone; insert the pyk knockout cassette into plasmid T2, and use T2-F/T2-R for colony PC R, positive transformants were obtained, and after DNA sequencing, the pyk knockout plasmid T2-△-pyk was successfully constructed and introduced into Bacillus licheniformis DW2 (or called Bacillus licheniformis DW2, CN112226437A), and the pyk knockout strain DH1 was obtained by homologous recombination; the tyrosine/phenylalanine transaminase gene hisC and aldehyde dehydrogenase gene dhaS knockout vector construction method was the same as pyk, and the strain DH3 with pyk, hisC and dhaS genes knocked out at the same time was obtained by iterative knockout;

Among them, the primer sequence required for pyk knockout is:

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

Primers for knocking out hisC

hisC-AF: ctgcagcccgggggatccaacggcgtcgtgttcagc

hisC-AR: gtaaaatgaggtgacagagaggtgatgattcagtca

hisC-BF:tgactgaatcatcacctctctgtcacctcattttac

hisC-BR:gatcttttctacgagctccttggcggtgaaatgaaa

hisC-YF:gtgcggaaagtggctgat

hisC-YR:acatgaacatcgtaaaagc

Primers for knocking out hisC

dhaS-AF:ctgcagcccgggggatccgtgcgggtgtatgttcaa

dhaS-AR:atttcacgtccgagtccctcgggtgagttgtcttgg

dhaS-BF: ccaagacaactcacccgagggactcggacgtgaaat

dhaS-BR:gatcttttctacgagctcaataccaggaaccgacaa

dhaS-YF:accgcagcaggatgttct

dhaS-YR:cgcatttgctaaaccttc

(2) Knockout of the alcohol dehydrogenase gene adhA: Using the genome of Bacillus licheniformis DW2 as a template, the upstream and downstream homology arms of the adhA fragment were amplified using primers adhA-AF/AR and adhA-BF/BR, respectively. The upstream and downstream homology arm fragments were fused to obtain the ahdA knockout cassette. The backbone was amplified using primers (T2-T5-F/R) and plasmid T2(2)-ori as a template. The ahdA knockout cassette was inserted into plasmid T2, and colony PCR was performed using T2-F/T2-R to obtain positive transformants. After DNA sequencing, the adhA knockout plasmid T2-△-adhA was successfully constructed and introduced into Bacillus licheniformis DH3. The adhA knockout strain DH4 was obtained by homologous recombination.

The primers used for adhA knockout are as follows

adhA-AF:ctgcagcccgggggatccagcagtgtagcacgataa

adhA-AR:gggaggcggaatctttccaatcatatgtaatacagagag

adhA-BF:ctctctgtattacatatgattggaaagattccgcctccc

adhA-BR:gatcttttctacgagctcccttaatggagggcggtcaa

adhA-YF:tcatgagtcctccgattc

adhA-YR:ttttatacgagcggtgac.

(3) TyrA ^fbr is integrated into the ldh site: Based on the sequence of ldh in the genomic DNA sequence of Bacillus licheniformis, primers (ldh-AF/AR, ldh-BF/BR) are designed to amplify the upstream and downstream homology arms of ldh; based on the sequence of Pylb in the DNA sequence of Bacillus subtilis, primers (Pylb-F/R) are designed to amplify the promoter Pylb; based on the sequence of tyrA in the DNA sequence of Escherichia coli and the sequence of the mutation site of tyrA (shown in SEQ ID NO.85), primers (tyrA-F/tyrA-R) are designed to amplify tyrA ^fbr ; based on the sequence of TamyL in the DNA sequence of Bacillus licheniformis, primers (TamyL-F and TamyL-R) are designed to amplify TamyL; the upstream and downstream homology arms of ldh, the Pylb promoter and the tyrA ^fbr fragment are fused with primers to construct tyrA ^fbr expression cassette; using primers (T2-T5-F/R) and plasmid T2(2)-ori as a template to amplify the backbone; inserting it into plasmid T2, using T2-F/T2-R for colony PCR, obtaining positive transformants, and successfully constructing tyrA ^fbr integration expression plasmid T2-ldh-tyrA ^fbr through DNA sequencing, and introducing it into Bacillus licheniformis DH4, and obtaining tyrA ^fbr integration expression strain DH5 through homologous recombination; the amino acid sequence encoded by the prephenate dehydrogenase TyrA ^fbr is shown in SEQ ID NO.86.

The introduction of tyrA ^fbr into the ldh locus was as follows:

ldh-AF:ctgcagcccgggggatccccgacctgtgatggagat

ldh-AR:ggagcgcgttcgacgatgcatattgtgcaatacttc

Pylb-F：gaagtattgcacaatatgcatcgtcgaacgcgctcc

Pylb-R: ggtcaattcagcaaccatacaaatctccccctttgt

TyrA-F: acaaagggggagattgtatggttgctgaattgacc

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:

Primers ParoK-AF/AR were used to amplify the 500bp sequence upstream and downstream of the aroK promoter, which were used as the upper and lower homology arms, respectively. The PbacA promoter sequence was amplified using the genomic DNA of Bacillus licheniformis DW2 as a template, and the upper homology arm, promoter and lower homology arm were fused. Primers (T2-T5-F and T2-T5-R) were designed, and the backbone was amplified using plasmid T2(2)-ori as a template. The fusion fragment and the linear plasmid fragment were connected using a one-step cloning kit and transformed into Escherichia coli DH5α competent cells. Recombinant plasmids T ₂ (2)-PbacA-aroK were obtained by PCR verification and sequencing analysis. The recombinant plasmids were electroporated into Bacillus licheniformis DH5 for homologous recombination exchange and screening and verification, and the promoter-replaced aroK promoter strain DH6 was obtained.

The method for constructing the aroA enhanced expression vector strain was the same as that for the aroK promoter replacement strain, and finally the DH7 strain was 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

Embodiment 2:

Construction of wild-type kivD and hpaBC co-expression strain

Step 1: According to the sequence of hpaBC in the genome of Escherichia coli BL21 (DE3), primers (P43-HpaBC-F and HpaBC-R) were designed to amplify the sequence of hpaBC, and according to the sequence of the P43 promoter in the genome of Bacillus subtilis 168 (shown in SEQ ID NO.92), primers (Amp-P43-F and P43-HpaBC-R) were designed to amplify the sequence of P43;

Among them, the sequences of Amp-P43-F, P43-HpaBC-R, P43-HpaBC-F, and HpaBC-R are:

Amp-P43-F：acttttcggggaaatgtctgataggtggtatgtttt

P43-HpaBC-R：gaaatcttctggtttcatgtgtacattcctctctta

P43-HpaBC-F：taagagaggaatgtacacatgaaaccagaagatttc

HpaBC-R:gtaaacttggtctgacagttaaatcgcagcttccattt

Step 2: P43 and hpaBC (shown in SEQ ID NO. 91) were linked together by overlap extension PCR (primers used were Amp-P43-F and HpaBC-R) to obtain a fusion fragment;

Step 3: Design primers (T5-amp-F and T5-amp-R) and amplify the backbone using plasmid pHY-PbacA-kivD (Zhan Y, et al. Efficient synthesis of 2-phenylethanol from L-phenylalanine by engineered Bacillus lichenif ormis using molasses as carbon source. Appl Microbiol Biotechnol. 2020. 104 (17): 7507-7520) as a template. The kivD sequence is shown in SEQ ID NO. 88.

Among them, the sequences of T5-amp-F and T5-amp-R are:

T5-amp-F：gacatttccccgaaaagtgccac

T5-amp-R:ctgtcagaccaagtttactcatata;

Step 4: Use a one-step cloning kit to connect the gene fragment obtained in steps (6) and (7) and the linear plasmid fragment, and transform the ligation product into Escherichia coli DH5α by calcium chloride transformation method. Screen the transformants in a medium containing tetracycline resistance at 37°C, and perform colony PCR verification on the plasmid of the transformants (primers used are: amp-YF and amp-YR). Screen the positive transformants, and verify the correctness of DNA sequencing, and name it as the co-expression vector pHY-PbacA-kivD-P43-hpaBC;

Among them, the sequences of amp-YF and amp-YR are:

amp-YF:ccctgatctcgacttcgt

amp-YR:ttaaggggtctgacgctc

Step 5: The pHY-PbacA-kivD-P43-hpaBC plasmid was electroporated into DH7, and the strain was verified by PCR to successfully obtain the recombinant strain HT1 that was successfully transformed with the recombinant plasmid pHY-PbacA-kivD-P43-hpaBC.

Embodiment 3:

Construction of a strain co-expressing mutant kivD (V461A or V461I) and hpaBC

Step 1: Using the vector pHY-PbacA-kivD-P43-hpaBC in Example 2 as a template, primers V461A-F/R and V461I-F/R were used to amplify the mutated backbone containing V461A and V461I, respectively.

V461A-F：gttatacagctgaaagagaaattcatggaccaaatcaaa

V461A-R：ctctttcagctgtataaccatcattattgataataaagc

V461I-F：gttatacaatcgaaagagaaattcatggaccaaatcaaa

V461I-R：ctctttcgattgtataaccatcattattgataataaagc

Step 2: Use a one-step cloning kit to self-ligate the linear backbones of V461A and V461I obtained in step (1), and transform the ligation product into Escherichia coli DH5α by calcium chloride transformation method, screen the transformants at 37°C in a medium containing tetracycline resistance, and perform colony PCR verification on the plasmids of the transformants (primers used are: pHY-YF and pHY-YR), and verify the successful mutation of the mutation site by DNA sequencing, and name the V461A and V461I mutant vectors pHY-PbacA-kivD ^V461A -P43-hpaBC and pHY-PbacA-kivD ^V461I -P43-hpaBC, respectively. The kivD ^V461A sequence is shown in SEQ ID NO.89, and the kivD ^V461I sequence is shown in SEQ ID NO.90.

Step 3: The pHY-PbacA-kivD ^V461A -P43-hpaBC and pHY-PbacA-kivD ^V461I -P43-hpaBC plasmids were electroporated into the recombinant strain DH7 to obtain HT2 and HT3 strains.

Embodiment 4:

Construction of recombinant strains expressing mutant kivD (V461A and V461I) and hpaBC with different promoter combinations

Step 1: Using the plasmids pHY-PbacA-kivD ^V461A -P43-hpaBC and pHY-PbacA-kivD ^V461I -P43-hpaBC in Example 3 as templates, and using PbacA (shown in SEQ ID NO.93), P43 and Pbay (shown in SEQ ID NO.94) as promoters, 18 different combinations of expression vectors were constructed. Taking the construction of pHY-P43-kivD ^V461I -P43-hpaBC as an example, the P43 fragment was used as a template, the P43 promoter was amplified by P43-kivD-F/R primers, the pHY-PbacA-kivD ^V461I -P43-hpaBC was used as a template, and the pHY-P43-kivD ^V461I -P43-hpaBC vector backbone was amplified by kivD-T5-F/R primers; the P43 promoter fragment and the pHY-P43-kivD ^V461I -P43-hpaBC linear backbone were connected using a one-step cloning kit, and the connection product was transformed into Escherichia coli DH5α by a calcium chloride transformation method, and the transformants were screened at 37°C in a culture medium containing tetracycline resistance to obtain transformants, which were named co-expression vector pHY-P43-kivD ^V461I -P43-hpaBC. The construction methods of the remaining 15 different combination expression vectors were similar to 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 expressing different combinations and the control vector pHY300 were electro-transformed into the recombinant strain DH7 to obtain 18 recombinant Bacillus licheniformis HT2-HT19 and HT0 control strains.

Embodiment 5:

Hydroxytyrosol fermentation by recombinant Bacillus licheniformis

1) Seed fermentation: Bacillus licheniformis DW2/pHY-300, HT0 and different recombinant Bacillus licheniformis HT1-HT19 were activated on the plate, and the bacteria were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of liquid LB, and cultured at 37° C. and 230 rpm for 13 h. Then, the inoculum was inoculated into the fermentation medium at a volume ratio of 2%; the culture conditions of the hydroxytyrosol fermentation medium were 37° C. and 230 rpm for 48 h, and the hydroxytyrosol yield was measured after the fermentation was completed.

The formula of the hydroxytyrosol fermentation medium is 10 g/L peptone, 5 g/L yeast powder, 10 g/L NaCl, 18.6 g/L K ₂ HPO ₄ , 5.2 g/L KH ₂ PO ₄ , 70 g/L glucose, pH 7.0, and the rest is water.

2) Fermentation broth pretreatment: The fermentation broth was diluted with deionized water, centrifuged to remove bacteria, and then filtered through a 0.22 μm aqueous phase filter membrane.

3) Hydroxytyrosol was detected by high performance liquid chromatography (HPLC). The specific conditions of the determination were: using an Agilent 1260 high performance liquid chromatograph, a C18 column (4.6mm ID×250mm, 5μm), a mobile phase of methanol: 0.1% formic acid = 2:8, a flow rate of 0.6mL/min, a column temperature of 30°C, an injection volume of 10.0μL, a detection wavelength of 224nm, and an elution time of 25min. The content of hydroxytyrosol in the fermentation broth was calculated based on the standard curve prepared with the hydroxytyrosol standard. The results showed that hydroxytyrosol was not detected in the control strains DW2/pHY-300 and DH0, while hydroxytyrosol was detected in all the recombinant strains, among which HT7 (pHY-P43-kivD ^V461A -Pbay-hpaBC) and HT16 (pHY-P43-kivD ^V461I -Pbay-hpaBC) strains had the highest yields of 5.55±0.37 and 5.49±0.33 g/L.

Table 1 Hydroxytyrosol production of different recombinant strains

Sequence Listing

<110> Hubei University

<120> A Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, its construction method and application

<160> 94

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ctgcagcccgggggatccgggatacagctacatccc 36

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<211> 37

<212> DNA

<213> Artificial Sequence

<400> 2

ttaaagtacg cttgcacgcg gcccaattgt acaaact 37

<210> 3

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 3

agtttgtaca attgggccgc gtgcaagcgt actttaa 37

<210> 4

<211> 36

<212> DNA

<213> Artificial Sequence

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gatcttttct acgagctcgc ggcagcctgc tttttc 36

<210> 5

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<212> DNA

<213> Artificial Sequence

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ggatcccccg ggctgcagga attc 24

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 6

gagctcgtag aaaagatcaa agga 24

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 7

atgtgataac tcggcgta 18

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 8

gcaagcagca gattacgc 18

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 9

ttctcggatt gatcatgg 18

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 10

aacggcttga cgactttc 18

<210> 11

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 11

ctgcagcccg ggggatccaa cggcgtcgtg ttcagc 36

<210> 12

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<213> Artificial Sequence

<400> 12

gtaaaatgag gtgacagaga ggtgatgatt cagtca 36

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<400> 13

tgactgaatc atcacctctc tgtcacctca ttttac 36

<210> 14

<211> 36

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<213> Artificial Sequence

<400> 14

gatcttttct acgagctcct tggcggtgaa atgaaa 36

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 15

gtgcggaaag tggctgat 18

<210> 16

<211> 19

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<213> Artificial Sequence

<400> 16

acatgaacat cgtaaaagc 19

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<211> 36

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ctgcagcccg ggggatccgt gcgggtgtat gttcaa 36

<210> 18

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 18

atttcacgtc cgagtccctc gggtgagttg tcttgg 36

<210> 19

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 19

ccaagacaac tcacccgagg gactcggacg tgaaat 36

<210> 20

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 20

gatcttttct acgagctcaa taccaggaac cgacaa 36

<210> 21

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 21

accgcagcag gatgttct 18

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 22

cgcatttgct aaaccttc 18

<210> 23

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 23

ctgcagcccg ggggatccag cagtgtagca cgataa 36

<210> 24

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 24

gggaggcgga atctttccaa tcatatgtaa tacagagag 39

<210> 25

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 25

ctctctgtat tacatatgat tggaaagatt ccgcctccc 39

<210> 26

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 26

gatcttttct acgagctccc ttaatggagg gcggtcaa 38

<210> 27

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 27

tcatgagtcc tccgattc 18

<210> 28

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 28

ttttatacga gcggtgac 18

<210> 29

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 29

ctgcagcccg ggggatcccc gacctgtgat ggagat 36

<210> 30

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 30

ggagcgcgtt cgacgatgca tattgtgcaa tacttc 36

<210> 31

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 31

gaagtattgc acaatatgca tcgtcgaacg cgctcc 36

<210> 32

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 32

ggtcaattca gcaaccatac aaatctcccc ctttgt 36

<210> 33

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 33

acaaaggggg agatttgtat ggttgctgaa ttgacc 36

<210> 34

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 34

tccgtcctct ctgctcttaa tgaaggtatt gggctg 36

<210> 35

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 35

cagcccaata ccttcattaa gagcagagag gacgga 36

<210> 36

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 36

aacagattcc caaacggacg caataatgcc gtcgca 36

<210> 37

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 37

tgcgacggca ttattgcgtc cgtttgggaa tctgtt 36

<210> 38

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 38

gatcttttct acgagctctc aagcctccca tctgtg 36

<210> 39

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 39

catatcagcg gaatcatc 18

<210> 40

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 40

ccgcttaata caaggaga 18

<210> 41

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 41

ctgcagcccg ggggatccgt aggctcattt gctgat 36

<210> 42

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 42

aatctcgccg aaatcgcagg ctatttccac cagtcgtcaa 40

<210> 43

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 43

ttgacgactg gtggaaatag cctgcgattt cggcgagatt 40

<210> 44

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 44

tctcattgcg gcattcatat aaaaattctc ctttttgat 39

<210> 45

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 45

atcaaaaagg agaattttta tatgaatgcc gcaatgaga 39

<210> 46

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 46

gatcttttct acgagctcctttcaagttgt ggaatg 36

<210> 47

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 47

aggcgttcag gcggaatt 18

<210> 48

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 48

gacacagcga tagaaaca 18

<210> 49

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 49

ctgcagcccg ggggatccga agtggacgca catttc 36

<210> 50

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 50

tctcgccgaa atcgcagggt tatccatcct ttcttt 36

<210> 51

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 51

aaagaaagga tggataaccc tgcgatttcg gcgaga 36

<210> 52

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 52

aagttcagtg ttgctcatat aaaaattctc cttttt 36

<210> 53

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 53

aaaaaggaga atttttatat gagcaacact gaactt 36

<210> 54

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 54

gatcttttct acgagctcca acacgcttta agattt 36

<210> 55

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 55

gttgacgcac gcttcgtt 18

<210> 56

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 56

attgatgaat tccttcag 18

<210> 57

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 57

acttttcggg gaaatgtctg ataggtggta tgtttt 36

<210> 58

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 58

gaaatcttct ggtttcatgt gtacattcct ctctta 36

<210> 59

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 59

taagagagga atgtacacat gaaaccagaa gatttc 36

<210> 60

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 60

gtaaacttgg tctgacagtt aaatcgcagc ttccattt 38

<210> 61

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 61

gacatttccc cgaaaagtgc cac 23

<210> 62

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 62

ctgtcagacc aagtttactc atata 25

<210> 63

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 63

ccctgatctc gacttcgt 18

<210> 64

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 64

ttaaggggtc tgacgctc 18

<210> 65

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 65

gttatacagc tgaaagagaa attcatggac caaatcaaa 39

<210> 66

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 66

ctctttcagc tgtataacca tcattattga taataaagc 39

<210> 67

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 67

gttatacaat cgaaagagaa attcatggac caaatcaaa 39

<210> 68

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 68

ctctttcgat tgtataacca tcattattga taataaagc 39

<210> 69

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 69

tttttataac aggaattctg ataggtggta tgtttt 36

<210> 70

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 70

atctcctact gtatacatgt gtacattcctctctta 36

<210> 71

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 71

tttttataac aggaattccc tgcgatttcg gcgaga 36

<210> 72

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 72

atctcctact gtatacatat aaaaattctc cttttt 36

<210> 73

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 73

tttttataac aggaattccc tgcgatttcg gcgaga 36

<210> 74

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 74

atctcctact gtatacatac aaatctcccc ctttgt 36

<210> 75

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 75

gaattcctgt tataaaaaaa ggatc 25

<210> 76

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 76

atgtatacag taggagatta ccta 24

<210> 77

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 77

acttttcggg gaaatgtctg ataggtggta tgtttt 36

<210> 78

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 78

gaaatcttct ggtttcatgt gtacattcct ctctta 36

<210> 79

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 79

acttttcggg gaaatgtccc tgcgatttcg gcgaga 36

<210> 80

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 80

gaaatcttct ggtttcatat aaaaattctc cttttt 36

<210> 81

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 81

acttttcggg gaaatgtccc tgcgatttcg gcgaga 36

<210> 82

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 82

gaaatcttct ggtttcatac aaatctcccc ctttgt 36

<210> 83

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 83

gacatttccc cgaaaagtgc cac 23

<210> 84

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 84

atgaaaccag aagatttccg 20

<210> 85

<211> 1122

<212> DNA

<213> 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 ggcgtttattt 1020

gacagtttcc gcaaggtgga gcactggttc ggcgattacg tacagcgttt tcagagtgaa 1080

agccgcgtgttattgcgtca ggcgaatgac aatcgccagt aa 1122

<210> 86

<211> 373

<212> PRT

<213> 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

<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

<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

<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

<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

<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

<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

<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

<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, comprising the steps of: The pyruvate kinase gene pyk , the tyrosine/phenylalanine aminotransferase gene hisC , the aldehyde dehydrogenase gene dhaS and the alcohol dehydrogenase gene adhA in bacillus licheniformis DW2 were sequentially knocked out to obtain bacillus licheniformis DH4 , and the tyrA ^fbr gene Integrate into the ldh site of Bacillus licheniformis DH4 to obtain Bacillus licheniformis DH5, and replace both the aroK promoter and aroA promoter in Bacillus licheniformis DH5 with PbacA to obtain Bacillus licheniformis DH7; The PbacA is derived from Bacillus licheniformis DW2, and the amino acid sequence encoded by the tyrA ^fbr gene is shown in SEQ ID NO.86; The 4-hydroxyphenylacetic acid-3-hydroxylase gene hpaBC in the Escherichia coli BL21 (DE3) genome was co-expressed with kivD ^V461A or kivD ^V461I , and then transferred into DH7. The sequence of kivD ^V461A is shown in SEQ ID NO.89, The sequence of kivD ^V461I is shown in SEQ ID NO.90, wherein the promoter of hpaBC is: PbacA, P43 or Pbay; the promoter of kivD ^V461A or kivD ^V461I is: PbacA, P43 or Pbay. 2. the application of bacillus licheniformis described in claim 1 in the synthesis of hydroxytyrosol.

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