CN114196697B

CN114196697B - Method for integrating, digesting and fermenting residual lactose by genome based on arabinose operon

Info

Publication number: CN114196697B
Application number: CN202111613984.7A
Authority: CN
Inventors: 刘振云; 倪磊
Original assignee: Huangshan Tongxi Biotechnology Co ltd
Current assignee: Huangshan Tongxi Biotechnology Co ltd
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2024-03-12
Anticipated expiration: 2041-12-27
Also published as: CN114196697A

Abstract

The invention belongs to the microbial fermentation technology, and particularly relates to a method for integrating, digesting and fermenting residual lactose by using a genome based on an arabinose operon. To be used forLacZGene and geneLacYGene replacement of the arabinose operon on the genes of the fermentation strainsaraAGene and genearaBGenes to obtain a replacement fermentation strain; then carrying out lactose fermentation by using the replacement fermentation strain; finally, adding arabinose for induction to complete the digestion of the residual lactose after fermentation. Experimental results show that the addition of the inducer arabinose after replacement has a great influence on lactose consumption, and lactose is consumed on a larger level.

Description

Method for integrating, digesting and fermenting residual lactose by genome based on arabinose operon

Technical Field

The invention belongs to the microbial fermentation technology, and particularly relates to a method for integrating, digesting and fermenting residual lactose by using a genome based on an arabinose operon.

Background

The breast milk oligosaccharide is a common breast milk oligosaccharide, such as 2 ' -FL, 3 ' -SL, 6 ' -SL, LNnt and the like, and the content of the milk and other raw milk is extremely low because the breast milk oligosaccharide is only largely present in the breast milk, and the functions of regulating intestinal flora, facilitating the growth of probiotics, preventing infection of respiratory system and urinary system, regulating immune system, improving organism immunity, promoting the brain development of infants and the like are excellent, so that the addition of the breast milk oligosaccharide into the infant milk powder becomes one of the items of hot hands in the market. BL21 (DE 3) is a commonly used E.coliOne of the bacterial fermentation strains. Lactose is a fermentation substrate commonly used in the fermentation engineering of breast milk oligosaccharide, and in the fermentation experiment of BL21 (DE 3) using lactose as a substrate, the strain per se is often usedLacZThe gene knockout or the activity of the lactose is eliminated, so that the strain can block or eliminate the lactose consumption path originally, thereby reducing the lactose consumption in the unnecessary path and affecting the expression of the target product. In addition, lactose is often excessively added in order to ensure the production of the product during fermentation, so that a large amount of undigested lactose remains in the fermentation broth, and lactose remaining in the fermentation tank greatly influences the subsequent purification work, and lactose decomposing enzyme is added in the current common lactose treatment method, so that the purification cost is increased.

Disclosure of Invention

In the prior art, lactose decomposing enzyme is added in the later period of lactose fermentation to digest residual lactose, so that the purification cost is increased; the invention processes lactose at the end of fermentation, re-excites the consumption of lactose by the strain, reduces the lactose residue in the subsequent fermentation process, and can reduce the purification cost and the required time to a certain extent.

The invention firstly constructs a replacement plasmid which contains beta-galactosidase capable of being editedLacZGene and synthesis permeaseLacYGene editing is performed on lactose-fermented strainsLacZGene and geneLacYGene editing into the arabinose operon region, the replacement plasmid will be the arabinose operonaraAGene and genearaBGene replacement intoLacZGene and geneLacYAnd (3) a gene. During the fermentation processLacZGene and geneLacYGenes are not expressed, but added with arabinose at the end of fermentation, inducedLacZGene and geneLacYThe strain can metabolize lactose remained in the tank body to reduce lactose residue.

The invention adopts the following technical scheme:

a method for digesting residual lactose by genome integration based on arabinose operon comprises the following stepsLacZGene and geneLacYGene replacement of the arabinose operon on the genes of the fermentation strainsaraAGene and genearaBGenes to obtain a replacement fermentation strain; then carrying out lactose fermentation by using the replacement fermentation strain; finally, adding arabinose for induction to complete the digestion of the residual lactose after fermentation. The arabinose promoter takes arabinose as inducer, and when no arabinose exists, the arabinose promoter is leftaraCGene expression, which results inAraCProteins, which distort DNA at the arabinose operon, prevent mRNA transcription, and are behind promotersaraAAnd (3) witharaBGenes are not expressed, but after the addition of arabinose, the arabinose activates the operator region upstream of the promoter, activates the RNA polymerase,araAand (3) witharaBThe gene starts to express. The invention constructs a replacement plasmidLacZGene and geneLacYGene and knockoutaraA、araBGene plasmid ligation to construct a replacement plasmid, i.eLacZGene and geneLacYGene replacement originalaraA、araBGene knockoutaraAGene and genearaBAfter the gene, arabinose cannot be consumed,LacZgene and geneLacYGenes will always be expressed and lactose will always be consumed. During the fermentation processLacZGene and geneLacYGenes are not expressed, but added with arabinose at the end of fermentation, inducedLacZGene and geneLacYThe strain can metabolize lactose remained in the tank body to reduce lactose residue.

In the present invention, CRISP/CAS9 gene editing technology is used toLacZGene and geneLacYGene replacement of the arabinose operon on the genes of the fermentation strainsaraAGene and genearaBAnd (3) a gene. Specifically, the N20 sequence and the sgRNA sequence are inserted on the plasmid, followed by insertionaraABase sequence before genearaBThe base sequence after the gene is inserted between the twoLacZGene and geneLacYGenes to obtain a replacement plasmid; and transferring the replaced plasmid into a fermentation strain by an electrochemical conversion method, culturing overnight, identifying, selecting a strain which is replaced successfully, carrying out plasmid loss, obtaining a replaced fermentation strain, and carrying out subsequent fermentation operation.

In the invention, the time of lactose fermentation is 22-65 hours; the induction time is 10-30 hours; in the induction, the amount of arabinose added is 0.2 to 0.8 mM mM, preferably 0.3 to 0.6mM, based on the final concentration.

In the present invention, the fermentation strain is a conventional lactose fermentation strain and the lactose metabolism gene is knocked out, such as bacteria, fungi, preferably Corynebacterium, brevibacterium, bacillus, saccharomyces, escherichia, especially Corynebacterium glutamicum, brevibacterium, most preferably beta-galactosidase(LacZ) E.coli of (E.coli).

In the prior art, lactose decomposing enzyme is added in the later period of lactose fermentation to digest residual lactose, so that the purification cost is increased; the invention processes lactose at the end of fermentation, re-excites the consumption of lactose by the strain, reduces the lactose residue in the subsequent fermentation process, and can reduce the purification cost and the required time to a certain extent. The invention firstly constructs a replacement plasmid which contains beta-galactosidase capable of being editedLacZGene and synthesis permeaseLacYGene editing is performed on lactose-fermented strainsLacZGene and geneLacYGene editing into the arabinose operon region, the replacement plasmid will be the arabinose operonaraAGene and genearaBGene replacement intoLacZGene and geneLacYAnd (3) a gene. During the fermentation processLacZGene and geneLacYGenes are not expressed, but added with arabinose at the end of fermentation, inducedLacZGene and geneLacYThe strain can metabolize lactose remained in the tank body to reduce lactose residue.

Drawings

FIG. 1 shows the results of monoclonal identification.

Detailed Description

Taking the synthesis of LNnt (lactosyl-N-neotetraose) as an example, the invention first constructs a replacement plasmid, which was identified in the case of pTarget plasmid, using CRISP/CAS9 gene editing technologyaraAOr (b)araBN20 sequence on gene fragment, insertion of N20 and sgRNA on plasmid, followed by insertion of fragment comprisingaraABase sequence of 634bp length before gene and its preparation methodaraBBase sequence 824bp length after gene and inserted between themLacZGene and geneLacYThe fragment size of the gene is 4377bp, thereby constructing a gene containingLacZAnd (3) withLacYThe replacement plasmid of the gene is subjected to the next replacement and fermentation; transferring the plasmid into the strain by electrochemical conversion, culturing overnight, identifying whether replacement is successful, if so, losing the plasmid, and confirming that the plasmid is lost, and then carrying out subsequent fermentation operation.

In the present invention, the gene sequences involved are as follows:

araA gene:

TTAGCGACGAAATCCGTAATACACTTCGTTCCAGCGCAGCGCGTCTTTAAACGCTGGCAGGCGGGTATCGTTATCAATCACCGTGATTTCAATGTCGTGCATCTCGGCGAACTGGCGCATATCGTTGAGGTTCAGCGCATGGCTGAAGACGGTATGGTGCGCGCCACCAGCGAGGATCCACGCTTCGGAAGCAGTTGGCAGATCCGGTTGCGCTTTCCACAGCGCATTCGCCACCGGCAGTTTCGGCAGGGAGTGCGGTGTTTTCACCGTGTCGATACAGTTAACCAGCAGACGGTAACGATCGCCGAGATCAATCAGGCTGGCGACAATCGCTGGACCGGTTTGGGTATTGAAGATCAGTCGGGCAGGATCGTCCTTACCACCAATACCGAGATGCTGAACGTCGAGGATCGGTTTCTCTTCTACGGCAATCGACGGGCAGACTTCCAGCATATGGGAGCCGAGCACCAGGTCATTACCTTTCTCGAAGTGATAGGTGTAGTCCTCCATAAAGGAGGTGCCGCCCTGCAGACCGGTTGACATCACCTTCATGATGCGAAGCAGGGCGGCGGTTTTCCAGTCGCCTTCGCCCGCAAAGCCGTAACCCTGCTGCATCAGACGCTGTACGGCCAGACCTGGAAGCTGTTTCAGACCGTGCAAATCTTCAAAGGTGGTGGTGAACGCGTGGAAGCCACCTTGTTCCAGGAAACGCTTCATCCCCAGCTCAATACGCGCCGCTTCCAGCACGTTCTGTCGTTTTTCGCCGTGGATTTGTGTTGCAGGCGTCATGGTGTAGCAGCTTTCGTACTCATCGACCAGCGCGTTAACATCGCCGTCGCTGATGGAGTTCACCACCTGCACCAGATCGCCAACCGCCCAGGTATTGACGGAGAAACCGAACTTGATCTGTGCGGCAACTTTATCACCATCGGTGACCGCCACTTCACGCATGTTATCGCCAAAACGGCAGACTTTCAGATGACGGGTATCCTGTTTAGAAACCGCCTGACGCATCCAGGAGCCGATACGCTCATGGGCTTGTTTATCCTGCCAGTGACCGGTAACGACGGCATGTTGCTGACGCATACGCGCGCCAATGAAGCCGAACTCGCGACCGCCATGTGCAGTCTGGTTCAGGTTCATAAAGTCCATATCGATACTGTCCCACGGCAGCGCCGCGTTGAACTGGGTGTGGAATTGCAGCAACGGTTTGTTGAGCATGGTCAGGCCGTTGATCCACATTTTGGCCGGGGAGAAGGTGTGCAGCCACACCACCAGACCAGCGCAACGATCGTCGTAATTCGCGTCGCGGCAAATAGCGGTGATTTCATCCGGCGTGGTGCCCAGCGGTTTCAACACCAGTTTGCAGGGCAGTTTCGCTTCCGTATTCAGCGCATTAACAACGTGCTCGGCATGTTGGGTGACCTGACGCAGGGTTTCCGGGCCATACAGATGCTGGCTGCCAATGACAAACCACACTTCATAATTATCAAAAATCGTCAT

araB gene:

TTATAGAGTCGCAACGGCCTGGGCAGCCTGTGCCGGGGCGGAAGTTGGAAGATAGTGTTGTTCGGCGCTCATCGCCCATTGCTGATAGCGGCGATAAAGCTGTTCAAAGCGTTGTGCCTGTTCGCTGCGCGGTTGCAGGGTTTTCTCTACCGCACTGGCCATTTTTTGCTGGGCTGATGGGATGTCTGCGTGCACTTTCGCGGCGACGGCAGCAAAAATCGCCGCACCGAGCGCACAGCACTGGTCAGAGGCAACAATTTGCAGCGGGCGATTCAGCACGTCGCAGCAGGCCTGCATAATGACCTGGTTTTTCCGCGCGATGCCGCCCAGCGCCATCACGTTATTGACGGCGATCCCCTGATCGGTAAAGCACTCCATGATTGCGCGTGCGCCAAAGGCGGTGGCAGCAATCAAACCGCCGAACAGCAGCGGAGCGTCGGTAGCGAGGTTAAGATCGGTAATCACCCCTTTCAGGCGTTGGTTAGCGTTTGGCGAGCGACGACCGTTAAACCAGTCGAGCACCACCGGCAGGTGATCCAGAGACGGATTTTTGGCCCATGCTTCGGTCAGCGCCGGAAGCAGTTGTTTCTGGCTGGCGTTGATTTGCGCTTTCAGTTCCGGATGCTGGGCGGCAAGCTGTTCCAGCGGCCAGCTGAGTACGCGACCGAACCAGGCGTAGATATCACCAAACGCCGATTGGCCTGCTTCCAGACCGATAAATCCAGGCACCACGCTGCCATCAACCTGACCGCAAATACCTTTAACTGCCCGCTCGCCAACGCTCTGTTTGTCGGCAATCAGAATGTCGCAGGTGGAAGTACCGATAACTTTTACCAGTGCGTTAGGCTGTGCGCCTGCGCCAACTGCGCCCATATGGCAGTCAAACGCGCCGCCGGAAATCACCACGCTTTCAGGCAGGCCGAGACGCTGCGCCCATTCCGGGCATAAGGTGCCCACCGGAATATCGGCAGTCCAGGTGTCAGTGAACAGCGGGGAAGGCAAATGGCGATTGAGGATCGGGTCCAGCTCATCAAAGAAACTGGCTGGCGGCAAGCCGCCCCAGCTTTCGTGCCACAGAGATTTATGCCCGGCGCTGCAACGTCCGCGACGAATATCCTGCGGGCGGGTGGTACCGGAAAGCAGAGCTGGCACCCAGTCGCACAGCTCAATCCACGATGCGGCAGATTGCGCCACGGCGCTGTCCTGGCGAGTCACATGCAGGATTTTTGCCCAGAACCATTCGCTGGAATAAATACCGCCAATATAGCGGGAGTAGTCAACATTGCCCGGCGCGTGGCACAAACGGGTAATCTCTTCCGCTTCTTCAACCGCAGTGTGGTCTTTCCACAATACGAACATCGCGTTCGGGTTTTCGGCAAACTCCGGGCGCAGCGCCAGCACGTTACCGTCGGCATCAATCGGTGCGGGCGTCGAGCCGGTACTGTCAACGCCAATCCCGACCACAGCTGCGCGCTGTTCGACGCTAAGCTCTGCAAGCACGGTTTTCAGTGCCGCTTCCATTGACTCAATGTAGTCACGCGGATGATGACGGAACTGGTTATTCGGGGCATCACAAAATTGCCCTTTTTGCCAACGGGGATACCACTCTACGCTGGTGGCGATCTCTTCACCGCTGGCGCAGTCCACCGCCAAAGCTCGCACAGAATCACTGCCAAAATCGAGGCCAATTGCAATCGCCAT

LacZ gene:

TTATTTTTGACACCAGACCAACTGGTAATGGTAGCGACCGGCGCTCAGCTGGAATTCCGCCGATACTGACGGGCTCCAGGAGTCGTCGCCACCAATCCCCATATGGAAACCGTCGATATTCAGCCATGTGCCTTCTTCCGCGTGCAGCAGATGGCGATGGCTGGTTTCCATCAGTTGCTGTTGACTGTAGCGGCTGATGTTGAACTGGAAGTCGCCGCGCCACTGGTGTGGGCCATAATTCAATTCGCGCGTCCCGCAGCGCAGACCGTTTTCGCTCGGGAAGACGTACGGGGTATACATGTCTGACAATGGCAGATCCCAGCGGTCAAAACAGGCGGCAGTAAGGCGGTCGGGATAGTTTTCTTGCGGCCCTAATCCGAGCCAGTTTACCCGCTCTGCTACCTGCGCCAGCTGGCAGTTCAGGCCAATCCGCGCCGGATGCGGTGTATCGCTCGCCACTTCAACATCAACGGTAATCGCCATTTGACCACTACCATCAATCCGGTAGGTTTTCCGGCTGATAAATAAGGTTTTCCCCTGATGCTGCCACGCGTGAGCGGTCGTAATCAGCACCGCATCAGCAAGTGTATCTGCCGTGCACTGCAACAACGCTGCTTCGGCCTGGTAATGGCCCGCCGCCTTCCAGCGTTCGACCCAGGCGTTAGGGTCAATGCGGGTCGCTTCACTTACGCCAATGTCGTTATCCAGCGGTGCACGGGTGAACTGATCGCGCAGCGGCGTCAGCAGTTGTTTTTTATCGCCAATCCACATCTGTGAAAGAAAGCCTGACTGGCGGTTAAATTGCCAACGCTTATTACCCAGCTCGATGCAAAAATCCATTTCGCTGGTGGTCAGATGCGGGATGGCGTGGGACGCGGCGGGGAGTGTCACGCTGAGGTTTTCAGCCAGACGCCACTGCTGCCAGGCGCTGATGTGTCCGGCTTCTGACCATGCGGTCGCGTTCGGTTGCACTACGCGTACTGTGAGCCAGAGTTGCCCGGCGCTCTCCGGCTGCGGTAGTTCAGGCAGTTCAATCAACTGTTTACCTTGTGGAGCGACATCCAGAGGCACTTCACCGCTTGCCAGCGGCTTACCATCCAGCGCCACCATCCAGTGCAGGAGCTCGTTATCGCTATGACGGAACAGGTATTCGCTGGTCACTTCGATGGTTTGCCCGGATAAACGGAACTGGAAAAACTGCTGCTGGTGTTTTGCTTCCGTCAGCGCTGGATGCGGCGTGCGGTCGGCAAAGACCAGACCGTTCATACAGAACTGGCGATCGTTCGGCGTATCGCCAAAATCACCGCCGTAAGCCGACCACGGGTTGCCGTTTTCATCATATTTAATCAGCGACTGATCCACCCAGTCCCAGACGAAGCCGCCCTGTAAACGGGGATACTGACGAAACGCCTGCCAGTATTTAGCGAAACCGCCAAGACTGTTACCCATCGCGTGGGCGTATTCGCAAAGGATCAGCGGGCGCGTCTCTCCAGGTAGCGAAAGCCATTTTTTGATGGACCATTTCGGCACAGCCGGGAAGGGCTGGTCTTCATCCACGCGCGCGTACATCGGGCAAATAATATCGGTGGCCGTGGTGTCGGCTCCGCCGCCTTCATACTGCACCGGGCGGGAAGGATCGACAGATTTGATCCAGCGATACAGCGCGTCGTGATTAGCGCCGTGGCCTGATTCATTCCCCAGCGACCAGATGATCACACTCGGGTGATTACGATCGCGCTGCACCATTCGCGTTACGCGTTCGCTCATCGCCGGTAGCCAGCGCGGATCATCGGTCAGACGATTCATTGGCACCATGCCGTGGGTTTCAATATTGGCTTCATCCACCACATACAGGCCGTAGCGGTCGCACAGCGTGTACCACAGCGGATGGTTCGGATAATGCGAACAGCGCACGGCGTTAAAGTTGTTCTGCTTCATCAGCAGGATATCCTGCACCATCGTCTGCTCATCCATGACCTGACCATGCAGAGGATGATGCTCGTGACGGTTAACGCCTCGAATCAGCAACGGCTTGCCGTTCAGCAGCAGCAGACCATTTTCAATCCGCACCTCGCGGAAACCGACATCGCAGGCTTCTGCTTCAATCAGCGTGCCGTCGGCGGTGTGCAGTTCAACCACCGCACGATAGAGATTCGGGATTTCGGCGCTCCACAGTTTCGGGTTTTCGACGTTCAGACGTAGTGTGACGCGATCGGCATAACCACCACGCTCATCGATAATTTCACCGCCGAAAGGCGCGGTGCCGCTGGCGACCTGCGTTTCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTTCAGCCTCCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTTGTGTAGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTCCAGCGCAGCACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTCAGGTCAAATTCAGACGGCAAACGACTGTCCTGGCCGTAACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATAATTCGCGTCTGGCCTTCCTGTAGCCAGCTTTCATCAACATTAAATGTGAGCGAGTAACAACCCGTCGGATTCTCCGTGGGAACAAACGGCGGATTGACCGTAATGGGATAGGTCACGTTGGTGTAGATGGGCGCATCGTAACCGTGCATCTGCCAGTTTGAGGGGACGACGACAGTATCGGCCTCAGGAAGATCGCACTCCAGCCAGCTTTCCGGCACCGCTTCTGGTGCCGGAAACCAGGCAAAGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATCCGTAATCATGGTCAT

LacY Gene:

TTAAGCGACTTCATTCACCTGACGACGCAGTAGAGAAAGCGGACCGGGGCCGCTAAGCGTGAACACGGAAATTAAGGTGAAGCCCAGCGCCACCAGACCCAGCACCAGATAAGCGCCCTGGAAACCGATGCTTTCATACATATTGCCCGCCAGTACAGACATAAAAATCATCGCCAGTTGCTTAAAGAAGCAGAAACAGACCAGATAAATCGTCGCTGAAAAACGCACTTCAAACTGGCTGGTAATATATTTAAAGCAGCCCACCAGCAGGAACGGTACTTCAAACATATGCAGCGTTTTCAGAATAACCACTTCCAGCGCTGAGGTGGCGAACGATGAGCCAATAATACGTACAGACATAATAGTGCCAGCCAGCAGCAGGGCGTTTTTCCCACCGATGCGATTAATGATCAGTGGCGCAAAGAACATAATCGAGGCGTTAAGTAATTCGCCCATTGTCGTTACGTAGCCAAATACCCGCGTACCCTGTTCACCGGTAGCAAAGAACGAAGTAAAGAAATTAGCAAACTGTTGGTCAAAAACATCGTAGGTGCAGGAAACGCCAATAACATACAGTGACAAAAACCACAGTTTTGGCTGTCTGAACAGTTCCAGTGCCAGCTTAAGGCTAAATGCCGAATGGTTGGCACCTACCGCATTGGCAACCGTGGCAGAAGAGGGCGCATCCGTTTTGGCGAAAAAGAGTAAAACGGCGAGGATGAGTGCACAGCCAGAGCCCAGCCAGAAAACAAACTGATTATTGATGGTGAACATGATGCCGACAATCGAGGCACACAGCGCCCAGCCAACACAGCCAAACATCCGCGCGCGACCAAATTCGAAATTACTGCGACGGCTGACTTTCTCAATAAATGCCTCTACTGCTGGCGCACCGGCGTTAAAACAAAAGCCTAGATAAATACCACCAACAATCGATCCTACTAAAATGTTGTATTGTAACAGTGGCCCGAAGATAAAAATAAAGAACGGCGCAAACATCACTAACATGCCGGTAATAATCCACAGCAGGTATTTGCGCAGCCCGAGTTTGTCAGAAAGCAGACCAAACAGCGGTTGGAATAATAGCGAGAACAGAGAAATAGCGGCAAAAATAATACCCGTATCACTTTTGCTGATATGGTTGATGTCATGTAGCCAAATCGGGAAAAACGGGAAGTAGGCTCCCATGATAAAAAAGTAAAAGAAAAAGAATAAACCGAACATCCAAAAGTTTGTGTTTTTTAAATAGTACAT

all of the above genes were from E.coli BL21 (DE 3) of the indigenous organism, and the LacZ gene thereof had been knocked out.

Examples

Transformation of strains

The strain used in this example was E.coli BL21 (DE 3), and the LacZ gene thereof had been knocked out and was conventionally modified into a fermentation strain, and it was confirmed that the objective product could be synthesized by fermentation test (this strain will be referred to as E.coli BL21 (DE 3) Y strain hereinafter). The plasmid replicative strains used were E.col DH 5. Alpha. Purchased from Videoside organisms, the pTarget plasmid and the pCas plasmid used were common plasmids sold on the market.

Experimental method

The strain construction of the experiment is to utilize CRISPR/Cas9 gene editing system to carry out beta-galactose nucleoside enzymeLacZGene and permeaseLacYArabinose metabolizing enzyme on gene replacement genomearaBGene and genearaAAnd (3) a gene. Published according to NCBI databaseEColl BL21 (DE 3) information lookuparaB、araA、LacZAndLacYThe gene sequences, primers of Table 1 were designed and all primer synthesis and sequencing work was done by Suzhou Jin Weizhi Co.

The genome integration steps are as follows:

(1) Enzyme metabolizing according to arabinosearaB、araAIs used to determine the position of the substitution and the N20 sequence (N20-1 is TTTTGGATGGAGTGAAACGA and N20-2 is CGCTGGAACGAAGTGTATTA) is determined on an N20 search site (http:// crispor. Tefor. Net/crispor. Py) using the pTargetF plasmid as templatepTS-CP-F/R，343-N20-F/RAfter obtaining the linear plasmid by PCRDH5a competent cells were transformed after ligation by Gibson Assembly Master Mix and plated with spectinomycin resistant plates and incubated overnight in an incubator at 37 ℃; selecting monoclonal shake bacteria and sequencing the next day, sequencing to successfully replace the plasmid of the N20 sequence as positive clone, selecting positive clone shake bacteria for culture and extracting the plasmid to obtain N20-343 plasmid;

(2) Using N20-343 plasmid as template, usingN20-CPF/RObtaining a linearized N20-343 carrier fragment by PCR as a primer;

(3) To be used forEThe coll BL21 genome is used as a template343-HL-F/R、343-HR-F/R、lacZ-CDS-F/RAs primers, upstream and downstream homology arms were obtained by PCRLacZYA sequence;

(4) The linear fragments of the two steps are connected through Gibson Assembly Master Mix, DH5a competent cells are transformed, a spectinomycin resistant plate is coated, the culture is carried out in a culture box at 37 ℃ for overnight, the monoclonal shaking bacteria are selected for identification and sequencing the next day, and the target plasmid is extracted after the sequencing is successful;

(5) Preparing electric transformation competence: when (when)EWhen OD value of the bacterial liquid of the coll BL21 (DE 3) Y reaches 0.8, adding 50 ug/mL kanamycin and 10mmol arabinose to induce for 4 hours, and preparing competence;

(6) Transferring the target plasmid into electrotransformation competence, coating a kanamycin and spectinomycin double-resistance plate on the strain subjected to electrotransformation, culturing overnight at 30 ℃, selecting a monoclonal PCR for identification the next day, and selecting a correct positive monoclonal;

11 single clones are selected for identification, each single clone is identified by using two pairs of primers (343-AS-F: TTATTCAGCAGCGTTTGCTG,343-AS-R1: GATCCTCGACGTTCAGCATC,343-AS-R2: TTTCTCTGTTCTCGCTATTA), if the left side is free of a band and the right side is provided with a band (343-AS-F/R1, the size is 2034 bp), the single clone proves successful replacement, and if the left side is provided with a band (343-AS-F/R2, the size is 1346 bp) and the right side is free of a band, the single clone proves unsuccessful replacement, wherein MARK is shown in the middle of the figure, 100bp, 250bp, 500bp, 750bp, 1000bp and 2000bp are sequentially arranged from bottom to top, and clone numbers 4 and 6 are successful replacement strains and positive clones can be found in the figure;

(7) The positive clone colony is picked to a 4mL LB liquid test tube, IPTG with the final concentration of 1mmol/L and 50 ug/mL kanamycin are added for culturing for 12 hours at 37 ℃, LB solid plates (kanamycin resistance) are streaked, and monoclonal verification is performed to remove the pTargetF plasmid;

(8) The single clone from which pTargetF plasmid was removed was picked up into an antibiotic-free LB liquid test tube, cultured at 42℃for 12 hours, streaked onto an antibiotic-free plate, and a single clone PCR (Cas 9-IS-F: TTATGTTGGTCCATTGGCGC, cas9-IS-R: AGCAAATCATGGTAGGTACC) was picked up to verify whether pCas plasmid was removed, and after successful removal of pTargetF and pCas plasmid by the strain successfully replacing the gene, glycerol bacteria were prepared and designated 343 and stored in a refrigerator at-80 ℃.

The PCR reaction system comprises:

the reaction procedure:

three identical sets of fermentation equipment were used for strain fermentation, labeled as replacement induced set (a, 343+ arabinose), replacement uninduced set (B, 343+ water), and non-replacement induced set (C, 343+), respectively. Inoculating strains into 70ml of culture medium according to the proportion of 1:100, culturing for 8 hours at 37 ℃, using a spectrophotometer to measure the OD value of bacterial liquid, inoculating the bacterial liquid into a 1.5L fermentation tank when the OD value is about 0.5, adding fermentation culture medium which is added before and sterilized together into the tank, not feeding the strains within 8 hours after inoculation, starting feeding after 8 hours, using the same fermentation operation for three strains, wherein the three strains need to use the same fermentation operation, in order to ensure that the three strains have the same physiological state when the arabinose is added later, in the process, keeping the fermentation at 37 ℃, starting to regulate the temperature when the OD value of fermentation reaches 60, then fermenting until the OD value reaches 80 (at least 1 hour needs to be kept at 30 ℃), adding 80g of lactose (lactose adding amount is 200ml,0.4 g/ml), the final concentration is 20g/L, the inducer IPTG (isopropyl-beta-D-thiogalactoside) is 8ml (0.1M), the final concentration is 0.2mM, detecting the lactose content in the liquid phase of the fermentation tank is 37g, and the lactose content of the liquid phase is 35 g when the lactose content reaches 37g/L, detecting the residual content is 35 g, and the residual content is 24 g, and the residual content is detected by the high-quality group of lactose is 35 g, and the residual content is detected by the liquid chromatography group of the lactose content of 35 g; subsequently, the group A fermenter was charged with the final arabinose concentration of 0.5mM for induction (40 ml, concentration of 0.5 g/ml), the group B fermenter was charged with pure water (40 ml), the group C fermenter was charged with the final arabinose concentration of 0.5mM for induction (40 ml, concentration of 0.5 g/ml), the fed-batch fermentation culture was continued, and a sample was taken after 6 hours, a sample was taken after 12 hours, a sample was taken after 24 hours, the residual amount of lactose was detected, and finally the residual lactose content of the group A fermenter was measured to be 0.002g/L, the lactose content of the group B was 3.625g/L, and the lactose content of the group C was 3.447g/L, see Table 2.

The test result shows that the addition of the inducer arabinose after replacement has a great influence on lactose consumption, lactose is consumed on a larger level, the lactose content after water addition is slightly reduced, the reduction amount is far smaller than that of an induction group added with the arabinose, the reduced part is estimated to be the lactose consumed by the target product of the fermentation of the strain per se by analysis on the lactose consumption amount, and the lactose content of the inducer added in the non-replacement (E.coli BL21 (DE 3) Y strain) is slightly reduced, and the reason for the reduction is estimated to be the same as that of the non-induction group.

Similar effects can be obtained by fermenting 2 ' -FL, 3 ' -SL, 6 ' -SL and other products in the same experiment, and the residual lactose is greatly consumed by adding the inducer arabinose after replacement, and the residual lactose is induced for 20 hours, which is within 0.005 g/L.

The existing method for removing residual lactose from fermentation liquor mainly focuses on adding lactose decomposing enzyme during purification, such as directly adding beta-galactosidase into fermentation liquor, and the beta-galactosidase has higher price, and the cost of fermentation is greatly increased when residual lactose is removed every time, and the purification is also greatly increasedCost and time. Construction of plasmids to eliminate also occurs by constructing a plasmid containingLacZPlasmids of genes by means of plasmidsLacZThe beta-galactosidase is synthesized by gene expression, and the beta-galactosidase can decompose lactose, so that the purpose of consuming residual lactose is achieved, but the scheme is often accompanied by the generation of antibiotics and can digest lactose in advance to affect the yield, so that the plasmids can exist in the strain and cannot be lost, antibiotics are often added, the subsequent purification work is also increased, and the purification cost is increased. Therefore, the lactose removing method is simple and quick, does not introduce new substances, and can greatly save the purifying cost. The current method on the market cannot achieve the problem of thoroughly reducing the purification cost, so a method which can not generate new byproducts and remove lactose is urgently needed in the market. The invention mainly aims to solve the problems of increased purification steps and increased cost caused by excessive lactose residue in the fermentation process. The most critical point of the invention is that the original is to beLacZIn the case of gene knockout willLacZGene and geneLacYThe gene is introduced into the strain and inserted into the arabinose operon region, and the genes are only inserted after the operon under the action of an arabinose inducerLacZGene and geneLacYThe gene is expressed and normal fermentation is not affected when arabinose is not added, so that lactose is ensured not to be consumed by the strain in the fermentation process. In the case of lactose as a fermentation substrate, the fermentation substrate willLacZGene knockout postgeneLacYThe region after the gene is inserted into the arabinose operon is continuously fermented for 60 hours on the premise of not influencing the fermentation of the strain, then the arabinose is added for induction, and along with the continuous increase of the fermentation time, lactose is continuously consumed in the process, and the consumption rate is far greater than that of the strain which is not induced or replaced. Therefore, the invention can play a huge role in treating lactose residue in the late fermentation period, and the rate of treating the lactose residue is higher.

Sequence listing

<110> Huangshan same-part Biotech Co., ltd

<120> a method for digesting residual lactose of fermentation by genome integration based on arabinose operon

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1503

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ttagcgacga aatccgtaat acacttcgtt ccagcgcagc gcgtctttaa acgctggcag 60

gcgggtatcg ttatcaatca ccgtgatttc aatgtcgtgc atctcggcga actggcgcat 120

atcgttgagg ttcagcgcat ggctgaagac ggtatggtgc gcgccaccag cgaggatcca 180

cgcttcggaa gcagttggca gatccggttg cgctttccac agcgcattcg ccaccggcag 240

tttcggcagg gagtgcggtg ttttcaccgt gtcgatacag ttaaccagca gacggtaacg 300

atcgccgaga tcaatcaggc tggcgacaat cgctggaccg gtttgggtat tgaagatcag 360

tcgggcagga tcgtccttac caccaatacc gagatgctga acgtcgagga tcggtttctc 420

ttctacggca atcgacgggc agacttccag catatgggag ccgagcacca ggtcattacc 480

tttctcgaag tgataggtgt agtcctccat aaaggaggtg ccgccctgca gaccggttga 540

catcaccttc atgatgcgaa gcagggcggc ggttttccag tcgccttcgc ccgcaaagcc 600

gtaaccctgc tgcatcagac gctgtacggc cagacctgga agctgtttca gaccgtgcaa 660

atcttcaaag gtggtggtga acgcgtggaa gccaccttgt tccaggaaac gcttcatccc 720

cagctcaata cgcgccgctt ccagcacgtt ctgtcgtttt tcgccgtgga tttgtgttgc 780

aggcgtcatg gtgtagcagc tttcgtactc atcgaccagc gcgttaacat cgccgtcgct 840

gatggagttc accacctgca ccagatcgcc aaccgcccag gtattgacgg agaaaccgaa 900

cttgatctgt gcggcaactt tatcaccatc ggtgaccgcc acttcacgca tgttatcgcc 960

aaaacggcag actttcagat gacgggtatc ctgtttagaa accgcctgac gcatccagga 1020

gccgatacgc tcatgggctt gtttatcctg ccagtgaccg gtaacgacgg catgttgctg 1080

acgcatacgc gcgccaatga agccgaactc gcgaccgcca tgtgcagtct ggttcaggtt 1140

cataaagtcc atatcgatac tgtcccacgg cagcgccgcg ttgaactggg tgtggaattg 1200

cagcaacggt ttgttgagca tggtcaggcc gttgatccac attttggccg gggagaaggt 1260

gtgcagccac accaccagac cagcgcaacg atcgtcgtaa ttcgcgtcgc ggcaaatagc 1320

ggtgatttca tccggcgtgg tgcccagcgg tttcaacacc agtttgcagg gcagtttcgc 1380

ttccgtattc agcgcattaa caacgtgctc ggcatgttgg gtgacctgac gcagggtttc 1440

cgggccatac agatgctggc tgccaatgac aaaccacact tcataattat caaaaatcgt 1500

cat 1503

<210> 2

<211> 1701

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ttatagagtc gcaacggcct gggcagcctg tgccggggcg gaagttggaa gatagtgttg 60

ttcggcgctc atcgcccatt gctgatagcg gcgataaagc tgttcaaagc gttgtgcctg 120

ttcgctgcgc ggttgcaggg ttttctctac cgcactggcc attttttgct gggctgatgg 180

gatgtctgcg tgcactttcg cggcgacggc agcaaaaatc gccgcaccga gcgcacagca 240

ctggtcagag gcaacaattt gcagcgggcg attcagcacg tcgcagcagg cctgcataat 300

gacctggttt ttccgcgcga tgccgcccag cgccatcacg ttattgacgg cgatcccctg 360

atcggtaaag cactccatga ttgcgcgtgc gccaaaggcg gtggcagcaa tcaaaccgcc 420

gaacagcagc ggagcgtcgg tagcgaggtt aagatcggta atcacccctt tcaggcgttg 480

gttagcgttt ggcgagcgac gaccgttaaa ccagtcgagc accaccggca ggtgatccag 540

agacggattt ttggcccatg cttcggtcag cgccggaagc agttgtttct ggctggcgtt 600

gatttgcgct ttcagttccg gatgctgggc ggcaagctgt tccagcggcc agctgagtac 660

gcgaccgaac caggcgtaga tatcaccaaa cgccgattgg cctgcttcca gaccgataaa 720

tccaggcacc acgctgccat caacctgacc gcaaatacct ttaactgccc gctcgccaac 780

gctctgtttg tcggcaatca gaatgtcgca ggtggaagta ccgataactt ttaccagtgc 840

gttaggctgt gcgcctgcgc caactgcgcc catatggcag tcaaacgcgc cgccggaaat 900

caccacgctt tcaggcaggc cgagacgctg cgcccattcc gggcataagg tgcccaccgg 960

aatatcggca gtccaggtgt cagtgaacag cggggaaggc aaatggcgat tgaggatcgg 1020

gtccagctca tcaaagaaac tggctggcgg caagccgccc cagctttcgt gccacagaga 1080

tttatgcccg gcgctgcaac gtccgcgacg aatatcctgc gggcgggtgg taccggaaag 1140

cagagctggc acccagtcgc acagctcaat ccacgatgcg gcagattgcg ccacggcgct 1200

gtcctggcga gtcacatgca ggatttttgc ccagaaccat tcgctggaat aaataccgcc 1260

aatatagcgg gagtagtcaa cattgcccgg cgcgtggcac aaacgggtaa tctcttccgc 1320

ttcttcaacc gcagtgtggt ctttccacaa tacgaacatc gcgttcgggt tttcggcaaa 1380

ctccgggcgc agcgccagca cgttaccgtc ggcatcaatc ggtgcgggcg tcgagccggt 1440

actgtcaacg ccaatcccga ccacagctgc gcgctgttcg acgctaagct ctgcaagcac 1500

ggttttcagt gccgcttcca ttgactcaat gtagtcacgc ggatgatgac ggaactggtt 1560

attcggggca tcacaaaatt gccctttttg ccaacgggga taccactcta cgctggtggc 1620

gatctcttca ccgctggcgc agtccaccgc caaagctcgc acagaatcac tgccaaaatc 1680

gaggccaatt gcaatcgcca t 1701

<210> 3

<211> 3075

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ttatttttga caccagacca actggtaatg gtagcgaccg gcgctcagct ggaattccgc 60

cgatactgac gggctccagg agtcgtcgcc accaatcccc atatggaaac cgtcgatatt 120

cagccatgtg ccttcttccg cgtgcagcag atggcgatgg ctggtttcca tcagttgctg 180

ttgactgtag cggctgatgt tgaactggaa gtcgccgcgc cactggtgtg ggccataatt 240

caattcgcgc gtcccgcagc gcagaccgtt ttcgctcggg aagacgtacg gggtatacat 300

gtctgacaat ggcagatccc agcggtcaaa acaggcggca gtaaggcggt cgggatagtt 360

ttcttgcggc cctaatccga gccagtttac ccgctctgct acctgcgcca gctggcagtt 420

caggccaatc cgcgccggat gcggtgtatc gctcgccact tcaacatcaa cggtaatcgc 480

catttgacca ctaccatcaa tccggtaggt tttccggctg ataaataagg ttttcccctg 540

atgctgccac gcgtgagcgg tcgtaatcag caccgcatca gcaagtgtat ctgccgtgca 600

ctgcaacaac gctgcttcgg cctggtaatg gcccgccgcc ttccagcgtt cgacccaggc 660

gttagggtca atgcgggtcg cttcacttac gccaatgtcg ttatccagcg gtgcacgggt 720

gaactgatcg cgcagcggcg tcagcagttg ttttttatcg ccaatccaca tctgtgaaag 780

aaagcctgac tggcggttaa attgccaacg cttattaccc agctcgatgc aaaaatccat 840

ttcgctggtg gtcagatgcg ggatggcgtg ggacgcggcg gggagtgtca cgctgaggtt 900

ttcagccaga cgccactgct gccaggcgct gatgtgtccg gcttctgacc atgcggtcgc 960

gttcggttgc actacgcgta ctgtgagcca gagttgcccg gcgctctccg gctgcggtag 1020

ttcaggcagt tcaatcaact gtttaccttg tggagcgaca tccagaggca cttcaccgct 1080

tgccagcggc ttaccatcca gcgccaccat ccagtgcagg agctcgttat cgctatgacg 1140

gaacaggtat tcgctggtca cttcgatggt ttgcccggat aaacggaact ggaaaaactg 1200

ctgctggtgt tttgcttccg tcagcgctgg atgcggcgtg cggtcggcaa agaccagacc 1260

gttcatacag aactggcgat cgttcggcgt atcgccaaaa tcaccgccgt aagccgacca 1320

cgggttgccg ttttcatcat atttaatcag cgactgatcc acccagtccc agacgaagcc 1380

gccctgtaaa cggggatact gacgaaacgc ctgccagtat ttagcgaaac cgccaagact 1440

gttacccatc gcgtgggcgt attcgcaaag gatcagcggg cgcgtctctc caggtagcga 1500

aagccatttt ttgatggacc atttcggcac agccgggaag ggctggtctt catccacgcg 1560

cgcgtacatc gggcaaataa tatcggtggc cgtggtgtcg gctccgccgc cttcatactg 1620

caccgggcgg gaaggatcga cagatttgat ccagcgatac agcgcgtcgt gattagcgcc 1680

gtggcctgat tcattcccca gcgaccagat gatcacactc gggtgattac gatcgcgctg 1740

caccattcgc gttacgcgtt cgctcatcgc cggtagccag cgcggatcat cggtcagacg 1800

attcattggc accatgccgt gggtttcaat attggcttca tccaccacat acaggccgta 1860

gcggtcgcac agcgtgtacc acagcggatg gttcggataa tgcgaacagc gcacggcgtt 1920

aaagttgttc tgcttcatca gcaggatatc ctgcaccatc gtctgctcat ccatgacctg 1980

accatgcaga ggatgatgct cgtgacggtt aacgcctcga atcagcaacg gcttgccgtt 2040

cagcagcagc agaccatttt caatccgcac ctcgcggaaa ccgacatcgc aggcttctgc 2100

ttcaatcagc gtgccgtcgg cggtgtgcag ttcaaccacc gcacgataga gattcgggat 2160

ttcggcgctc cacagtttcg ggttttcgac gttcagacgt agtgtgacgc gatcggcata 2220

accaccacgc tcatcgataa tttcaccgcc gaaaggcgcg gtgccgctgg cgacctgcgt 2280

ttcaccctgc cataaagaaa ctgttacccg taggtagtca cgcaactcgc cgcacatctg 2340

aacttcagcc tccagtacag cgcggctgaa atcatcatta aagcgagtgg caacatggaa 2400

atcgctgatt tgtgtagtcg gtttatgcag caacgagacg tcacggaaaa tgccgctcat 2460

ccgccacata tcctgatctt ccagataact gccgtcactc cagcgcagca ccatcaccgc 2520

gaggcggttt tctccggcgc gtaaaaatgc gctcaggtca aattcagacg gcaaacgact 2580

gtcctggccg taaccgaccc agcgcccgtt gcaccacaga tgaaacgccg agttaacgcc 2640

atcaaaaata attcgcgtct ggccttcctg tagccagctt tcatcaacat taaatgtgag 2700

cgagtaacaa cccgtcggat tctccgtggg aacaaacggc ggattgaccg taatgggata 2760

ggtcacgttg gtgtagatgg gcgcatcgta accgtgcatc tgccagtttg aggggacgac 2820

gacagtatcg gcctcaggaa gatcgcactc cagccagctt tccggcaccg cttctggtgc 2880

cggaaaccag gcaaagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 2940

ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 3000

aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatc 3060

cgtaatcatg gtcat 3075

<210> 4

<211> 1254

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ttaagcgact tcattcacct gacgacgcag tagagaaagc ggaccggggc cgctaagcgt 60

gaacacggaa attaaggtga agcccagcgc caccagaccc agcaccagat aagcgccctg 120

gaaaccgatg ctttcataca tattgcccgc cagtacagac ataaaaatca tcgccagttg 180

cttaaagaag cagaaacaga ccagataaat cgtcgctgaa aaacgcactt caaactggct 240

ggtaatatat ttaaagcagc ccaccagcag gaacggtact tcaaacatat gcagcgtttt 300

cagaataacc acttccagcg ctgaggtggc gaacgatgag ccaataatac gtacagacat 360

aatagtgcca gccagcagca gggcgttttt cccaccgatg cgattaatga tcagtggcgc 420

aaagaacata atcgaggcgt taagtaattc gcccattgtc gttacgtagc caaatacccg 480

cgtaccctgt tcaccggtag caaagaacga agtaaagaaa ttagcaaact gttggtcaaa 540

aacatcgtag gtgcaggaaa cgccaataac atacagtgac aaaaaccaca gttttggctg 600

tctgaacagt tccagtgcca gcttaaggct aaatgccgaa tggttggcac ctaccgcatt 660

ggcaaccgtg gcagaagagg gcgcatccgt tttggcgaaa aagagtaaaa cggcgaggat 720

gagtgcacag ccagagccca gccagaaaac aaactgatta ttgatggtga acatgatgcc 780

gacaatcgag gcacacagcg cccagccaac acagccaaac atccgcgcgc gaccaaattc 840

gaaattactg cgacggctga ctttctcaat aaatgcctct actgctggcg caccggcgtt 900

aaaacaaaag cctagataaa taccaccaac aatcgatcct actaaaatgt tgtattgtaa 960

cagtggcccg aagataaaaa taaagaacgg cgcaaacatc actaacatgc cggtaataat 1020

ccacagcagg tatttgcgca gcccgagttt gtcagaaagc agaccaaaca gcggttggaa 1080

taatagcgag aacagagaaa tagcggcaaa aataataccc gtatcacttt tgctgatatg 1140

gttgatgtca tgtagccaaa tcgggaaaaa cgggaagtag gctcccatga taaaaaagta 1200

aaagaaaaag aataaaccga acatccaaaa gtttgtgttt tttaaatagt acat 1254

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gttttagagc tagaaatagc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

gctagcatta tacctaggac 20

<210> 7

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ctaggtataa tgctagcttt tggatggagt gaaacgagtt ttagagctag aaatagc 57

<210> 8

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

atttctagct ctaaaactaa tacacttcgt tccagcggct agcattatac ctaggac 57

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

tctagaacta gtctgcaggg 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gaattcaata gatctaagct 20

<210> 11

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ttagatctat tgaattctga taccattcgc gagcctc 37

<210> 12

<211> 66

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tccgtaatca tggtcatttt ttataacctc cttagagctc gaattcccaa aaaaacgggt 60

atggag 66

<210> 13

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aggtgaatga agtcgcttaa gtagtcgcat caggtgt 37

<210> 14

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

tgcagactag ttctagagtg agaatggacc aggacgc 37

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

atgaccatga ttacggattc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

agcgacttca ttcacctgac 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ttttggatgg agtgaaacga 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

cgctggaacg aagtgtatta 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

ttattcagca gcgtttgctg 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

gatcctcgac gttcagcatc 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 21

tttctctgtt ctcgctatta 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 22

ttatgttggt ccattggcgc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

agcaaatcat ggtaggtacc 20

Claims

1. Genome integration eliminator based on arabinose operonA method for converting residual lactose from fermentation, characterized byLacZGene and geneLacYGene replacement of the arabinose operon on the genes of the fermentation strainsaraAGene and genearaBGenes, resulting in a replacement fermentation strain BL21 (DE 3); then carrying out lactose fermentation by using the replacement fermentation strain; finally, adding arabinose for induction to complete the digestion of residual lactose after fermentation; the fermentation strain is conventional lactose fermentation strain and knocks out lactose metabolism genes; during induction, the addition amount of the arabinose is 0.2-0.8 mM; the lactose fermentation time is 22-65 hours; the induction time is 10-30 hours; the alternative fermentation strain is prepared by determining the N20 sequence on the araA or araB gene fragment using CRISP/CAS9 gene editing techniques with the pTarget plasmid, inserting the N20 sequence and the sgRNA sequence on the plasmid, followed by insertionaraABase sequence before genearaBThe base sequence after the gene is inserted between the twoLacZGene and geneLacYGenes to obtain a replacement plasmid; transferring the replaced plasmid into a fermentation strain by an electrochemical conversion method, culturing overnight, identifying, and selecting a strain which is replaced successfully to lose the plasmid to obtain a replaced fermentation strain; the gene sequences involved are as follows:

araA gene:

araB gene:

LacZ gene:

LacY Gene:

TTAAGCGACTTCATTCACCTGACGACGCAGTAGAGAAAGCGGACCGGGGCCGCTAAGCGTGAACACGGAAATTAAGGTGAAGCCCAGCGCCACCAGACCCAGCACCAGATAAGCGCCCTGGAAACCGATGCTTTCATACATATTGCCCGCCAGTACAGACATAAAAATCATCGCCAGTTGCTTAAAGAAGCAGAAACAGACCAGATAAATCGTCGCTGAAAAACGCACTTCAAACTGGCTGGTAATATATTTAAAGCAGCCCACCAGCAGGAACGGTACTTCAAACATATGCAGCGTTTTCAGAATAACCACTTCCAGCGCTGAGGTGGCGAACGATGAGCCAATAATACGTACAGACATAATAGTGCCAGCCAGCAGCAGGGCGTTTTTCCCACCGATGCGATTAATGATCAGTGGCGCAAAGAACATAATCGAGGCGTTAAGTAATTCGCCCATTGTCGTTACGTAGCCAAATACCCGCGTACCCTGTTCACCGGTAGCAAAGAACGAAGTAAAGAAATTAGCAAACTGTTGGTCAAAAACATCGTAGGTGCAGGAAACGCCAATAACATACAGTGACAAAAACCACAGTTTTGGCTGTCTGAACAGTTCCAGTGCCAGCTTAAGGCTAAATGCCGAATGGTTGGCACCTACCGCATTGGCAACCGTGGCAGAAGAGGGCGCATCCGTTTTGGCGAAAAAGAGTAAAACGGCGAGGATGAGTGCACAGCCAGAGCCCAGCCAGAAAACAAACTGATTATTGATGGTGAACATGATGCCGACAATCGAGGCACACAGCGCCCAGCCAACACAGCCAAACATCCGCGCGCGACCAAATTCGAAATTACTGCGACGGCTGACTTTCTCAATAAATGCCTCTACTGCTGGCGCACCGGCGTTAAAACAAAAGCCTAGATAAATACCACCAACAATCGATCCTACTAAAATGTTGTATTGTAACAGTGGCCCGAAGATAAAAATAAAGAACGGCGCAAACATCACTAACATGCCGGTAATAATCCACAGCAGGTATTTGCGCAGCCCGAGTTTGTCAGAAAGCAGACCAAACAGCGGTTGGAATAATAGCGAGAACAGAGAAATAGCGGCAAAAATAATACCCGTATCACTTTTGCTGATATGGTTGATGTCATGTAGCCAAATCGGGAAAAACGGGAAGTAGGCTCCCATGATAAAAAAGTAAAAGAAAAAGAATAAACCGAACATCCAAAAGTTTGTGTTTTTTAAATAGTACAT；

the N20 sequences were TTTTGGATGGAGTGAAACGA and CGCTGGAACGAAGTGTATTA.

2. An alternative fermentation strain BL21 (DE 3) for integrating residual lactose from digestion fermentation, which is characterized byLacZGene and geneLacYGene replacement of the arabinose operon on the genes of the fermentation strainsaraAGene and genearaBGenes to obtain a replacement fermentation strain; the fermentation strain is conventional lactose fermentation strain and knocks out lactose metabolism genes; the alternative fermentation strain is prepared by determining the N20 sequence on the araA or araB gene fragment using CRISP/CAS9 gene editing techniques with the pTarget plasmid, inserting the N20 sequence and the sgRNA sequence on the plasmid, followed by insertionaraABase sequence before genearaBThe base sequence after the gene is inserted between the twoLacZGene and geneLacYGenes to obtain a replacement plasmid; transferring the replaced plasmid into a fermentation strain by an electrochemical conversion method, culturing overnight, identifying, and selecting a strain which is replaced successfully to lose the plasmid to obtain a replaced fermentation strain; the gene sequences involved are as follows:

araA gene:

araB gene:

LacZ gene:

LacY Gene:

the N20 sequences were TTTTGGATGGAGTGAAACGA and CGCTGGAACGAAGTGTATTA.