阅读说明：本技术 一种增加基因组突变率的碱基编辑系统及碱基编辑方法 (Base editing system and base editing method for increasing mutation rate of genome ) 是由 连佳长 潘颖佳 董昌 夏思杨 于 2021-07-07 设计创作，主要内容包括：本发明公开了一种增加基因组突变率的碱基编辑系统及碱基编辑方法,所述碱基编辑系统包括编码胞嘧啶脱氨酶的基因和编码特异性单链DNA结合蛋白的基因。利用本发明构建的碱基编辑系统,对酿酒酵母基因组进行进化,在操作上具有简单,效率高等优点,在功能上,能提高β-胡萝卜素的产量、提高对有机试剂的耐受性。并且本发明构建的基因组碱基编辑系统在毕赤酵母、大肠杆菌以及罗尔斯通氏菌等大部分工业菌株中同样适用。所以,使用本发明碱基编辑系统能够用于提高菌株的基因组突变率,基因组中突变碱基累积,造成菌体性状和功能的多样性,根据工业生产要求,可对某一性状进行连续性状筛选和定向进化。(The invention discloses a base editing system and a base editing method for increasing the mutation rate of a genome. The basic group editing system constructed by the invention is used for evolving the saccharomyces cerevisiae genome, has the advantages of simplicity in operation, high efficiency and the like, and can functionally improve the yield of beta-carotene and the tolerance to organic reagents. The genome base editing system constructed by the invention is also applicable to most industrial strains such as pichia pastoris, escherichia coli, ralstonia and the like. Therefore, the base editing system can be used for improving the genome mutation rate of the strain, accumulating the mutant bases in the genome and causing the diversity of the characteristics and functions of thalli, and can carry out continuous characteristic screening and directed evolution on a certain characteristic according to the requirements of industrial production.)

1. A base editing system for increasing the mutation rate of genome, which comprises a gene coding cytosine deaminase and a gene coding specific single-stranded DNA binding protein.

2. The base editing system of claim 1, wherein the gene encoding a cytosine deaminase is mammalian cytosine deaminase APOBEC 1.

3. The base editing system of claim 2, wherein the gene encoding cytosine deaminase is rat cytosine deaminase APOBEC1, and the gene sequence is shown in SEQ ID No. 1.

4. The base editing system according to claim 1, wherein the gene encoding the specific single-stranded DNA binding protein is one of:

(1) at least one of 3 subunits RFA1, RFA2 and RFA3 of DNA single-strand binding protein of saccharomyces cerevisiae, wherein the gene sequences of RFA1, RFA2 and RFA3 are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;

(2) the gene sequence of DNA primer enzyme PRI1 of Saccharomyces cerevisiae is shown in SEQ ID No. 5;

(3) the gene sequence of helicase HCS1 of Saccharomyces cerevisiae is shown in SEQ ID No. 6;

(4) the gene sequence of topoisomerase TOP1 of Saccharomyces cerevisiae is shown in SEQ ID No. 7;

(5) the gene sequence of the single-chain binding protein subunit RPA of the pichia pastoris is shown in SEQ ID No. 8;

(6) the gene sequence of the DNA single-strand binding protein ssb of the escherichia coli is shown as SEQ ID No. 9;

(7) the gene sequence of the single-chain binding protein h16_ A0402 of the ralstonia is shown as SEQ ID No. 10.

5. The base editing system of claim 1, wherein a gene encoding cytosine deaminase and a gene encoding a specific single-stranded DNA binding protein are cloned into the same expression vector for co-expression.

6. Use of the base editing system of any one of claims 1 to 5 to increase the mutation rate of a strain's genome.

7. Use of the base editing system of any one of claims 1 to 5 for strain trait screening and directed evolution.

8. A base editing method for increasing the mutation rate of a genome, which is characterized in that a gene coding cytosine deaminase and a gene coding specific single-stranded DNA binding protein in the base editing system according to any one of claims 1 to 5 are simultaneously transferred into a host cell for exogenous expression to obtain a recombinant expression cell.

9. The base editing method of claim 8, wherein the gene encoding cytosine deaminase is rat cytosine deaminase APOBEC1, and the gene sequence is shown in SEQ ID No. 1.

10. The base editing method according to claim 8,

when the host cell is Saccharomyces cerevisiae, the gene encoding a specific single-stranded DNA binding protein is one of:

(1) at least one of 3 subunits RFA1, RFA2 and RFA3 of DNA single-strand binding protein of saccharomyces cerevisiae, wherein the gene sequences of RFA1, RFA2 and RFA3 are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;

(2) the gene sequence of DNA primer enzyme PRI1 of Saccharomyces cerevisiae is shown in SEQ ID No. 5;

(3) the gene sequence of helicase HCS1 of Saccharomyces cerevisiae is shown in SEQ ID No. 6;

(4) the gene sequence of topoisomerase TOP1 of Saccharomyces cerevisiae is shown in SEQ ID No.7,

when the host cell is Pichia pastoris, the gene for coding the specific single-stranded DNA binding protein is the single-stranded binding protein subunit RPA of the Pichia pastoris, the gene sequence is shown as SEQ ID No.8,

when the host cell is escherichia coli, the gene for coding the specific single-stranded DNA binding protein is the DNA single-stranded binding protein ssb of the escherichia coli, and the gene sequence is shown as SEQ ID No. 9;

when the host cell is Ralstonia, the gene encoding the specific single-stranded DNA binding protein is the single-stranded binding protein h16_ A0402 of Ralstonia, and the gene sequence is shown in SEQ ID No. 10.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a base editing system and a base editing method for increasing the mutation rate of a genome.

Background

Saccharomyces cerevisiae (Saccharomyces cerevisiae) is one of the first eukaryons with whole genome sequencing applied to brewing, food and other aspects, and is now widely applied to the large-scale production of various foods, chemical products and medicines. The saccharomyces cerevisiae has the advantages of clear genetic background, strong genetic operability, good fermentation performance and the like, and is an excellent metabolic engineering chassis cell. With the development of synthetic biology, various microorganisms, including Pichia pastoris (Pichia pastoris), Escherichia coli (Escherichia coli), Ralstonia eutropha, and the like, have been developed as cell factories for producing natural products.

However, due to the complexity of biological systems, complex phenotypes regulated by multiple genes, such as stress tolerance, it is difficult for a single genetic engineering approach to achieve the intended goal. To overcome this major limitation, a powerful multifunctional tool, genome mutation, was constructed in the microbial cell factory to meet the industrial production requirements (Xia Si Yang; Jianghong; Cai Hai, Huang Lei, Xu Zhi nan; Lijiachang, research progress on the evolution of Saccharomyces cerevisiae genome. synthetic biology 2020, 1(05), 556 one 569.).

Apolipoprotein B mRNA editing enzyme catalyzes polypeptide-1 (APOBEC1) to mediate cytosine deamination, but the catalysis of ApoB mRNA C-to-U requires an auxiliary protein binding specificity recognition sequence, fixes ApoB mRNA, and brings a DNA single strand and an APOBEC1 catalytic activity center close to each other to finally exert deamination activity ([1] Komor, A.C. 2020; Kim, Y.B.; Packer, M.S.; Zuris, J.A.; Liu, D.R. promoter, promoter of a target base in genomic DNA with a gene-derived DNA restriction. Nature 2016, 533(7603), 420-4.; [2] Sui, Y.; Qi, L.; Zhang, K.; N.J. Ackok.C. promoter, J.S. promoter, expression of nucleic acids, J.S. C.S. J.S. J. C.S. J. C.S. J. C.S. J. C., 117(17),201922472.). When the APOBEC1 is combined with the specific single-stranded DNA binding protein, the APOBEC1 can be randomly positioned on the single-stranded DNA to realize the deamination reaction of the monocytosine, so that the base mutation is caused, the characters and functions of the chassis cells are changed along with the accumulation of the random mutation in the genome, and the strains after the genome mutation are subjected to character screening and directed evolution according to the industrial production requirements.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a base editing system for increasing the genome mutation rate and application thereof, and the genome mutation rate can be simply and efficiently increased.

A base editing system for increasing the rate of genomic mutations comprising a gene encoding a cytosine deaminase and a gene encoding a specific single stranded DNA binding protein. Preferably, the gene encoding cytosine deaminase is mammalian cytosine deaminase APOBEC 1. More preferably, the gene coding cytosine deaminase is rat cytosine deaminase APOBEC1, and the gene sequence is shown as SEQ ID No. 1. Preferably, the gene encoding a specific single-stranded DNA binding protein is one of:

(1) at least one of 3 subunits RFA1, RFA2 and RFA3 of DNA single-strand binding protein of saccharomyces cerevisiae, wherein the gene sequences of RFA1, RFA2 and RFA3 are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;

(2) the gene sequence of DNA primer enzyme PRI1 of Saccharomyces cerevisiae is shown in SEQ ID No. 5;

(3) the gene sequence of helicase HCS1 of Saccharomyces cerevisiae is shown in SEQ ID No. 6;

(4) the gene sequence of topoisomerase TOP1 of Saccharomyces cerevisiae is shown in SEQ ID No. 7;

(5) the gene sequence of the single-chain binding protein subunit RPA of the pichia pastoris is shown in SEQ ID No. 8;

(6) the gene sequence of the DNA single-strand binding protein ssb of the escherichia coli is shown as SEQ ID No. 9;

(7) the gene sequence of the single-chain binding protein h16_ A0402 of the ralstonia is shown as SEQ ID No. 10.

Preferably, the base editing system clones a gene encoding cytosine deaminase and a gene encoding a specific single-stranded DNA binding protein into the same expression vector for co-expression.

The invention also provides application of the base editing system in improving the mutation rate of the strain genome.

The invention also provides the application of the base editing system in strain character screening and directed evolution.

The invention also provides a base editing method for increasing the genome mutation rate, wherein a gene coding cytosine deaminase and a gene coding specific single-stranded DNA binding protein in the base editing system are simultaneously transferred into a host cell for exogenous expression to obtain a recombinant expression cell. Preferably, the gene coding the cytosine deaminase is rat cytosine deaminase APOBEC1, and the gene sequence is shown as SEQ ID No. 1. Preferably, when the host cell is Saccharomyces cerevisiae, the gene encoding a specific single-stranded DNA binding protein is one of:

(1) at least one of 3 subunits RFA1, RFA2 and RFA3 of DNA single-strand binding protein of saccharomyces cerevisiae, wherein the gene sequences of RFA1, RFA2 and RFA3 are respectively shown as SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;

(2) the gene sequence of DNA primer enzyme PRI1 of Saccharomyces cerevisiae is shown in SEQ ID No. 5;

(3) the gene sequence of helicase HCS1 of Saccharomyces cerevisiae is shown in SEQ ID No. 6;

(4) the gene sequence of topoisomerase TOP1 of Saccharomyces cerevisiae is shown in SEQ ID No.7,

when the host cell is Pichia pastoris, the gene for coding the specific single-stranded DNA binding protein is the single-stranded binding protein subunit RPA of the Pichia pastoris, the gene sequence is shown as SEQ ID No.8,

when the host cell is escherichia coli, the gene for coding the specific single-stranded DNA binding protein is the DNA single-stranded binding protein ssb of the escherichia coli, and the gene sequence is shown as SEQ ID No. 9;

when the host cell is Ralstonia, the gene encoding the specific single-stranded DNA binding protein is the single-stranded binding protein h16_ A0402 of Ralstonia, and the gene sequence is shown in SEQ ID No. 10.

The basic group editing system constructed by the invention is used for evolving the saccharomyces cerevisiae genome, has the advantages of simplicity in operation, high efficiency and the like, and can functionally improve the yield of beta-carotene and the tolerance to organic reagents. The genome base editing system constructed by the invention is also applicable to most industrial strains such as pichia pastoris, escherichia coli, ralstonia and the like.

Therefore, the base editing system can be used for improving the genome mutation rate of the strain, accumulating the mutant bases in the genome and causing the diversity of the characteristics and functions of thalli, and can carry out continuous characteristic screening and directed evolution on a certain characteristic according to the requirements of industrial production.

Drawings

FIG. 1 is a schematic diagram of a base editing system for increasing the mutation rate of a genome.

FIG. 2 is a plasmid map of p 415-APOBEC.

FIG. 3 is a plasmid map of pGAP-APOBEC-RPA.

FIG. 4 is a plasmid map of pGEX-APOBEC-ssb.

FIG. 5 is a plasmid map of pBBR1-APOBEC-h16_ A0402.

FIG. 6 shows the results of gene mutation rates caused by different genomic mutation systems in Saccharomyces cerevisiae.

FIG. 7 shows the growth of the mutant Saccharomyces cerevisiae strains in different concentrations of isobutanol.

FIG. 8 growth of a continuous evolved strain of the Saccharomyces cerevisiae genome in medium containing 8% and 9% isobutanol.

FIG. 9 shows the growth of the Saccharomyces cerevisiae genomic mutant strains in media containing different concentrations of acetate.

FIG. 10 shows the differences in the production of beta-carotene caused by different genomic mutation systems in Saccharomyces cerevisiae.

FIG. 11 growth of the Pichia genome mutant in a medium containing 7% methanol.

FIG. 12 growth of Ralstonia genome mutant strain in medium containing 5g/L sodium formate.

FIG. 13 shows the growth of the E.coli genomic mutant in a medium containing 1% sodium acetate.

Detailed Description

Plasmid p415 was purchased from SnapGene. Coli DH5 a used for plasmid construction was purchased from Biotech, Inc., of Ongbenaceae, Beijing. Saccharomyces cerevisiae BY4741, Pichia pastoris GS115, Escherichia coli DH5 alpha and Ralstonia eutropha H16 were purchased from the China center for Industrial culture Collection of microorganisms (CICC). DNApolymerase, restriction enzyme and T4 ligase were purchased from NEB. The plasmid extraction kit was purchased from AXYGEN ltd, and the PCR product nucleic acid purification kit was purchased from Thermo Scientific.

The Escherichia coli culture medium adopts an LB culture medium, and the formula is as follows: 10g of peptone, 5g of yeast extract, 10g of NaCl and 1L of deionized water.

The culture medium of the saccharomyces cerevisiae uses an SCD-LEU culture medium, and the formula is as follows: amino-free yeast nitrogen source (Difco, Boom, The Netherlands)1.7g, ammonium sulfate 5g, CMS-LEU (MP Biomedicals, Solon, Ohio)0.69g, glucose 20g, agar powder 20g, deionized water 1L.

The culture medium of the saccharomyces cerevisiae uses an SCD-LEU-ARG + canavanine culture medium, and the formula is as follows: 1.7g of non-amino yeast nitrogen source (Difco, Boom, The Netherlands), 0.02g of histidine, 0.02g of methionine, 0.02g of uracil, 0.05g of phenylalanine, 0.05g of tyrosine, 5g of ammonium sulfate, 20g of glucose, 60mg of canavanine, 20g of agar powder and 1L of deionized water.

The Ralstonia uses MM culture medium, and the formula is as follows: 9g/LNa₂HPO₄·12H₂O，1.5g/L KH₂PO₄，1g/L(NH₄)₂SO₄，80mg/L MgSO₄·7H₂O，1mg/L CaSO₄·2H₂O，0.56mg/L NiSO₄·7H₂O, 0.4mg/L ferric citrate, 200mg/L NaHCO₃1mL/L microelement, and 10mg/mL fructose is used as carbon source.

The formula of the SED culture medium used by the pichia pastoris is as follows: 1.7g/LYNB, 1.107g/L sodium glutamate and 20g/L glucose as carbon source. The formula of the SEM culture medium used by the pichia pastoris is as follows: 1.7g/L YNB, 1.107g/L sodium glutamate and 6% methanol as carbon sources.

FIG. 1 is a schematic diagram of a base editing system for increasing the genomic mutation rate according to the present invention. The primer sequences used in the following examples or application examples are shown in Table 1, and the gene sequences used in the present invention are shown in Table 2.

TABLE 1 primer sequence Listing used in the present invention

Primer name	Sequence (5 '-3')
		APOBEC-FOR	atgagctcagagactggccca
APOBEC-REV	ggatccaccagaagaaccaccagaactttcgg
		GAP-FOR	tttttgtagaaatgtcttggt
GAP-REV	atagttgttcaattgattgaa
		pGEX-FOR	GAATACTGTTTCCTGTGTGAA
pGEX-REV	GCCACCGCTGAGCAATAACTA
		RFA1-FOR	ccgaaagttctggtggttcttctggtggatccatgagcagtgttcaactttcg
RFA1-REV	ataaatcataagaaattcgcctcggtttctcgagttaagctaacaaagccttggataac
		RFA2-FOR	NNNNNggatccatggcaacctatcaaccatataacgaatattc
RFA2-REV	NNNNNctcgagtcatagggcaaagaagttattgtc
		RFA3-FOR	NNNNNggatccatggccagcgaaacaccaag
RFA3-REV	NNNNNctcgagctagtatatttctgggtatttcttacatag
		PRI1-FOR	ccgaaagttctggtggttcttctggtggatccATGACCAATTCAGTAAAGACT
PRI1-REV	AatcataaatcataagaaattcgcctcggtttctcgagTCAGAAATCTAAAGAAGCCGG
		HCS1-FOR	ccgaaagttctggtggttcttctggtggatccATGAACAAAGAATTGGCTTCT
HCS1-REV	cataaatcataagaaattcgcctcggtttctcgagTTACAAATAATCGTCAATGTT
		top1-FOR	ccgaaagttctggtggttcttctggtggatccATGACTATTGCTGATGCTTCC
top1-REV	cataaatcataagaaattcgcctcggtttctcgagTTAAAACCTCCAATTTTCATC
		ssb-FOR	ccgaaagttctggtggttcttctggtggatccATGGCCAGCAGAGGCGTAAAC
ssb-REV	cataaatcataagaaattcgcctcggtttctcgagTCAGAACGGAATGTCATCATC
		RPA-FOR	ccgaaagttctggtggttcttctggtggatccATGACAGAGTTCAGTAAAGGT
RPA-REV	cataaatcataagaaattcgcctcggtttctcgagTTAATTAACCTTGACACCCTC
		h16_A0402-FOR	ccgaaagttctggtggttcttctggtggatccGCATCGGTCAACAAAGTCATT
h16_A0402-REV	cataaatcataagaaattcgcctcggtttctcgagTCAGAACGGGATATCGTCGTC

TABLE 2 Gene sequence Listing used in the present invention

In the following and in the accompanying figures, y415 represents s.cerevisiae transformed with plasmid p415 as control 1; yAPOBEC represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC as control 2; yRFA1 represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC-RFA 1; yRFA2 represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC-RFA 2; yRFA3 represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC-RFA 3; yPRI1 represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC-PRI 1; yHCS1 represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC-HCS 1; yTOP1 represents Saccharomyces cerevisiae transformed with plasmid p415-APOBEC-TOP 1.

Example 1: base editing system for increasing genome mutation rate and application

1. The genome expression vector is prepared by the following steps:

(a) amplifying an APOBEC sequence (the gene sequence is shown as SEQ ID No. 1):

the PCR reaction system is as follows: the 50. mu.L reaction system contained 25. mu.L of Q5 DNA polymerase mix, 2.5. mu.L of each of the forward primer and the reverse primer, 1. mu.L of the template, and ddH₂O19. mu.L. The PCR cycle program is pre-denaturation at 98 ℃ for 2 min; denaturation at 98 deg.C for 10s, annealing at 55 deg.C for 10s, extension at 72 deg.C for 4min, and circulation for 31 times; the extension was carried out at 72 ℃ for 2min and stored at 4 ℃. The PCR cycle program of the specific DNA single-strand binding protein amplification is pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 10s, extension at 72 ℃ for 2kb/min, and circulation for 30 times; the extension was carried out at 72 ℃ for 2min and stored at 4 ℃. And purifying and recycling the fragment obtained by PCR by a PCR product purification kit.

The primers used For the amplification were APOBEC-For (see Table 1 For primers used in the present invention) and APOBEC-Rev, the template was xCas9(3.7) -BE4 (http:// www.addgene.org/search/catalog/plasmids/.

(b) And (2) amplifying a gene sequence coded by a corresponding specific DNA single-strand binding protein by taking the genome of each strain as a template:

the PCR reaction system was the same as above. In Saccharomyces cerevisiae, primers For RFA1 (gene sequence is shown as SEQ ID No. 2) amplification are RFA1-For and RFA 1-Rev; the primers used For RFA2 (the gene sequence is shown as SEQ ID No. 3) amplification are RFA2-For and RFA 2-Rev; the primers used For RFA3 (the gene sequence is shown as SEQ ID No. 4) amplification are RFA3-For and RFA 3-Rev; primers For amplification of PRI1 (gene sequence is shown as SEQ ID No. 5) are PRI1-For and PRI 1-Rev; the primers used For HCS1 (the gene sequence is shown as SEQ ID No. 6) amplification are HCS1-For and HCS 1-Rev; primers For amplification of TOP1 (the gene sequence is shown as SEQ ID No. 7) are TOP1-For and TOP 1-Rev;

the primers For amplifying the Pichia pastoris RPA (the gene sequence is shown as SEQ ID No. 8) are RPA-For and RPA-Rev.

The primers used For amplifying the Escherichia coli ssb (the gene sequence is shown as SEQ ID No. 9) are ssb-For and ssb-Rev.

The primers used For amplifying Ralstonia h16_ A0402 (gene sequence is shown as SEQ ID No. 10) are h16_ A0402-For and h16_ A0402-Rev.

(c) Preparing a plasmid skeleton suitable for each strain:

a plasmid backbone for saccharomyces cerevisiae base editing; taking a plasmid p415 as a template, carrying out double enzyme digestion by using restriction enzymes Pst1 and Sca1, and separating and purifying a skeleton fragment through gel electrophoresis, namely a plasmid skeleton applied to saccharomyces cerevisiae by the system;

amplifying a plasmid backbone for pichia pastoris base editing; based on a yellow fluorescent expression vector containing a panARS replicon constructed in Gu et al (Construction of a series of epidemic plasmids and the same application in the degradation of an efficacy CRISPR/Cas9 system in Pichia pastoris, May2019, World Journal of Microbiology and Biotechnology 35(6), 10.1007/s11274-019-2654-5), the AOX1 promoter and the yellow fluorescent protein gene are removed by BglII and NotI, and a GAP promoter obtained by PCR amplification from the Pichia pastoris genome is inserted, and the used primers are FOR and GAP-REV, namely the plasmid skeleton applied to the Pichia pastoris by the system;

designing primers pGEX-FOR and pGEX-REV, and amplifying a plasmid skeleton FOR basic group editing of escherichia coli; taking the existing plasmid pGEX-6P-1 in a laboratory as a template, and carrying out PCR amplification on a plasmid skeleton containing elements such as ori replication initiation sites, ampicillin resistance genes AmpR, galactose operon and the like, namely the plasmid skeleton applied to escherichia coli by the system;

amplifying a plasmid skeleton for the base editing of the ralstonia, taking an existing plasmid pBBR1-Para-rfp in a laboratory as a template, performing double enzyme digestion by using XhoI and BamHI, and separating and purifying a skeleton fragment by gel electrophoresis, namely the plasmid skeleton applied to the ralstonia by the system;

(d) and (3) enzymatically connecting a plasmid skeleton and the APOBEC, and performing homologous recombination and connection on the fragments by utilizing a Gibson technology to obtain the genome base editing expression vector.

Purified APOBEC1 (obtained in step a), specific DNA single strand binding protein (obtained in step b) and plasmid backbone (obtained in step c) were purified as 0.03 pmol: 0.03 pmol: 0.03pmol was added to the Gibson Assembly reaction system, 15. mu.L of Gibson Assembly mix was added, and ddH was added₂O makes the final reaction system 20 μ L; incubate at 50 ℃ for 60 min. Transformation of e.coli DH5 α competent cells by heat shock; evenly coating on LB solid culture medium of 100 mug/mL ampicillin, and carrying out inverted culture at 37 ℃ for 16 h; and selecting positive clones, and extracting plasmids, namely the genome base editing expression vector. The expression vector for Saccharomyces cerevisiae is shown in FIG. 2, the expression vector for Pichia pastoris is shown in FIG. 3, the expression vector for Escherichia coli is shown in FIG. 4, and the expression vector for Ralstonia is shown in FIG. 5.

2. Functional verification of genomic base editing system

The genomic base editing process is as follows: extracting genome base editing system expression plasmid and converting saccharomyces cerevisiae, culturing and screening to obtain a transformant;

a. extracting expression plasmids of a genome base editing system and a control group from E.coli, respectively transforming the expression plasmids into Saccharomyces cerevisiae BY4741, and screening the transformed Saccharomyces cerevisiae on an SCD-LEU solid culture medium;

b. selecting 3 single colonies on each solid culture medium, respectively inoculating the single colonies into an SCD-LEU liquid culture medium for culture, and carrying out subculture when the bacterial liquid grows to a stable stage;

c. continuously culturing the bacterial liquid for 2 generations until the bacterial liquid grows to a stable period, and measuring OD of each sample₆₆₀Value, and diluting the bacterial liquid to 10 per ml⁷Taking 1mL of bacterial liquid from each cell, wherein 5 mu L of bacterial liquid is diluted to 10 per mL⁵Coating the cells on 5 mu L of bacterial liquid to an SCD-LEU solid culture medium, centrifuging 995 mu L of bacterial liquid, then suspending the bacterial liquid in 100 mu L of sterilized water, coating the bacterial liquid on an SCD-LEU-ARG + canavanine solid culture medium, and culturing the inoculated culture medium in an incubator at 30 ℃ for 3-7 days;

d. the number of single colonies growing on the SCD-LEU and SCD-LEU-ARG + canavanine culture media of each sample is counted respectively, and the gene mutation rate is calculated.

e. Single colonies on the SCD-LEU-ARG + canavanine culture medium are picked, the CAN1 gene of the single colonies is sequenced, and the mutation type is verified.

f. After three replicates of the experiment, statistical analysis of the data was performed.

The experimental results are shown in fig. 6, the mutation rate of the CAN1 gene is significantly improved by the base editing system, and compared with the yAPOBEC, the yRFA1 and the yHCS1 are improved by 6 times, the yRFA2 and the yTOP1 are improved by 12 times, and the yRFA3 and the yPRI1 are respectively improved by 21 times and 25 times. Therefore, the system can be proved to be capable of obviously improving the mutation rate of the saccharomyces cerevisiae genome.

Application example 1: tolerance test for isobutanol by genomic mutant strains

A base editing system for increasing the mutation rate of a genome accelerates the directed evolution of a saccharomyces cerevisiae genome and improves the tolerance of the saccharomyces cerevisiae genome to isobutanol.

a. Taking each sample of 2.c (example 1, section 2. c) and continuously carrying out 2 generations of culture solution and growing stable culture solution, inoculating equal amount of culture solution to SCD-LEU liquid culture medium containing isobutanol with different concentrations for culture, wherein the concentration of isobutanol in the culture medium is respectively as follows: 0%, 1%, 2%, 3%, 4%, 5%;

b. measuring the OD of the bacterial liquid every 24h₆₆₀Comparing the growth of different strains in culture media containing different concentrations of isobutanol;

the experimental result shows that when the concentration of isobutanol in the culture medium is 0-2%, the growth rate of different strains is not different, and when the concentration is 3%, the growth rate of y415 is lower than that of other strains as shown in figure 7; when the concentration of isobutanol was 4%, as shown in fig. 7, the growth rates of the strains differed, wherein the fastest growth was yRFA2 and yrop 1, followed by yRFA3 and yrri 1, and the two grew more stably, while the growth rates of yRFA1, yHCS1 and yippobec were slower, and y415 growth was most limited; at 5% isobutanol, as shown in figure 7, strains yTOP1, yffa 3, yPRI1, and yHCS1 grew at higher rates, followed by yffa 2, whereas yappobec and yffa 1 grew more restricted, and y415 failed to grow at all. Taken together, the strains yTOP1, yRFA3 and yPRI1 were the best resistant to isobutanol, while the mutation rates of these three strains were relatively highest among the strains, assuming that in saccharomyces cerevisiae, a high mutation rate favors the evolution of isobutanol resistance.

Application example 2: tolerance test for isobutanol by genomic continuous evolution strains

a. According to the test result and the deduction of the application example 1, the yRFA3 bacterial liquid which has the highest mutation rate and is high in growth rate in the culture medium containing 5% of isobutanol is selected and inoculated to the culture medium containing higher concentration of isobutanol for subculture, and the inoculation amount is 1%.

b. Inoculation into medium containing higher concentrations of isobutanol was continued until the strain failed to grow, after continued evolution the yRFA3 strain tolerated isobutanol to a maximum concentration of 9%.

c. Preserving a strain liquid with highest tolerance to isobutanol, namely saccharomyces cerevisiae yRFA3 after continuous evolution of genome, separating a single strain on a solid culture medium, and simultaneously separating the transformed 2.a on the solid culture mediumRespectively selecting 3 single bacterial colonies of yRNA3 single bacterial strains without subculture, namely saccharomyces cerevisiae strains before and after continuous evolution of genome, inoculating the single bacterial colonies into a liquid culture medium containing isobutanol, setting the concentration of the isobutanol to be 0%, 3%, 6%, 8% and 9%, and measuring the OD (origin-to-destination) of the bacterial solution every 24h₆₆₀Value, comparing the tolerance and tolerance stability of saccharomyces cerevisiae to isobutanol before and after continuous evolution of the genome. 3 single colonies of y415 and yAPOBEC were inoculated simultaneously as controls.

The experimental results show that the control group y415 and the yAPOBEC can not survive in the culture medium with higher concentration of isobutanol; at isobutanol concentrations of 8-9%, the unexplained strain of yRFA3 failed to survive, as shown in fig. 8; the experimental group, the strain of yRFA 3' after continuous evolution, was tolerant and fast growing.

Application example 3: tolerance test for acetate by genomic mutant strains

A base editing system for increasing the mutation rate of a genome accelerates the directed evolution of a saccharomyces cerevisiae genome and improves the tolerance of the saccharomyces cerevisiae genome to acetate.

a. Taking each sample of 2.c (example 1, section 2. c) and continuously carrying out 2 generations of culture solution and growing stable culture solution, inoculating equal amount of culture solution to SCD-LEU liquid culture medium containing acetate with different concentrations for culture, wherein the concentrations of the acetate in the culture medium are respectively as follows: 2g/L, 4g/L, 6g/L, 8g/L, 10g/L and 12 g/L;

b. measuring the OD of the bacterial liquid every 24h₆₆₀Values, growth of different strains in media containing different concentrations of acetate were compared.

The experimental result shows that the growth rate of each strain is not obviously different in the culture medium with low-concentration acetate; at acetate concentrations in the medium of 6-8g/L, as shown in FIG. 9, the bacterial liquid concentrations of yHCS1, yRFA2, yRFA3, and yRFA1 were highest, with the growth rates of yPRI1, yAPOBEC, and yTOP1 being lower, while the growth onset of y415 was limited; when acetate concentration in the medium reached 10g/L, as shown in fig. 9, growth rates were highest for yfra 3, yfra 2, yRyFA1, and yHCS1, growth of yPRI1, yTOP1, and yappobec was inhibited, and y415 was completely inhibited; when the acetate concentration reached 12g/L, as shown in fig. 9, the best tolerated were yHCS1, yRFA2, and yRFA1, followed by yRFA3, and the growth of the other strains was significantly inhibited. Taken together, the strains yHCS1, yRFA2 and yRFA1 were the best resistant to acetate, while the mutation rates of these three strains were at moderate levels in each strain, suggesting that in saccharomyces cerevisiae, moderate mutation rates favor the evolution of acetate resistance.

Application example 4: effect of the mutant strains on the production of beta-carotene

A base editing system for increasing the mutation rate of a genome accelerates the directed evolution of the saccharomyces cerevisiae genome and improves the yield of intracellular beta-carotene of a strain.

a. Extracting expression plasmids of a genome base editing system and a control group from E.coli, respectively transforming the expression plasmids into Saccharomyces cerevisiae BY4741 with a genome integrated with beta-carotene synthetic genes (three exogenous genes of CrtE, CrtYB and CrtI), respectively inoculating the transformed strains into an SCD-LEU liquid culture medium for culture, and carrying out subculture when the bacterial liquid grows to a stable stage.

c. Continuously culturing the bacterial liquid for 2 generations until the bacterial liquid grows to a stable period, diluting the bacterial liquid to the same concentration, coating the diluted bacterial liquid on a solid culture medium of SCD-LEU, and culturing the culture medium in an incubator at 30 ℃ for 48 hours.

d. Single colonies with darker orange color were screened on SCD-LEU solid medium and transferred to SCD-LEU liquid medium for 48 h.

e. 1mL of the bacterial solution was taken from each sample, and the OD thereof was measured₆₆₀And extracting intracellular beta-carotene.

f. Standards of beta carotene were measured by HPLC and standard curves were prepared with mobile phases of 50% acetonitrile + 30% methanol + 20% isopropanol.

g. The absorbance peak at 450nm of each sample was measured by HPLC, and the yield of beta-carotene was calculated from the standard curve.

The experimental results are shown in FIG. 10, where the β -carotene yields of control y415 and yRFA1 strains were 104.71 and 108.94mg/L, the average yield of yRFA2 was 1.63 times that of p415, the average yield of yHCS1 was 1.87 times that of y415, and the average yield of yTOP1 was 2.42 times that of y 415. In conclusion, the yield of B-carotene was significantly improved by yTOP1, yHCS1 and yRFA2, and it is speculated that a moderate mutation rate in the genome favors the synthetic pathway for the evolution of carotene in Saccharomyces cerevisiae.

Experiments prove that the genome base editing system constructed by the invention is used for modifying the genome of the saccharomyces cerevisiae, has the advantages of simplicity, high efficiency and the like, and can be quickly and efficiently used for modifying the saccharomyces cerevisiae, so that the tolerance of the saccharomyces cerevisiae to organic reagents is improved, and the yield of target products is improved.

Application example 5: the base editing system is used for testing and applying different industrial microorganisms

A base editing system for increasing random mutation rate of genome and application thereof. The genome base editing system constructed by the invention has universality, can be used for continuous evolution of most industrial strains, and can obviously improve the robustness and the production performance of the industrial strains. The base editing system is used for carrying out system construction and function verification in pichia pastoris, escherichia coli and ralstonia.

a. The construction of a base editing system to increase the random mutation rate of genome in different industrial strains is the same as in example 1.

b. Testing of a class of base editing systems that increase random mutation rates of genomes in pichia pastoris: 30 mu L of a pichia pastoris (MT) bacterial solution transformed with a genome mutation system is inoculated into 3mL of SED culture medium, and 2 generations of culture are continuously carried out, wherein the inoculation amount of each passage is 1%. Respectively inoculating 1% of Pichia pastoris wild bacteria (WT) and evolved strains into SEM culture medium containing 7% of methanol, setting 3 times of each strain for repetition, culturing at 30 ℃ for 20h, and testing OD of bacterial liquid₆₀₀The value is obtained.

The results of the experiments are shown in FIG. 11, the OD of the cell of the Pichia pastoris genome mutant strain is 7% methanol concentration₆₀₀The value was 2.10 times that of the wild strain. The growth of pichia pastoris wild strains is inhibited, while the strains with mutated genomes have higher methanol tolerance.

c. Testing of a class of base editing systems that increase random mutation rates of genomes in ralstonia: inoculating 30 μ L of Ralstonia bacterium (MT) transformed with genome mutation system into 3mL of MM medium, and continuously culturing for 2 generationsThe amount of inoculation of (2) was 1%. Respectively inoculating 1% of wild type strain (WT) and evolved strain into inorganic culture medium MM, setting 3 repeats of each strain with 5g/L sodium formate as unique carbon source, culturing at 30 deg.C for 48 hr, and testing OD of bacterial liquid₆₀₀The value is obtained.

As shown in FIG. 12, the cell OD of the mutant strain of Ralstonia genome was found at a sodium formate concentration of 5g/L₆₀₀The value was 3.56 times that of the wild strain. The growth of wild bacteria of ralstonia is inhibited, while the strains with mutated genomes have higher tolerance of sodium formate.

d. Testing of a class of base editing systems that increase random mutation rates of genomes in E.coli: 30 mu L of the Escherichia coli (MT) transformed with the genome mutation system is inoculated into 3mL of LB culture medium, and the culture is carried out for 2 generations continuously, wherein the inoculum concentration of each passage is 1%. Respectively inoculating 1% of Escherichia coli wild bacteria (WT) and evolved strains into LB culture medium containing 0.1% of sodium acetate, setting 3 times of each strain, culturing at 37 deg.C for 48 hr, and testing OD of bacterial liquid₆₀₀The value is obtained.

As shown in FIG. 13, the OD of the cells of the E.coli genome mutant strain at 0.1% sodium acetate concentration was determined₆₀₀The value was 2.72 times that of the wild strain. The growth of E.coli wild-type bacteria is inhibited, whereas the genomically mutated strains have a higher methanol tolerance.

Experimental results show that the base editing system for increasing the random mutation rate of the genome improves the tolerance of strains such as pichia pastoris, escherichia coli, ralstonia sp and the like to organic reagents, and verifies the applicability of the base editing system for increasing the random mutation rate of the genome in most industrial strains.

Sequence listing

<110> Hangzhou international scientific center of Zhejiang university

<120> base editing system and base editing method for increasing mutation rate of genome

<160> 34

<170> SIPOSequenceListing 1.0

<210> 1

<211> 687

<212> DNA

<213> rat (Rattus norvegicus)

<400> 1

atgagctcag agactggccc agtggctgtg gaccccacat tgagacggcg gatcgagccc 60

catgagtttg aggtattctt cgatccgaga gagctccgca aggagacctg cctgctttac 120

gaaattaatt gggggggccg gcactccatt tggcgacata catcacagaa cactaacaag 180

cacgtcgaag tcaacttcat cgagaagttc acgacagaaa gatatttctg tccgaacaca 240

aggtgcagca ttacctggtt tctcagctgg agcccatgcg gcgaatgtag tagggccatc 300

actgaattcc tgtcaaggta tccccacgtc actctgttta tttacatcgc aaggctgtac 360

caccacgctg acccccgcaa tcgacaaggc ctgcgggatt tgatctcttc aggtgtgact 420

atccaaatta tgactgagca ggagtcagga tactgctgga gaaactttgt gaattatagc 480

ccgagtaatg aagcccactg gcctaggtat ccccatctgt gggtacgact gtacgttctt 540

gaactgtact gcatcatact gggcctgcct ccttgtctca acattctgag aaggaagcag 600

ccacagctga cattctttac catcgctctt cagtcttgtc attaccagcg actgccccca 660

cacattctct gggccaccgg gttgaaa 687

<210> 2

<211> 1866

<212> DNA

<213> Saccharomyces cerevisiae

<400> 2

atgagcagtg ttcaactttc gaggggcgat tttcatagca tcttcaccaa taagcaaagg 60

tacgataatc ccaccggtgg cgtttatcaa gtttataaca ccaggaaatc tgatggggct 120

aacagcaaca gaaagaattt gatcatgatt tccgatggta tttaccatat gaaggctctg 180

ttgagaaacc aagctgcatc caagttccag tcaatggaac tacaaagggg tgatatcatt 240

cgcgtgataa ttgcagaacc tgctattgtc agggaaagaa agaaatacgt tcttttagta 300

gatgactttg agttggtcca gtcgcgtgct gatatggtca accaaactag tacttttttg 360

gataactatt tctcagagca tccaaatgaa accttaaaag acgaagatat aactgacagt 420

ggtaatgttg ccaatcaaac aaacgccagc aatgctggtg tccctgatat gctgcattca 480

aactcaaact tgaatgcaaa tgagagaaaa ttcgccaatg aaaaccctaa ttcgcaaaaa 540

accagaccaa tttttgccat cgaacaactg tctccatacc aaaacgtttg gactatcaaa 600

gcaagagttt cctacaaggg agaaattaaa acgtggcaca atcaaagagg tgatggtaaa 660

ctattcaatg tcaacttctt ggatacctct ggagaaatcc gagccacggc gtttaatgat 720

tttgctacaa aatttaacga aattttacaa gaaggcaaag tatactatgt atcaaaggca 780

aaactccaac cagctaagcc ccaatttact aatctaacac acccttatga actgaatttg 840

gatagagaca ctgttataga agaatgtttc gatgaaagta atgttccgaa aacccatttc 900

aatttcatca aactagatgc tattcagaac caggaagtaa attccaacgt agacgtcctc 960

ggtattatcc aaactataaa cccacatttt gagctaactt caagggctgg gaagaaattc 1020

gatcgtcgtg acatcacaat tgttgacgac tctgggtttt ctatctctgt tggcctatgg 1080

aatcagcaag cccttgattt caaccttcct gaaggttctg ttgctgccat taaaggtgtt 1140

cgtgtgacgg attttggtgg caaatctttg tctatgggat tttctagtac cctgattccg 1200

aatccagaaa ttcctgaggc atatgcctta aagggttggt atgattccaa gggccgcaac 1260

gcaaacttca tcactttaaa gcaagaaccc ggtatgggtg gtcaatcggc tgctagctta 1320

acaaaattca ttgctcagcg tattactatt gctagagctc aagctgaaaa tctaggaaga 1380

agcgagaaag gtgacttttt tagtgttaaa gctgctataa gtttcttaaa agttgataat 1440

tttgcatatc ctgcctgttc taatgagaat tgtaataaga aagttctgga acagcctgat 1500

ggtacttgga gatgtgagaa gtgcgacacc aataatgcaa ggccaaattg gagatacatc 1560

ttgacaatat caattattga cgaaaccaat caactatggc tcactttatt tgacgaccaa 1620

gctaaacaat tattgggtgt tgatgctaat acattaatgt ctttgaagga agaagacccc 1680

aacgaattca caaaaattac tcaaagtatc caaatgaacg aatatgactt taggattaga 1740

gcgcgtgagg atacatacaa tgatcaaagc agaattagat ataccgttgc taacctacac 1800

agcttgaatt acagggctga agccgactat cttgccgatg agttatccaa ggctttgtta 1860

gcttaa 1866

<210> 3

<211> 930

<212> DNA

<213> Saccharomyces cerevisiae

<400> 3

atggcaagta tgtattagtg ctaggaattg gaggatttgc tcttttctgc atcatttttt 60

ttatttttat tggcgaatgt taaaattact aacaatggat gggattccaa tacagcctat 120

caaccatata acgaatattc atcagtaacg ggcggtggct ttgagaactc tgagtcccgc 180

ccaggtagtg gggagtcgga aactaacact agagttaaca ccttgacacc tgtgacgatc 240

aaacaaattc tagagtccaa acaggatatt caggacggcc ccttcgtttc gcataaccaa 300

gaacttcatc acgtttgttt tgtaggtgtg gtgagaaaca ttacagacca tactgcaaat 360

atttttttaa ctattgagga tggaactggt caaatagaag tgagaaaatg gagcgaagat 420

gcaaatgact tggctgccgg taacgatgac tcttctggta aaggttatgg ttcgcaagtc 480

gcccaacaat ttgaaattgg cggttacgta aaagtttttg gtgctttgaa agagtttggt 540

ggtaagaaaa atatacagta tgcggtgatt aagcccatag attcattcaa tgaagtgttg 600

acgcatcact tggaagtcat caaatgtcat tccatagcca gtggaatgat gaaacaacct 660

ttggagagtg catccaacaa caatgggcaa tcattatttg tcaaggatga taacgataca 720

tcttccggct ccagtccgtt acaaagaatt ctagaatttt gtaagaagca atgtgagggc 780

aaagacgcta attcattcgc tgttcccatt ccattgatct cgcaatcctt gaatttggat 840

gaaactaccg tcagaaactg ctgtacgacc ttgactgacc agggttttat ctacccaact 900

tttgatgaca ataacttctt tgccctatga 930

<210> 4

<211> 369

<212> DNA

<213> Saccharomyces cerevisiae

<400> 4

atggccagcg aaacaccaag agttgacccc acagaaatct ccaacgtcaa tgctcctgtg 60

tttaggataa tagcgcaaat caaatcacaa cccactgaat ctcagttgat tttacaatcg 120

ccaaccatat catcgaaaaa cggcagcgag gttgaaatga ttacgttgaa caatattcgt 180

gtatctatga ataaaacatt tgagatcgac tcgtggtatg agtttgtttg caggaacaat 240

gatgacggcg agctaggatt tttgattcta gacgctgtac tatgcaaatt caaggagaac 300

gaagatttaa gcttaaacgg tgtggttgct ttacagagac tatgtaagaa atacccagaa 360

atatactag 369

<210> 5

<211> 1230

<212> DNA

<213> Saccharomyces cerevisiae

<400> 5

atgaccaatt cagtaaagac taatgggcca agctcctcag acatggagta ctactataag 60

tcgctctatc cattcaaaca cattttcaac tggctgaatc attcgcccaa accttctagg 120

gatatgatta acagagaatt cgctatggcg ttcaggtcag gtgcttacaa aaggtataat 180

tcttttaact ctgtgcaaga tttcaaagca caaatagaaa aagccaaccc agacagattt 240

gaaatcggtg ccatatataa caaaccgcca agagaacgtg atactctatt aaaaagcgaa 300

ttgaaagctt tagagaagga gttggtgttt gatattgata tggatgatta cgatgccttc 360

agaacgtgtt gctccggagc ccaagtttgc tctaagtgct ggaaatttat atctttggca 420

atgaaaatta cgaacacggc cctaagagag gattttggtt ataaggactt catctgggta 480

ttttcaggta gacgtggtgc tcattgttgg gtaagcgaca aacgggcacg ggctttgacg 540

gatgtacaac gaagaaacgt tttagattat gtcaacgtga ttagagacag aaatactgac 600

aagcgattgg ctttgaagag gccttatcac cctcatcttg ctcgttcttt ggagcaattg 660

aaacctttct ttgtcagcat tatgctcgaa gagcaaaatc cttgggaaga tgaccaacat 720

gctatccaaa ctttattgcc tgcattatat gataaacaat tgattgattc attaaagaag 780

tattggctgg acaatccaag gaggtcaagc aaagagaagt ggaatgatat agatcagata 840

gctacatcgc tcttcaaagg ccccaagcaa gactctcaca taattaagtt acgtgaatgt 900

aaggaagatc tcgtattgat gactctttat ccgaagctgg atgtggaagt tacaaagcaa 960

acaattcatt tgttaaaggc ccctttttgt attcatcctg ctacggggaa tgtctgtgtg 1020

cctattgatg aatcctttgc acctgaaaaa gcacctaagc taattgatct tcaaacagaa 1080

atggagaaaa ataatgatgt ttcattaaca gctttacaac cttttatcaa tcagttccaa 1140

gcatatgtga gttctctttt gaaaaatgaa ctgggttcag tgaaaagaga acgtgaagat 1200

gatgatgaac cggcttcttt agatttctga 1230

<210> 6

<211> 2052

<212> DNA

<213> Saccharomyces cerevisiae

<400> 6

atgaacaaag aattggcttc taagttttta tcaagcatca agcatgaacg tgaacaagac 60

attcaaacaa cttctaggct attaacaacg ctatctatcc aacaattggt gcaaaatggc 120

ttggcaataa acaacataca tctagaaaac ataagatccg gtctcattgg caaattatat 180

atggaactgg gacctaactt ggcggttaac gacaaaattc aaaggggtga tattaaggtt 240

ggtgatattg ttttggtacg ccctgcaaaa accaaagtaa ataccaagac taagcccaaa 300

gtcaaaaagg tgtctgaaga ctccaatggc gagcaagcgg aatgctcagg cgttgtctac 360

aaaatgagtg atactcaaat caccatagct ctagaagaat ctcaagatgt tattgctacc 420

acattttatt cttatagcaa actttacatt ttaaagacta ccaatgtcgt cacgtataat 480

agaatggaat ccacaatgag aaaactgtct gaaattagtt cacccataca agacaaaatt 540

atacaatact tggtaaacga acgccccttc atccccaata caaacagctt tcaaaacatt 600

aaatcttttt taaacccgaa tctgaatgac tcccagaaaa ctgccattaa ttttgccatt 660

aacaatgact tgaccattat acatggtcct cctggtacgg gtaaaacatt cacattaatt 720

gaattgatcc agcaattgct aattaaaaat cctgaggaga gaatcttaat ttgtgggcct 780

tccaatattt ctgtggatac gattctggag aggctaacgc ctcttgtgcc gaataattta 840

ttattaagaa tcggtcatcc tgctaggcta ttagactcta ataaaagaca ctctcttgat 900

atacttagta aaaagaatac tattgtgaag gacatttccc aggagattga caaattaatt 960

caggagaata aaaaactaaa aaactataag caacgtaaag aaaactggaa cgaaattaaa 1020

ctgctgcgca aagatttaaa gaaaagagag ttcaaaacca ttaaggactt aataatacaa 1080

tcgagaatag tcgtcaccac tttacacggt tcatcatcga gagaactatg ctccctttat 1140

agagatgatc caaatttcca gcttttcgat accttgatca tcgatgaagt ctcacaggcc 1200

atggaaccac aatgctggat cccactaatt gcacatcaaa atcagttcca caaactagtc 1260

cttgctggtg acaataaaca attgccaccc acaatcaaaa cagaagacga caaaaatgtc 1320

attcacaatt tggaaaccac actatttgac agaataatca aaatattccc gaaaagggat 1380

atggtaaaat ttcttaacgt tcaatacagg atgaatcaaa aaattatgga atttccatcg 1440

cactcaatgt ataatgggaa actattggcc gatgcaacgg tcgcgaacag acttttgata 1500

gacctaccga ccgtcgatgc tacgccatct gaggatgatg acgatacaaa aattccttta 1560

atttggtatg atacgcaagg tgatgaattt caagagactg cagatgaagc tactatcctt 1620

ggatctaagt ataatgaggg cgaaattgcc attgtaaaag aacacattga gaatttaagg 1680

tcattcaacg tcccggagaa ctctataggt gttatttctc catacaatgc acaagtttct 1740

catctgaaaa aattgatcca tgatgaatta aaattaactg atattgaaat atcaactgta 1800

gatgggttcc agggccgtga aaaagatgtt atcatattga gtttagttcg tagcaatgaa 1860

aaatttgaag ttggtttcct taaggaagaa cgaagactga acgtcgccat gacaagaccc 1920

agaaggcaac tagttgttgt tggcaatata gaagttctgc aaaggtgcgg taacaagtac 1980

ctaaaaagtt ggtcagaatg gtgtgaagag aacgctgacg taaggtaccc caacattgac 2040

gattatttgt aa 2052

<210> 7

<211> 2310

<212> DNA

<213> Saccharomyces cerevisiae

<400> 7

atgactattg ctgatgcttc caaagttaat catgagttgt cttctgatga cgatgacgat 60

gtgccattat ctcaaacttt aaaaaaaaga aaggtggcgt ccatgaactc tgcctctctt 120

caagacgaag cggaacctta tgatagtgat gaggcaatct ctaagatttc caagaaaaag 180

actaagaaaa taaagaccga accagtgcaa tcgtcgtcat taccatcgcc tccagcaaag 240

aaaagcgcga catcaaagcc taaaaaaatc aagaaagaag atggtgatgt aaaggtaaaa 300

acaactaaaa aggaagaaca ggagaacgaa aaaaagaaac gagaagaaga agaagaggag 360

gacaagaaag cgaaggagga ggaggaagaa tataaatggt gggaaaaaga aaacgaagat 420

gacaccataa aatgggtcac actgaagcat aacggtgtta tattccctcc accataccag 480

cccttaccat ctcacatcaa attatattac gatgggaagc cagtagattt acctccgcaa 540

gctgaagaag tagccgggtt ctttgctgcc ctattagaga gtgatcatgc caaaaatcct 600

gttttccaaa agaacttctt caatgatttc ttgcaagtac tgaaagaaag tggtggtccc 660

ctcaatggaa ttgagataaa ggaattttct cgttgcgatt tcaccaaaat gtttgattac 720

ttccagttac aaaaagaaca gaaaaagcaa ctgacttccc aagaaaagaa acagattcgt 780

ttggaaagag aaaaattcga ggaagattat aaattctgtg aattagatgg cagaagggaa 840

caagtaggga atttcaaggt tgaacctcct gatctattta gaggtcgtgg cgctcaccca 900

aaaacaggca aattgaagag aagagtgaat cctgaggata tcgttttaaa tctaagtaaa 960

gacgcacccg ttccgccagc cccagaaggg cacaagtggg gtgaaatcag acacgacaat 1020

accgttcaat ggttagccat gtggagagag aatattttca actcattcaa atacgtcaga 1080

ttggcagcga actcttcatt gaagggtcaa agtgactaca agaagtttga aaaggcgaga 1140

caattgaaat cctatatcga tgccatcaga agggattaca cgagaaattt gaaaagcaaa 1200

gttatgctag agcgccaaaa ggccgtagcc atttatttga tcgatgtatt cgctttaaga 1260

gccggtggtg aaaaatccga agatgaagcc gatactgtgg gttgttgttc attgcgatat 1320

gagcatgtta ctttgaaacc tccgaatact gttatctttg atttcttagg taaggattct 1380

attagatttt atcaagaggt agaagttgac aaacaagttt tcaaaaattt gacaattttt 1440

aaaaggccgc ccaaacagcc aggacatcaa ctgtttgatc gtctagatcc atctatactg 1500

aacaaatatc tacaaaacta catgccggga ttgactgcta aagttttccg tacatataat 1560

gcttccaaaa caatgcaaga tcaactggat ctaattccaa ataaaggatc tgtcgcagag 1620

aaaatattga agtacaacgc agcaaataga actgtagcca tcctatgtaa ccatcaaagg 1680

actgtcacga aggggcatgc acaaacagtg gaaaaggcca ataatagaat acaagagttg 1740

gaatggcaaa agattcgttg caagagggcc attttacaat tggataagga tcttttaaag 1800

aaagagccaa aatatttcga agaaatcgac gatttgacga aagaagatga agccaccatt 1860

cacaagagaa ttattgatag agaaattgaa aaatatcagc gaaaatttgt tagggagaac 1920

gataagagga aatttgaaaa ggaagaatta ttgccggaaa gtcaattgaa ggaatggttg 1980

gagaaagtcg acgaaaagaa acaagaattc gaaaaggaat tgaaaaccgg tgaagtggaa 2040

ctgaaatcaa gttggaattc agtcgaaaaa ataaaagcac aagtagagaa attagaacag 2100

cgtatccaaa ctagttccat tcagttgaaa gataaagagg aaaactccca ggtttcactg 2160

ggcacttcca aaatcaatta tatagaccct agactttctg tggtattttg caaaaagtat 2220

gatgttccga ttgaaaagat ttttacaaaa accctaagag aaaaattcaa atgggccata 2280

gaatcggtag atgaaaattg gaggttttaa 2310

<210> 8

<211> 1866

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 8

atgacagagt tcagtaaagg tagtttggtt gaaatcttcc agaagggcta taaaggtgga 60

ctaaaacctt tgacggttca agttttgaat ttgaaggcaa ttcccaacaa tacgggcaaa 120

aggcttcgac tcgccctttg tgatggtttg tataatgcca acgctgttat taggcctgaa 180

tcggtcgaaa aggccgaagc tcaaggaatc aagaagggaa gcattgtcca attgctcgag 240

tacaaagctt caatgataag tcctgtcaaa cacgtcttga tcatcgataa tcttcaagtt 300

ttgggatttc aagaggagaa aatcaaccca tccccaacta gtgtagacca atatttcagc 360

aaccactctg gagaaagcaa cgaagacttg ttgggcacct caatgaattc tccagcgcct 420

caagagcctg cgcagaaagc gcaatcacat caccaagaag atgcaaagcc aaaactgtca 480

gcacaggtta cgtccaaacc ccaacagacc aattcaagta ccgctaaatt tccaaatatc 540

catgcaattg atcaactaaa cccctatcag aataattgga ccattaaggc tcgggtttct 600

tataaatctg atatgagaaa atggtccaac cagaggggtg aaggtcagct cttcaatgtg 660

aatcttttag atgaaactaa tgaaatcaga gcaactgctt tcaacgatgt tgcagataaa 720

tattacgacc tcctacaaga gggcaaggtt tactacatca gcaaagcacg tattcagcca 780

gcaaaacctc aattttccaa ccttacccat acttacgaat tagctttgga tcgggacact 840

cagatcatcg aagcagagga tgcatctgat gtcccttctc ttcattttaa ttttgttaaa 900

ctcaacaagg tacaggatct tgacgcaaat gccattattg acgttattgg tgtcatcaaa 960

gtggtgaatc ctgctttcca aatcgtggcc aagtcaaccg gaagaccatt tgacaggaga 1020

gatattgaag ttgtagataa tactggattt gctataactg tagggttgtg gaataacact 1080

gctcttgagt ttgacattcc tgttggatca gtagttgcgt ttaaaggtgc taaagttcaa 1140

gattttggtg gcagaagtct ttcattgact caatcagcta ccataatcac taatccagac 1200

tctccagagg cttatcagtt gaaagcatgg tatgatcaac aaggtggctc caatcaggag 1260

tttaagtccc tgaaaaacga agtatcttcc aacagtggtt tgaatacgaa acaagacatt 1320

cagtcccgca aaaccatttt gcaagctcag tctgaagaac tcggtaagaa tgacaagccg 1380

gactatttct ccattaaagc ttacatcagt tatattagga cagaaaactt ttcgtatcca 1440

gcttgtgcat ccgagggttg caatagaaaa gtcatccaac agagtgacga tacttggagg 1500

tgtgaaaagt gtgatgtcaa ccaccccaaa ccgaaccatc gttatatatt gacattgtct 1560

gtggtagacc acacaggaca actatgggtc acattatttg acgaccaagc acaacagctc 1620

ttgggacaat cagctggtga attgatcgac ttgaaagaga atgacatgtc tgaaaataac 1680

catgcattcc agcaagtatt caacaggata caaatgaagg agttttcgtt cagagtaaag 1740

gcatctccgg attcttacaa gggtcaaacc cgtattcggt ataatgccgt gtctctggcc 1800

aaactggatt tcgctttgga agcagatgca ttggcagatt attttgaggg tgtcaaggtt 1860

aattaa 1866

<210> 9

<211> 537

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 9

atggccagca gaggcgtaaa caaggttatt ctcgttggta atctgggtca ggacccggaa 60

gtacgctaca tgccaaatgg tggcgcagtt gccaacatta cgctggctac ttccgaatcc 120

tggcgtgata aagcgaccgg cgagatgaaa gaacagactg aatggcaccg cgttgtgctg 180

ttcggcaaac tggcagaagt ggcgagcgaa tatctgcgta aaggttctca ggtttatatc 240

gaaggtcagc tgcgtacccg taaatggacc gatcaatccg gtcaggatcg ctacaccaca 300

gaagtcgtgg tgaacgttgg cggcaccatg cagatgctgg gtggtcgtca gggtggtggc 360

gctccggcag gtggcaatat cggtggtggt cagccgcagg gcggttgggg tcagcctcag 420

cagccgcagg gtggcaatca gttcagcggc ggcgcgcagt ctcgcccgca gcagtccgct 480

ccggcagcgc cgtctaacga gccgccgatg gactttgatg atgacattcc gttctga 537

<210> 10

<211> 537

<212> DNA

<213> Ralstonia sp.)

<400> 10

gcatcggtca acaaagtcat tctcgtcggc aacctcggcg cagacccgga aacccgctac 60

ctgcccagcg gcgacgccgt gaccaatatc cgcctggcga ccaccgatcg ctacaaggac 120

aagcagagcg gcgagatgaa ggaggccacc gaatggcacc gcgtctcgtt cttcggcaag 180

atcgccgaaa tcgccggcca gtacctgcgc aagggatcgt cggtctatat cgaaggccgc 240

atccgcaccc gcaagtggca ggaccagtcg ggccaggaca agtactccac tgaaatcgtt 300

gccgaccaga tgcagatgct gggctcgcgc cagggtggtg gcggcggtgg cggtgacgaa 360

ggcggctacg gtggcggcgc aggcggcggt ggtggctaca gccgcgaagc gtcgggcggc 420

ggctacggcg gtggccgcgg cggccagggc ggcggccaga gcggtggcgc tgcgcgccgg 480

ccgcagcagc ccgcctcgaa tggtttcgag gatatggacg acgatatccc gttctga 537

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgagctcag agactggccc a 21

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggatccacca gaagaaccac cagaactttc gg 32

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tttttgtaga aatgtcttgg t 21

<210> 14

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atagttgttc aattgattga a 21

<210> 15

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gaatactgtt tcctgtgtga a 21

<210> 16

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gccaccgctg agcaataact a 21

<210> 17

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ccgaaagttc tggtggttct tctggtggat ccatgagcag tgttcaactt tcg 53

<210> 18

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ataaatcata agaaattcgc ctcggtttct cgagttaagc taacaaagcc ttggataac 59

<210> 19

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(5)

<223> n is a, t, c or g.

<400> 19

nnnnnggatc catggcaacc tatcaaccat ataacgaata ttc 43

<210> 20

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(5)

<223> n is a, t, c or g.

<400> 20

nnnnnctcga gtcatagggc aaagaagtta ttgtc 35

<210> 21

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(5)

<223> n is a, t, c or g.

<400> 21

nnnnnggatc catggccagc gaaacaccaa g 31

<210> 22

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(5)

<223> n is a, t, c or g.

<400> 22

nnnnnctcga gctagtatat ttctgggtat ttcttacata g 41

<210> 23

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ccgaaagttc tggtggttct tctggtggat ccatgaccaa ttcagtaaag act 53

<210> 24

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

aatcataaat cataagaaat tcgcctcggt ttctcgagtc agaaatctaa agaagccgg 59

<210> 25

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ccgaaagttc tggtggttct tctggtggat ccatgaacaa agaattggct tct 53

<210> 26

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cataaatcat aagaaattcg cctcggtttc tcgagttaca aataatcgtc aatgtt 56

<210> 27

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccgaaagttc tggtggttct tctggtggat ccatgactat tgctgatgct tcc 53

<210> 28

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cataaatcat aagaaattcg cctcggtttc tcgagttaaa acctccaatt ttcatc 56

<210> 29

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ccgaaagttc tggtggttct tctggtggat ccatggccag cagaggcgta aac 53

<210> 30

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cataaatcat aagaaattcg cctcggtttc tcgagtcaga acggaatgtc atcatc 56

<210> 31

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccgaaagttc tggtggttct tctggtggat ccatgacaga gttcagtaaa ggt 53

<210> 32

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

cataaatcat aagaaattcg cctcggtttc tcgagttaat taaccttgac accctc 56

<210> 33

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ccgaaagttc tggtggttct tctggtggat ccgcatcggt caacaaagtc att 53

<210> 34

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

cataaatcat aagaaattcg cctcggtttc tcgagtcaga acgggatatc gtcgtc 56