CN117778440A - Gene editing system of candida viscidosa, gene editing method and application thereof - Google Patents
Gene editing system of candida viscidosa, gene editing method and application thereof Download PDFInfo
- Publication number
- CN117778440A CN117778440A CN202211201273.3A CN202211201273A CN117778440A CN 117778440 A CN117778440 A CN 117778440A CN 202211201273 A CN202211201273 A CN 202211201273A CN 117778440 A CN117778440 A CN 117778440A
- Authority
- CN
- China
- Prior art keywords
- gene
- segment
- editing
- candida
- gene editing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000010362 genome editing Methods 0.000 title claims abstract description 239
- 241000222120 Candida <Saccharomycetales> Species 0.000 title claims abstract description 159
- 238000000034 method Methods 0.000 title claims description 41
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 250
- 238000012216 screening Methods 0.000 claims abstract description 90
- 239000012634 fragment Substances 0.000 claims abstract description 80
- 230000014509 gene expression Effects 0.000 claims abstract description 68
- 108091033409 CRISPR Proteins 0.000 claims abstract description 48
- 210000000349 chromosome Anatomy 0.000 claims abstract description 46
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 45
- 102000053642 Catalytic RNA Human genes 0.000 claims abstract description 42
- 108090000994 Catalytic RNA Proteins 0.000 claims abstract description 42
- 108091092562 ribozyme Proteins 0.000 claims abstract description 42
- 230000008685 targeting Effects 0.000 claims abstract description 28
- 230000004807 localization Effects 0.000 claims abstract description 7
- TVIDDXQYHWJXFK-UHFFFAOYSA-N dodecanedioic acid Chemical compound OC(=O)CCCCCCCCCCC(O)=O TVIDDXQYHWJXFK-UHFFFAOYSA-N 0.000 claims description 226
- 238000000855 fermentation Methods 0.000 claims description 29
- 230000004151 fermentation Effects 0.000 claims description 29
- SNRUBQQJIBEYMU-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 23
- 210000004027 cell Anatomy 0.000 claims description 20
- 101150092742 FAO2 gene Proteins 0.000 claims description 14
- 230000006798 recombination Effects 0.000 claims description 14
- 238000005215 recombination Methods 0.000 claims description 14
- QFGCFKJIPBRJGM-UHFFFAOYSA-N 12-[(2-methylpropan-2-yl)oxy]-12-oxododecanoic acid Chemical compound CC(C)(C)OC(=O)CCCCCCCCCCC(O)=O QFGCFKJIPBRJGM-UHFFFAOYSA-N 0.000 claims description 12
- 101150059359 POX2 gene Proteins 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 8
- 230000006801 homologous recombination Effects 0.000 claims description 8
- 238000002744 homologous recombination Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- 241001661539 Kregervanrija fluxuum Species 0.000 claims description 5
- 239000011159 matrix material Substances 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- 210000004899 c-terminal region Anatomy 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 241000222157 Candida viswanathii Species 0.000 claims description 3
- 101150117587 pos5 gene Proteins 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 description 45
- 230000003197 catalytic effect Effects 0.000 description 28
- 102000004190 Enzymes Human genes 0.000 description 27
- 108090000790 Enzymes Proteins 0.000 description 27
- 238000010586 diagram Methods 0.000 description 23
- 238000010276 construction Methods 0.000 description 19
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 15
- 239000000047 product Substances 0.000 description 14
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 11
- 101710198130 NADPH-cytochrome P450 reductase Proteins 0.000 description 11
- 230000037361 pathway Effects 0.000 description 11
- 238000007254 oxidation reaction Methods 0.000 description 10
- 101100168397 Debaryomyces hansenii CYP52A13 gene Proteins 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 101100173277 Lotus japonicus FAO2 gene Proteins 0.000 description 7
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 7
- 101100137242 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) POS5 gene Proteins 0.000 description 7
- 230000037353 metabolic pathway Effects 0.000 description 7
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 241000724709 Hepatitis delta virus Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 230000012010 growth Effects 0.000 description 6
- 230000003647 oxidation Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 208000037262 Hepatitis delta Diseases 0.000 description 5
- 101000579123 Homo sapiens Phosphoglycerate kinase 1 Proteins 0.000 description 5
- KJWZYMMLVHIVSU-IYCNHOCDSA-N PGK1 Chemical compound CCCCC[C@H](O)\C=C\[C@@H]1[C@@H](CCCCCCC(O)=O)C(=O)CC1=O KJWZYMMLVHIVSU-IYCNHOCDSA-N 0.000 description 5
- 102100028251 Phosphoglycerate kinase 1 Human genes 0.000 description 5
- 150000001335 aliphatic alkanes Chemical class 0.000 description 5
- 208000029570 hepatitis D virus infection Diseases 0.000 description 5
- 239000013612 plasmid Substances 0.000 description 5
- 239000006227 byproduct Substances 0.000 description 4
- 101150038500 cas9 gene Proteins 0.000 description 4
- 230000004186 co-expression Effects 0.000 description 4
- LQZZUXJYWNFBMV-UHFFFAOYSA-N dodecan-1-ol Chemical compound CCCCCCCCCCCCO LQZZUXJYWNFBMV-UHFFFAOYSA-N 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 208000031448 Genomic Instability Diseases 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 238000004520 electroporation Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 2
- 101150028074 2 gene Proteins 0.000 description 2
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 2
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 2
- 101150095763 CYP52A13 gene Proteins 0.000 description 2
- 241000222173 Candida parapsilosis Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 101710140859 E3 ubiquitin ligase TRAF3IP2 Proteins 0.000 description 2
- 102100026620 E3 ubiquitin ligase TRAF3IP2 Human genes 0.000 description 2
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 2
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 2
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 2
- 101100084403 Homo sapiens PRODH gene Proteins 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 101150053659 POX4 gene Proteins 0.000 description 2
- 208000020584 Polyploidy Diseases 0.000 description 2
- 102100028772 Proline dehydrogenase 1, mitochondrial Human genes 0.000 description 2
- 241000251131 Sphyrna Species 0.000 description 2
- 101000910035 Streptococcus pyogenes serotype M1 CRISPR-associated endonuclease Cas9/Csn1 Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 101100029251 Zea mays PER2 gene Proteins 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 229940055022 candida parapsilosis Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 150000001991 dicarboxylic acids Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005805 hydroxylation reaction Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- LSKONYYRONEBKA-UHFFFAOYSA-N 2-Dodecanone Natural products CCCCCCCCCCC(C)=O LSKONYYRONEBKA-UHFFFAOYSA-N 0.000 description 1
- YDZIJQXINJLRLL-UHFFFAOYSA-N 2-hydroxydodecanoic acid Chemical compound CCCCCCCCCCC(O)C(O)=O YDZIJQXINJLRLL-UHFFFAOYSA-N 0.000 description 1
- 101150096316 5 gene Proteins 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- -1 CYP52a13 Chemical class 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 101710151348 D-3-phosphoglycerate dehydrogenase Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 101150040622 Dp gene Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000643374 Homo sapiens Serrate RNA effector molecule homolog Proteins 0.000 description 1
- 108030003884 NADH kinases Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000235343 Saccharomycetales Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000012824 chemical production Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- LXHHROBUZABRSD-UHFFFAOYSA-N dodecane Chemical compound CCCCCCCCCCCC.CCCCCCCCCCCC LXHHROBUZABRSD-UHFFFAOYSA-N 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 230000007457 establishment of nucleus localization Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 101150042777 flp gene Proteins 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 238000012269 metabolic engineering Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 235000018343 nutrient deficiency Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a candida viscidosa gene editing system, which comprises candida viscidosa, a first gene editing segment and a second gene editing segment. The first gene editing segment comprises a first homology arm and a screening gene. The second gene editing fragment comprises a second homology arm, a Cas9 expression cassette, and a sgRNA cassette. The Cas9 expression cassette comprises a Cas9 promoter, a Cas9 gene and a trinuclear localization sequence. The sgRNA cassette comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a scaffold sequence, and a second ribozyme. The first gene editing segment and the second gene editing segment form a linear segment for gene editing of the chromosome of candida viscidosa. Thus, each set of target chromosomes of candida viscidosa can be conveniently edited, and stable transformants can be obtained under no specific conditions.
Description
[ field of technology ]
The invention relates to a gene editing technology of microorganisms, in particular to a gene editing system of candida viscidosa and application thereof.
[ background Art ]
Candida viscidosa (Candida viswanathii) is a budding yeast that spontaneously converts long chain alkanes to the corresponding acids/diacids and grows by the beta-oxidation pathway as a carbon source for consumption. The genetically edited strain after metabolic engineering improvement is a promising cell factory in industrial application, for example, can block catalytic enzymes participating in related oxidative metabolic pathways, but simultaneously retain and/or strengthen key catalytic enzymes for converting alkanes into acids/diacids, so that the alkanes can be stably and continuously consumed, and the carbon flux in the body can be more intensively put into the production of acid/diacid chemicals.
However, the current genetic editing engineering of candida viscidosa is still carried out in a high-pressure selective homologous recombination mode, so that the recombination probability is low, only one gene can be edited in one genetic editing process, and the genetic editing engineering is also very limited by the size and editing position of the editing fragments which are removed/embedded, and the genetic editing engineering is very laborious and time-consuming. Furthermore, candida viscidosa is a diploid yeast with a plurality of homologous chromosomes, and due to the characteristic of quasistigmentation, the candida viscidosis is given an opportunity to gradually lose part of chromosomes in a plurality of mitosis and restore to original ploidy instead of meiosis in the process of reproduction, so that the deletion or recombination of editing fragments is caused, and the instability and the operation difficulty of the strain in gene editing engineering are greatly increased.
The CRISPR-Cas9 gene editing technology is widely applied to various types of organisms in recent years, and the accurate and traceless targeted editing technology of the chromosome overcomes the editing efficiency and the editing technology bottleneck faced by the related fields of biology. In contrast, although CRISPR-Cas9 gene editing technology has excellent cleavage capability, the Cas9 protein expression cassette and sgRNA for targeted guiding of Cas9 protein cleavage are quite large, and if other expression cassettes of exogenous genes to be embedded together are integrated together, the whole editing system is too large, and editing effect is affected. Furthermore, despite years and many improvements in the CRISPR-Cas9 system, it has not been successfully established in candida vista. Therefore, it is an important issue faced in the related art to establish a gene editing technique for efficiently editing genes in Candida vista and stably existing an editing target in a transformant.
[ invention ]
In view of the above, an object of the present invention is to provide a gene editing system and a gene editing method for candida vista, which overcome the difficulty in gene editing and the low recombination rate of candida vista having diploid by introducing a CRISPR-Cas9 system into candida vista, and by selecting a combination of a promoter, a first ribozyme and a second ribozyme and optimizing a recombination strategy of homologous arms. Furthermore, by using the gene editing system and the gene editing method of candida viscidosis, not only can the time course of gene editing be shortened, but also the success rate and the accuracy of gene editing are greatly improved, and gene editing such as rejecting, mutating, replacing or inserting fragments can be conveniently and rapidly realized without being limited by the size of the fragments or the inserting positions, so that stable transformants of candida viscidosis can be obtained.
Further, another object of the present invention is to provide a transformant for producing dodecanedioic acid and a method for producing dodecanedioic acid, by which a transformant for stably producing dodecanedioic acid having an exogenous gene in a chromosome thereof, which is a catalytic enzyme required for converting dodecane into dodecanedioic acid, can be edited by the gene editing system of candida vissii of the present invention. Furthermore, the transformant for producing dodecanedioic acid can continuously and efficiently ferment to produce the high-purity dodecanedioic acid, antibiotics or inducers are not required to be added in the fermentation process, and the fermentation product has little other byproducts except the dodecanedioic acid, so that the transformant is very suitable for mass industrialized production economically, can reduce the culture and purification cost of biological fermentation, and can achieve environmental-friendly recycling economy in the field of chemical production.
In one aspect, the present invention provides a gene editing system for candida viscidosa, comprising candida viscidosa, a first gene editing segment and a second gene editing segment. The first gene editing segment comprises a first homology arm and a screening gene which are arranged in sequence. The second gene editing segment is connected to the C end of the first gene editing segment, and the second gene editing segment comprises a second homology arm, a Cas9 expression cassette and a sgRNA cassette which are sequentially arranged, wherein the Cas9 expression cassette comprises a Cas9 promoter, a Cas9 gene and a trinuclear positioning sequence which are sequentially arranged, the sgRNA cassette comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a bracket sequence and a second ribozyme which are sequentially arranged, and the first gene editing segment and the second gene editing segment form a linear segment for gene editing of a chromosome of candida vinis disclosed. Wherein the first homology arm and the second homology arm correspond to a specific fragment of a gene on the chromosome of the candida viscidosa, respectively, and the targeting sequence corresponds to a specific sequence of the gene on the chromosome of the candida viscidosa.
According to the above-mentioned gene editing system of candida vista, the screening gene of the first gene editing segment may further comprise a first screening gene segment, the second gene editing segment may further comprise a second screening gene segment of the screening gene at the N-terminus of the second homology arm, and the first screening gene segment and the second screening gene segment may have homologous segments, respectively, through which the first gene editing segment and the second gene editing segment are recombinantly linked into the linear segment.
According to the above-mentioned gene editing system of candida viscidosa, the first gene editing segment and/or the second gene editing segment may further comprise at least one expression cassette, wherein the at least one expression cassette comprises a foreign gene promoter and a foreign gene.
Another aspect of the present invention is to provide a method for editing a gene of Candida viscidosa, comprising constructing a first gene editing segment, constructing a second gene editing segment, performing a transformation step, and performing a transformant culture step. The first gene editing segment comprises a first homology arm and a screening gene which are arranged in sequence. The second gene editing segment is connected to the C end of the first gene editing segment, and the second gene editing segment comprises a second homology arm, a Cas9 expression cassette and a sgRNA cassette which are sequentially arranged, wherein the Cas9 expression cassette comprises a Cas9 promoter, a Cas9 gene and a trinuclear positioning sequence which are sequentially arranged, the sgRNA cassette comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a bracket sequence and a second ribozyme which are sequentially arranged, the first gene editing segment and the second gene editing segment form a linear segment for carrying out gene editing on a chromosome of candida vinsis, and the first homology arm and the second homology arm respectively correspond to a specific segment of a gene on the chromosome of candida vinsis, and the targeting sequence corresponds to a specific sequence of the gene on the chromosome of candida vinsis. A transformation step is performed in which the first gene-editing fragment and the second gene-editing fragment are transformed into the Candida viscidosa to obtain a transformant. A transformant culturing step is performed in which the transformant is cultured in a screening culture at an editing temperature for an editing time, wherein the Cas9 expression cassette expresses Cas9 gene and the sgRNA cassette expresses a targeting sequence, the first homology arm and the second homology arm are homologous recombined with specific fragments, respectively, and the first gene editing fragment and the second gene editing fragment located at the portion between the first homology arm and the second homology arm are embedded in the gene of the transformant.
According to the above method for editing genes in candida viscidosis, the screening genes of the first gene editing segment may further comprise a first screening gene segment, the second gene editing segment may further comprise a second screening gene segment of the screening genes at the N-terminus of the second homology arm, the first screening gene segment and the second screening gene segment may have homologous segments, respectively, and the first gene editing segment and the second gene editing segment are connected into the linear segment by recombination of the homologous segments.
According to the above-mentioned method for editing genes in candida viscidosa, the first gene editing segment and/or the second gene editing segment may further comprise at least one expression cassette, wherein the at least one expression cassette comprises a foreign gene promoter and a foreign gene.
In still another aspect, the present invention provides a transformant for producing dodecanedioic acid, comprising a host cell and at least two exogenous genes. The host cell is candida one-dimensional (Candida viswanathii). The at least two exogenous genes comprise a CYP52A19 gene and a CPRb gene, wherein the at least two exogenous genes are embedded on a chromosome of the host cell using the gene editing system of Candida viscidosa as described in the preceding paragraph.
The dodecanedioic acid production transformant according to the foregoing, wherein the at least two exogenous genes may comprise the CYP52A18 gene.
The transformant producing dodecanedioic acid according to the above, wherein the at least two exogenous genes can be inserted into the POX2 gene of the chromosome of the host cell.
The dodecanedioic acid production transformant according to the foregoing, wherein the at least two exogenous genes may comprise a FAO2 gene.
The dodecanedioic acid production transformant according to the foregoing, wherein the at least two exogenous genes may comprise a POS5 gene.
In another aspect, the present invention provides a method for producing dodecanedioic acid, comprising providing a reaction substrate and performing a fermentation step. The reaction matrix comprises dodecane. A fermentation step is carried out by inoculating the dodecanedioic acid-producing transformant described in the preceding paragraph into a reaction substrate and culturing under a fermentation condition for a fermentation time to obtain a fermentation substance, wherein the fermentation substance comprises dodecanedioic acid.
[ description of the drawings ]
The foregoing and other objects, features, advantages and embodiments of the invention will be apparent from the following description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram showing a linear segment of a gene editing system of Candida viscidosa according to an embodiment of the present invention;
FIG. 2A is a schematic diagram showing a first gene editing segment and a second gene editing segment of a gene editing system of Candida vista according to another embodiment of the present invention;
FIG. 2B is a schematic diagram showing a first gene editing segment and a second gene editing segment of a gene editing system of Candida vista according to still another embodiment of the present invention;
FIG. 3 is a flowchart showing the steps of a method for editing genes in Candida viscidosa according to another aspect of the present invention;
FIG. 4 is a flow chart showing the steps of a method for producing dodecanedioic acid according to still another aspect of the present invention;
FIG. 5A is a schematic diagram of the construction of examples 1 to 3 of the combination of the first and second ribozymes of the screening sgRNA cassette of the present invention;
FIG. 5B is a graph of colony results of examples 1 to 3 of the present invention;
FIG. 5C is a graph of colony phenotype ratio results of FIG. 5B;
FIG. 5D is a graph of the results of an assay to verify example 2 of the present invention with colony PCR;
FIG. 5E is a graph of colony results from the nutrient deficiency test of example 2 of the invention;
FIG. 6A is a graph of colony results validating the optimized homologous recombination strategy of the present invention;
FIG. 6B is a graph of colony phenotype ratio results of FIG. 6A;
FIG. 6C is a graph of the results of analysis of colonies verifying the optimized homologous recombination strategy of the present invention by colony PCR;
FIG. 6D is a graph of the results of analysis of colony PCR to verify the stability of the transformants of the present invention;
FIG. 7 is a graph showing the results of analysis of the marker-free transformants of the present invention by colony PCR;
FIG. 8 shows a schematic diagram of the metabolic pathway for producing dodecanedioic acid in Candida dodecanone Yu Weisi;
FIG. 9A is a schematic diagram showing the construction of examples 4 to 22 for screening an expression cassette for a dodecanedioic acid producing catalytic enzyme according to the present invention;
FIG. 9B is a graph showing the results of the production of dodecanedioic acid by the shake flask method according to examples 4 to 22 of the present invention;
FIG. 10A is a schematic diagram showing the construction of examples 23 to 27 for screening embedded bits according to the present invention;
FIG. 10B is a graph showing the results of the production of dodecanedioic acid by the shake flask method according to examples 23 to 27 of the present invention;
FIG. 10C is a schematic diagram showing the construction of examples 26 and 28 to 33 for screening an expression cassette for a catalytic enzyme optimized for dodecanedioic acid production according to the present invention;
FIG. 10D is a graph showing the results of the shake flask production of dodecanedioic acid according to examples 26 and 28 to 33 of the present invention;
FIG. 10E is a graph showing the results of fermentation production of dodecanedioic acid by Candida viscidosa;
FIG. 10F is a graph showing the results of fermentation production of dodecanedioic acid by the P-19C strain of the present invention;
FIG. 11A is a schematic diagram showing the construction of examples 34 to 36 of the expression cassette for screening a catalytic enzyme for the medium chain alcohol oxidation pathway according to the present invention;
FIG. 11B is a schematic diagram showing the construction of examples 34 to 36 in Candida Visiae by the gene editing system of Candida Visiae of the present invention;
FIG. 11C is a graph showing the results of the production of dodecanedioic acid by the shake flask method according to examples 34 to 36 of the present invention;
FIG. 11D is a graph showing the results of fermentation production of dodecanedioic acid by the PF19C strain of the present invention;
FIG. 12A is a schematic diagram showing the construction of examples 37 to 41 of the expression cassette for screening NADPH-regenerated catalytic enzymes of the present invention;
FIG. 12B is a graph showing the results of the production of dodecanedioic acid by the shake flask method according to examples 37 to 41 of the present invention;
FIG. 13A is a schematic diagram showing construction of the PFP19C strain of the present invention;
FIG. 13B is a schematic diagram of the edited chromosome of the PFP19C strain of the present invention; and
FIG. 13C is a graph showing the results of fermentation production of dodecanedioic acid by the PFP19C strain of the present invention.
[ detailed description ] of the invention
[ Gene editing System of Candida Vissiana ]
The gene editing system of candida viscidosis of the present invention comprises candida viscidosis, a first gene editing segment 110 and a second gene editing segment 120, wherein the second gene editing segment 120 is connected to the C-terminal of the first gene editing segment 110, and the first gene editing segment 110 and the second gene editing segment 120 form a linear segment 100 for performing gene editing on a chromosome of candida viscidosis.
Referring to FIG. 1, a schematic diagram of a linear segment 100 of a gene editing system of Candida viscidosa according to an embodiment of the invention is shown. In fig. 1, a linear segment 100 comprises a first gene editing segment 110 and a second gene editing segment 120. The first gene editing segment 110 comprises a first homology arm 111 and a screening gene 112 in a sequential arrangement. The second gene editing segment 120 is connected to the C-terminal end of the first gene editing segment 110, and the second gene editing segment 120 comprises a second homology arm 121, a Cas9 expression cassette 130 and a sgRNA cassette 140 which are sequentially arranged, wherein the Cas9 expression cassette 130 comprises a Cas9 promoter 131, a Cas9 gene 132 and a trinuclear localization sequence 133 which are sequentially arranged, the sgRNA cassette 140 comprises a sgRNA promoter 141, a first ribozyme 142, a targeting sequence 143, a scaffold sequence 144 and a second ribozyme 145 which are sequentially arranged, and the first gene editing segment 110 and the second gene editing segment 120 form a linear segment 100 for gene editing of a chromosome of candida vista. Wherein the first homology arm 111 and the second homology arm 121 correspond to a specific fragment of a gene on the chromosome of candida viscidosa, respectively, and the targeting sequence 143 corresponds to a specific sequence of the gene on the chromosome of candida viscidosa.
Referring to FIG. 2A, a schematic diagram of a first gene editing segment 200 and a second gene editing segment 300 of a gene editing system of Candida vista according to another embodiment of the invention is shown. The first gene editing segment 200 comprises a first homology arm 210 and a first screening gene segment 230 of a screening gene (not numbered) in sequence. The second gene editing segment 300 comprises a second screening gene segment 310, a second homology arm 330, a Cas9 expression cassette 340 and a sgRNA cassette 350 of the screening genes which are sequentially arranged, wherein the Cas9 expression cassette 340 comprises a Cas9 promoter 341, a Cas9 gene 342 and a trinuclear localization sequence 343 which are sequentially arranged, the sgRNA cassette 350 comprises a sgRNA promoter 351, a first ribozyme 352, a targeting sequence 353, a scaffold sequence 354 and a second ribozyme 355 which are sequentially arranged, and the first gene editing segment 200 and the second gene editing segment 300 form a linear segment for gene editing of a chromosome of candida vista. The first homology arm 210 and the second homology arm 330 correspond to a specific fragment of a gene on the chromosome of candida viscidosa, respectively, and the targeting sequence 353 corresponds to a specific sequence of the gene on the chromosome of candida viscidosa.
Specifically, the screening gene (not numbered) of the first gene-editing segment 200 may further comprise a first screening gene segment 230, the second gene-editing segment 300 may further comprise a second screening gene segment 310 of the screening gene at the N-terminus of the second homology arm 330, and the first screening gene segment 230 and the second screening gene segment 310 may have a homology segment 231 and a homology segment 311, respectively, and the first gene-editing segment 200 and the second gene-editing segment 300 may be recombinantly connected into linear segments by the homology segment 231 and the homology segment 311.
In detailThe homologous segment 231 and the homologous segment 311 may be homologous sequences with a size of about 400bp, thereby improving the success rate and accuracy of the recombinant connection between the first gene editing segment 200 and the second gene editing segment 300, which is not limited in this disclosure. Furthermore, the sgRNA promoter 351 and Cas9 promoter 341 may be selected from the group consisting of TDH1 promoter (P TDH1 ) PGK1 promoter (P) PGK1 ) ACT1 promoter (P) ACT1 ) Or ADH1 promoter (P) ADH1 ) Preferably, sgRNA promoter 351 may be P TDH1 While Cas9 promoter 341 may be P PGK1 The disclosure is not limited thereto. Furthermore, wherein the first ribozyme 352 may be selected from Hammerhead (Ham) or tRNA Ala Preferably, the first ribozyme 352 may be a tRNA Ala The disclosure is not limited thereto. Further, the second ribozyme 355 may be selected from Hepatitis Delta Virus (HDV) or tRNA Gly Preferably, the second ribozyme 355 may be an HDV, which is not limited in this disclosure. In addition, the trinuclear localization sequence may be SV40NLS, which is not limited in this disclosure. The screening gene may be an antibiotic resistance gene, preferably, the screening gene may be anti-Noralserin (Nrs) R ) The genes, the disclosure is not limited thereto.
In addition, the Frt sequence 220 may be inserted upstream of the first selectable gene segment 230 and the Frt sequence 320 may be inserted downstream of the second selectable gene segment 310, so that subsequent de-labelling projects to reject the selectable gene may be performed as desired. Thus, a high-efficiency gene editing technique of Candida avermitilis can be achieved without being limited by the position of gene editing and the size of the gene editing fragment.
Referring to FIG. 2B, a schematic diagram of a first gene editing segment 200a and a second gene editing segment 300a of a gene editing system of Candida vista according to another embodiment of the invention is shown. In fig. 2B, a first gene-editing segment 200a and a second gene-editing segment 300a are similar to the first gene-editing segment 200 and the second gene-editing segment 300, except that the first gene-editing segment 200a comprises at least one expression cassette 240 and the second gene-editing segment 300a comprises at least one expression cassette 360. Other similar technical details are not described again.
The first gene editing segment 200a comprises, in order, a first homology arm 210a, at least one expression cassette 240, and a first screening gene segment 230a of a screening gene (not numbered). At least one expression cassette 240 comprises an exogenous gene promoter 241 and an exogenous gene 242.
The second gene-editing segment 300a comprises a second selectable gene segment 310a of the selectable gene, at least one expression cassette 360, a second homology arm 330a, a Cas9 expression cassette 340a, and an sgRNA cassette 350a, in sequence. At least one expression cassette 360 comprises an exogenous gene promoter 361 and an exogenous gene 362.Cas9 expression cassette 340a comprises Cas9 promoter 341a, cas9 gene 342a and trinuclear localization sequence 34a in a sequential order, sgRNA cassette 350a comprises sgRNA promoter 351a, first ribozyme 352a, targeting sequence 353a, scaffold sequence 354a and second ribozyme 355a in a sequential order, and first gene editing fragment 200a and second gene editing fragment 300a constitute a linear fragment for gene editing of a chromosome of candida vista. The first homology arm 210a and the second homology arm 330a correspond to a specific fragment of a gene on the chromosome of candida viscidosis, respectively, and the targeting sequence 353a corresponds to a specific sequence of the gene on the chromosome of candida viscidosis.
Specifically, the screening gene (not numbered) of the first gene editing segment 200a may further comprise a first screening gene segment 230a, the second gene editing segment 300a may further comprise a second screening gene segment 310a of the screening gene at the N-terminus of the second homology arm 330a, and the first screening gene segment 230a and the second screening gene segment 310a may have a homologous segment 231a and a homologous segment 311a, respectively, and the first gene editing segment 200a and the second gene editing segment 300a may be recombinantly linked into linear segments by the homologous segment 231a and the homologous segment 311 a.
Although the number of the at least one expression cassette is two in fig. 2B and is located in the first gene editing segment 200a and the second gene editing segment 300a, respectively, the number and the position of the at least one expression cassette can be adjusted according to the editing requirement in practical application, and the disclosure is not limited thereto.
In addition, the Frt sequence 220a may be inserted upstream of the first screening gene fragment 230a and the Frt sequence 320a may be inserted downstream of the second screening gene fragment 310a, so that a label removal process for deleting the screening gene may be performed later as needed.
[ Gene editing method of Candida Vissimae ]
Referring to FIG. 3, a flowchart of a method 400 for editing genes of Candida viscidosa according to another aspect of the present invention is shown. In fig. 3, the gene editing method 400 of candida viscidosis includes steps 410, 420, 430 and 440.
Step 410 is constructing a first gene editing segment. The first gene editing segment comprises first homology arms and screening genes which are arranged in sequence.
Step 420 is to construct a second gene editing segment. The second gene editing fragment is connected to the C end of the first gene editing fragment, and the second gene editing fragment comprises a second homology arm, a Cas9 expression cassette and a sgRNA cassette which are sequentially arranged, wherein the Cas9 expression cassette comprises a Cas9 promoter, a Cas9 gene and a trinuclear positioning sequence which are sequentially arranged, the sgRNA cassette comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a bracket sequence and a second ribozyme which are sequentially arranged, and the first gene editing fragment and the second editing fragment form a linear fragment for carrying out gene editing on a chromosome of candida visas. Wherein the first homology arm and the second homology arm correspond to a specific fragment of a gene on the chromosome of candida viscidosa, respectively, and the targeting sequence corresponds to a specific sequence of the gene on the chromosome of candida viscidosa.
Further, wherein the first gene editing segment and/or the second gene editing segment may comprise at least one expression cassette comprising a foreign gene promoter and a foreign gene. In practical application, the number and position of at least one expression cassette can be adjusted according to editing requirements. In addition, a Frt sequence can be inserted into each of the upstream of the first screening gene fragment and the downstream of the second screening gene fragment, so that the label removing engineering of removing the screening genes can be performed later according to the requirement.
Step 430 is a conversion step. The first gene-editing fragment and the second gene-editing fragment were transformed into candida viscidosa to obtain a transformant.
In detail, when the first homology arm and the second homology arm perform homologous recombination on the specific fragment of candida viscidosis, a fragment of about 1kb can be removed from the gene at the same time, so as to improve the efficiency of complete embedding, which is not limited in the present disclosure.
Step 440 is a transformant culturing step. Culturing the transformant in a screening culture at an editing temperature for an editing time, wherein the Cas9 expression cassette expresses Cas9 gene and the sgRNA cassette expresses the targeting sequence, the first homology arm and the second homology arm are homologous recombined with the specific fragment, respectively, and a first gene editing fragment and a second gene editing fragment located at a portion between the first homology arm and the second homology arm are embedded in the gene of the transformant.
Further, the screening gene of the first gene editing segment may further comprise a first screening gene segment, the second gene editing segment may further comprise a second screening gene segment of the screening gene at the N-terminus of the second homology arm, and the first screening gene segment and the second screening gene segment may have a homology segment, respectively, and the first gene editing segment and the second gene editing segment are connected into a linear segment by homologous segment recombination. That is, the first gene editing segment and the second gene editing segment may be the same linear segment and transformed into candida viscidosa cell for gene editing, or may be transformed into candida viscidosa cell for recombination connection, and the timing of using recombination connection is determined according to the editing requirement, which is not limited in the disclosure. Thus, a high-efficiency gene editing technique of Candida avermitilis can be achieved without being limited by the position of gene editing and the size of the gene editing fragment.
[ transformant for producing dodecanedioic acid ]
The dodecanedioic acid-producing transformant of the present invention comprises a host cell and at least two exogenous genes. Wherein the host cell is candida one-dimensional. The at least two exogenous genes comprise a CYP52A19 gene and a CPRb gene, wherein the at least two exogenous genes are embedded on the chromosome of the host cell using the gene editing system of candida viscidosis of the invention. Wherein the at least two exogenous genes are embedded in the chromosome of the host cell using the gene editing system of candida viscidosis of the present invention as shown in fig. 2B.
Further, the at least exogenous gene may include a CYP52A18 gene, a FAO2 gene, and a POS5 gene, whereby the production rate, purity, and molar conversion of dodecanedioic acid may be increased. Furthermore, the at least exogenous gene may be embedded in a POX2 gene of a chromosome of the host cell.
[ method for producing dodecanedioic acid ]
Referring to FIG. 4, a flow chart of steps of a method 500 for producing dodecanedioic acid according to still another aspect of the present invention is shown. In fig. 4, a method 500 of producing dodecanedioic acid comprises steps 510 and 520.
Step 510 provides a reaction matrix comprising dodecane.
Step 520 is a fermentation step of inoculating the dodecanedioic acid producing transformant of the present invention into the reaction substrate and culturing under a fermentation condition for a fermentation time to obtain a fermentation material, wherein the fermentation material comprises dodecanedioic acid.
The present invention is further illustrated by the following specific examples, which are presented to facilitate a person of ordinary skill in the art to which the invention pertains and to make and practice the invention without undue interpretation, and are not to be construed as limiting the scope of the present invention, but are presented to illustrate how the materials and methods of the present invention can be practiced.
Test example
1. Assessing the editability of a CRISPR-Cas9 system in candida vista
Referring to FIG. 5A, the construction of the first and second ribozymes of the present invention for screening sgRNA cassettes are shown in examples 1 to 3. Since there is no way to dateApplication of CRISPR-Cas9 System to the literature on Yu Weisi candida, to assess the ability of the CRISPR-Cas9 system to embed foreign genes into the candida vista genome, a major gene editing ability assessment was experimentally performed with the construction of a linear fragment (denoted as Cas-HRAde in FIG. 5A), wherein the Cas9 promoter is P of the sequence shown in SEQ ID NO:1 PGK1 To drive the triple nuclear localization sequence (denoted by SV40NLS in FIG. 5A) of the Cas9 gene (denoted by SpCas9 in FIG. 5A) of the sequence denoted by SEQ ID NO:2 in the Cas9 expression cassette. The sgRNA promoter is P with the sequence shown in SEQ ID NO. 4 TDH1 To drive the sgRNA cassette, and the Cas-HRAde has Nrs with the sequence shown in SEQ ID NO:5 R The gene is used as a screening gene and is shown in Nrs R The gene was inserted upstream and downstream with a Frt sequence (shown as Frt in FIG. 5A) of the sequence shown in SEQ ID NO:6, respectively, and Cas-HRAde was transformed into wild-type Candida Violae ATCC20962 (hereinafter abbreviated as "Candida Violae") by electroporation. A first homology arm (shown as HRL in FIG. 5A) of the sequence shown as SEQ ID NO. 7 and a second homology arm (shown as HRR in FIG. 5A) of the sequence shown as SEQ ID NO. 8 were experimentally designed for a specific fragment of the Ade2 gene on the Ying Weisi Candida chromosome, whereas the targeting sequence (shown as N20:: ade2 in FIG. 5A) may correspond to a specific sequence of the Ade2 gene on the Candida velutipes chromosome. The Ade2 gene is a reporter gene which, when disrupted on the chromosome of Candida viscidosa, causes the colonies on YPD plates to appear pink. The sgRNA consists of a targeting sequence (N20:: ade 2) and a scaffold sequence of the sequence shown in SEQ ID NO:9, driving the P of the sgRNA cassette TDH1 Belongs to a PolII promoter, and the processing of sgRNA can be completed only after the targeting sequence and the bracket sequence are subjected to resultant cis-cleavage by utilizing a first ribozyme and a second ribozyme, so that the first ribozyme is expressed upstream of the targeting sequence and the second ribozyme is expressed downstream of the bracket sequence in a test, and the first ribozyme, the Hammerhead (Ham) of the sequence shown in SEQ ID NO:10, the Hepatitis Delta Virus (HDV) of the sequence shown in SEQ ID NO:11, and the tRNA of the sequence shown in SEQ ID NO:12 derived from candida parapsilosis (Candida parapsilosis) are utilized Ala And SEQ ID NO. 13 derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae)tRNA Gly A combination comprising three different sets of first and second ribozymes was constructed as an example, ham-HDV (example 1, HH), tRNA, respectively Ala HDV (example 2, TH) and tRNA Ala -tRNA Gly (example 3, TTg). In addition, two groups of sgRNAs, namely sgRNA1 and sgRNA2, are designed in the experiment, wherein the targeting sequence of the sgRNA1 is N20 shown in SEQ ID NO. 14, ade2-1, the targeting sequence of the sgRNA2 is N20 shown in SEQ ID NO. 15, ade2-2 is used for targeting different specific sequences on the Ade2 gene, and linear fragments (shown in Ctrl in FIG. 5A) which only express the SpCas9 gene but do not have the targeting sequence, the scaffold sequence, the first ribozyme and the second ribozyme are used as a control group.
Referring to fig. 5B and 5C, fig. 5B is a graph showing colony results of examples 1 to 3 of the present invention, and fig. 5C is a graph showing colony phenotype ratio results of fig. 5B. Upon selection of Noralserin (Nrs), pink colonies were generated using both the sgRNA1 and example 1 (HH) through example 3 (TTg) using the sgRNA2, representing successful Nrs binding using the CRISPR-Cas9 system R The gene is embedded in the Ade2 gene. Specifically, the pink colony ratio of example 1 (HH) was about 2.5-8.8%, the pink colony ratio of example 3 (TTg) was about 10.8-13.2%, and the pink colony ratio of example 2 (TH) was 21.2-23.1%, which is at least one time higher than that of examples 3 (TTg) and 1 (HH), indicating tRNA in example 2 (TH) Ala And HDV is used as a first ribozyme and a second ribozyme respectively and added on two sides of the sgRNA, so that processing and cutting of the sgRNA can be more effectively performed in candida viscidosa.
From the above results, it is known that the editing system of candida vista and the application thereof of the present invention verify that the CRISPR-Cas9 system can indeed perform gene editing on candida vista, and that the success rate of gene editing can be improved by adjusting the combination of the first ribozyme and the second ribozyme.
2. Optimizing the editability of CRISPR-Cas9 systems in candida vista
Although the CRISPR-Cas9 system has been experimentally demonstrated to be effective in gene editing of Candida vini, some white colonies were present among the colonies of example 2 (TH), so four sets of primers (P1 of the sequence shown by SEQ ID NO:16, P2 of the sequence shown by SEQ ID NO:17, P3 of the sequence shown by SEQ ID NO:18 and P4 of the sequence shown by SEQ ID NO: 19) were experimentally designed for the endogenous genome on the Candida vini chromosome, and 6 pink colonies and 2 white colonies were randomly picked and the edited genome of the colonies was confirmed by colony (colly) PCR.
Referring to FIG. 5D, FIG. 5D is a graph showing the results of colony PCR analysis in example 2 of the present invention. P1 and P4 are respectively designed on the endogenous genome for the upstream positive sequence and the downstream negative sequence of the Ade2 gene, and P2 and P3 are respectively designed on Nrs R The front end reverse sequence and the tail end forward sequence of the gene. In the case of the endogenous genome of Candida vista or the control group, PCR was performed with the primers P1+P4 to obtain a PCR product of about 3.5kb in size. If Nrs is successful R The genome after editing the gene inserted into the Ade2 gene can obtain a PCR product of about 1.8kb after PCR by the primer P1+P2, a PCR product of about 2.2kb after PCR by the primer P3+P4, and a PCR product of about 5.2kb after PCR by the primer P1+P4. In FIG. 5D, the PCR products obtained from pink colonies via each set of primers all fit the size of the PCR products of the edited genome, indicating successful Nrs R The gene is embedded into the Ade2 gene, while the white colony has the size of the PCR product of the endogenous genome and the edited genome, which indicates that the white colony contains Nrs R The edited genome of the gene-embedded Ade2 gene may also retain the unedited endogenous genome. In addition, referring to fig. 5E, fig. 5E is a graph showing colony results of the nutrition deficiency test of example 2 of the present invention. White colonies and pink colonies were simultaneously redrawn on YPD plates (Nrs plates) and mineral culture plates (MM plates) containing Nrs, and 40 selected pink colonies grew well on the Nrs plates and grew poorly on the MM plates, whereas white colonies grew well on both the Nrs plates and the MM plates as in the control group. As can be seen from the above results, nrs was successfully established in pink colonies R Two sets of fully edited transformants of the Ade2 gene of candida viscidosa (Ade 2) -/- ) While white colonies were unsuccessful in binding Nrs R Two of gene-embedded candida viscidosaIncompletely edited transformant harboring Ade2 gene (Ade 2 +/- )。
Referring to fig. 6A to 6C, fig. 6A is a graph of colony results for verifying the optimal homologous recombination strategy of the present invention, fig. 6B is a graph of colony phenotype ratio results for fig. 6A, and fig. 6C is a graph of analysis results for colonies for verifying the optimal homologous recombination strategy of the present invention by colony PCR. To achieve the effect of complete editing, a linear fragment (represented by TH-Del in fig. 6A to 6C, and its structure is not shown) was experimentally constructed, and the structure of the linear fragment is the same as that of example 2 (TH), which is to increase the efficiency of embedding by merely changing the positions of the specific fragments on the Ade2 gene to which the first homology arm and the second homology arm correspond, respectively, so that the first homology arm and the second homology arm generate about 1kb fragment knockout when homologous recombination is performed with the Ade2 gene via the specific fragments. In addition, the electroporated Candida vista was recovered by first using 2 XYPD broth supplemented with essential amino acids and adenine under a culture condition (4 hours, 30 ℃ C., 250 rpm) to slow down the growth pressure and promote the growth, and after an additional 25mg/L of Nrs was added to promote the progress of gene editing, and further cultivation was continued for 20 hours, colonies were painted on the Nrs plate containing adenine. The results showed that, by optimizing the design of the first and second homology arms and electroporation recovery procedure described above, more than 60% pink colonies Ade2 were produced on the Nrs plate -/- The pink colony ratio was increased by approximately three times compared to example 2 (TH). Furthermore, 24 randomly selected pink colonies after TH-Del editing were subjected to colony PCR to obtain 4.2kb PCR products, confirming that the genomes were all fully edited Ade2 -/- . From the above results, it was found that, although Candida Violae has a polyploid chromosome, gene editing by the gene editing system of Candida Violae of the present invention successfully embeds a foreign gene into a target gene and into the polyploid chromosome of Candida Violae at one time, and that the gene editing of Candida Violae and its application of the present invention have high efficiency in completely editing Candida Violae.
Referring to FIG. 6D, a graph of the results of analysis of colony PCR to verify the stability of the transformants of the present invention is shown. The 24 pink bacteriaFall Ade2 -/- After ten times of subculture, each colony can still obtain a 4.2kb PCR product after colony PCR, and the transformant edited by the gene editing and application of the candida viscidosis provided with extremely high genome stability.
It should be noted that, in order to reduce the cost and consideration required for practical application, such as adding antibiotics to maintain strain stability, replacing screening genes according to development requirements, etc., a plasmid pHyg-Flp (not shown) may be constructed experimentally, comprising Hyg of the sequence shown in SEQ ID NO:20 R The gene and the Flp gene expressing the sequence shown in SEQ ID NO. 21 recognize and excise the Frt sequence (shown as Frt in FIGS. 6C and 7) on the DNA. Referring to FIG. 7, a graph of the analysis results of colony PCR for verifying the unlabeled transformants of the present invention is shown. The results show that, in the transformation of pHyg-Flp into Ade2 +/ -or Ade2 -/- After incubation, all randomly selected colonies gave a 2.5kb PCR product after colony PCR, indicating Nrs in their genome R The gene has been successfully knocked out, and the CRISPR-Cas9 system established by the gene editing system of the candida viscidosis provided with the invention can successfully edit the marker-free gene in the candida viscidosis.
From the above results, it is understood that the gene editing system and application of the candida viscidosis of the present invention not only has high efficiency of completely editing candida viscidosis, but also has extremely high genome stability of the transformant edited by the gene editing and application of the candida viscidosis of the present invention, and can construct a label-free transformant according to the practical application requirements.
3. Construction of a transformant stably producing dodecanedioic acid and a method for producing dodecanedioic acid to verify the feasibility of application of a gene editing system of Candida vista
3.1 screening of catalytic enzymes for the production of dodecanedioic acid
Referring to FIG. 8, a schematic diagram of the metabolic pathway for producing dodecanedioic acid in Candida dodecandrum Yu Weisi is shown. Cytochrome P450 monooxygenases (CYP) and NADPH cytochrome reductase (CPR) are families of catalytic enzymes capable of catalyzing the production of dicarboxylic acids from various medium alkanes, such as CYP52a13, CYP52a15, CYP52a18, CYP52a19 and CPRb, dodecane (Dodecane) can be taken up intracellular by candida vista and converted to Dodecanol (1-Dodecanol), dodecanoic Acid (DA), hydroxydodecanoic acid (HDA) via a cascade of oxidative pathways, and finally converted to dodecanedioic acid (DDA) by CYP, CPR and other catalytic enzymes.
Referring to FIG. 9A, the construction of the expression cassettes for screening the catalytic enzymes for dodecanedioic acid production of the present invention, examples 4 (pC 13), 5 (pP 13), 6 (pT 13), 7 (p 15), 8 (pC 15), 9 (pP 15), 10 (pT 15), 11 (p 18), 12 (pC 18), 13 (pP 18), 14 (pT 18), 15 (p 19), 16 (pC 19), 17 (pP 19), 18 (pT 19), 19 (pC), 20 (pCC), 21 (pPC) and 22 (pTC), is schematically shown in dotted lines, which are the promoters of the exogenous genes in the expression cassettes of the respective examples, and the exogenous genes of the catalytic enzymes for dodecanedioic acid production. To construct transformants which stably produce dodecanedioic acid, examples 4 to 22 were experimentally constructed with Nrs R Plasmid pPro-Enz of the gene, plasmid pPro-Enz contains ARS2 gene with sequence shown in SEQ ID NO. 22, colE1 gene with sequence shown in SEQ ID NO. 23, ap with sequence shown in SEQ ID NO. 24 R Gene, nrs R A gene and an expression cassette for screening a catalytic enzyme suitable for converting dodecane into DDA in the metabolic pathway of candida viscidosis, the expression cassette comprising an exogenous gene promoter and an exogenous gene for the catalytic enzyme to be expressed, wherein the catalytic enzyme encoded by the exogenous gene is selected from endogenous CYP or CPRb of candida viscidosis, for example: CYP52A13 gene of the sequence shown by SEQ ID NO. 25 (shown as CYP52A13 in FIG. 9A), CYP52A15 gene of the sequence shown by SEQ ID NO. 26 (shown as CYP52A15 in FIG. 9A), CYP52A18 gene of the sequence shown by SEQ ID NO. 27 (shown as CYP52A18 in FIG. 9A), CYP52A19 gene of the sequence shown by SEQ ID NO. 28 (shown as CYP52A19 in FIG. 9A) and CPRb gene of the sequence shown by SEQ ID NO. 29 (shown as CPRb in FIG. 9A), the exogenous gene promoter is selected from the endogenous promoters of the endogenous CYP/CPRb (for example, the sequence shown by SEQ ID NO. 30)P CYP52A15 P of the sequence shown in SEQ ID NO. 31 CYP52A18 P of the sequence shown in SEQ ID NO. 32 CYP52A19 And P of the sequence shown in SEQ ID NO. 33 CPRb ) Dodecane inducible promoter (P of the sequence shown in SEQ ID NO: 34) CYP52A13 ) Or P TDH1 And P PGK1 The control group was pNrsR, which did not express the expression cassette. After the plasmids of the above examples were electroporated into wild-type Vis pseudoyeast, DDA was produced by shake flask culture of examples 4 to 22, and the supernatant was analyzed for DDA production by gas chromatography-flame ionization detector (GC-FID).
Referring to FIG. 9B, the results of the shaking flask process for producing dodecanedioic acid according to examples 4 to 22 of the present invention are shown. All of examples 4 to 22 produced DDA with only minimal by-product HDA and DA. In detail, in example 4 (pC 13), example 5 (pP 13), example 6 (pT 13), example 7 (P15), example 8 (pC 15), example 9 (pP 15) and example 10 (pT 15) expressing the CYP52A13 gene (represented by CYP52A13 in FIG. 9A) and the CYP52A15 gene (represented by CYP52A15 in FIG. 9A), the DDA yield was only comparable to or even lower than that of the control group, but in example 19 (pC), example 20 (pCC), example 21 (pPC) and example 22 (pTC) expressing the CPRb gene (represented by CPRb in FIG. 9A), only P was used CPRb The DDA yield of example 19 (pC) of (C) was slightly higher than that of the control. In contrast, DDA yields of example 11 (p 18), example 12 (pC 18), example 15 (p 19) and example 16 (pC 19) were all significantly improved relative to the control group.
From the foregoing results, it is shown that the editing system of Candida viscidosa and its application use endogenous promoter or P CYP52A13 And the CYP52A18 gene, the CYP52A19 gene or the CPRb gene is driven to be expressed, so that the DDA yield can be effectively improved.
Please refer to fig. 10A and fig. 10B. FIG. 10A is a schematic diagram showing the construction of examples 23 (Δcyp13/14), 24 (Δcyp15/16), 25 (p-C18), 26 (p-C19) and 27 (p-C) for screening embedded bits according to the present invention. FIG. 10B is a graph showing the results of the production of dodecanedioic acid by the shake flask method according to examples 23 to 27 of the present invention. To further increase DDA yield, a linear fragment (denoted Cas-GOI in fig. 10A) was experimentally constructed comprising an editing template, examples 23 to 27 were constructed based on the linear fragment (denoted Cas-GOI in fig. 10A), with the difference that the first homology arm, the second homology arm and the expression cassette represented by the editing template were constructed, wherein the first homology arm and the second homology arm are different specific fragments of different genes on the chromosome of candida Ying Weisi for rejecting the catalytic enzymes (CYP 52a13/14 or CYP52a 15/16) in candida vista that might compete with the conversion of dodecane to DDA, such as SEQ ID NO:35 (shown in FIG. 10A as CYP52A 13-HRL) and the second (shown in FIG. 10A as CYP52A 13-HRR) of the sequence shown in SEQ ID NO:36, and the first (shown in FIG. 10A as CYP52A 15-HRL) and the second (shown in FIG. 10A as CYP52A 15-HRR) of the sequence shown in SEQ ID NO:37 of example 24 (Δcyp15/16) or the second (shown in FIG. 10A as CYP52A 15-HRR) of the sequence shown in SEQ ID NO: 38), or the expression cassette for converting dodecane to DDA is inserted simultaneously with the removal of the competitive pathway catalytic enzyme (POX 2), such as example 25 (p-C18), A first homology arm of the sequence shown in SEQ ID NO:39 (shown as POX2-HRL in FIG. 10A) and a second homology arm of the sequence shown as SEQ ID NO:40 (shown as POX2-HRR in FIG. 10A) in example 26 (p-C19) and example 27 (p-C). The Spacer sequence (Spacer) on the Cas-GOI is the target of the gene to be inserted in each example, and the position of the corresponding target sequence is reserved, for example, N20 of the sequence shown in SEQ ID NO. 41 used in example 23, CYP52A13/14, N20 of the sequence shown in SEQ ID NO. 42 used in example 24, CYP52A15/16, N20 of the sequence shown in SEQ ID NO. 43 used in example 25 and example 26, POX2. After electroporation of the above examples to Vis pseudoyeast, the transformants were cultured in shake flask to produce DDA, and the supernatant was analyzed for DDA yield by GC-FID. After 24 hours of shake flask cultivation, the DDA yields of example 23 (Δcyp13/14), example 24 (Δcyp15/16) and example 27 (p-C) were all comparable to the control group in which DDA was produced by Candida vista, while the DDA yields of example 25 (p-C18) and example 26 (p-C19) were 90% higher than those of the control group.
From the foregoing results, it is again demonstrated that the Wis of the present inventionEditing system of candida and application thereof to P CYP52A13 Driving expression of the CYP52A18 gene or CYP52A19 gene and simultaneously blocking catalytic enzyme (POX 2 gene) in the omega-hydroxylation pathway can remarkably improve the yield of DDA.
Please refer to fig. 10C and fig. 10D. FIG. 10C is a schematic diagram showing the construction of the expression cassettes for screening of the catalytic enzymes optimized for dodecanedioic acid production of the present invention, examples 26 (p-C19), 28 (p-1819), 29 (p-19 n 18), 30 (p-19 trC), 31 (p-18 trC), 32 (p-19C) and 33 (p-18C). FIG. 10D is a graph showing the results of the shake flask production of dodecanedioic acid according to examples 26 and 28 to 33 of the present invention. As is known from the above results, the selection of the exogenous gene promoter and the CYP or CPRb encoded by the exogenous gene in the expression cassette significantly affects DDA production, and in order to further enhance the ability of the transformant to produce DDA, the test was experimentally constructed to test the production of DDA in examples 28 to 33 together with example 26 to obtain the preferred expression cassette for the conversion of dodecane into DDA, wherein examples 28 (p-1819), 29 (p-19 n 18), 32 (p-19C) and 33 (p-18C) were the CYP52A18 gene (CYP 52A18 in FIG. 10C), the CYP52A19 gene (CPRb 19 in FIG. 10C) or CPRb gene (CPRb in FIG. 10C), and examples 30 (p-19 trC) and 31 (p-18 trC) were the CYP52A18-CPRb gene (CPRb 52A 52 in FIG. 10C) or CPRb 45 in which the sequence shown in SEQ ID NO:44 was expressed in a single expression cassette. The result shows that the method is equal to P CYP52A13 DDA yields (11.6 g/L) of example 26 (p-C19) in which the CYP52A19 gene (represented by CYP52A19 in FIG. 10C) was expressed alone were compared, and DDA yields (8.3-10.9 g/L) of example 28 (p-1819) and example 29 (p-19 n 18) in which the CYP52A18 gene (represented by CYP52A18 in FIG. 10C) or the CYP52A19 gene (represented by CYP52A19 in FIG. 10C) were lower than those of example 26 (p-C19), respectively. In contrast, the CYP52A18 gene (represented by CYP52A18 in FIG. 10C) or the CYP52A19 gene (represented by CYP52A19 in FIG. 10C) or the CPRb gene (represented by CPRb in FIG. 10C) is expressed alone or the CYP52A18-CPRb gene (represented by CYP52A18-CPRb in FIG. 10C) or the CYP52A19-CPRb gene is expressed in fusionThe DDA yields (12.9-15.1 g/L) of examples 30 (P-19 trC) to 33 (P-18C) (shown by CYP52A19-CPRb in FIG. 10C) were all greater than those of example 26 (P-C19), and the transformants constructed in example 32 (P-19C) in particular (P-19C strain) produced the highest DDA yield of 15.1g/L in 24 hours.
Referring to FIGS. 10E and 10F, FIG. 10E is a graph showing the results of fermentation of Candida vista to produce dodecanedioic acid, and FIG. 10F is a graph showing the results of fermentation of P-19C strain of the present invention to produce dodecanedioic acid. To evaluate the potential of the P-19C strain for large scale production of DDA, the P-19C strain was inoculated and pre-cultured in a 3L fermenter, dodecane (3.6 g/h) and a feed solution (containing urea and glucose) were added in a fed-batch mode after 16 hours to induce DDA production, and Candida vista was cultured and induced in the same manner as a control group. The results showed that the P-19C strain grew at a similar rate to the final CDW (65 g/L) but produced DDA (48 g/L) at a faster rate during the first 48 hours, as compared to the control group. The DDA yield increased almost linearly thereafter and reached 156g/L at 120 hours. The corresponding productivity (1.3 g/L/h) and molar conversion (59%) were about 41% higher than those of Candida victima.
3.2 screening of catalytic enzymes optimized for production of dodecanedioic acid and construction of transformants producing dodecanedioic acid
In candida vista, a fatty pure oxidase (FAO 2) system catalyzes a step of regenerating a medium chain alcohol into a corresponding dicarboxylic acid through a series of oxidation reactions, and expression of FAO2 may be induced by long chain alkanes, but then is rapidly down-regulated, which affects metabolic pathways for producing dicarboxylic acids. Thus, a linear fragment was experimentally constructed for co-expression of the FAO2 gene, CYP52A19 gene and CPRb gene in order to optimize DDA production. However, since the aforementioned linear fragment of example 32 (p-19C) having the highest DDA yield is large (about 18.9 kb), it is very challenging to integrate the FAO2 gene into example 32 (p-19C) for co-insertion of the CYP52A19 gene, the CPRb gene and the FAO2 gene. In order to solve this problem, a first gene-editing segment and a second gene-editing segment are experimentally designed, and the gene-editing system different from the above-described Candida viscidosa completes the recombination ligation before transformation, where the recombination ligation is performed when the first gene-editing segment and the second gene-editing segment are co-transformed into the Candida viscidosa cell.
Referring to FIGS. 11A and 11B, FIG. 11A is a schematic diagram showing the construction of the expression cassettes of examples 34 (s-pF), 35 (s-17F) and 36 (s-F) for screening a catalytic enzyme of the medium chain alcohol oxidation pathway according to the present invention, and FIG. 11B is a schematic diagram showing the construction of examples 34 to 36 in Candida vista by the gene editing system of Candida vista according to the present invention. Examples 34 to 36 were experimentally constructed as a first gene editing fragment comprising, in order, a first homology arm (represented by POX2-HRL in FIGS. 11A and 11B), an expression cassette optimizing the medium chain alcohol oxidation pathway, and a first screening gene fragment (represented by l-Nrs in FIGS. 11A and 11B) R Representation). In the expression cassette for optimizing the medium chain alcohol oxidation pathway, three different exogenous gene promoters can be used for driving FAO2 gene (shown as FAO2 in FIG. 11A and FIG. 11B) with sequence shown as SEQ ID NO:46, and the exogenous gene promoters are selected from P with sequence shown as SEQ ID NO:47 POX4 P of the sequence shown in SEQ ID NO. 48 CYP52A17 Or P of the sequence shown in SEQ ID NO. 49 FAO2 The composition is respectively P POX4 Example 34 (s-pF), P for driving FAO2 Gene (FAO 2 in FIGS. 11A and 11B) CYP52A17 Example 35 (s-17F) for driving FAO2 Gene (denoted as FAO2 in FIGS. 11A and 11B), or P FAO2 Example 36 (s-F) in which the FAO2 gene (denoted as FAO2 in FIGS. 11A and 11B) was driven. The second gene editing fragment (represented by s-19C in FIG. 11A) was similar to example 32 (p-19C), but the first homology arm (represented by POX2-HRL in FIGS. 11A and 11B) was set to Nrs R The fragment of the gene was replaced with the fragment of the second selected gene (r-Nrs in FIGS. 11A and 11B R Represented by r-Nrs in FIGS. 11A and 11B) of the second selected gene fragment R Represented) has a sum of parts Nrs R A homologous fragment (not shown) of the gene, and the homologous fragment is about 400bp.
Whereby the and part Nrs is shared by the first screening gene fragment and the second screening gene fragment R Homologous fragments of the genes are homologous, thus when the first and second gene-editing fragments are electroporated simultaneously into WeissWhen in candida, will cause the first gene editing fragment and the second gene editing fragment to pass through the and part Nrs R The CRISPR-Cas9 system can be used for simultaneously embedding the expression cassettes for expressing CYP52A19 gene, CPRb gene and FAO2 gene into POX2 gene of candida vista for gene editing, so that the problem of overlarge gene editing fragments to be embedded can be solved.
Referring to fig. 11C and 11D, fig. 11C is a graph showing the results of the shake flask production of dodecanedioic acid according to examples 34 to 36 of the present invention, and fig. 11 is a graph showing the results of the fermentation production of dodecanedioic acid by the PF19C strain of the present invention. After 24 hours of shake flask culture, the three transformants constructed in example 34 (s-pF), example 35 (s-17F) and example 36 (s-F) gave a higher DDA yield (16.9-17.9 g/L), especially the transformant of example 34 (s-pF) (PF 19C strain) which had a DDA yield (17.9 g/L) increased by 13% over that of the P-19C strain, as compared to the DDA yield (15.8 g/L) produced by the P-19C strain expressing only the CYP52A19 gene (shown as CYP52A19 in FIG. 10C) and the CPRb gene (shown as CPRb in FIG. 10C). To evaluate the potential of PF19C strain for large-scale DDA production, PF19C strain was experimentally cultured in a 3L fermenter. The PF19C strain grew at a similar rate compared to the P-19C strain, 170g/LDDA was produced at 120 hours of fermentation production and intermediate HDA was hardly detected, the molar conversion was slightly increased to 63%, and the yield reached about 1.42g/L/h.
From the foregoing results, it is shown that the editing system of candida vista and the application thereof of the present invention combine the co-expression of exogenous genes (CYP 52a19 gene and CPRb gene) of the catalytic enzyme for producing dodecanedioic acid and exogenous genes (FAO 2 gene) of the catalytic enzyme for the medium chain alcohol oxidation pathway, and can significantly enhance the oxidation reaction step for converting HDA into DDA. From the above results, it is understood that the gene editing of candida viscidosis and the application thereof according to the present invention can be easily applied to the screening of catalytic enzymes to establish a stable dodecanedioic acid-producing transformant, and can further efficiently embed large fragments into the genes of candida viscidosis by the isolated CRISPR-Cas9 system.
Please refer to fig. 12A and 12B. FIG. 12A is a schematic illustration of the present inventionThe construction scheme of example 37 (ptG), example 38 (ptGD), example 39 (ptG), example 40 (P17P) and example 41 (P17 Pab) for screening NADPH regenerated catalytic enzyme. FIG. 12B is a graph showing the results of the production of dodecanedioic acid by the shake flask method according to examples 37 to 41 of the present invention. The transformants for producing dodecanedioic acid established in this experimental example are all constructed based on the extension of the metabolic pathway catalyzed by CYP and CPRb, however, the consumption of the cofactor NADPH in the catalytic process of CYP and CPRb is almost irreversible and continuous to pull the reaction downstream of the metabolic pathway, so that it is necessary to co-express a catalytic enzyme that enhances the NADPH regeneration reaction if a strain for producing dodecanedioic acid more stably and continuously is to be established. In Candida vista, NADPH can be regenerated by activating the endogenous glucose 6-phosphate dehydrogenase (G6 DP gene of the sequence shown in SEQ ID NO:50, in FIG. 12A, as G6 DP), glucose 6-phosphate dehydrogenase (6 PGDH gene of the sequence shown in SEQ ID NO:51, in FIG. 12A, as 6-PGDH) or NADH kinase (POS 5 gene of the sequence shown in SEQ ID NO:52, in FIG. 12A, as POS 5), or by exogenous glyceraldehyde 3-phosphate dehydrogenase (GDP 1 gene of the sequence shown in SEQ ID NO:53, in FIG. 12A, as GDP 1) or transhydrogenase (PNTA gene of the sequence shown in FIG. 12A, as PNTA gene of the sequence shown in SEQ ID NO:54, in FIG. 12A, as PNTB) thus constructing a plasmid template pNADPH-reg to express a gene promoter and a catalytic enzyme as described above and converting to a strain of FIG. 12A, as example, and a strain of a more preferred strain 37 to implement, to drive the expression of the exogenous gene and the more preferred strain of the promoter to regenerate the exogenous gene by selecting the promoter and the more preferred strain to express the HyH gene by using the promoter R Gene (Hyg in FIG. 12A) R Indicated) but not the expression cassette, was used as a control. After 24 hours of incubation in shake flask, the cells were incubated with P as compared to the DDA yield of the control group (15.9 g/L) CYP52A17 The DDA yield of example 40 (P17P) expressing the POS5 gene (represented by POS5 in FIG. 12A) was significantly improved (18.9 g/L), while that of the other examples was comparable to that of the control group only.
From the above results, it is shown that the editing system of candida viscidosis and application thereof in P CYP52A17 Driving expression of the POS5 gene can effectively catalyze the regeneration of NADPH and further improve DDA production.
Referring to fig. 13A to 13C, fig. 13A is a schematic diagram of the construction of the PFP19C strain of the present invention, fig. 13B is a schematic diagram of the edited chromosome of the PFP19C strain of the present invention, and fig. 13C is a graph of the yield of dodecanedioic acid produced by fermentation of the PFP19C strain of the present invention. In view of the above results, it was further experimentally constructed by simultaneously embedding the CYP52A19 gene (represented by CYP52A19 in FIGS. 13A and 13B), the CPRb gene (represented by CPRb in FIGS. 13A and 13B), the FAO2 gene (represented by FAO2 in FIGS. 13A and 13B) and the POS5 gene (represented by POS5 in FIGS. 13A and 13B) into the POX2 gene (represented by POX2 in FIG. 13B) of Candida vista to construct a transformant (PFP 19C strain) producing dodecanedioic acid, and culturing it in a 3L fermenter to produce DDA. Unlike the previously described candida viscidosa, which undergoes lag phase and slow growth, the PFP19C strain can grow faster in 120 hours to 78g/L Cell Dry Weight (CDW) and produce up to 224g/LDDA at 120 hours at a nearly linear growth rate. It is worth mentioning that the molar conversion of the PFP19C strain was also significantly increased to 83% and no by-products were detectable, while the corresponding productivity was also significantly increased to 1.87g/L/h. The PFP19C strain accumulates biomass required for growth faster in the fermenter than candida viscidosa and produces approximately 102% more DDA than candida viscidosa at a faster production rate.
From the above results, it was found that the transformant for producing dodecanedioic acid constructed by gene editing and application of the candida vista of the present invention not only inserts a large gene editing fragment of about 13.6kb into the genome of candida vista, but also significantly improves the growth rate of the transformant for producing dodecanedioic acid by co-expression of POS5 gene, and further improves the yield, productivity, product purity and molar conversion rate of dodecane produced by oxidation reaction, and the potential and feasibility of the gene editing system of candida vista and the gene editing method thereof of the present invention applied to construct a transformant for producing dodecanedioic acid were verified.
In addition, referring to Table I, the comparison of the dodecanedioic acid production transformant of the present invention and the fermentation production of dodecanedioic acid by Candida vista established in this test example was made.
As can be seen from the results of Table I, the gene editing system of Candida vista and the gene editing method thereof according to the present invention are achieved by using endogenous promoter or P CYP52A13 Driving expression of CYP52A18 gene or CYP52A19 gene and expression thereof CPRb The co-expression of CPRb gene can be driven to promote the obvious improvement of the DDA yield of candida viscidosa. Furthermore, the P-19C strain constructed by embedding the above exogenous gene into POX2 gene of Candida viscidosa to block omega-hydroxylation pathway can increase DDA yield from 111g/L to 156g/L and achieve productivity of 1.3g/L/h and 59 mol% conversion rate relative to Candida viscidosa. To further increase the yield, product purity, molar conversion and productivity of DDA production. In addition, the gene editing system and the gene editing method of candida viscidosis can support different optimization strategies to further construct a transformant for producing dodecanedioic acid without being limited by the size of a gene editing target, such as a PF19C strain in which a FAO2 gene, a CYP52A19 gene and a CPRb gene are jointly embedded into a POX2 gene of candida viscidosis, an oxidation pathway for enhancing the conversion of medium chain alcohol into dicarboxylic acid, and a PFP19C strain in which a POS5 gene, a FAO2 gene, a CYP52A19 gene and a CPRb gene are jointly embedded into a POX2 gene of candida viscidosis used for further improving NADPH regeneration. As a result, the dodecanedioic acid-producing transformant of the present invention was able to efficiently convert 200g/L dodecane to 224g/LDDA with a productivity of 1.87g/L/h and a molar conversion of 83%, and showed little detectable by-product HDA, demonstrating the gene editing system by Candida vista of the present invention The transformant for producing dodecanedioic acid and the method for producing dodecanedioic acid, which are established by the system, have high genome stability, high product selectivity and high matrix conversion rate, and the gene editing system of candida viscidosis and the gene editing field of candida viscidosis applying Yu Weisi and the potential of continuously producing DDA are also practically verified.
In summary, the gene editing system and the gene editing method of candida viscidosis disclosed by the invention are that firstly, a CRISPR-Cas9 system is established in candida viscidosis, through screening exogenous gene promoters, adjusting the combination of a first ribozyme and a second ribozyme and optimizing the recombination strategy of homologous arms, the operation difficulty and low recombination rate which are encountered when the traditional candida viscidosis with diploid are effectively overcome, so that the success rate and the accuracy of the gene editing of candida viscidosis can be greatly improved to 60%, and the gene editing means such as rejection, mutation, replacement or fragment insertion are conveniently and rapidly realized without being limited by the size of fragments or the insertion positions, thereby obtaining the gene editing strain of candida viscidosis capable of stable subculture. Thus, the gene editing system and method using candida vista of the present invention can be further applied to construct a transformant for producing chemicals as a loop for a cell factory to participate in the perpetual economy of chemicals.
While the present invention has been described with reference to the embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
[ symbolic description ]
100 linear segment
110,200 a first Gene editing fragment
111,210 a first homology arm
112 screening genes
120,300 a second Gene editing fragment
121,330 a second homology arm
130,340,340a:cas9 expression cassettes
131,341, 3411 a:cas9 promoter
132,342,352 a:cas9 gene
133,343, 34a: nuclear positioning sequence
140,350,350a: sgRNA cassettes
141,351,351a sgRNA promoters
142,352,352a: first ribozyme
143,353, 35a: targeting sequences
144,354 a. Scaffold sequences
145,355 a, second ribozyme
220,220a,320 a: frt sequence
230,230a first selection Gene fragment
231,231a,311 a homologous fragments
240,360 expression cassettes
241,361 exogenous gene promoter
242,362 exogenous genes
310,310a second screening Gene fragment
400 Gene editing method for candida viscidosa
410,420,430,440 step
500 Process for producing dodecanedioic acid
510,520 step
Claims (12)
1. A gene editing system of candida viscidosa (Candida viswanathii), comprising:
candida one-dimensional;
a first gene editing segment comprising a first homology arm and a screening gene arranged in sequence; and
a second gene editing segment connected to the C-terminal of the first gene editing segment, wherein the second gene editing segment comprises a second homology arm, a Cas9 expression cassette and a sgRNA cassette which are sequentially arranged, the Cas9 expression cassette comprises a Cas9 promoter, a Cas9 gene and a trinuclear localization sequence which are sequentially arranged, the sgRNA cassette comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a scaffold sequence and a second ribozyme which are sequentially arranged, and the first gene editing segment and the second gene editing segment form a linear segment for gene editing of a chromosome of candida vista;
wherein the first homology arm and the second homology arm respectively correspond to a specific fragment of a gene on the chromosome of the candida viscidosa, and the targeting sequence corresponds to a specific sequence of the gene on the chromosome of the candida viscidosa.
2. The system of claim 1, wherein the screening gene of the first gene-editing segment further comprises a first screening gene segment, the second gene-editing segment further comprises a second screening gene segment of the screening gene at the N-terminus of the second homology arm, and the first screening gene segment and the second screening gene segment have a homology segment, respectively, and the first gene-editing segment and the second gene-editing segment are joined into the linear segment by recombination of the homology segments.
3. The system of claim 2, wherein the first gene editing segment and/or the second gene editing segment further comprises at least one expression cassette comprising a foreign gene promoter and a foreign gene.
4. A method for editing a gene of candida viscidosa, comprising:
constructing a first gene editing segment, wherein the first gene editing segment comprises a first homology arm and a screening gene which are sequentially arranged;
constructing a second gene editing segment, wherein the second gene editing segment is connected to the C-terminal of the first gene editing segment, and the second gene editing segment comprises a second homology arm, a Cas9 expression cassette and a sgRNA cassette which are sequentially arranged, wherein the Cas9 expression cassette comprises a Cas9 promoter, a Cas9 gene and a trinuclear localization sequence which are sequentially arranged, the sgRNA cassette comprises a sgRNA promoter, a first ribozyme, a targeting sequence, a scaffold sequence and a second ribozyme which are sequentially arranged, the first gene editing segment and the second gene editing segment form a linear segment for carrying out gene editing on a chromosome of the candida vinis, and the first homology arm and the second homology arm respectively correspond to a specific segment of a gene on the chromosome of the candida vinis correspondingly provided;
Performing a transformation step of transforming the first gene editing fragment and the second gene editing fragment into the candida viscidosa to obtain a transformed strain; and
and (3) performing a transformant culture step, namely culturing the transformant with a screening culture at an editing temperature for an editing time, wherein the Cas9 expression cassette expresses the Cas9 gene and the sgRNA cassette expresses the targeting sequence, the first homology arm and the second homology arm are respectively subjected to homologous recombination with the specific fragment, and the first gene editing fragment and the second gene editing fragment positioned at the part between the first homology arm and the second homology arm are embedded into the gene of the transformant.
5. The method of claim 4, wherein the screening gene of the first gene-editing segment further comprises a first screening gene segment, the second gene-editing segment further comprises a second screening gene segment of the screening gene at the N-terminus of the second homology arm, and the first screening gene segment and the second screening gene segment have a homologous segment, respectively, and the first gene-editing segment and the second gene-editing segment are joined into the linear segment by recombination of the homologous segments.
6. The method according to claim 4, wherein the first gene editing segment and/or the second gene editing segment further comprises at least one expression cassette comprising a foreign gene promoter and a foreign gene.
7. A transformant for producing dodecanedioic acid, comprising:
a host cell, wherein the host cell is candida one-dimensional; and
at least two exogenous genes comprising a CYP52A19 gene and a CPRb gene, wherein the at least two exogenous genes are embedded in a chromosome of the host cell using the gene editing system of candida viscidosa of claim 3.
8. The dodecanedioic acid producing transformant of claim 7, wherein the at least two exogenous genes comprise the CYP52a18 gene.
9. The dodecanedioic acid producing transformant according to claim 7, wherein the at least exogenous gene is embedded in the POX2 gene of the chromosome of the host cell.
10. The dodecanedioic acid producing transformant of claim 7, wherein the at least two exogenous genes comprise a FAO2 gene.
11. The dodecanedioic acid producing transformant of claim 7, wherein the at least two exogenous genes comprise POS5 genes.
12. A method of producing dodecanedioic acid, comprising:
providing a reaction matrix, wherein the reaction matrix comprises dodecane; and
a fermentation step is carried out by inoculating the dodecanedioic acid-producing transformant according to any one of claims 7 to 11 into the reaction substrate and culturing under a fermentation condition for a fermentation time to obtain a fermentation substance, wherein the fermentation substance contains dodecanedioic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211201273.3A CN117778440A (en) | 2022-09-29 | 2022-09-29 | Gene editing system of candida viscidosa, gene editing method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211201273.3A CN117778440A (en) | 2022-09-29 | 2022-09-29 | Gene editing system of candida viscidosa, gene editing method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117778440A true CN117778440A (en) | 2024-03-29 |
Family
ID=90393305
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211201273.3A Pending CN117778440A (en) | 2022-09-29 | 2022-09-29 | Gene editing system of candida viscidosa, gene editing method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117778440A (en) |
-
2022
- 2022-09-29 CN CN202211201273.3A patent/CN117778440A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK2305826T3 (en) | Materials and methods for the efficient production of lactic acid | |
| JP5810143B2 (en) | Yeast cells with disrupted pathway from dihydroxyacetone phosphate to glycerol | |
| JP5121070B2 (en) | L- or D-lactate: Lactic acid-producing yeast in which ferricytochrome C oxidoreductase gene does not function | |
| US7141410B2 (en) | Methods and materials for the production of organic products in cells of Candida species | |
| KR101616171B1 (en) | Use of monascus in organic acid production | |
| JP2019513408A (en) | Lactic acid production method | |
| CN107287144B (en) | Metabolically-modified bacillus subtilis biotransformation cell and preparation method and application thereof | |
| EP2357222B1 (en) | Scyllo-inositol-producing cell and scyllo-inositol production method using said cells | |
| CN117778440A (en) | Gene editing system of candida viscidosa, gene editing method and application thereof | |
| WO2024020252A1 (en) | A genetically engineered yeast producing lactic acid with improved lactic acid with improved lactic acid export | |
| TW202413626A (en) | Gene editing system of candida viswanathii, gene editing method thereof, transformant for producing dodecanedioic acid, and metohd for producing dodecanedioic acid | |
| US20240110208A1 (en) | Gene editing system of candida viswanathii, gene editing method thereof, transformant for producing dodecanedioic acid and method for producing dodecanedioic acid | |
| JP2022078003A (en) | Synthetic promoter based on acid-resistant yeast gene | |
| CN105593368B (en) | Recombinant microorganism having increased ability to produce 2,3-butanediol and method for producing 2,3-butanediol using same | |
| US20140302577A1 (en) | Enhanced yeast fermentation platform using yeast lacking mitochondrial dna and containing growth improving mutations | |
| US20200131538A1 (en) | Microorganism with stabilized copy number of functional dna sequence and associated methods | |
| KR102613937B1 (en) | Yeast strain in which all genes involved in galactose utilization are deleted and method for producing recombinant protein using the same | |
| CN110872595A (en) | Acid-resistant expression cassette and application thereof in organic acid production by fermentation | |
| Sánchez et al. | Microbial Synthesis of Secondary Metabolites and Strain Improvement: Current Trends and Future Prospects | |
| US20240052382A1 (en) | Process control for 3-hydroxypropionic acid production by engineered strains of aspergillus niger | |
| EP4375378A1 (en) | Novel metabolic pathway for producing itaconic acid and method for producing itaconic acid using same | |
| CN115960802A (en) | Engineering bacterium of vibrio natriegens for high yield recombination protein and its application | |
| CN115678866A (en) | Application of catalase in preparation of LCDA (calcium-phosphate-dehydrogenase) and genetic engineering bacteria for over-expressing catalase | |
| CN115992161A (en) | Construction method of actinobacillus succinogenes CRISPRi system | |
| US20110129894A1 (en) | Secretion optimized microorganism |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |