CN107384968B

CN107384968B - Yeast engineering strain for chromosome fusion modification

Info

Publication number: CN107384968B
Application number: CN201710729269.7A
Authority: CN
Inventors: 覃重军; 邵洋洋; 薛小莉; 鲁宁
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2017-08-23
Filing date: 2017-08-23
Publication date: 2021-06-08
Anticipated expiration: 2037-08-23
Also published as: CN107384968A

Abstract

The invention relates to a yeast engineering strain modified by chromosome fusion. The inventor artificially fuses 16 natural chromosomes of yeast with each other to obtain a series of strains in which chromosome fusion occurs, wherein the strains comprise 16 linear or circular chromosomes fused into 1. The yeast engineering strain of the invention can be used as a eukaryotic heterologous gene expression and large-fragment DNA cloning host.

Description

Yeast engineering strain for chromosome fusion modification

Technical Field

The invention belongs to the fields of microbial synthetic biology, genome engineering and molecular biology, and particularly relates to a yeast engineering strain for chromosome fusion modification.

Background

Saccharomyces cerevisiae is a unicellular eukaryote, the first sequenced eukaryotic model organism, has a genome size of 12Mb, and contains 16 linear chromosomes. Saccharomyces cerevisiae has long been used in alcohol brewing and food fermentation, and various studies in genetics, molecular biology, physiology and other aspects show that Saccharomyces cerevisiae wild strains are safe for human beings. The saccharomyces cerevisiae is easy to culture, clear in genetic background and relatively simple in genetic operation, and has a protein post-translational processing and modifying system of eukaryotes, so that the saccharomyces cerevisiae becomes a common host bacterium for heterologous higher organism gene expression and has important application in the aspects of industry, medicine, energy and the like. In recent years, with the rapid development of synthetic biology technology, saccharomyces cerevisiae has been used as an artificial cell factory to ferment and produce precursor substances of natural product drugs of plant origin, such as artemisinin, paclitaxel and ginsenoside, and the like, and the yield of the rare drugs is greatly improved.

At present, Yeast Artificial Chromosomes (YACs) capable of cloning extra-large fragments of DNA of complex genomes of higher organisms have been developed in the field by utilizing autonomous replication regions, centromere and telomere of Saccharomyces cerevisiae chromosomes. YAC vectors can accommodate 300kb to 1Mb of oversized heterologous DNA in Saccharomyces cerevisiae. Corresponding YAC libraries have been constructed from the plant to mouse to human genomes. Various YAC libraries constructed in the saccharomyces cerevisiae greatly facilitate the function research of complex genomes of higher organisms and the positioning of disease genes.

However, the 16-line chromosome naturally occurring in Saccharomyces cerevisiae, which is a host for heterologous gene and large fragment cloning, has a size ranging from 230kb to 1500kb, which results in a linear expression or cloning plasmid carrying large fragment DNA (>200kb) that is difficult to separate from the natural linear chromosome. In addition, a plurality of repetitive sequences exist near telomeres of saccharomyces cerevisiae chromosomes, so that homologous recombination is easy to occur, and the stability of the saccharomyces cerevisiae as a heterologous gene expression host or a cloning host of extra-large fragment DNA is influenced. For example, libraries of YAC clones carrying complex genomic DNA from higher organisms are susceptible to recombinant deletion or chimeric clones in Saccharomyces cerevisiae. These deficiencies of s.cerevisiae limit its use as a host for heterologous gene expression and large fragment DNA cloning. Therefore, there is a need in the art to construct new saccharomyces cerevisiae host bacteria with simplified genome and genetically stable.

Disclosure of Invention

The invention aims to provide a saccharomyces cerevisiae engineering strain with chromosome fusion, simplified genome and genetic stability.

In the first aspect of the invention, a yeast engineering strain with fused chromosomes is provided, wherein two or more chromosomes in the engineering strain are fused, and the engineering strain has 1-15 centromeres and 0-30 telomere regions; and, the multiple copy repeat sequence at the proximal plasmid of the engineered strain is deleted to a single copy sequence.

In a preferred embodiment, the multicopy repeat sequence is a sequence greater than 2kb, such as 2-30 kb; more particularly 2.7-26.2 kb.

In another preferred embodiment, the repetitive sequence is selected from the group consisting of:

repeat sequence set 1: chromosome VIII 525437-539926; 13089-; i: 203448-219229;

repeat sequence set 2: chromosome I219230-229411; VIII, 539927-543610, 549638-556001;

repeat sequence group 7: chromosome XIII 917474-923540; XV 1078061-1083736; 7409-13083;

repeat sequence group 9: chromosome XV 10454-13126; IX, 8286-10347; 8269-10330;

repeat sequence set 10: chromosome XV 22397-27006; 16656-25250; 16639-21229;

repeat sequence group 3: chromosome VII 1076381-1083886; 805133-812631;

repeat sequence group 4: chromosome XIV 7429-17224; VI 5531-14039;

repeat sequence set 5: chromosome XI 658572-665429; 4327-11225;

repeat sequence group 6: chromosome VII 5545-9584; IX 430983-4367;

repeat sequence group 8: chromosome XV 1073988-1078544; XVI: 12601-17099;

repeat sequence group 11: chromosome V18067-23447; XIV 772693-;

repeat sequence set 12: chromosome IV 905-18681; x727164-;

repeat sequence group 13: chromosome XII, 1059296-; 303903-308316;

repeat sequence set 14: chromosome IX 21251-25254; 27007-30676; and/or

Repeat sequence set 15: chromosome IX 10348-16655; x10331-16638.

In another preferred embodiment, in the repeated sequence, the following deletion is performed:

repeat sequence set 1: 13089-;

repeat sequence set 2: keeping VIII, 539927-;

repeat sequence group 7: 7409-13083 is reserved, and other 2 strips are deleted;

repeat sequence group 9: 8286-10347 is reserved, and the other 2 strips are deleted;

repeat sequence set 10: 16656 and 25250, and deleting the other 2 strips;

repeat sequence group 3: the 805133 and 812631 are reserved, and another 1 is deleted;

repeat sequence group 4: VI is reserved, 5531 and 14039, and another 1 is deleted;

repeat sequence set 5: keeping 4327-11225, and deleting another 1;

repeat sequence group 6: reserving IX 430983-;

repeat sequence group 8: 12601-;

repeat sequence group 11: 772693-777126, and deleting another 1;

repeat sequence set 12: reserving X727164 and 744901 and deleting another 1 strip;

repeat sequence group 13: 303903-308316, and deleting another 1 strip;

repeat sequence set 14: 21251-; and/or

Repeat sequence set 15: IX 10348-16655 was reserved and another 1 was deleted.

In another preferred embodiment, the engineered strain is selected from the group consisting of:

SY15, which is based on SY14, and deletes the left telomere of chromosome XVI and the right telomere of chromosome X;

SY14, which is based on SY13, and has deleted the center granule of chromosome IV, the right telomere of XI and the left telomere of repetitive sequence 5 and I;

SY13, which is characterized in that right telomere of chromosome XIV, center granule of XVI and left telomere of chromosome XIII are deleted on the basis of SY 12;

SY12, which is prepared by deleting right telomere of chromosome IV, left telomere of chromosome VII and central particle on the basis of SY 11;

SY11, which is characterized in that on the basis of SY10, the center granule, the right end granule and the repetitive sequence 13 of chromosome XII, the left telomere of XV, are deleted;

SY10, which is characterized in that on the basis of SY9, the center granule of chromosome I, the right telomere of chromosome II and the left telomere of chromosome III are deleted;

SY9, which is characterized in that on the basis of SY8, right end granule of chromosome VIII, left telomere of chromosome IX and central granule of chromosome X are deleted;

SY8, which is based on SY7, and has right end grain and repetitive sequences 8 and 7 of chromosome XV, central grain and left telomere of XI deleted;

SY7, which is characterized in that right end granule of chromosome V, left telomere of VI and central granule of XIV are deleted on the basis of SY 6;

SY6, which is characterized in that right telomere and central granule of chromosome III, left telomere of chromosome IV and repetitive sequence 12 are deleted on the basis of SY 5;

SY5, which is characterized in that right telomere and central granule of chromosome IX, left telomere of X and repetitive sequences 9, 10 and 15 are deleted on the basis of SY 4;

SY4, which is characterized in that right telomere, left telomere and central grain of chromosome XVI are deleted on the basis of SY 3;

SY3, which is characterized in that right telomere and central granule of chromosome VI, left telomere of XIV and repetitive sequence 4 are deleted on the basis of SY 2;

SY2, which is based on SY1, and has right telomere and central grain deleted from chromosome I,

repetitive sequences

1 and 2 and chromosome II;

SY1, which is characterized in that on the basis of SY0, right end grain of chromosome XIII, repetitive sequence 7, central grain and left telomere of chromosome XII are deleted;

SY0, which is based on natural Saccharomyces cerevisiae and has deleted the repetitive sequence 11, VII right telomere and

repetitive sequences

6 and 3 in chromosome V, the left telomere, the central particle and repetitive sequence 1 in chromosome VIII, and the repetitive sequences 9, 10 and 14 in XV.

In another preferred example, the engineering strain is SY15 strain, and telomeres at both ends of fused chromosome in the strain are deleted and cyclized.

In another aspect of the present invention, there is provided a fused chromosome isolated from said engineered yeast strain

In another aspect of the present invention, there is provided a method for preparing a chromosome fused engineered yeast strain, the method comprising:

(1) fusing chromosomes VII and VIII on the basis of natural saccharomyces cerevisiae; deleting the repeated sequence 11, the right telomere VII and the repeated

sequences

6 and 3 in the chromosome V, the left telomere, the centromere and the repeated sequence 1 in the chromosome VIII, and the repeated sequences 9, 10 and 14 in the chromosome XV; obtaining a SY0 strain;

(2) fusing chromosomes XIII and XII on the basis of SY 0; deleting right telomere of chromosome XIII, repetitive sequence 7, central telomere and left telomere of chromosome XII; obtaining a SY1 strain;

(3) fusing chromosomes I and II on the basis of SY 1; deleting the right telomere and the

repetitive sequences

1 and 2 of the chromosome I and the left telomere and the central grain of the chromosome II; obtaining a SY2 strain;

(4) fusing chromosome VI and XIV based on SY 2; deleting right and central grains of chromosome VI, left telomere of XIV and repetitive sequence 4; obtaining a SY3 strain;

(5) fusing chromosome XVI and V on the basis of SY 3; deleting the right telomere, the left telomere and the center grain of the chromosome XVI; obtaining a SY4 strain;

(6) fusing chromosome IX and X on the basis of SY 4; deleting right and central grains of chromosome IX, left telomere of X and repetitive sequences 9, 10 and 15; obtaining a SY5 strain;

(7) fusing chromosomes III and IV on the basis of SY 5; deleting right end grain and central grain of chromosome III, left telomere of chromosome IV and repetitive sequence 12; obtaining a SY6 strain;

(8) fusing previously fused chromosomes XVI-V and VI-XIV on the basis of SY 6; deleting right telomere of chromosome V, left telomere of chromosome VI and central grain of chromosome XIV; obtaining a SY7 strain;

(9) fusing XV and XI based on SY 7; deleting right end grain and repetitive sequences 8 and 7 of chromosome XV, and central grain and left telomere of XI; obtaining a SY8 strain;

(10) fusing the previously fused chromosomes VII-VIII and IX-X on the basis of SY 8; deleting right telomere of chromosome VIII, left telomere of chromosome IX and central particle of chromosome X; obtaining a SY9 strain;

(11) fusing the previously fused chromosomes I-II and III-IV on the basis of SY 9; deleting the center granule of the chromosome I, the right telomere of the chromosome II and the left telomere of the chromosome III; obtaining a SY10 strain;

(12) fusing previously fused chromosomes XIII-XII and XV-XI on the basis of SY 10; deleting the center granule, the right end granule and the repetitive sequence 13 of the chromosome XII, and the left telomere of XV; obtaining a SY11 strain;

(13) fusing the previously fused chromosomes I-II-III-IV and VII-VIII-IX-X on the basis of SY 11; deleting right telomere of chromosome IV, left telomere of chromosome VII and central particle; obtaining a SY12 strain;

(14) fusing previously fused chromosomes XVI-V-VI-XIV and XIII-XII-XV-XI on the basis of SY 12; and deleting the right telomere of chromosome XIV, the center telomere of XVI, and the left telomere of XIII; obtaining a SY13 strain;

(15) fusing the previously fused chromosomes XVI-V-VI-XIV-XIII-XII-XV-XI and I-II-III-IV-VII-VIII-IX-X on the basis of SY 13; deleting the center granule of chromosome IV, the right telomere of XI, the repeat sequence 5 and the left telomere of I; obtaining a SY14 strain;

(16) deleting the left telomere of the chromosome XVI and the right telomere of the chromosome X on the basis of SY 14; the SY15 strain was obtained.

In another preferred example, the chromosome is edited by using a CRISPR/Cas9 method, and the target sequence is deleted.

In another preferred example, when the CRISPR/Cas9 method is used, the target site of the sgRNA on the genome is selected from:

when the chromosomes VII and VIII are fused in the step (1), the target is to the sites shown by SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3;

when the chromosomes XIII and XII are fused in the step (2), the chromosomes XIII and XII are targeted to the sites shown by SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;

when the chromosomes I and II are fused in the step (3), the target is at the sites shown by SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9;

when the chromosomes VI and XIV are fused in the step (4), the target is to the sites shown by SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12;

when the chromosomes XVI and V are fused in the step (5), the target is the sites shown by SEQ ID NO. 13, SEQ ID NO. 14 and SEQ ID NO. 15;

when the chromosomes IX and X are fused in the step (6), the target is the sites shown by SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO: 18;

when the chromosomes III and IV are fused in the step (7), the target is the site shown by SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21;

when chromosomes XVI-V and VI-XIV are fused in the step (8), the target is shown in the sites of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO: 24;

when the chromosomes XV and XI are fused in the step (9), the chromosomes are targeted to the sites shown by SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27;

when chromosomes VII-VIII and IX-X are fused in the step (10), the target genes are shown as SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO: 30;

when the chromosomes I-II and III-IV are fused in the step (11), the target is the sites shown by SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33;

when chromosomes XIII-XII and XV-XI are fused in the step (12), the sites shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36 are targeted;

when the chromosomes I-II-III-IV and VII-VIII-IX-X are fused in the step (13), the chromosomes are targeted to the sites shown by SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO: 39;

when chromosomes XVI-V-VI-XIV and XIII-XII-XV-XI are fused in step (14), the target sites shown in SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42 are reached;

targeting the sites shown in SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45 when chromosomes XVI-V-VI-XIV-XIII-XII-XV-XI and I-II-III-IV-VII-VIII-IX-X are fused in step (15); or

In step (16), the sites shown in SEQ ID NO:46 and SEQ ID NO:47 are targeted.

In another aspect of the present invention, there is provided a kit comprising the engineered yeast strain with any of the above chromosome fusions.

In another preferred embodiment, the kit contains the yeast engineering strain with chromosome fusion of SY0-SY 15.

In another aspect of the invention, the use of said engineered yeast strain or said chromosome is provided as cloning host for eukaryotic heterologous gene expression and large fragment nucleic acids.

In another aspect of the invention, the use of said kit is provided for the expression of eukaryotic heterologous genes or for the cloning of large fragments of nucleic acids.

In another aspect of the invention, a kit for establishing a chromosome fusion yeast engineering strain is provided, wherein the kit contains the part or all of the chromosome fusion yeast engineering strain described in SEQ ID NO. 1-SEQ ID NO. 47. The portions are defined as a set of sequences selected according to the chromosome type to be fused, for example, when the chromosome VII is fused to VIII, the portions are defined as the small sequences of SEQ ID NO 1 to SEQ ID NO 3, and the other cases can be determined according to the corresponding classification in Table 3.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, construction of a series of engineered strains of Saccharomyces cerevisiae chromosomal fusion.

FIG. 2, the genome of the saccharomyces cerevisiae engineering strain SY0-SY10 with chromosome fusion is verified by pulse electrophoresis.

FIG. 3, the genome of the saccharomyces cerevisiae engineering strain SY11-SY15 with chromosome fusion is verified by pulse electrophoresis.

FIG. 4, co-linear circle diagram of 1 large linear chromosome (left) of strain SY14 with the reference genome of 16 s.cerevisiae chromosomes (right).

FIG. 5 shows the growth curves of SY11-13(A), SY14 and SY15(B) of the engineering strain of Saccharomyces cerevisiae with chromosome fusion.

FIG. 6, pulsed field gel electrophoresis detecting the enzyme cutting map of SY14 and SY15 genome growing 0 generation and 100 generation.

Fig. 7, a method for efficiently fusing chromosomes in yeast by using CRISPR/Cas9 system, and a principle and a flow chart of the method.

Figure 8, plasmid map of Cas9 expression vector.

Fig. 9, plasmid map of sgRNA expression vector.

Detailed Description

The inventor carries out mutual artificial fusion on 16 natural chromosomes of yeast through a large number of constructions, identifications and tests and design optimization to obtain a series of bacterial strains with chromosome fusion, wherein the bacterial strains comprise 16 linear or circular chromosomes with 1 chromosome fusion.

Term(s) for

In the present invention, the "nucleic acid" may refer to DNA or RNA. The term "nucleic acid sequence" as used herein may refer to a sequence of DNA or RNA. The nucleic acid sequence may be circular. The DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. For example, a coding region sequence may be a degenerate variant of a particular coding sequence. As used herein, "degenerate variants" refer to variants of a nucleic acid that encode the same amino acid sequence as a reference nucleic acid, but differ in nucleic acid sequence from the reference nucleic acid.

In the present invention, the terms "vector" and "expression vector" are used interchangeably and refer to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. These vectors are capable of replication and stability in a host. An important feature of these vectors is that they usually contain an origin of replication, a promoter, a marker gene and translation control elements.

In the present invention, the "centromere knockout element" refers to a nucleic acid comprising a series of elements for knocking out an excess centromere when performing chromosome fusion, so that the fused chromosome contains only one centromere. The centromere knockout component contains homologous sequences (homologous arms) on both sides of the centromere to be knocked out and a screening marker. The "centromere knockout element" also contains the necessary "regulatory elements" so that it can be introduced into and replicated and expressed in a cell.

In the present invention, the "chromosome fusion module" refers to a nucleic acid containing a series of elements for fusing chromosomes to be fused after they are recognized and cleaved at appropriate sites (usually, at telomere regions); the "chromosome fusion module" contains the homologous sequence of the proximal plasmid region of the chromosome to be fused and a selection marker. The "chromosomal fusion module" also contains the necessary "regulatory elements" that can be introduced into the cell and replicated and expressed in the cell.

In the invention, the "sgRNA targeted cleavage module" refers to a nucleic acid for generating sgRNA targeting a specific site in a cell, and the generated sgRNA can be cleaved between two homologous sequences of a centromere and at two homologous sequences of a proximal region, and is matched with a "centromere knockout module" and a "chromosome fusion module" to realize chromosome fusion. The sgRNA targeted cleavage module also contains necessary "regulatory elements" so that it can be introduced into cells and replicated and expressed in cells.

In the present invention, the "regulatory element" may include a promoter, an enhancer, a response element, a signal peptide, an internal ribosome entry sequence, a polyadenylation signal, a terminator, or an inducible element that regulates the expression (e.g., transcription or translation) of a nucleic acid.

The term "genome" as used herein refers to the entire genetic information contained in the DNA (part of the virus is RNA) of an organism. The genome includes both genetic and non-coding DNA. Generally, the genome of an organism refers to the complete DNA sequence in a set of chromosomes. For example, a diploid in a somatic cell of an individual organism consists of two sets of chromosomes, wherein one set of DNA sequences is a genome. The term genome may particularly denote nuclear DNA (e.g. nuclear genome), but also organelle genomes comprising self DNA sequences, such as mitochondrial genomes or chloroplast genomes.

As used herein, "knockout" or "deletion" refers to a technique for deleting a gene of interest from a genome.

Fusion method

The inventor deeply researches yeast chromosomes, comprehensively considers the factors according to the gene distribution characteristics, the sequence repeatability characteristics, the centromere position, the telomere region characteristics and the like on 16 yeast chromosomes, and designs the fusion method and the fusion strain.

In the process of constructing the strain, the inventor adopts a gradual fusion method, simultaneously determines a plurality of large-fragment repetitive sequences, and properly deletes a plurality of copies of the repetitive sequences in the fusion process, thereby reducing the length of the fused chromosome as much as possible and simplifying the fused chromosome as much as possible under the condition of ensuring that the construction is successful and the expected function is not damaged.

As a preferred mode of the present invention, the method comprises: (1) fusing chromosomes VII and VIII; (2) fusing chromosomes XIII and XII; (3) fusing chromosomes I and II; (4) fusing chromosomes VI and XIV; (5) fusing chromosomes XVI and V; (6) fusing chromosome IX and X; (7) fusing chromosomes III and IV; (8) fusing the previously fused chromosomes XVI-V and VI-XIV; (9) fusing XV and XI; (10) fusing the previously fused chromosomes VII-VIII and IX-X; (11) fusing the previously fused chromosomes I-II and III-IV; (12) fusing previously fused chromosomes XIII-XII and XV-XI; (13) fusing the previously fused chromosomes I-II-III-IV and VII-VIII-IX-X; (14) fusing previously fused chromosomes XVI-V-VI-XIV and XIII-XII-XV-XI; (15) fusing the previously fused chromosomes XVI-V-VI-XIV-XIII-XII-XV-XI and I-II-III-IV-VII-VIII-IX-X; (16) the left telomere of chromosome XVI and the right telomere of X are deleted. In each step, strains which are further subjected to chromosome fusion on the basis of the strains obtained in the previous step are obtained and are gradually increased.

In the invention, preferably, the method for efficiently fusing chromosomes in yeast by using the CRISPR/Cas9 system is shown in the principle and flow chart of the method in FIG. 7 (taking chromosomes Chr.VII and Chr.VIII as examples). Through yeast protoplast transformation technology, the sgRNA plasmid containing homologous sequences on both sides of the centromere and a component of a selection marker, homologous sequences of two near telomere regions of a chromosome involved in fusion and a component of a selection marker, and two homologous sequences between the two homologous sequences of the centromere and the two homologous sequences of the near telomere regions can be cut once and transformed into a yeast cell. After the chromosome is cut near the centromere and the telomere, a centromere knockout component and a chromosome fusion component containing homologous sequences on the chromosome are integrated on the chromosome through homologous recombination in a yeast body, and a needed transformant is screened out through markers on a sgRNA vector, a Cas9 vector, the centromere component and the chromosome fusion component.

More specifically, as shown in fig. 7, the method of fusing chromosome a (e.g., chr.vii) and chromosome B (e.g., chr.viii) according to the present invention includes: a centromere knockout module (such as a module marked with DR1 and ura in the figure), a chromosome fusion module (such as a module marked with DR2 and ura in the figure), a sgRNA targeting cleavage module (such as pCas9 in the figure) and a Cas9 expression module are introduced into the yeast, so that the yeast with the fused chromosome A and chromosome B is obtained.

The centromere knockout module is used for knocking out the centromere of one of two chromosomes, and comprises homologous sequences and a screening marker on both sides of the centromere (taking the centromere of Chr.VII as an example in figure 7). When the targeted sgRNA (S1) is cleaved on one side of the centromere, the centromere knockout element is integrated into the chromosome by homologous exchange. To select for the exchanged cells, it is often necessary to add a selection marker (e.g., ura) to the centromeric knockout module. If desired, the selectable marker may be a resistance gene or a readily identifiable nucleic acid fragment. As a preferred mode of the invention, in the centromeric knock-out module, a direct repeat sequence (e.g., DR1) is provided between the homologous sequence and the selectable marker in the module; the sequence is the sequence of homologous sequence on the side of chromosome next to the centromere, and the forward repeat sequence is arranged to be beneficial to further recombination operation in the section to eliminate the screening marker, so as to obtain traceless fused chromosome.

The chromosome fusion module aims at telomere regions to be fused of two chromosomes, and contains homologous sequences of a chromosome A (such as Chr.VII) proximal region, homologous sequences of a chromosome B (such as Chr.VIII) proximal region and a screening marker. After the targeted sgRNA is cut in telomere regions to be fused of two chromosomes respectively, the chromosome fusion components are integrated to the chromosomes through homologous exchange, and the two chromosomes are connected together. In order to select for cells in which chromosomal fusion occurs, it is often necessary to add a selection marker (e.g., ura) to the centromeric knockout module. In a preferred embodiment of the present invention, a direct repeat sequence (e.g., DR2) is provided in the chromosomal fusion module between the homologous sequence and the selectable marker; the sequence is the sequence of homologous sequence next to the telomere near region of one chromosome.

The sgRNA targeted cleavage module contains sgRNAs capable of cleaving between two homologous sequences of the centromere and two homologous sequences of the proximal centromere region. In practical practice, multiple sgrnas can be connected in series in one plasmid for expression at a time to simplify the operation.

Compared with the conventional method, the method disclosed by the invention omits the tedious operations of introducing the inactivated centromere component, transferring the inactivated centromere component into the chromosome fusion component, inducing the inactivation of the centromere and the like when fusing the two chromosomes, and is quicker and more efficient than the traditional method. And the method can also fuse three chromosomes at a time and fuse two groups of two chromosomes at a time. Compared with the traditional method, the method has the advantages that the operation steps are simpler and more convenient, the experiment time is greatly shortened, a larger number of chromosomes can be fused at one time, and the chromosome fusion efficiency is remarkably improved.

Chromosome fused strain

The invention provides a yeast engineering strain with fused chromosomes, which is characterized in that two or more chromosomes in the engineering strain are fused, and the engineering strain has 1-15 central granules and 0-30 telomere areas; and, the large repeats at the proximal grain of the engineered strain are deleted as a single copy. As a preferred mode of the present invention, 1 copy of each set of the repetitive sequences is retained based on the information of the repetitive sequences listed in Table 1, thereby obtaining a simplified, chromosomally fused, active strain.

In the specific embodiment of the invention, saccharomyces cerevisiae is used as a starting strain, and in the process of chromosome fusion, the natural centromere of the yeast chromosome is gradually deleted from 16 to 1, and the natural telomere region is gradually deleted from 32 to 2 (SY 0-SY 14). On the basis of the above, 2 telomeres are further removed to become a strain SY15 containing an artificially cyclized chromosome. In the artificial fusion process, 16 large repetitive sequences (2.7-26.2 kb) at the telomere-proximal position of the natural chromosome are deleted at the same time.

The series of engineering strains constructed by the invention have simplified and genetically stable genomes and good growth, so the engineering strains are novel eukaryotic heterologous gene expression and large-fragment DNA cloning hosts and have important application in the aspects of industry, medicine, energy and the like.

The engineering strain constructed by the invention has obvious advantages in cloning identification compared with a natural strain due to the simplified chromosome. For example, when cloning exogenous fragments into the engineered strain, recombinant cloning events are more easily distinguished.

The invention also provides a kit, wherein the yeast engineering strain containing the chromosome fusion can be one or more or all of the strains selected from the invention. Preferably, the yeast engineering strain contains chromosome fusion of SY0-SY 15.

Generally, the kit further comprises instructions for proper use of the yeast engineered strain containing the chromosome fusion of the invention by those skilled in the art.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

Construction of Cas9 expression vector

The plasmid map of the Cas9 expression vector is shown in figure 8. Plasmids were constructed which replicated high copies in E.coli (origin of replication origin from pBR322, selection marker Ampicillin (Ampicillin) -resistant gene), replicated single copies in yeast (origin of vector replication region from CEN6ARS4, selection marker LEU2) and were capable of constitutive expression of Cas 9. PCR amplification LEU2 marker (using Saccharomyces cerevisiae S288c genome as template), screening marker MET14(Zhou, J., et al., Nucleic Acids Res,2016.44(14): p.e124) homologous sequence of 50bp on both sides of plasmid pMetcas9 carried on 5' end of forward and reverse primers, after PCR product purification, transferring into yeast together with pMetcas9, screening on leucine single-deficiency culture medium, after PCR and sequencing verification, transforming into Escherichia coli DH10B, and extracting plasmid for use.

sgRNA expression vector construction

The plasmid map of the sgRNA expression vector is shown in fig. 9. Plasmids were constructed which were high-copy replication in E.coli (origin of replication origin from pBR322, selection marker ampicillin resistance gene), multicopy replication in yeast (origin of vector replication region from 2 μm, selection marker HIS3) and capable of constitutive expression of sgRNA. The sgRNA includes SNR52 promoter, handle Cas9 sequence, 20bp cleavage site.

The fusion of ChrVII and ChrVIII requires three cleavage sites S1, 2, 3 (i.e., SEQ ID NOS: 1-3), located near the centromere, right telomere and left telomere of ChrVIII, respectively.

Construction of centromere knock-out modules

And (3) selecting 50bp sequences on the left side and the right side of the centromere as a left homologous arm and a right homologous arm of the knockout component BY taking the BY4742 genome as a template, and taking a 200bp sequence on one side of the centromere as a forward repeat sequence on the targeting component (so that recombination at the sequence is generated in the future to eliminate a screening marker, and a traceless fused chromosome is obtained). PCR URA3 gene was used as a selection marker using the S288C genome as a template. The 50bp homologous arm sequence is carried on a primer, PCR forward repeat sequence and a screening marker are respectively carried out, and a centromere knockout component is obtained through fusion PCR.

Construction of chromosome fusion modules

A BY4742 genome is used as a template, a 400bp sequence on the left side of a right telomere of one chromosome is used as a homologous arm of a chromosome fusion assembly in PCR, and a 200bp sequence near one telomere of the PCR is used as a forward repeat sequence on the fusion assembly (so that recombination elimination screening markers occur at the sequence in the future to obtain a traceless fused chromosome). PCR LYS2 gene was used as a selection marker using the S288C genome as a template. The four segments are provided with 40bp homologous sequences through primers, and a chromosome fusion assembly is obtained through fusion PCR.

Saccharomyces cerevisiae chromosomal fusion

1. Taking out the strain to be fused from-70 ℃, scratching SC-leu (pleuCas9 auxotrophic mark) solid culture medium, and culturing for 2 days at 30 ℃ to grow a monoclonal;

2. selecting single clone to be inoculated in 4mL SC-leu liquid culture medium, and culturing overnight at 30 ℃;

3. transfer of overnight inoculum to 25mL SC-leu broth, starting OD_λ＝600nmCultured at 220rpm at 0.2 ℃ to the final OD_λ＝600nm＝0.8-1.0；

Centrifuging at 5,000rpm at 4.20 deg.C for 5min to collect bacteria;

5. discard the supernatant and add 20mL sterile ddH₂O heavy suspension, centrifugation at 20 ℃ and 5,000rpm for 5min for strain collection, and repeating the steps once;

6. with 500. mu.L sterile ddH₂O, resuspending, and subpackaging 5 tubes for later use;

7. the yeast was centrifuged at 13,000g for 30 seconds and the supernatant was discarded. Adding the following components in sequence (the mixture except the DNA fragment can be prepared, and then the DNA fragments can be added respectively):

note: DNA fragment 1. mu.g fusion module (URA3), 1. mu.g centromere knockout module (URA3), 1. mu.g gRNA-p426(HIS 3); positive control: circular yeast-e.coli shuttle plasmid pXX11(URA 3); negative control: only add ddH₂O 34μL。

8. Re-suspending the yeast with the mixed solution;

heat shock at 9.42 ℃ for 25 minutes;

10.13,000 g centrifuge for 1 minute to remove supernatant and use 1mLddH₂O resuspending yeast, and coating 200 and 800 μ L of the suspension on SC-Ura-His-Leu three-lacking plate;

culturing at 11.30 deg.C for 2-3 days.

12. After a transformant grows out, performing multiple PCR verification;

marking a square on an SC-Ura-His-Leu three-lacking plate (purification and amplification culture) by PCR (polymerase chain reaction) to verify the correct strain, and culturing at 30 ℃ for overnight;

14. the square-picking bacteria were inoculated into 2mL galactose induction medium SC-Leu + Gal + Raff with an initial OD of about 0.3 and incubated overnight (12-16 hours) at 30 ℃;

15. taking 100 mu L of overnight-induced bacterial liquid, coating the liquid on an SC-leu plate containing 1mg/mL 5-FOA, and culturing for 2-3 days at 30 ℃;

16. the grown transformants are marked with small squares on single-lacking plates of SC-His, SC-Ura and SC-leu in sequence for primary screening to confirm that URA3 marker and gRNA-p426 vector are lost;

17. the deletion of the URA3 marker was confirmed by PCR again;

18. the truffle on SC-leu with correct PCR was inoculated to SC-leu, cultured overnight at 30 ℃ and prepared for the next day of switching for the next set of chromosome fusion.

Pulsed field gel electrophoresis

1. Selecting a new-drawn yeast plate, inoculating a single clone to an YPAD culture medium, and culturing at 30 ℃ overnight at 240 rpm;

2. yeast cultured overnight in vitro were transferred to 50mL YPAD medium and OD initiated_λ＝600nm＝0.1；

Cultured at 3.30 ℃ and 240rpm to OD_λ＝600nmCollecting bacteria at 20 ℃ and 5,000rpm when the temperature is 1.0;

4. 50mL ddH₂o resuspending the yeast, collecting the yeast at 20 ℃ and 5,000 rpm;

5.10 mL pH8.0, 10mM EDTA heavy suspension yeast, 20 degrees C, 5,000rpm harvest;

6.750 μ L pH7.2, 10mM Tris.HCl heavy suspension, transferred to 1.5mL EP tube, 20 degrees, 5,000rpm bacteria collection;

7.150 μ L Tris.HCl (pH 7.2, 10 mM) was resuspended and equilibrated in a 50 ℃ water bath;

8. adding 150mL of Zymolyse-20T solution (20mg/mL of Zymolyse-20T, 50% glycerol, 2.5% glucose, 50mM pH8.0Tris.HCl) and 225. mu.L of 2% TE 25S-dissolved low-melting agarose (prepared in advance, equilibrated in a 50 ℃ water bath), mixing, pouring into a mold, and cooling in a refrigerator at 4 ℃;

after 9.30 min, the gel was removed, 5mL of lyticase buffer (pH7.5, 10mM Tris.HCl and pH8.0, 50mM EDTA) and 500. mu.L of Zymolyse-20T were added, and incubated at 37 ℃ for 3 hours;

10. 25mLddH₂o washing the gel lump once, and then washing once with wash buffer (pH7.5, 20mM Tris.HCl and pH8.0, 50mM EDTA);

11. 5mL of protease reaction solution (pH8.0, 100mM EDTA, 0.2% sodium deoxyholate, 1% sodium laurylsarcosine, 1mg/mL proteinase K) was added to each 1mL of the gel block and digested at 50 ℃ for 36 hours;

12.50 mL wash buffer washes the gel block 4 times, gently shaking at room temperature for 30-60 minutes each time.

13. Chromosomes of the SY0-SY10 series of strains were separated using a Bio-Rad CHEFRII instrument on a 1% agarose gel in 0.5 TBE buffer at 12.5 ℃. The switching angle was set to 120 deg., the voltage was set to 6V/cm, and the electrophoresis was carried out for 22 hours at 60s switching time and 12 hours at 90s switching time.

Chromosomes of the SY11-SY115 series of strains were separated using a Bio-Rad chefdii instrument on a 0.8% agarose gel in 1 × TAE buffer at 6 ℃. The conversion angle was set to 106 °^.Setting voltage at 1.5V/cm, electrophoresis for 27 hr at 1800s, and setting the conversion angle at 100 °^.Voltage is set to 2V/cm, switching time is 1500s electrophoresis time for 27 hours, and switching angle is set to 96 °^.The voltage was set at 2.5V/cm and the switching time 1200s electrophoresis was carried out for 27 hours.

Growth Curve determination

1. Freezing the strain on SC-leu plate at-70 deg.C, and culturing in 30 deg.C incubator for 2-3 days;

2. selecting a monoclonal antibody, inoculating the monoclonal antibody into a test tube containing YPAD liquid culture medium, and culturing overnight at 30 ℃ at the rotating speed of 240 rpm;

3. transfer overnight-cultured broth to 50ml YPAD medium to starting OD_λ＝600nmEach sample was transferred to three flasks and incubated at 30 ℃ and 240rpm, 0.1;

4. samples were taken at two hour intervals and OD determined on a spectrophotometer_λ＝600nmYPAD medium was selected as a reference, and OD was measured by sampling every 1 hour after 4 hours_λ＝600nm；

5. To measure OD twice consecutively_λ＝600nmThe overnight OD was determined after the values did not change_λ＝600nmStopping the measurement;

6. drawing a strain growth curve chart by using the recorded data;

saccharomyces cerevisiae chromosome enzyme digestion verification

10. 25mLddH₂o Wash the gel lump once and then wash once with wash buffer (pH7.5, 20mM Tris. HCl and pH8.0, 50mM EDTA)；

11. 5mL of protease reaction solution (pH8.0, 100mM EDTA, 0.2% sodium deoxyholate, 1% sodium lauryl sarcosine, 1mg/mL proteinase K) was added to each 1mL of the gel block and digested at 50 ℃ for 36 hours;

13. Cutting 1/3 gel blocks into a 1.5mL EP tube, adding 1mL wash buffer and 1mM PMSF, placing in an ice box, and shaking for 30-60 minutes;

14. pouring out the wash buffer, adding 1mL of TE ice, standing for 30 minutes, and reversing and mixing uniformly for multiple times;

15. adding 1mL of TE on ice for 30min, and reversing and mixing uniformly for multiple times;

16. the TE solution was carefully aspirated off with a 1mL pipette and the tube was flicked to allow the agarose block to fall to the bottom of the tube. Adding 500 mu L of enzyme digestion reaction buffer solution, standing on ice for 30-60 minutes, and balancing the reaction system;

17. carefully absorbing the enzyme digestion reaction buffer solution by using a 1mL pipette gun, adding 10 mu L of the enzyme digestion reaction buffer solution and 22U of FseI endonuclease, and digesting for 2 hours at 37 ℃;

18. after the enzyme digestion reaction, the centrifuge tube was placed on ice and the enzyme and buffer were aspirated. Adding 1mL of electrophoresis buffer solution into the tube, balancing on ice for 15-30min, and turning over the EP tube when not needed;

19. the digested gel was separated on a Bio-Rad CHEFRII instrument on a 1% agarose gel in 0.5 TBE buffer at 8 ℃. The conversion angle is set to 120 °^.The voltage was set at 6V/cm, electrophoresis was carried out for 23 hours at 60s switching time and for 12.5 hours at 90s switching time.

Saccharomyces cerevisiae genome de novo sequencing

The Saccharomyces cerevisiae wild type starting strain BY4742 (containing 16 natural chromosomes) and the engineering strain SY14 of 'chromosome 16 in 1' are sent to Wuhan Feisha gene information limited company for de novo sequencing of genome. PacBio platform pooling was performed on each sample, and 1 large fragment library of 20kb was constructed for each sample. Using the PacBio sequential platform data acquisition model, 1 cell was sequenced per sample DNA library, with single cell data volumes ranging from 4-5G. And performing genome assembly after removing joints, pollution sequences and low-quality reads on the original data.

Example 1Construction of the strains

The starting strain of the series of chromosome fusion engineering strains constructed BY the invention is a derivative strain BY4742 of a Saccharomyces cerevisiae wild strain S288C, and the genotype is MAT alpha his3 delta 1 leu2 delta 0 lys2 delta 0 ura3 delta 0(Brachmann, C.B. et al (1998); Yeast, 14, 115-.

The 16 natural chromosome telomere-proximal regions of the strain BY4742 have 13 main repeated sequences (Table 1), wherein the repeated

sequences

1, 7, 9 and 10 are 3 copies, and the rest 11 repeated sequences are 2 copies. The length of the repeated sequences is 2.7-26.2kb, the repeated sequences are easy to become hot spots of chromosome homologous recombination, further influence the stability of the whole genome, and can interfere the site-directed homologous recombination of chromosome fusion. To eliminate the main telomeric repeat, the inventors deleted altogether 19 repeats, including 2 copies of

repeat

1, 7, 9, 10 and 1 copy of

repeat

2, 3, 4, 5, 6, 8, 11, 12, 13, 14, 15.

TABLE 1 information of the repeat sequences of the telomeric near region of the Saccharomyces cerevisiae native chromosomes

The construction process of a series of chromosome fused engineering strains is shown in figure 1. A high-efficiency chromosome fusion method mediated BY a CRISPR-Cas9 system utilizing DNA double-strand fixed-point cutting is carried out, and a strain SY0(14 natural chromosomes and 1 '2 in 1' fused chromosome) is obtained BY deleting 5 repetitive sequences close to a chromosome telomere region and fusing 2 natural chromosomes VII and VIII from a strain BY 4742. Deletion of the repeat sequence at 11 was additionally achieved during chromosome fusion by deleting telomeres of the chromosome to be fused (Table 2).

As shown in FIG. 1, the natural chromosomes XIII and XII, I and II, VI and XIV, IVI and V, IX and X, III and IV are fused on the basis of SY0 to obtain strains SY1(12 natural chromosomes and 2 fused chromosomes of 2 in 1), SY2(10 natural chromosomes and 3 fused chromosomes of 2 in 1), SY3(8 natural chromosomes and 4 fused chromosomes of 2 in 1), SY4(6 natural chromosomes and 5 fused chromosomes of 2 in 1), SY5(4 natural chromosomes and 6 fused chromosomes of 2 in 1) and SY6(2 natural chromosomes and 7 fused chromosomes of 2 in 1). On the basis of SY6, 2 '2 in 1' fusion chromosomes XVI-V and VI-XIV are fused to obtain SY7(2 natural chromosomes, 5 '2 in 1' fusion chromosomes, and 1 '4 in 1' fusion chromosome). SY8(6 "2 in 1" fusion chromosomes and 1 "4 in 1" fusion chromosome) is obtained after the last 2 natural chromosomes XV and XI of SY7 are fused. Cumulative fusion of the "2 in 1" fused chromosomes VII-VIII and IX-X, I-II and III-IV, XIII-XII and XV-XI on the basis of SY8 gives strains SY9(4 "2 in 1" fused chromosomes and 2 "4 in 1" fused chromosomes), SY10(2 "2 in 1" fused chromosomes and 3 "4 in 1" fused chromosomes), SY11(4 "4 in 1" fused chromosomes). Cumulative fusion of the "4 in 1" fusion chromosomes I-II-III-IV and VII-VIII-IX-X, XVI-V-VI-XIV and XIII-XII-XV-XI on the basis of SY11 gave the strain SY12(2 "4 in 1" fusion chromosomes and 1 "8 in 1" fusion chromosome), SY13(2 "8 in 1" fusion chromosomes). 2 "8 in 1" fusion chromosomes XVI-V-VI-XIV-XIII-XI-XV-XI and I-II-III-IV-VII-VIII-IX-X of SY13 were fused into 1 "16 in 1" fusion chromosome, resulting in strain SY 14. The chromosomes of the obtained strain SY0-SY14 are all linear chromosomes. The strain SY15 containing only 1 large circular chromosome is obtained after 1 large '16 in 1' linear chromosome of the strain SY14 is cyclized. The detailed information of the saccharomyces cerevisiae engineering strain constructed by the invention is shown in table 2.

TABLE 2 details of the engineered strains for chromosomal fusions

TABLE 3 Targeted cleavage sites at chromosomal fusions

Pulsed Field Gel Electrophoresis (PFGE) is used for identifying the chromosome size of a series of saccharomyces cerevisiae engineering strains constructed by the invention. As shown in FIG. 2, the change of chromosomal bands of Saccharomyces cerevisiae strains SY0-SY10 detected by PFGE was in line with the theoretical calculation (Table 2). About 10⁸Individual cells were collected separately and used to prepare corresponding low melting agarose gel blocks. The gel block is treated by muramidase and proteinase K and then subjected to pulse electrophoresis. The separation was performed using a Bio-Rad CHEFRII instrument at 12.5 ℃ in 0.5 XTBE buffer. The electrophoresis conditions were such that the switching angle was set to 120 ℃ and the voltage was set to 6V/cm, the electrophoresis was carried out for 22 hours at 60s switching time and for 12 hours at 90s switching time.

The fusion chromosome of the strain SY11-SY15 is large (2.5-11.8 Mb), so that the PFGE condition needs to be changed for carrying out chromosome separation and identification (figure 3). The theoretical size of 4 fused chromosomes of strain SY11 is 2523, 2747, 2821 and 3682 kb; the theoretical size of the 3 fused chromosomes of strain SY12 was 2523, 3682 and 5558 kb; the theoretical size of 2 fused chromosomes of strain SY13 was 5558 and 6191 kb. As shown in FIG. 3, the chromosome size of strain SY11-SY13 is consistent with theoretical calculations. The theoretical size of the 1-line chromosome of strain SY14 is 11737kb, and it can be seen in FIG. 3 that a large chromosome of SY14 is well above the 5.7Mb DNA standard band. The theoretical size of the 1 circular chromosome of strain SY15 was 11727kb, but the large circular chromosome failed to escape the rubber block (chromosomal DNA remained in the spotted well), so SY15 did not escape the visible DNA band in fig. 3.

About 10 for each strain⁸Individual cells were collected separately and used to prepare corresponding low melting agarose gel blocks. The gel block is treated by muramidase and proteinase K and then subjected to pulse electrophoresis. The separation was performed using a Bio-Rad CHEFRII instrument in 1 XTAE buffer at 6 ℃. The electrophoresis conditions were that the switching angle was set to 106 °, the voltage was set to 1.5V/cm, the switching time was 1800s, electrophoresis was carried out for 27 hours, the switching angle was set to 100 °, the voltage was set to 2V/cm, the switching time was 1500s, electrophoresis was carried out for 27 hours, the switching angle was set to 96 °, the voltage was set to 2.5V/cm, and electrophoresis was carried out for 27 hours, the switching time was 1200 s.

Genomic de novo sequencing showed that the chromosome of SY14 had good colinearity with the reference genome of saccharomyces cerevisiae (fig. 4), and that the chromosome fusion sequence was consistent with the design. The results of the genomic sequencing of strain SY14 are reported in the appendix.

Example 2Strain performance

Growth of the Strain

The saccharomyces cerevisiae engineering strains with the chromosome fusion constructed by the invention have good growth. The one-by-one fusion of the linear natural chromosomes does not affect the growth of the strain. The growth curves in FIG. 5 show that the growth of strains SY11-SY13 (FIG. 5, A) and SY14 (FIG. 5, B) is almost identical to the wild-type starting strain BY 4742. While the cyclization of the chromosome resulted in a slightly slower growth of strain SY15 compared to the wild-type strain BY4742 (FIG. 5, B).

Example 3Stability of genome

The saccharomyces cerevisiae engineering strains with the chromosome fusion constructed by the invention have genetically stable genomes. FIG. 6 shows the restriction enzyme digestion verification diagram of the genome of SY14 and SY15 of the strain after 0 generation growth and 100 successive generations growth. The strain SY14 chromosome can theoretically cut 23 bands of 1569, 1446, 1125, 1050, 892, 891, 778, 587, 548, 414, 412, 381, 363, 318, 258, 210, 141, 133, 82, 78, 27, 25.7 and 0.9kb by using a restriction enzyme FseI. The chromosome of the strain SY15 is cyclized to the chromosome SY14, so that the FseI enzyme cutting map of SY15 is reduced by 1125 and 82kb compared with SY 14. The two bands are combined into one in SY15, which is a new 1193kb difference band.

The de novo sequencing whole genome sequence of the strain SY14 is shown as SEQ ID NO: 48.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

1. The yeast engineering strain with fused chromosomes is characterized in that more than two chromosomes in the engineering strain are fused, and the engineering strain is provided with 1-15 central granules and 0-30 telomere areas; and, the multiple copy repeat sequence at the proximal plasmid of the engineered strain is deleted to a single copy sequence; the yeast is saccharomyces cerevisiae;

the repetitive sequence is selected from:

repeat sequence set 1: chromosome VIII 525437-539926; 13089-; i: 203448-219229; wherein I is reserved at 13089-;

repeat sequence set 2: chromosome I219230-229411; VIII, 539927-543610, 549638-556001; wherein, the VIII is preserved as 539927-;

repeat sequence group 7: chromosome XIII 917474-923540; XV 1078061-1083736; 7409-13083; wherein, the XVI is preserved 7409-13083, and the other 2 strips are deleted;

repeat sequence group 9: chromosome XV 10454-13126; IX, 8286-10347; 8269-10330; wherein IX 8286-10347 is reserved, and the other 2 strips are deleted;

repeat sequence set 10: chromosome XV 22397-27006; 16656-25250; 16639-21229; wherein IX 16656 and 25250 are reserved, and the other 2 strips are deleted;

repeat sequence group 3: chromosome VII 1076381-1083886; 805133-812631; wherein 805133 and 812631 are reserved, and another 1 is deleted;

repeat sequence group 4: chromosome XIV 7429-17224; VI 5531-14039; wherein VI is reserved for 5531-14039 and another 1 is deleted;

repeat sequence set 5: chromosome XI 658572-665429; 4327-11225; wherein, the III is reserved 4327-11225, and the other 1 is deleted;

repeat sequence group 6: chromosome VII 5545-9584; IX 430983-4367; wherein IX is reserved 430983-;

repeat sequence group 8: chromosome XV 1073988-1078544; XVI: 12601-17099; wherein, the XVI:12601-17099 is reserved, and the other 1 is deleted;

repeat sequence group 11: chromosome V18067-23447; XIV 772693-; wherein 772693-777126 is reserved, and another 1 is deleted;

repeat sequence set 12: chromosome IV 905-18681; x727164-; wherein, X is 727164-;

repeat sequence group 13: chromosome XII, 1059296-; 303903-308316; wherein, the step III is reserved for 303903-308316, and the other 1 is deleted;

repeat sequence set 14: chromosome IX 21251-25254; 27007-30676; wherein, the IX:21251-25254 is reserved, and the other 1 is deleted; and/or

Repeat sequence set 15: chromosome IX 10348-16655; 10331 and 16638; wherein IX:10348-16655 is reserved and another 1 is deleted.

2. The engineered yeast strain of claim 1, wherein the engineered strain is selected from the group consisting of:

SY2, which is based on SY1, and has right telomere and central grain deleted from chromosome I, repetitive sequences 1 and 2 and chromosome II;

SY0, which is based on natural Saccharomyces cerevisiae and has deleted the repetitive sequence 11, VII right telomere and repetitive sequences 6 and 3 in chromosome V, the left telomere, the central particle and repetitive sequence 1 in chromosome VIII, and the repetitive sequences 9, 10 and 14 in XV.

3. The engineered yeast strain of claim 2, wherein the engineered strain is selected from the group consisting of:

SY15, which is characterized in that on the basis of SY14, chromosomes XVI:1-7700, X:744639-745751 are deleted;

SY14, which is based on SY13 and has the deletion of chromosome IV 449662-449840, XI 658549-666816, I1-3294;

SY13, which is characterized in that on the basis of SY12, chromosomes XIV, 779113, 784333, XVI, 555957, 556549 and XIII, 1-9319 are deleted;

SY12, which is characterized in that on the basis of SY11, chromosomes IV 1522800-1531933, VII:1-1240 and 496756-497069 are deleted;

SY11, which has deleted chromosome XII:150828-150947, 1059256-1078177, XV:1-3709 on the basis of SY 10;

SY10, which is characterized in that on the basis of SY9, chromosomes I, 151221, 151933, II, 809896, 813184 and III, 1-1500 are deleted;

SY9, which is based on SY8 and has the deletion of chromosome VIII 552000-562643, IX 1-11214, X436229-436425;

SY8, which is characterized in that on the basis of SY7, chromosomes XV:1073966-1091291, XI:439551-440264 and 1-3182 are deleted;

SY7, which is characterized in that on the basis of SY6, chromosomes V, 569325-576874, VI, 1-8380, XIV, 628734-629219 are deleted;

SY6, which is based on SY5 and has the deletion of chromosomes III 313621-316620, 114297-114969, IV 1-19188;

SY5, which is based on SY4 and has the deletion of chromosomes IX 436361-439888, 355607-356006 and X1-21750;

SY4, which is characterized in that on the basis of SY3, chromosomes XVI 941976-948066, V1-8079 and 151829-152588 are deleted;

SY3, which is characterized in that on the basis of SY2, chromosomes VI, 269616, 148460, 148774 and XIV, 1-17790, are deleted;

SY2, which is characterized in that on the basis of SY1, chromosomes I, i.e. 203183-230218, II, i.e. 1-9089, 237784-238794 are deleted;

SY1, which has the deletion of chromosomes XIII 916869-924431, 268013-268803 and XII 1-14474 on the basis of SY 0;

SY0, which has the deletion of chromosomes V18067-23447, VII 5545-9584, 1076068-1090940, VIII 524501-541100, 1-8217, 105447-106013, XV 10454-13126 and 21791-30776 on the basis of native Saccharomyces cerevisiae.

4. The engineered yeast strain of claim 3, wherein the engineered strain is SY15 strain, and telomeres at both ends of fused chromosome are deleted and cyclized.

5. A fused chromosome isolated from the engineered yeast strain of any one of claims 1 to 4; the yeast is saccharomyces cerevisiae.

6. The chromosome of claim 5, wherein the chromosome is isolated from the engineered yeast strain of claim 3.

7. A method for preparing a yeast engineering strain with chromosome fusion, wherein the yeast is Saccharomyces cerevisiae, and the method comprises the following steps:

(1) fusing chromosomes VII and VIII on the basis of natural saccharomyces cerevisiae; deleting the repeated sequence 11, the right telomere VII and the repeated sequences 6 and 3 in the chromosome V, the left telomere, the centromere and the repeated sequence 1 in the chromosome VIII, and the repeated sequences 9, 10 and 14 in the chromosome XV; obtaining a SY0 strain;

(3) fusing chromosomes I and II on the basis of SY 1; deleting the right telomere and the repetitive sequences 1 and 2 of the chromosome I and the left telomere and the central grain of the chromosome II; obtaining a SY2 strain;

(16) deleting the left telomere of the chromosome XVI and the right telomere of the chromosome X on the basis of SY 14; obtaining a SY15 strain;

wherein, the repetitive sequence 1: chromosome VIII 525437-539926; 13089-; i: 203448-219229;

repeat sequence 2: chromosome I219230-229411; VIII, 539927-543610, 549638-556001;

repeat sequence 3: chromosome VII 1076381-1083886; 805133-812631;

repeat sequence 4: chromosome XIV 7429-17224; VI 5531-14039;

repeat sequence 5: chromosome XI 658572-665429; 4327-11225;

repeat sequence 6: chromosome VII 5545-9584; IX 430983-4367;

repeat sequence 7: chromosome XIII 917474-923540; XV 1078061-1083736; 7409-13083;

repeat sequence 8: chromosome XV 1073988-1078544; XVI: 12601-17099;

repeat sequence 9: chromosome XV 10454-13126; IX, 8286-10347; 8269-10330;

repeat sequence 10: chromosome XV 22397-27006; 16656-25250; 16639-21229;

repeat sequence 11: chromosome V18067-23447; XIV 772693-;

repeat sequence 12: chromosome IV 905-18681; x727164-;

repeat sequence 13: chromosome XII, 1059296-; 303903-308316;

repeat sequence 14: chromosome IX 21251-25254; 27007-30676; and/or

Repeat sequence 15: chromosome IX 10348-16655; x10331-16638.

8. The method of claim 7, wherein the chromosome is edited by the CRISPR/Cas9 method to delete the target sequence.

9. The method of claim 7 or 8, wherein when the CRISPR/Cas9 method is used, the sgRNA target site on the genome is selected from the group consisting of:

In step (16), the sites shown in SEQ ID NO:46 and SEQ ID NO:47 are targeted.

10. A kit comprising the engineered yeast strain fused with the chromosome of any one of claims 1 to 4.

11. The kit of claim 10, wherein the engineered yeast strain comprises a chromosomal fusion of SY0-SY 15 as follows:

12. Use of the engineered yeast strain of claims 1-4 or the chromosome of claims 5 or 6 as cloning host for eukaryotic heterologous gene expression.

13. Use of the kit of claim 10 or 11 for the expression of a eukaryotic heterologous gene.

14. A kit for establishing a chromosome fusion yeast engineering strain is characterized by comprising a targeted cleavage site sequence SEQ ID NO 1-47 during chromosome fusion; the yeast is saccharomyces cerevisiae.