Saccharomyces cerevisiae engineered strain with single-chromosome large fragment overlapping deletion and application thereof

Abstract

The present application relates to a kind of single-chromosome large fragment superimposed deletion engineering strains of saccharomyces cerevisiae, it is IV number chromosome large fragment superimposed deletion saccharomyces cerevisiae, the chassis of the saccharomyces cerevisiae is wild type saccharomyces cerevisiae strain CEN.PK113-5D;The IV number chromosome large fragment superimposed deletion includes at least two large fragments of large fragment Chr4-1, Chr4-10, Chr4-11 in IV number chromosome of wild type saccharomyces cerevisiae strain CEN.PK113-5D, and the superimposed deletion of large fragment, thereby obtaining the genome simplified engineering strain, its growth phenotype is significantly better than wild type strain;The present application realizes the optimization of saccharomyces cerevisiae growth performance by single-chromosome large fragment combination deletion strategy, and the obtained engineering strain can be widely used in industrial production scenarios such as ethanol fermentation, biosynthetic chemical synthesis, to provide efficient strain resources and technical support for improving microbial fermentation efficiency and reducing production cost.

Description

Technical Field

[0001] This invention belongs to the field of microbial genome engineering and synthetic biology technology, specifically relating to an engineered strain of Saccharomyces cerevisiae with a large superimposed deletion of a single chromosome segment and its application. Background Technology

[0002] Saccharomyces cerevisiae is one of the most commonly used host bacteria in eukaryotic genetic engineering, and it has irreplaceable application value in fermented foods, bioenergy, and biomedicine. Targeted optimization of its growth performance is a core requirement for reducing industrial fermentation costs and improving production efficiency. However, its 16 natural chromosomes contain a large number of non-essential gene clusters and redundant fragments. These fragments increase the metabolic burden on the genome, interfere with target metabolic pathways, and limit the efficiency of the strain's application in industrial production. The maturity of CRISPR / Cas9 gene editing technology has provided an efficient tool for precise genome modification. Further research on the minimal genome of *Saccharomyces cerevisiae* (e.g., Kaboli et al., 2014 report, Genome-wide mapping of unexplored essential regions in the *Saccharomyces cerevisiae* genome: evidence for hidden syntheticlethal combinations in a genetic interaction network, Nucleic Acids Research, 2014, 1 doi: 10.1093 / nar / gku576, 565-0871) has revealed that there are 110 large regions containing only non-essential genes in the entire *Saccharomyces cerevisiae* genome. Chromosome IV (approximately 1532 kb in length) contains multiple deleteable segments (such as Chr4-1, Chr4-2, Chr4-7, Chr4-10, Chr4-11, etc.), and some regions contain synthetic lethal combinations that were not detected by SGA technology. This provides important theoretical and experimental evidence for large-segment deletion modification of chromosome IV. Although existing research has laid the foundation for genome simplification, there are still unresolved technical gaps in the targeted growth optimization of Saccharomyces cerevisiae chromosome IV. Specifically: First, most modification methods are "single-point deletion" or "scattered fragment deletion," lacking an efficient and systematic strategy for "continuous large-fragment superposition deletion" of a single chromosome, resulting in incomplete genome simplification. Second, for Saccharomyces cerevisiae chromosome IV (approximately 1532kb in length), current modifications can only achieve limited fragment deletion, failing to reach the goal of simplification to a smaller or even minimal genome. A large number of redundant chromosome fragments remain, failing to maximize the release of the strain's metabolic potential. Third, large-fragment deletions can easily lead to slowed growth and decreased environmental adaptability, making it difficult to balance "genome simplification" and "functional integrity." Fourth, there is a lack of systematic characterization of strains with continuous deletions of multiple fragments on a single chromosome, and the correlation between the number of deleted fragments and strain performance (growth, tolerance) remains unclear.

[0003] Current technologies have not yet achieved the superposition and deletion of large, continuous segments of chromosome IV, making it impossible to construct a smaller genome for this chromosome, nor can they meet the industrial production requirements for host bacteria with "high precision, high stability, and high adaptability." Therefore, developing single-chromosome directed large-segment superposition and deletion technology to construct engineered strains with higher precision on chromosome IV has significant application value. Summary of the Invention

[0004] The technical problem to be solved by this invention is that existing technologies cannot meet the industrial production requirements for host bacteria with "high precision, high stability, and high adaptability". This invention provides an engineered strain of Saccharomyces cerevisiae with a large superimposed deletion of a single chromosome and its application. This engineered strain of Saccharomyces cerevisiae can meet the industrial production requirements for host bacteria with "high precision, high stability, and high adaptability" and can achieve the goal of constructing a smaller genome of the chromosome.

[0005] The first aspect of the present invention provides an engineered strain of Saccharomyces cerevisiae with a large superimposed deletion on a single chromosome, wherein the Saccharomyces cerevisiae is a wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

[0006] According to the present invention, the large fragment superposition deletion of chromosome IV includes the superposition deletion of at least two large fragments of Chr4-1, Chr4-10, and Chr4-11 on chromosome IV of wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

[0007] In this invention, the large fragment Chr4-1 is the fragment from position 906 to 31829 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D, with a length of 30924 bp.

[0008] In this invention, the large fragment Chr4-10 is the fragment from position 1474773 to 1487620 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D, with a length of 12848 bp.

[0009] In this invention, the large fragment Chr 4-11 is the fragment at positions 1502166-1524625 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D, with a length of 22460bp.

[0010] In some specific preferred embodiments of the present invention, the large fragment superimposed deletion of chromosome IV is the superimposed deletion of two large fragments, Chr4-1 and Chr4-10, on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

[0011] In some other preferred embodiments of the present invention, the large fragment superposition deletion of chromosome IV is the superposition deletion of three large fragments Chr4-1, Chr4-10 and Chr4-11 on chromosome IV of wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

[0012] According to the present invention, the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D has no missing telomeres on chromosome IV, specifically the large segments Chr4-11 and Chr4-11.

[0013] The second aspect of this invention provides the application of the above-mentioned engineered strain of Saccharomyces cerevisiae in the expression of eukaryotic heterologous genes.

[0014] In some specific embodiments of the present invention, the applications include, but are not limited to, pharmaceutical protein synthesis, natural product precursor synthesis, and bio-based chemical synthesis.

[0015] The advantages of this invention are:

[0016] (1) Innovation of modification goals: For the first time, the construction of a smaller or even smallest genome of Saccharomyces cerevisiae chromosome IV was achieved. Through the triplet continuous large fragment superposition deletion, the chromosome reduction rate was increased to 4.3%, breaking through the reduction limit of existing modifications;

[0017] (2) Modification strategy system: Abandon the "dispersed deletion" mode and adopt the continuous stacking deletion strategy. The genome simplification is more controllable, more thorough and faster, and maximizes the removal of redundant fragments;

[0018] (3) Performance balance optimization: While achieving the minimum genome construction of chromosome IV, ensure that the growth rate and nutrient utilization efficiency of the strain are comparable to or even better than those of the wild type, and significantly improve environmental tolerance (ethanol, drug stress);

[0019] (4) High technical reproducibility: The standardized process of sgRNA targeted design, homologous arm construction and step-by-step screening can be directly extended to minimal genome modification of other chromosomes or even other species;

[0020] (5) Wide range of applications: The high-precision genome reduces metabolic redundancy and can be used as a highly efficient host bacteria for heterologous gene expression (such as drug proteins, natural product precursors) and industrial fermentation (such as ethanol and organic acid production).

[0021] (6) The present invention provides a host bacterium that can meet the industrial production requirements for “high precision, high stability and high adaptability”. It is beneficial to improve replication / division efficiency, reduce metabolic load, enhance genetic stability, improve the expression efficiency of exogenous genes, and facilitate rapid analysis of gene functions. It can achieve the goal of constructing a smaller genome of the chromosome. Attached Figure Description

[0022] The present invention will now be described in further detail with reference to the accompanying drawings:

[0023] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0024] Figure 1 This demonstrates the validation results of the engineered strain successfully knocking out the large fragment 4-1.

[0025] Figure 2 This demonstrates the validation results of the engineered strain successfully knocking out the large fragment 4-10.

[0026] Figure 3 This demonstrates the validation results of the engineered strain successfully knocking out the large fragment 4-11.

[0027] Figure 4 This diagram shows the location of the large fragment knocked out by the engineered strain.

[0028] Figure 5 The growth curve of the engineered strain in YPD liquid medium is shown.

[0029] Figure 6 This is the growth curve of the engineered strain in SC liquid medium.

[0030] Figure 7 The specific growth rate of the engineered strain is shown.

[0031] Figure 8 The specific growth rate of the engineered strain is shown.

[0032] Figure 9 The images show electron microscope images of the engineered strains; the left image is the wild-type control strain CEN.PK113-5D of Saccharomyces cerevisiae; the right image is the engineered strain 5D△Chr.IV-1-10-11.

[0033] Figure 10 The images show the drop plate images of the engineered strains; the top image shows the drop plate images in YPD medium and SC complete medium at 22℃, 30℃, and 37℃; the bottom image shows the drop plate images under different tolerance conditions. Detailed Implementation

[0034] To facilitate understanding of the present invention, it will be described in detail below with reference to the accompanying drawings and embodiments. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0035] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0036] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0037] I. Terminology

[0038] The symbol “Δ” used in this invention represents deletion.

[0039] II. Implementation Plan

[0040] As mentioned earlier, although existing research has laid the foundation for genome simplification, there are still unresolved technical gaps in the targeted growth optimization of Saccharomyces cerevisiae chromosome IV. Specifically, these gaps are: First, most modification methods involve "single-point deletion" or "dispersed fragment deletion," lacking an efficient and systematic strategy for "continuous large-fragment superimposed deletion" of a single chromosome, resulting in incomplete genome simplification. Second, for Saccharomyces cerevisiae chromosome IV (approximately 1532kb in length), current modifications can only achieve limited fragment deletion, failing to reach the goal of simplification to a smaller or even minimal genome. Redundant chromosome fragments remain in large quantities, failing to maximize the release of the strain's metabolic potential. Third, large-fragment deletions can easily lead to slowed growth and decreased environmental adaptability, making it difficult to balance "genome simplification" and "functional integrity." Fourth, there is a lack of systematic characterization of strains with continuous deletions of multiple fragments on a single chromosome, and the correlation between the number of deleted fragments and strain performance (growth, tolerance) remains unclear. Existing technologies have not yet achieved the superposition and deletion of large consecutive segments of chromosome IV, which cannot achieve the goal of constructing a smaller genome of this chromosome, nor can they meet the industrial production requirements for host bacteria with "high precision, high stability, and high adaptability". In view of this, the inventors have conducted extensive and in-depth research on the engineering technology of Saccharomyces cerevisiae.

[0041] The inventors discovered that by superimposing and deleting large segments of a single chromosome in a brewer's yeast chassis strain, genome simplification can be achieved, resulting in an engineered brewer's yeast strain with improved strain performance. This invention was thus obtained.

[0042] Specifically, to achieve the present invention, an engineered strain of *Saccharomyces cerevisiae* with a large superimposed deletion on a single chromosome was constructed. The inventors used the wild-type strain *Saccharomyces cerevisiae* CEN.PK113-5D as the starting strain (i.e., the chassis strain), and targeted the longest chromosome IV of *Saccharomyces cerevisiae*. Using CRISPR / Cas9-mediated homologous recombination technology, a "stepwise superposition and continuous deletion" modification strategy was designed to precisely delete non-essential continuous large segments on chromosome IV, constructing a core engineered strain (achieving the construction of a smaller genome on chromosome IV). At the same time, its growth and environmental adaptability were systematically characterized to obtain *Saccharomyces cerevisiae* with a large superimposed deletion on chromosome IV.

[0043] More specifically, the engineered strain obtained by superimposing and deleting at least two large segments from Chr4-1, Chr4-10, and Chr4-11 on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D exhibited a significantly improved specific growth rate.

[0044] In this invention, the large fragment Chr4-1 is a fragment located at positions 906-31829 on chromosome IV, with a length of 30924 bp. Its sequence is shown in SEQ ID No. 1, and it is referred to as "large fragment IV:4-1" in this invention.

[0045] Specifically, the elements contained in the above interval and the corresponding genome sequences of each element include:

[0046] Two ARS: ARS400_ARS, 906-1392, and ARS403_ARS, 15493-15739;

[0047] 1 ACS: ARS403_ARS_consensus_sequence, 15666-15682;

[0048] 14 CDSs: YDL248W_CDS, 1802 - 2953; YDL247W-A_CDS, 3762 - 3836; YDL247W_CDS, 5985 - 7814; YDL246C_CDS, 8683 - 9756; YDL245C_CDS, 11657 - 13360; YDL244W_CDS, 16204 - 17226; YDL243C_CDS, 17577 - 18566; YDL242W_CDS, 18959 - 19312; YDL241W_CDS, 20635 - 21006; YDL240C-A_CDS, 22471 - 22608; YDL240W_CDS, 22823 - 25876; YDL239C_CDS, 26403 - 28775; YDL238C_CDS, 28985 - 30454; YDL237W_CDS, 30657 - 31829;

[0049] 14 genes: YDL248W, 1802 - 2953; YDL247W-A, 3762 - 3836; YDL247W, 5985 - 7814; YDL246C, 8683 - 9756; YDL245C, 11657 - 13360; YDL244W, 16204 - 17226; YDL243C, 17388 - 19288; YDL242W, 18959 - 19312; YDL241W, 20553 - 21443; YDL240C-A, 22471 - 22608; YDL240W, 22657 - 26119; YDL239C, 26403 - 28775; YDL238C, 28894 - 30503; YDL237W, 30609 - 31829;

[0050] 18 mRNAs: YDL248W_mRNA, 1802-2953, YDL247W-A_mRNA, 3762-3836, YDL247W_mRNA, 5985-7814, YDL246C_mRNA, 8683-9756, YDL245C_mRNA, 11657-13360, YDL244W_mRNA, 16204-17226, YDL243C_id001, 17388-19288, YDL243C_id003, 17461-19280, YDL242W_mRNA, 18959-19312, YD L241W_id001, 20553-21443, YDL241W_id004, 20555-21335, YDL240C-A_mRNA, 22471-22608, YDL240W_id001, 22657-26119, YDL239C_mRNA, 26403-28775, YDL238C_id001, 28894-30503, YDL238C_id006, 28919-30495, YDL237W_id001, 30609-31829, YDL237W_id002, 30625-31829;

[0051] Fourteen unannotated sequences: 1392-1802, 2953-3762, 3836-5985, 7814-8683, 9756-11657, 13360-15493, 15739-16204, 17226-17388, 19312-20553, 21443-22471, 22608-22657, 26119-26403, 28775-28894, and 30503-30609.

[0052] In this invention, the large fragment Chr4-10 is a fragment located at positions 1474773-1487620 on chromosome IV, with a length of 12848 bp. Its sequence is shown in SEQ ID No. 2, and it is referred to as "large fragment IV:4-10" in this invention.

[0053] Specifically, the elements contained in the above interval and the corresponding genome sequences of each element include:

[0054] 1 ARS: ARS446, 1486918-1487161;

[0055] 1 ACS: ARS446_ARS_consensus_sequence, 1487076-1487092;

[0056] 9 CDSs: YDR516C_CDS, 1474974 - 1476476; YDR517W_CDS, 1477239 - 1478357; YDR518W_CDS, 1478608 - 1480161; YDR519W_CDS, 1480425 - 1480832; YDR520C_CDS, 1481085 - 1483403; YDR521W_CDS, 1483141 - 1483476; YDR522C_CDS, 1483795 - 1485303; YDR523C_CDS, 1485566 - 1487038; YDR524C_CDS, 1487542 - 1487620; 10 genes: YDR515W, 1474773 - 1474873; YDR516C, 1474842 - 1476588; YDR517W, 1476623 - 1478460; YDR518W, 1478467 - 1480211; YDR519W, 1479966 - 1482129; YDR520C, 1480938 - 1483487; YDR521W, 1483141 - 1483476; YDR522C, 1483795 - 1485303; YDR523C, 1485514 - 1487263; YDR524C, 1487035 - 1487620;

[0057] 16 mRNAs: YDR515W_id001, 1474773-1474873, YDR516C_id001, 1474842-1476588, YDR516C_id003, 1474854-1476588, YDR517W_id001, 1476623-1 478460, YDR517W_id004, 1476818-1478460, YDR518W_id001, 1478467-1480211, YDR518W_id002, 1478467-1480210, YDR519W_id001, 1480370-148 2129, YDR519W_id002, 1479966-1481057, YDR520C_id001, 1480938-1483487, YDR520C_id002, 1480950-1483485, YDR521W_mRNA, 1483141-14834 76. YDR522C_mRNA, 1483795-1485303, YDR523C_id001, 1485514-1487263, YDR524C_id001, 1487035-1487620, YDR524C_id002, 1487060-1487620;

[0058] Five unannotated sequences: 1476588-1476623, 1478460-1478467, 1483487-1483795, 1485303-1485514, and 1487263-1487542.

[0059] In this invention, the large fragment Chr 4-11 is a fragment located at positions 1502166-1524625 on chromosome IV, with a length of 22460 bp. Its sequence is shown in SEQ ID No. 3, and it is referred to as "large fragment IV: 4-11" in this invention.

[0060] Specifically, the elements contained in the above interval and the corresponding genome sequences of each element include:

[0061] 9 CDSs: YDR534C_CDS, 1503314 - 1504900; YDR535C_CDS, 1506606 - 1507058, 1507315 - 1507362; YDR536W_CDS, 1508005 - 1509714; YDR538W_CDS, 1510902 - 1511630; YDR537C_CDS, 1510856 - 1511461; YDR539W_CDS, 1512094 - 1513605; YDR540C_CDS, 1517136 - 1517675; YDR541C_CDS, 1519664 - 1520698; YDR542W_CDS, 1523249 - 1523611;

[0062] 10 genes: YDR533C, 1502166 - 1502385; YDR534C, 1503065 - 1505484; YDR535C, 1506606 - 1507362; YDR536W, 1507861 - 1509860; YDR538W, 1509972 - 1511919; YDR537C, 1510856 - 1511461; YDR539W, 1512000 - 1513955; YDR540C, 1516282 - 1518078; YDR541C, 1519437 - 1520919; YDR542W, 1523249 - 1523611;

[0063] 15 mRNAs: YDR533C_id001, 1502166-1502385, YDR533C_id004 1502166-1502324, YDR534C_id001, 1503065-1505484, YDR535C_mRNA 1506606-1507362, YDR536W_id001, 1507861-1509860, YDR538W_id001 1509972-1511919, YDR538W_id003, 1509993-1511861, YDR537C_mRNA 1510856-1511461, YDR539W_id001, 1512000-1513953, YDR539W_id0031512051-1513955, YDR540C_id001, 1516282-1517758, YDR540C_id002 1516834-1518078, YDR541C_id001, 1519437-1520919, YDR541C_id002 1519465-1520919, YDR542W_mRNA, 1523249-1523611;

[0064] One intron: YDR535C_intron, 1507059-1507314;

[0065] 1 LTR: YDRWdelta31, 1518484-1518807;

[0066] Ten unannotated sequences: 1502385-1503065, 1505484-1506606, 1507362-1507861, 1509860-1509972, 1511919-1512000, 1513955-1516282, 1518078-1518484, 1518807-1519437, 1520919-1523249, and 1523611-1524625.

[0067] It is worth mentioning that the study found that the loss of telomeres leads to the inability of engineered Saccharomyces cerevisiae strains with large deletions of single chromosome segments to express normally. Therefore, the large segments Chr4-1 and Chr4-11 on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D do not lack telomeres. That is, when the large segments Chr4-1 and Chr4-11 are knocked out, the large segments Chr4-1 and Chr4-11 on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D retain telomeres; while the large segment Chr4-10 is located internally and has no telomeres, so it is completely deleted.

[0068] In some specific preferred embodiments of the present invention, the large fragment superimposed deletion of chromosome IV is the superimposed deletion of two large fragments, Chr4-1 and Chr4-10, on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

[0069] In some other preferred embodiments of the present invention, the large fragment superposition deletion of chromosome IV is the superposition deletion of three large fragments Chr4-1, Chr4-10 and Chr4-11 on chromosome IV of wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

[0070] In some specific embodiments of the present invention, the present invention first provides an engineered strain of *Saccharomyces cerevisiae* with a large deletion, which is an engineered strain of *Saccharomyces cerevisiae* with a large deletion of the Chr4-1 fragment on chromosome IV of the wild-type *Saccharomyces cerevisiae* strain CEN.PK113-5D (named 5D△Chr.IV-1 in this invention); the deletion sequence group 1 of this engineered strain is: chromosome IV: 4-1, 906-31829; the specific deletion includes the following elements: 2 ARS, 1 ACS, 14 CDS, 14 genes, 18 mRNAs, and 14 unannotated sequences. The total deletion length of 5D△Chr.IV-1 compared to the wild type is 30924 bp.

[0071] In other specific embodiments of the present invention, a large-fragment superimposed deletion engineered strain of *Saccharomyces cerevisiae* is provided, which is an engineered strain with superimposed deletions of two large fragments, Chr4-1 and Chr4-10, on chromosome IV of the wild-type *Saccharomyces cerevisiae* strain CEN.PK113-5D (named 5D△Chr.IV-1-10 in this invention). It is obtained by further deleting sequence group 2 from the engineered strain 5D△Chr.IV-1: chromosome IV: 4-10, 1474773-1487620. The specific deletions of 5D△Chr.IV-1-10 compared to the wild-type strain include the following elements: 3 ARS, 2 ACS, 23 CDS, 24 genes, 34 mRNAs, and 19 unannotated sequences. The total deletion length of 5D△Chr.IV-1-10 compared to the wild-type strain is 43772 bp.

[0072] In some specific embodiments of the present invention, another engineered strain of *Saccharomyces cerevisiae* with large-fragment superimposed deletions is provided. This strain is an engineered strain with superimposed deletions of three large fragments, Chr4-1, Chr4-10, and Chr4-11, on chromosome IV of the wild-type *Saccharomyces cerevisiae* strain CEN.PK113-5D (named 5D△Chr.IV-1-10-11 in this invention). It is further modified by deleting sequence group 3 on chromosome IV: 4-11, 1502166-1524625, based on the engineered strain 5D△Chr.IV-1-10. The specific deletions of 5D△Chr.IV-1-10-11 compared to the wild-type strain include the following elements: 3 ARS, 2 ACS, 32 CDS, 34 genes, 49 mRNA, 1 intron, 1 LTR, and 29 unannotated sequences. The total deletion length of 5D△Chr.IV-1-10-11 compared to the wild-type is 66232 bp. The engineered strain achieved a chromosome IV reduction rate of 4.3%, realizing the smallest genome size among existing modifications of the Saccharomyces cerevisiae chromosome IV.

[0073] This invention relates to the construction method and application of engineered strains of *Saccharomyces cerevisiae* with large-fragment superposition deletions on a single chromosome. Using *Saccharomyces cerevisiae* CEN.PK 113-5D as the starting strain, this invention employs CRISPR / Cas9-mediated gene editing technology to precisely delete three pre-defined large fragments (4-1, 4-10, and 4-11) on chromosome IV of *Saccharomyces cerevisiae*, constructing an engineered strain with a simplified genome due to large-fragment combination deletions. Through droplet culture observation and quantitative determination of specific growth rate, an engineered strain with a growth phenotype significantly superior to the wild type was obtained. This invention optimizes the growth performance of *Saccharomyces cerevisiae* through a single-chromosome large-fragment combination deletion strategy. The resulting engineered strain can be widely applied in industrial production scenarios such as ethanol fermentation and bio-based chemical synthesis, providing efficient strain resources and technical support for improving microbial fermentation efficiency and reducing production costs.

[0074] The second aspect of this invention provides the application of the above-mentioned engineered strain of Saccharomyces cerevisiae in the expression of eukaryotic heterologous genes.

[0075] In some specific embodiments of the present invention, the applications include, but are not limited to, pharmaceutical proteins, natural product precursors, and the synthesis of bio-based chemicals.

[0076] III. Examples

[0077] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are all conventional laboratory methods. Unless otherwise specified, the experimental materials described below can be obtained from commercial channels (e.g., conventional biochemical reagent companies) or by conventional methods. In the quantitative experiments in the following embodiments, three replicate experiments were set up, and the results were averaged.

[0078] Experimental materials and methods used in the following examples

[0079] 1. Strains and plasmids

[0080] The strains and plasmids constructed in this invention are shown in Table 8, and the primers used in the construction process are shown in Table 9. All primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0081] This experiment used *Escherichia coli* stable (Uri-Co (Shanghai) Life Science Co., Ltd., Shanghai Fuyu Biotechnology Co., Ltd.) and *Saccharomyces cerevisiae* CEN.PK113-5D (MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2) (BioVector plasmid vector strain cell protein antibody gene depositation center - NTCC typical culture depositation center) as host bacteria. *Saccharomyces cerevisiae* CEN.PK113-5D is a modified strain that cannot express the URA3 gene after frameshift mutation in the yeast genome. URA3 can be used as a marker gene during transformation.

[0082] 2. Main instruments and equipment for the experiment

[0083] Table 1 Main Experimental Instruments

[0084]

[0085] 3. Culture medium

[0086] The culture media used in this invention include: Luria-Bertani (LB) medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride (NaCl) 10 g / L, autoclaved at 121℃ for 20 min before use; Yeast Extract-Tryptone-Dextrose (YPD) medium: yeast extract 10 g / L, tryptone 20 g / L; SC (Synthetic Complete) complete medium: yeast nitrogen base 1.7 g / L, ammonium sulfate ((NH4)2SO4) 5 g / L, amino acid mixture (AA-Ura) 1.913 g / L, uracil (Ura) 0.086 g / L; SC (Synthetic Complete) auxotrophic medium: yeast nitrogen base 1.7 g / L, ammonium sulfate ((NH4)2SO4) 5 g / L, amino acid mixture (AA-Ura) 1.913 g / L, uracil (Ura) 0.086 g / L; 5 g / L amino acid mixture (AA-Ura) 1.827 g / L; 5-FOA medium: yeast nitrogen base 1.7 g / L, ammonium sulfate ((NH4)2SO4), 5 g / L, amino acid mixture (AA-Ura) 1.827 g / L, uracil (Ura) 0.086 g / L. The above-mentioned culture media contain 0.2% 5-fluoroorotic acid (5-FOA) at a concentration of 20 g / L. These media are primarily used for the cultivation of *Escherichia coli* and *Saccharomyces cerevisiae*. YPD medium, SC complete medium, SC auxotrophic medium, and 5-FOA medium all contain 20 g / L glucose. The additional function of 5-FOA medium is to remove plasmids labeled with URA3. Furthermore, all solid culture media require the addition of a certain amount of agar powder to the liquid medium formulation. Generally, 15 g of agar powder is added to 1 L of liquid medium, and 25 g of agar powder is added to 5-FOA medium. A rotor is added during sterilization to facilitate thorough mixing of glucose and culture medium later. Finally, the media are autoclaved at 121°C for 20 minutes.

[0087] 4. Donor DNA PCR

[0088] In this experiment, the donor DNA was annealed using NEB's Q5 enzyme for PCR. Compared to other PCR polymerases, it requires a higher annealing temperature; the basic principle is to anneal at Tm+3℃ for 10-30 seconds from the lowest Tm. Two primers, upstream and downstream, were used for the donor in the PCR design. The Tm value of the repeat portion between the two primers was generally around 50-55℃, and the donor length was 60-200 bp.

[0089] The specific reaction system and experimental procedure settings are shown in Tables 2 and 3.

[0090] Table 2. Components of the PCR system for NEB Q5 enzyme

[0091]

[0092] Table 3. PCR reaction procedure for NEB Q5 enzyme.

[0093]

[0094] 5. Construction of plasmids using the GoldenGate method

[0095] Multi-fragment plasmids were constructed using the goldenGate method. The specific system is as follows:

[0096] Table 4 Components of the GoldenGate System

[0097]

[0098] Table 5. Reaction Procedure for GoldenGate

[0099]

[0100] Plasmids were constructed using the Golden Gate method. First, the target site to be edited was identified, followed by gRNA sequence design. The website https: / / www.atum.bio / eCommerce / cas9 / input was accessed, and the species *Saccharomyces cerevisiae*, "NGG", and "wildtype-Cas9" were selected. SnapGene was then used to design an upstream primer containing the guide RNA sequence corresponding to the target deletion sequence and a BsaI restriction site, and a downstream primer containing the homologous sequence with the URA3 selection tag and a BsaI restriction site. This constructed a plasmid with the insertion site guide, Cas9, and selection tag sequences. Golden Gate assembly technology only requires the participation of Bsa1 and T4 ligases to construct plasmids. Bsa1 recognizes and cleaves the cleavage site, forming sticky ends, while the cleavage site detaches. The T4 ligase ligates the sticky ends, leaving no cleavage site after ligation, preventing further cleavage by Bsa1. The PCR product was then transformed into *E. coli*, and single clones were selected for sequencing.

[0101] The pCas-LacZ vector is used, where the LacZ sequence is flanked by the SNR52 promoter and gRNA backbone, with BsaI cleavage sites at the ends of the SNR52 promoter (GATC) and the gRNA backbone (GTTT). To assemble multiple gRNAs on a single plasmid, the lacZ sequence is removed by BsaI and replaced with a PCR-generated fragment. The PCR template is a plasmid containing a gRNA scaffold with tRNA sequences or a gRNA backbone with selectable markers. For the first and last gRNAs, 20 bp gRNA targeting sequences are designed on primers, and these two sites can be ligated to the pCas vector. Four bp sequences from the remaining 20 bp gRNA targeting sequences are used as Golden Gate ligation sites to assemble different fragments. Primers used are designed to be no more than 60 bp in length.

[0102] The strains and plasmids constructed in this invention are shown in Table 8, and the primers used in the construction process are shown in Table 9.

[0103] 6. Saccharomyces cerevisiae transformation, genome extraction and validation procedures

[0104] Yeast transformation can be performed using either electroporation or chemical transformation, both methods generally yielding sufficient transformants. During transformation, Cas9 plasmids carrying different gRNAs and donors (PCR products recovered via ethanol precipitation) are simultaneously transformed into yeast strains. The plasmid dosage is 300-500 ng, and the donor dosage is 10,000-20,000 ng. Transformed yeast cells are plated on SC-URA plates, and after transformant enrichment, genome extraction is performed, followed by PCR verification of the knockout results. Streaking is done on 5-FOA plates to remove the Cas9 plasmid. This study used the defective medium SC-URA for preliminary screening of the constructed strains; however, this screening step cannot determine whether gene manipulation is complete, only screening for strains whose plasmids have been electroporated into yeast cells. Therefore, this study designed appropriate primers for PCR verification.

[0105] Yeast genome extraction: Yeast cell clones were enriched and cultured on SC-URA plates; yeast cells were collected by centrifugation after being suspended in 100 μL of sterile water. The yeast cells should occupy approximately 5-10 μL of the plate; the yeast cells were suspended in 100 μL of 200 mM LiOAc 1% SDS solution and placed in a metal bath at 70°C for 15 min; 70% ethanol was added to a final concentration (either 300 μL of 96% ethanol or 233 μL of 100% ethanol) and centrifuged at the highest speed for 3 min; the supernatant was discarded, and the precipitate was resuspended in 100 μL of sterile water and centrifuged again. The supernatant was then transferred to a new EP tube; generally, 0.5 μL of the supernatant was used as a template in a 25 μL PCR validation system for subsequent PCR validation.

[0106] 7. Validate PCR

[0107] This study used TIANGEN's 2×TaqPCRMasterMix for cloning PCR verification. The annealing time of the PCR reaction was set according to the Tm value of the primers, and the extension time was set according to the size of the target gene. All gene editing results in the experiments were verified using this enzyme.

[0108] Table 6. Components of TIANGEN's 2×TaqPCRMasterMixPCR system

[0109]

[0110] Table 7. Reaction procedures for TIANGEN's 2×TaqPCRMasterMixPCR.

[0111]

[0112] 8. Growth characterization methods for deletion strains

[0113] This technique involves the analysis of the growth characteristics of engineered Saccharomyces cerevisiae strains, applicable to the comparison of growth differences between gene-deleted strains and wild-type strains, and the quantitative characterization of specific growth rates. First, single colonies are picked and activated on plates. Saccharomyces cerevisiae is inoculated onto SC complete solid medium containing 20 g / L glucose or YPD solid medium containing 20 g / L glucose plates and incubated statically at 30°C for 2-3 days until distinct single colonies appear on the plates. A single colony is picked using a sterile inoculation loop and transferred to a shaker containing the appropriate liquid medium (e.g., SC complete medium or YPD medium containing 20 g / L glucose). The culture is then incubated overnight at 30°C and 250 rpm to obtain the seed culture.

[0114] Growth curve: The culture medium was transferred to 50 mL of SC complete medium containing 20 g / L glucose or YPD medium containing 20 g / L glucose in 100 mL shake flasks at an OD600 of 0.1. The entire process was carried out at a constant temperature of 30℃, and dissolved oxygen level was maintained by shaking at a frequency of 250 rpm. Samples were taken every 2-12 hours, and the OD600 value was measured using a UV spectrophotometer (zeroing was done with blank medium). The data were recorded, and the culture period was set to 70 hours.

[0115] Specific growth rate: At an OD600 of 0.01, the culture was transferred to 100 mL shake flasks containing either 20 mL of SC complete medium (20 g / L glucose) or YPD medium (20 g / L glucose). The entire process was conducted at a constant temperature of 30°C, with dissolved oxygen levels maintained by shaking at 250 rpm. Samples were taken every 1-3 hours, and the OD600 value was measured using a UV spectrophotometer (zeroing the culture medium). Data were recorded, and the logarithmic growth phase (OD600 0.2~1.5) data were selected. The result was calculated using the formula μ = (lnOD600) / (OD600). t μ is calculated as μ = lnOD0) / (t-t0), where h is the specific growth rate. - ¹; OD0 is the logarithmic initial OD value, OD t The OD value is the logarithmic value at a certain moment (t0 and t are the corresponding time points), and the average of the three parallel sets is taken as the final result.

[0116] By comparing the growth curves, logarithmic phase duration, and specific growth rates of the deleted strain and the wild-type strain, the promoting or inhibiting effect of the large deleted region on the strain's growth can be determined.

[0117] 9. Methods for tolerance analysis of deletion strains

[0118] YPD solid medium with different pH values ​​(4.0, 6.0, 9.0), and YPD and SC solid medium were prepared at different temperatures (22℃, 30℃, 37℃) to simulate environmental stress during growth. The growth and survival of the missing strain and the wild-type strain were compared to quantitatively assess their tolerance threshold.

[0119] By adding different concentrations of stress factors such as ethanol, lactic acid, glycerol, and rapamycin to YPD solid medium, environmental stress during the growth process was simulated. The growth and survival of the missing strain and the wild-type strain were compared, and their tolerance threshold was quantitatively evaluated.

[0120] If the growth inhibition rate of the deleted strain under a certain environmental condition or concentration of a certain stress factor is significantly lower than that of the wild type (or the survival rate is significantly higher than that of the wild type), it indicates that the deletion of the gene improves the strain's tolerance to the stress factor; otherwise, it indicates a decrease in tolerance.

[0121] Example 1:

[0122] Three different expression plasmids were constructed using the Golden Gate method (1.5): 4-1 (IV-1), 4-10 (IV-1-10), and 4-11 (IV-1-10-11). These plasmids were then serially transformed into *E. coli* for amplification, and then co-transformed with the donor into *Saccharomyces cerevisiae* strain CEN.PK113-5D, resulting in the construction of the engineered *Saccharomyces cerevisiae* strains 5D△Chr.IV-1, 5D△Chr.IV-1-10, and 5D△Chr.IV-1-10-11, respectively.

[0123] The strains and plasmids constructed in this invention are shown in Table 8, and the primers used in the construction process are shown in Table 9.

[0124] Table 8. Plasmids constructed in this invention

[0125]

[0126] Table 9 Primers used in this invention

[0127]

[0128] The results show that:

[0129] (1) Validation of knockout of large fragments in engineered strains

[0130] Methods 1.5 and 1.7 were used to perform agarose gel electrophoresis to verify the knockout results. The control group consisted of genomic bands with no large deleted fragments, while the bands with successfully deleted large fragments were set at approximately 500 bp. The specific primer list is shown in Table 9.

[0131] Figure 1 , Figure 2 and Figure 3 The bands represent the deletion results of different engineered strains obtained by successfully knocking out large fragments 4-1, 4-10, and 4-11 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D. Figure 4 A schematic diagram showing the location where large fragments were knocked out in the engineered strain.

[0132] (2) Growth curve of engineered strains

[0133] Figure 5 The growth curves of different engineered strains in YPD liquid medium at 30℃ are shown. Compared with the control group CEN.PK113-5D, the growth status of engineered strains 5D△Chr.IV-1 and 5D△Chr.IV-1-10 decreased, while the growth of 5D△Chr.IV-1-10-11 was significantly improved.

[0134] Figure 6The growth curves of different engineered strains in SC liquid medium at 30℃ are shown. Compared with the control group CEN.PK113-5D, the growth status of engineered strain 5D△Chr.IV-1 did not change significantly, the growth status of 5D△Chr.IV-1-10 decreased slightly, and the growth status of 5D△Chr.IV-1-10-11 decreased.

[0135] (3) Specific growth rate of engineered strains

[0136] Figure 7 The specific growth rates of different engineered strains in YPD liquid medium at 30°C were compared with those of the control group CEN.PK113-5D. The specific growth rates of engineered strains 5D△Chr.IV-1 and 5D△Chr.IV-1-10 decreased, while the specific growth rate of 5D△Chr.IV-1-10-11 increased significantly.

[0137] Figure 8 The specific growth rates of different engineered strains in SC liquid medium at 30°C were compared with those of the control group CEN.PK113-5D. The specific growth rates of the engineered strains 5D△Chr.IV-1, 5D△Chr.IV-1-10, and 5D△Chr.IV-1-10-11 were all slightly lower.

[0138] (4) Colony status of engineered strains

[0139] Figure 9 The image shows an electron microscope image of the engineered strain 5D△Chr.IV-1-10-11. The colonies are significantly smaller than those of the wild type. It is speculated that the absence of yeast 5D△Chr.IV-1-10-11 may lead to the loss of some important nutrient sources, resulting in this phenomenon.

[0140] (5) Tolerance analysis of engineered strains

[0141] Figure 10 This is a drop plate diagram showing the tolerance of the engineered strain. Under SC 22℃ (22°C), pH 4.0, and lactic acid conditions, 5D△Chr.IV-1-10-11, with the three large fragments removed, could not grow normally, exhibiting strong tolerance to ethanol, pH 9.0, and rapamycin.

[0142] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. An engineered strain of *Saccharomyces cerevisiae* with a large superimposed deletion on a single chromosome, wherein the substrate strain of *Saccharomyces cerevisiae* is a wild-type strain CEN.PK113-5D.

2. The engineered strain of *Saccharomyces cerevisiae* according to claim 1, characterized in that, The large fragment overlap deletion on chromosome IV includes the overlap deletion of at least two large fragments from Chr4-1, Chr4-10, and Chr4-11 on chromosome IV of wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

3. The engineered strain of *Saccharomyces cerevisiae* according to claim 2, characterized in that, The large fragment Chr4-1 is the fragment from position 906 to 31829 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D, with a length of 30924 bp.

4. The engineered strain of *Saccharomyces cerevisiae* according to claim 2, characterized in that, The large fragment Chr4-10 is the fragment from position 1474773 to 1487620 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D, with a length of 12848 bp.

5. The engineered strain of *Saccharomyces cerevisiae* according to claim 2, characterized in that, The large fragment Chr 4-11 is the fragment from position 1502166 to 1524625 of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D, with a length of 22460 bp.

6. The engineered strain of *Saccharomyces cerevisiae* according to any one of claims 2-5, characterized in that, The large fragment superposition deletion on chromosome IV refers to the superposition deletion of two large fragments, Chr4-1 and Chr4-10, on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

7. The engineered strain of *Saccharomyces cerevisiae* according to any one of claims 2-5, characterized in that, The large fragment superposition deletion on chromosome IV refers to the superposition deletion of three large fragments, Chr4-1, Chr4-10, and Chr4-11, on chromosome IV of the wild-type Saccharomyces cerevisiae strain CEN.PK113-5D.

8. The engineered strain of *Saccharomyces cerevisiae* according to any one of claims 3-7, characterized in that, The wild-type Saccharomyces cerevisiae strain CEN.PK113-5D did not have telomeres missing on large segments of Chr4-1 and Chr4-11 on chromosome IV.

9. The use of the engineered strain of *Saccharomyces cerevisiae* as described in any one of claims 1-8 in the expression of eukaryotic heterologous genes.

10. The application according to claim 9, characterized in that, The applications include pharmaceutical protein synthesis, natural product precursor synthesis, and bio-based chemical synthesis.

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