Method for improving yield of valarene by using saccharomyces cerevisiae rearrangement technology
Through synthetic chromosomal rearrangement technology and yeast Mating mating, a diploid valenne production strain was constructed, which solved the problem of low valenne production in Saccharomyces cerevisiae haploid strain, and achieved a significant increase in valenne production.
Patent Information
- Application Number
- CN202510338866.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-21
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, the Saccharomyces cerevisiae haploid strain is less efficient in valenne production, and there is no effective rearrangement technology to improve valenne production.
The Saccharomyces cerevisiae rearrangement technology was used to construct a library of Saccharomyces cerevisiae rearrangement strains, and the haploid strains were mated with valenne haploid production strains through yeast Mating technology to generate diploid valenne production strains, achieving genomic evolution.
Through the fermentation of diploid valenone production strains, the valenone production increased by 18.5%, reaching 194.14 mg/L, significantly improving the production capacity of Saccharomyces cerevisiae.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a method for increasing the yield of valencene by rearrangement technology of brewer's yeast. Background Art
[0002] Saccharomyces cerevisiae rearrangement technology is a type of engineering strategy that optimizes metabolic pathways, enhances cell tolerance or improves product synthesis efficiency by artificially intervening in genome structure or expression regulatory networks. The core of Saccharomyces cerevisiae rearrangement technology is to break the static limitations of the natural genome and build a more efficient "cell factory" by rearranging, combining or regulating genetic elements. Commonly used technologies include genome shuffling, synthetic chromosome shuffling (SCRaMbLE), and dynamic regulatory system design. For example, genome shuffling quickly screens out strains with optimized phenotypes through recursive protoplast fusion or transposon-mediated random recombination; while synthetic chromosome shuffling is based on artificially designed synthetic yeast genomes (such as the Sc2.0 project), using LoxP sites to induce large-scale chromosome recombination and generate a diverse mutant library. These technologies can obtain high-yield strains through high-throughput screening without pre-analyzing complex metabolic networks, significantly accelerating the process of metabolic engineering.
[0003] In terms of increasing the output of cell factories, rearrangement technology has shown significant results. Taking the production of terpenoids as an example, the JBEI Institute team in the United States used synthetic chromosome rearrangement technology to integrate the amorphadiene synthase gene into the synthetic chromosome of Saccharomyces cerevisiae. By inducing chromosome breakage and recombination, they screened mutants with increased flux of the terpene precursor farnesyl pyrophosphate (FPP), and finally increased the production of artemisinic acid to 2.5g / L, an 8-fold increase over the original strain. Another typical example is the biosynthesis of resveratrol: the Chinese Academy of Sciences team used genome rearrangement combined with adaptive laboratory evolution to obtain a yeast strain that tolerates high concentrations of malonyl-CoA, and increased the production of resveratrol from 32mg / L to 415mg / L by removing the competitive inhibition of precursors. In addition, in the production of amino acids, Novozymes used global regulatory factor rearrangement technology to perform combined mutations on transcription factors such as LEU3 and GCN4, successfully removing the feedback inhibition of the leucine synthesis pathway, and increasing the production of L-leucine by 12 times.
[0004] The rearrangement technology is particularly suitable for the collaborative optimization of complex traits. For example, in bioethanol production, it is necessary to simultaneously increase the glycolytic flux and ethanol tolerance. The University of California, Berkeley integrated multiple beneficial mutations of heat-resistant strains and high-yield strains through iterative genome rearrangement, and finally obtained an industrial strain with an ethanol yield of 90% of the theoretical value at 42°C. In the synthesis of the drug intermediate sclareol, the researchers adopted a dynamic regulation rearrangement strategy to decouple the expression of the rate-limiting enzyme HMG-CoA reductase from the cell growth stage, and activated product synthesis in the late stage of biomass accumulation through a self-regulating promoter, increasing the yield from 0.8 g / L to 5.2 g / L. These examples show that the rearrangement technology can break through the bottleneck of traditional rational design by systematically reconstructing the cell metabolic network, providing a powerful tool for constructing efficient microbial cell factories. Summary of the Invention
[0005] Currently, the biosynthesis of valencene mainly uses haploid strains of Saccharomyces cerevisiae for production, and the relevant literature has not reported using yeast rearrangement strategies to increase valencene production. Saccharomyces cerevisiae has extremely high operability at the genomic scale, and the phenotype of Saccharomyces cerevisiae can be optimized through rearrangement and Mating techniques, which is more conducive to the production of valencene.
[0006] One of the objectives of the present invention is to provide a method for increasing valencene production by using a Saccharomyces cerevisiae rearrangement technology.
[0007] Another objective of the present invention is to provide a method for constructing a diploid valencene-producing strain.
[0008] Another objective of the present invention is to provide a diploid valencene-producing strain.
[0009] Another objective of the present invention is to provide an application of a diploid valencene-producing strain in the fermentation preparation of valencene.
[0010] In order to achieve the above-mentioned invention objectives, the present invention is realized through the following technical solutions:
[0011] A method for increasing valencene production by using a Saccharomyces cerevisiae rearrangement technology, comprising the following steps:
[0012] S1: Construct a Saccharomyces cerevisiae rearrangement strain library using synthetic chromosome rearrangement technology;
[0013] S2: Mate the haploid strains in the a-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with α-type valencene haploid production strains to generate diploid valencene production strains; or, mate the haploid strains in the α-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with a-type valencene haploid production strains to generate diploid valencene production strains;
[0014] S3: Ferment the diploid valencene-producing strain obtained in S2 to obtain valencene products.
[0015] Further, S1 includes: using Saccharomyces cerevisiae as a chassis strain, constructing a starting strain with mating type a or α and synthetic chromosomes V and X by synthetic biology means; culturing, inducing, and rearranging the starting strain in sequence to cause genome evolution and obtain a Saccharomyces cerevisiae rearrangement strain library.
[0016] Preferably, the chassis strain is BY4741 strain or BY4742 strain.
[0017] A method for constructing a diploid valencene-producing strain, comprising the following steps:
[0018] S1: Construct a Saccharomyces cerevisiae rearrangement strain library using synthetic chromosome rearrangement technology;
[0019] S2: Mate the haploid strains in the a-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with α-type valencene haploid-producing strains to generate diploid valencene-producing strains; or mate the haploid strains in the α-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with a-type valencene haploid-producing strains to generate diploid valencene-producing strains;
[0020] Further, S1 includes: using Saccharomyces cerevisiae as a chassis strain, constructing a starting strain with mating type a or α and synthetic chromosomes V and X by synthetic biology means; culturing, inducing, and rearranging the starting strain in sequence to cause genome evolution and obtain a Saccharomyces cerevisiae rearrangement strain library.
[0021] Preferably, the chassis strain is BY4741 strain or BY4742 strain.
[0022] The diploid valencene-producing strain obtained by the above method for constructing a diploid valencene-producing strain.
[0023] The application of the above diploid valencene-producing strain in fermenting and preparing valencene.
[0024] The beneficial effects of the present invention are:
[0025] The present invention constructs a Saccharomyces cerevisiae rearrangement strain library using the Synthetic Chromosome Rearrangement (SCRaMbLE) technology, and mates the haploid strains in the Saccharomyces cerevisiae rearrangement strain library with the haploid strains for valencene production through yeast Mating to generate diploid valencene-producing strains. These strains and the α-type haploid valencene-producing strains (control) are subjected to fermentation verification. Finally, the yield of the control strain is 163.81 mg / L, and the highest yield of the strains in the diploid yeast rearrangement library is 194.14 mg / L, with a yield increase of 18.5%, proving that the present invention improves the valencene production ability. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 It is a graph showing the yield results of the haploid chassis strain and the diploid valencene-producing strain in the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION
[0027] The following combines the drawings and examples to describe the specific implementation of the present invention in more detail, so as to better understand the solution of the present invention and the advantages of its various aspects. It should be noted that the following described specific implementation manners and examples are for illustrative purposes only and are not limitations on the present invention.
[0028] Example 1
[0029] I. Construction of yeast rearrangement library:
[0030] Using Saccharomyces cerevisiae BY4741 as the chassis strain, synthetic biological means are used to construct a starting strain with synthetic chromosomes V and X and mating type a. The starting strain is cultured, induced, and rearranged to undergo directed genome evolution of the yeast flexible chassis.
[0031] The specific process is as follows:
[0032] 1. Inoculate a single colony in 2 mL of SC-His and culture overnight.
[0033] 2. Wash twice with ddH2O the next day.
[0034] 3. Resuspend in SGal-His medium containing 1 μM estradiol (Sigma-Aldrich) and 2% galactose (the content of galactose, i.e., mass / volume) to an OD 600 = 0.2. Induce the strain at 30 °C for 8 hours to express the recombinase in the cells and start SCRaMbLE.
[0035] 4. Take 1 mL of the culture and centrifuge to separate the yeast cells, wash twice with ddH2O, and resuspend in 1 mL of SC-His medium. Dilute the bacteria washed in the previous step to an OD 600= 0.2, dilute it 10-fold with SC-His glucose medium -3 、5 * 10 -4 and 10 -4 times, then coat it on the YPD glucose agar plate, and culture the strain at 30 °C until yeast colonies grow out (24 hours to 36 hours).
[0036] 5. Select yeast colonies from them. Streak culture on YPD glucose agar, and inoculate 1 mL of YPD glucose medium into a 96-well plate, then culture it with shaking at 30 °C for 12 hours.
[0037] 6. Subculture the rearranged bacteria, and lose the pGal-Cre413 plasmid through subculture. Take 100 μL of the bacterial liquid from the previous generation every 12 hours and transfer it to 1 mL of YPD medium, then culture it in a 96-well plate. After subculturing four generations, the rearranged library strains that can be mated with the corresponding strains are obtained.
[0038] II. Saccharomyces cerevisiae mating method:
[0039] Pick a single colony of the α-type valencene haploid production strain into 3 mL of SC-ura liquid medium, culture it at 30 °C and 250 rpm for 16 hours, then inoculate it into a 250 mL conical flask containing 50 mL of SC-ura liquid medium at an initial OD 600 = 0.2, and culture it at 30 °C and 250 rpm for 16 h. At the same time, inoculate 192 yeast strains from the a-type yeast rearrangement library into a 96-deep well plate containing 600 μL of YPD liquid medium per well, and culture it at 30 °C and 250 rpm for about 24 hours.
[0040] Take another two sterile 96-deep well plates, place 600 μL of YPD medium in each well, and add 100 μL of the valencene synthesis strain bacterial liquid and 100 μL of the corresponding α-type yeast rearrangement library bacterial liquid respectively. After culturing at 30 °C and 250 rpm for 8 h, use a pipette tip to pick the bacterial liquid and streak it on the SC-ura solid plate, then place it in an incubator at 30 °C for 24 h. Then pick the single colony with the largest relative size on the plate for boiling bacteria and colony PCR to verify the mating type, and obtain the diploid Saccharomyces cerevisiae strain that can produce valencene.
[0041] The verification primers are: VRF-mat-F, VRF-mat-A, VRF-mat-alpha. The sequences of these 3 primers from 5'→3' are: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 respectively. When using the primers for diploid screening, the primer pairs selected are: VRF-mat-F, VRF-mat-A and VRF-mat-F, VRF-mat-alpha. If only the first pair of primers amplifies a positive band, the strain is a-type haploid yeast. If only the second pair of primers amplifies a positive band, the strain is α-type haploid yeast. If both pairs of primers amplify positive bands, the strain is diploid yeast.
[0042] III. Fermentation of diploid rearrangement library strains:
[0043] (1) Primary seed preparation: Pick an appropriate amount of cells from the solid plate and inoculate them into a 10 mL shaking flask containing 3 mL of YPD liquid medium. Culture at 30 °C and 250 rpm for 16 hours.
[0044] (2) Transfer the primary seed liquid to a new 10 mL shaking flask at an initial concentration of OD 600 = 0.2. Each flask contains 5 mL of YPD liquid medium and is placed in a shaking incubator at 30 °C and 250 rpm for culture;
[0045] (3) After culturing for 12 h, add 1 mL of isopropyl myristate (IPM) and 500 μL of 100 g / L galactose mother liquor to each flask, and culture in a shaking incubator at 30 °C and 250 rpm for 72 hours to obtain the valencene fermentation product.
[0046] After fermentation, process the organic phase sample and perform gas chromatography-tandem mass spectrometry detection.
[0047] Using Saccharomyces cerevisiae BY4741 as the chassis strain to construct an a-type yeast rearrangement library, mate the α-type valencene-producing strain with 192 strains in the a-type Saccharomyces cerevisiae rearrangement library, boil the bacteria and verify the mating type by colony PCR to obtain 52 diploid Saccharomyces cerevisiae strains that can produce valencene. Perform two-phase fermentation in a 10 mL shaking flask, and determine the fermentation yields of the 52 diploid valencene-producing strains by gas chromatography-tandem mass spectrometry, as Figure 1 shown. The results show that the yield of the valencene haploid-producing strain is 163.81 mg / L, the highest yield of the diploid valencene-producing strain is 194.14 mg / L, and the yield has increased by 18.5% compared to the original strain (val_1). The yields of the other diploid valencene-producing strains are all lower than that of the haploid-producing strain.
[0048] Although the preferred embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many specific transformations in various forms without departing from the spirit of the invention and the scope protected by the claims. All of these fall within the protection scope of the present invention.
Claims
1. A method for increasing the yield of valencene by a Saccharomyces cerevisiae rearrangement technique, characterized in that, It includes the following steps: S1: Using synthetic chromosome rearrangement technology on the chassis strain to construct a Saccharomyces cerevisiae rearrangement strain library; S2: Mating the haploid strains in the a-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with the α-type valencene haploid production strains to generate diploid valencene production strains; or mating the haploid strains in the α-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with the a-type valencene haploid production strains to generate diploid valencene production strains; S3: Fermenting the diploid valencene production strains obtained in S2 to obtain valencene products.
2. A method for increasing the yield of valencene by using a Saccharomyces cerevisiae rearrangement technique according to claim 1, characterized in that, S1 includes: Using Saccharomyces cerevisiae as the chassis strain, constructing a starting strain with mating type a or α and synthetic chromosomes V and X by synthetic biology means; successively culturing, inducing, and rearranging the starting strain to cause genomic evolution and obtain a Saccharomyces cerevisiae rearrangement strain library.
3. A method for increasing the yield of valencene by a Saccharomyces cerevisiae rearrangement technique according to claim 2, characterized in that, The chassis strain uses BY4741 strain or BY4742 strain.
4. A method for constructing a diploid valencene-producing strain, characterized in that, It includes the following steps: S1: Using synthetic chromosome rearrangement technology to construct a Saccharomyces cerevisiae rearrangement strain library; S2: Mating the haploid strains in the a-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with the α-type valencene haploid production strains to generate diploid valencene production strains; or mating the haploid strains in the α-type Saccharomyces cerevisiae rearrangement strain library obtained in S1 with the a-type valencene haploid production strains to generate diploid valencene production strains.
5. The construction method of a diploid valencene-producing strain according to claim 4, characterized in that, S1 includes: Using Saccharomyces cerevisiae as the chassis strain, constructing a starting strain with mating type a or α and synthetic chromosomes V and X by synthetic biology means; successively culturing, inducing, and rearranging the starting strain to cause genomic evolution and obtain a Saccharomyces cerevisiae rearrangement strain library.
6. The construction method of a diploid valencene-producing strain according to claim 4, characterized in that, The chassis strain uses BY4741 strain or BY4742 strain.
7. A diploid valencene-producing strain, characterized in that, Obtained by the construction method according to any one of claims 4-6.
8. An application of fermenting a diploid valencene production strain as claimed in claim 7 to prepare valencene.