Application of MIC19 gene and ECM15 gene in improving yield of geraniol

Abstract

The invention provides a method for producing saccharomycetes with high geraniol yield or a method for improving the geraniol yield in the saccharomycetes, which comprises the following steps: reducing the expression of an MIC19 gene and / or improving the expression of an ECM15 gene in the saccharomycetes; wherein the saccharomycetes have geraniol synthase genes. The invention finds that two yeast endogenous targets, namely the MIC19 gene and the ECM15 gene, play an important role in regulating and controlling synthesis of geraniol, and the yield of geraniol of a yeast strain can be increased by regulating and controlling the expression of the two genes.

Description

Technical Field

[0001] The invention relates to geraniol production, and in particular to application of MIC19 gene and ECM15 gene in improving geraniol yield. Background Art

[0002] Geraniol, widely used in the cosmetics and perfume industries for its mild, sweet rose scent [1] It is also considered a promising biofuel due to its high energy content, low hygroscopicity and low volatility compared to ethanol. [2] In addition, geraniol, as a precursor for the biosynthesis of monoterpene indole alkaloids, also plays an important role in the production of well-known anticancer drugs such as vinblastine and vincristine. [3] At present, the use of microorganisms such as Saccharomyces cerevisiae to synthesize geraniol has become a potential solution for the large-scale supply of this compound with market prospects. However, yeast biosynthesis of geraniol currently faces three problems. First, the synthetic precursors provided by yeast cells are insufficient. [3] Second, the efficiency of the heterologous synthesis pathway of geraniol in yeast is low. [4] , thirdly, the accumulation of geraniol in cells is toxic to the strain [5] These three points restrict the increase of geraniol production in yeast, so it is urgent to study ways to increase geraniol production.

[0003] Existing research focuses on the breakthrough of increasing the yield of geraniol in the biosynthesis process of Saccharomyces cerevisiae. Figure 1 As shown. The first part is the endogenous mevalonate (MVA) pathway of yeast, which starts with the carbon source glucose in the culture medium and generates geranyl diphosphate (GPP) through endogenous metabolism. Through the study of genes in the MVA pathway, it was found that geranyl diphosphate synthase (ERG20), 3-hydroxy-3-methylglutaryl CoAreductase (3-hydroxy-3-methylglutaryl CoAreductase, HMG1), and isopentenyl pyrophosphate isomerase (IDI) have a key influence on the level of GPP synthesis. Ignea et al. obtained an ERG20 mutant with GPP synthase preference activity by protein modification of ERG20. K197E[6] 、ERG20 K197G[7] 、ERG20 N96W-N127W[8] etc.; Ro et al. [9]By protein modification of HMG1, the N-terminal amino acid was removed to obtain a form of tHMG1 that was not inhibited by the product; Liu et al.

[10] The pathway distribution of GPP and farnesyl pyrophosphate was optimized by overexpression of IDI. In addition, the genes ERG1, ERG8, ERG12, and the global transcription factor UPC2-1 in the MVA pathway were [9] , competitive pathway negative regulator MAF1

[10] Overexpression of the above factors can affect the strength of the MVA pathway to varying degrees. Researchers often use a combined multi-gene target metabolic engineering approach to maximize the MVA pathway metabolism to produce GPP, thereby breaking through the limiting factor of insufficient sources of synthetic precursor GPP provided by yeast cells. The second part of the geraniol synthesis pathway is based on the GPP produced by the MVA pathway. The biosynthesis of geraniol can be achieved by introducing plant-derived geraniol synthase (GES). Therefore, the existing technology starts from optimizing the activity of GES enzyme to increase the yield of geraniol. For example, by comparing the activity differences of GES from different plant sources in microorganisms, the best GES homologous gene can be selectively used. [4] ; or obtain mutant GES with enhanced GES activity through protein modification F418Q

[11] ; or remove the plant plastidial targeting signal peptide (PTS) at the N-terminus of GES to adapt to expression in yeast, etc. [5] In addition, in recent years, there have been optimization strategies for increasing the spatial proximity of enzymes and proteins in the synthesis pathway from various angles, including subcellular synthesis such as mitochondria and peroxisomes. [12,13] , Saccharomyces cerevisiae cell surface display

[14] Etc., thereby solving the problem of low efficiency of the heterologous synthesis pathway of geraniol in yeast.

[0004] Since the geraniol synthesis pathway in yeast is very clear, the above method focuses on each link of the geraniol synthesis pathway and optimizes the gene targets on the synthesis pathway or directly related to the synthesis pathway by means of metabolic engineering modification, which improves the geraniol production to a certain extent and becomes a common method for yeast to efficiently synthesize geraniol.

[0005] The existing technology partially solves the limiting factors one and two of geraniol biosynthesis by focusing on the modification of gene targets on the synthesis pathway, that is, it increases the synthetic precursor GPP provided by yeast cells to a certain extent and improves the effectiveness of the synthesis pathway. However, in fact, if the overall metabolism of the strain is not taken into consideration, only the modification of excessive gene targets that increase the production of geraniol will cause metabolic stress and growth defects to the strain. In addition, operations such as multi-gene knockout or overexpression are also time- and labor-intensive, and the required cycle is long. On the other hand, the chassis strain has incompatibility problems with the synthesis of non-strain natural compounds, and limiting factor three also shows that excessive accumulation of geraniol will also produce cytotoxicity. The incompatibility and intolerance of chassis strains with heterologous compound synthesis are more related to the regulation of endogenous metabolic networks. These regulatory targets are more genes outside the geraniol synthesis pathway. Therefore, the existing technology is limited to the modification of genes on the synthesis pathway. It is of great significance to systematically improve the adaptability of strains to the geraniol synthesis pathway, coordinate the overall metabolism of strains while increasing geraniol production, and discover new targets for endogenous metabolic regulation related to the increase in geraniol production. Summary of the invention

[0006] The present invention relates to the field of synthetic biology, and uses a saccharomyces cerevisiae containing an artificial synthetic chromosome as a starting strain, wherein a recombination site (symmetric locus of X-over P1, loxPsym) is added to the synthetic chromosome during design and synthesis.

[15] These recombination sites can induce large-scale rearrangement of the yeast genome under the implementation of the loxP-mediated synthetic chromosome rearrangement and modification by LoxP-mediated evolution (SCRaMbLE) system, which can produce a variety of genotypes and evolved strains with improved geraniol production.

[16] .

[0007] The present invention uses the adaptive evolution of strains to improve the adaptability and tolerance of strains to the geraniol synthesis pathway, which takes into account the systematic improvement of strains compared to the metabolic engineering transformation method, and saves time and effort. At the same time, compared with the directed evolution of synthetic pathway proteases in the prior art, the disturbance at the genome level can be increased, and the mutation involves large fragments of DNA, not limited to base mutations. In the absence of prior knowledge, new endogenous regulatory targets that contribute to geraniol production can be discovered in an irrational way for the complex yeast endogenous metabolism and regulatory network system, increasing the understanding of geraniol production-related regulation, supplementing existing gene targets and providing new ideas for metabolic transformation.

[0008] The technical solution of the present invention is as follows Figure 2The starting strain used in the present invention is a Saccharomyces cerevisiae (syn2369R) containing multiple artificial synthetic chromosomes, namely, synthetic right-arm chromosomes II, III, VI, and IX, wherein there are 473 loxPsym recombination sites on the synthetic chromosomes (synII contains 267, synIII contains 98, synVI contains 65, and synIXR contains 43). [17-20] . After combining with Cre recombinase (Cyclization Recombination Enzyme, Cre), deletion, flipping, duplication, or translocation of DNA fragments can occur between any two loxPsym sites, resulting in a greater degree of genome rearrangement than a single synthetic chromosome. The truncated geraniol synthase GES gene from Catharanthus roseus in the PTS region is integrated into the syn2369R strain genome, so that the synthetic strain has the ability to produce geraniol. Then, the key targets of the MVA pathway ERG20, HMGR, and IDI are optimized and integrated into the genome, so that the GPP and geraniol of the yeast are at a higher level, and a synthetic yeast strain that produces geraniol is obtained. The present invention induces the strain by SCRaMbLE, which promotes the synthetic genome rearrangement of the strain to produce a large number of genome evolution strains. Through the enzyme-coupled geraniol in vitro detection method, geraniol high-yield strains are quickly and efficiently screened out from a large number of post-SCRaMbLE strains. By performing whole genome sequencing analysis on the geraniol high-yield strain, it was found that the relevant gene variations included the deletion of the MIC19 gene and the duplication of the ECM15 gene. After reconstruction in a control strain (e.g., syn2369R_Cr MVA), it was verified that the knockout of the MIC19 gene, or the duplication of the ECM15 gene, or the knockout of the MIC19 gene superimposed on the duplication of the ECM15 gene can significantly increase the geraniol yield of the strain. The purpose of the present invention is to obtain a high-yield strain of geraniol through genome rearrangement evolution, and to explore metabolic regulation targets associated with geraniol production in the yeast endogenous metabolic network, that is, to discover new applications of the MIC19 and ECM15 genes. A method for transforming yeast for producing geraniol is also proposed, that is, the knockout of MIC19 or the duplication of the ECM15 gene can improve the synthesis of geraniol.

[0009] Specifically, the present invention relates to the following technical solutions:

[0010] 1. A method for producing a high-geraniol-producing yeast or a method for increasing the geraniol yield in a yeast, comprising reducing the expression of the MIC19 gene and / or increasing the expression of the ECM15 gene in the yeast; wherein the yeast has a geraniol synthase gene.

[0011] 2. The method according to item 1, wherein the reducing the expression of the MIC19 gene comprises deleting the MIC19 gene, or adding or deleting one or more bases in the nucleotide sequence of the MIC19 gene, or causing nucleotide substitution resulting in a missense mutation so that the expression of the MIC19 gene is reduced or absent, or gene silencing technology is used to achieve the reduced expression of the MIC19 gene; wherein the increasing the expression of the ECM15 gene comprises tandemly repeating the ECM15 gene or introducing a recombinant plasmid containing the ECM15 gene.

[0012] 3. The method according to item 1, wherein the geraniol synthase gene is introduced from an exogenous source.

[0013] 4. The method according to item 1, wherein the geraniol synthase gene is a codon-optimized sequence (preferably as shown in SEQ ID NO: 1) and the PTS region sequence thereof is removed;

[0014] Preferably, the HMG1, ERG20 and / or IDI genes in the yeast are also optimized (for example, the HMG1 gene is truncated, the ERG20 gene is mutated to ERG20 N96W-N127W , and / or codon optimization of the IDI gene).

[0015] 5. The method according to item 1, wherein the yeast is haploid yeast or diploid yeast, preferably haploid yeast;

[0016] Wherein, the haploid yeast includes Saccharomyces cerevisiae and Saccharomyces pastorianus.

[0017] 6. The method according to any one of items 1 to 5, wherein the yeast is a yeast containing a synthetic chromosome; preferably, it is a cerevisiae yeast containing a synthetic chromosome; more preferably, it is a haploid cerevisiae yeast containing 3.5 synthetic chromosomes in total, including the right arms of artificially synthesized chromosomes II, III, VI and IX.

[0018] 7. A geraniol high-producing yeast, wherein the MIC19 gene in the yeast is deleted or silenced, or the nucleotide sequence of the MIC19 gene contains the addition or deletion of one or more bases in the wild-type MIC19 gene, or a nucleotide substitution resulting in a missense mutation, such that the expression of the MIC19 gene is reduced or absent; and / or the ECM15 gene in the yeast is repeated in tandem, or the yeast contains a recombinant plasmid containing the ECM15 gene.

[0019] 8. A kit comprising compounds or reagents for reducing MIC19 gene expression and / or increasing ECM15 gene expression in yeast; preferably, the kit is used for producing geraniol high-yielding yeast or increasing geraniol production in yeast.

[0020] 9. The kit according to item 8, wherein the compound or agent for reducing the expression of the MIC19 gene in yeast comprises a compound or agent for yeast homologous recombination, or siRNA against the MIC19 gene.

[0021] 10. The kit according to item 8, wherein the compound or agent for increasing the expression of the ECM15 gene in yeast comprises a recombinant plasmid containing the ECM15 gene.

[0022] In a specific embodiment of the present invention, the SCRaMbLE refers to the artificial yeast genome project (Sc2.0 project) published by Professor Jef. Boeke of Johns Hopkins University in Nature, and its mechanism of action is to guide the recombination site LoxP, and under the action of Cre recombinase, the genome undergoes various types of structural variations such as deletion, inversion, and duplication. The SCRaMbLE effect can achieve the variability of the artificial yeast genome, and after a short period of induction, yeast strains containing a large number of different genotypes and phenotypes can be obtained for subsequent research.

[0023] In a specific embodiment of the present invention, the term "genome rearrangement" refers to gene rearrangement based on SCRaMbLE, i.e., a synthetic genome rearrangement system based on LoxPsym site-specific recombination. Generally, yeast strains with SCRaMbLE are mostly synthetic chromosomes, especially in Saccharomyces cerevisiae (Saccharomyces cerevisiae Synthetic Genome Project), such as the synthetic synIXR chromosome Saccharomyces cerevisiae in 2011, the synthetic synIII chromosome Saccharomyces cerevisiae in 2014, and the synthetic synV chromosome Saccharomyces cerevisiae in 2017. Under the action of Cre recombinase, any two loxPsym sites may interact with each other, resulting in random deletion and inversion of chromosome fragments in the region between the two sites, and at the same time, cells in a replication state under the action of recombination will randomly repeat chromosome fragments to achieve gene rearrangement.

[0024] In a specific embodiment of the present invention, the "YeastFab" method is disclosed in GUO Y, DONG J, ZHOU T, et al. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae [J]. Nucleic acids research, 2015, 43 (13): e88-e, which is a molecular cloning strategy for constructing the structure of Saccharomyces cerevisiae based on Golden-Gate assembled plasmids.

[0025] The present invention can obtain a large number of evolved strains in a short period of time, i.e., within 24 hours, by SCRaMbLE-induced strain evolution of a synthetic cerevisiae strain capable of producing geraniol. By coupling a rapid screening method and fermentation product detection, it was found that the geraniol production of 22 evolved strains was increased, with an increase range of 12.5%-69.0%. By performing whole genome sequencing on it, two new regulatory gene targets were found. By reconstructing these two gene targets in a control strain, the knockout of the MIC19 gene increased the geraniol production by 1.40 times, and the duplication of the ECM15 gene increased the geraniol production by 1.50 times. By combining the knockout of the MIC19 gene and the duplication of the ECM15 gene, the geraniol production was increased by 1.71 times, indicating that these two yeast endogenous targets play an important role in regulating the synthesis of geraniol. Therefore, the present invention obtains a strain with a 1.71-fold increase in geraniol production, discovers new applications of the MIC19 and ECM15 genes, and provides a method for transforming a geraniol-producing strain, namely, knocking out the MIC19 gene and / or overexpressing the ECM15 gene, so that the geraniol production of the transformed yeast strain is increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The synthesis pathway of geraniol in Saccharomyces cerevisiae is shown; solid arrows indicate one-step enzyme catalysis, and dashed arrows indicate multi-step enzyme catalysis. Abbreviations: HMG1, 3-hydroxy-3-methylglutaryl-CoA reductase; IDI, isopentenyl pyrophosphate isomerase; ERG20, geranyl diphosphate synthase; GPP, geranyl diphosphate; GES, geraniol synthase.

[0027] Figure 2 The technical scheme diagram of the present invention is shown.

[0028] Figure 3 Schematic diagram of the rapid detection of geraniol coupled to an enzyme is shown.

[0029] Figure 4The results show that the OD340 of the SCRaMbLE was detected by a microplate reader to screen the high-producing strains of geraniol; 340 = rearrangement strain ΔOD 340 Value / starting strain ΔOD 340 The high-yield strain was determined by the relative ΔOD 340 >1. Error bars represent mean ± SD of three replicate samples.

[0030] Figure 5 The GC-MS detection of the fermentation products of the strains shows the high-producing strains of geraniol after rescreening SCRaMbLE; wherein, relative geraniol yield = geraniol yield of the rearranged strain / geraniol yield of the starting strain, and the criterion for judging high-producing strains is relative geraniol yield> 1. The error bars represent the mean ± standard deviation of three replicate samples.

[0031] Figure 6 The geraniol production of Saccharomyces cerevisiae with MIC19 gene knockout or ECM15 gene duplication is shown; syn2369R_Cr MVA is the starting strain of SCRaMbLE; SCR(4)P4-B4 and SCR(4)P5-B9 are rearranged strains after SCRaMbLE; delMIC19 and dupECM15 are reconstructed strains. Error bars represent the mean ± standard deviation of three replicate samples. The significance test was performed using the Student's two-sided t test (***P<0.001).

[0032] Figure 7 A graph showing the geraniol production of Saccharomyces cerevisiae with potential metabolic target combinations; error bars represent the mean ± standard deviation of three replicate samples. Significance was tested using Student's two-sided t-test (**P<0.01). DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0034] The methods used in the following examples are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0035] Example 1: Construction of synthetic geraniol-producing yeast

[0036] 1) Construction of synthetic yeast syn2369R_Cr expressing tCrGES

[0037] This experiment used artificially synthesized II

[17] 、III

[18] VI

[19] IX right arm

[20] , a haploid yeast (syn2369R) with a total of 3.5 synthetic chromosomes is used as the chassis strain, which is obtained from Jef D. Boeke laboratory, or the chassis strain can also be constructed by prior art methods from 4 strains known in the prior art that contain synthetic chromosomes II, III, VI, or IX, respectively (construction method see Zhao Y, Coelho C, Hughes AL, et al. Debugging and consolidating multiple synthetic chromosomes reveals combinatorial genetic interactions[J]. bioRxiv, 2022: 2022.04.11.486913). First, a geraniol synthesis pathway was constructed in a synthetic chassis strain. The construction process of the synthetic pathway expression plasmid tCrGES-POT1 is as follows: First, the base sequence of geraniol synthase (CrGES, Genebank: AFD64744.1) from Catharanthus roseus was optimized according to the codon preference of Saccharomyces cerevisiae (the optimized sequence is shown in SEQ ID NO: 1 in Table 1), and it was synthesized by BGI. The PTS region (2-87aa) of the protein was removed by PCR (PCR primers are shown in Table 3) to obtain a truncated tCrGES sequence. The truncation is for better expression and activity in yeast. Using the YeastFab method

[21] The truncated tCrGES sequence was constructed into the HcKan_O vector (for detailed information on the construction method or map of the vector, please refer to reference

[21] ), specifically: after BsaI (NEB, R3733L) digestion, it was ligated to the HcKan_O vector using T4 DNA ligase (NEB, M0202L). After the promoter (TDH3p-HcKan) and terminator (ADH1t-HcKan) were selected (the information of the promoter TDH3p and the terminator ADH1t can be found in the literature Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, Wang L, Shen Y, Luo Y, Zheng Y, Lin J, Chen GQ, Wu Q, Cai Y, Dai J. YeastFab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015 Jul 27; 43 (13): e88.), the protein was digested with BsmBI (NEB, R0739L) and purified by T4 DNA ligase assembles the gene transcription unit in the order of promoter, tCrGES coding sequence, and terminator sticky end, and connects it to the POT1 vector (for detailed information on the construction method or map of the vector, please refer to reference

[21] ) to obtain the plasmid tCrGES-POT1 that can express tCrGES.

[0038] The plasmid was used as a template to amplify the tCrGES transcription unit, as well as 285 bp of the upstream homology arm of the YPL062W gene, the URA3 nutritional tag, and 320 bp of the downstream homology arm of the YPL062W gene (primer sequences are shown in Table 3). According to conventional operations in the prior art, the tCrGES transcription unit and the URA3 nutritional tag can be integrated into the ypl062w site of the yeast genome using the homologous recombination mechanism of Saccharomyces cerevisiae, and the SC was coated. -Ura The screening plate (medium preparation see Table 6) was cultured at 30°C for 3 days to obtain a synthetic yeast (syn2369R_Cr) capable of producing geraniol.

[0039] 2) Integration of tHMG1, IDI and ERG20 N96W-N127W Construction of the geraniol-producing strain syn2369R_Cr MVA

[0040] Furthermore, in order to eliminate the factors affecting geraniol synthesis and promote the production of geraniol, the inventors optimized the three metabolic targets of the known MVA pathway, HMG1, IDI and ERG20. The MVA pathway transformation plasmid MVA-pRS415 was constructed as follows: using the yeast genome as a template, PCR amplified the truncated tHMG1 gene (removing the N-terminal 2-530aa) and ERG20 gene (HMG1, Genbank: M22002.1; ERG20, Genbank: NM_001181600.1), and then using ERG20 as a template, PCR introduced point mutations to obtain mutant ERG20 N96W-N127W(Primer sequences are shown in Table 3). The IDI gene (GenBank: AF119715.1) from Escherichia coli was selected and the base sequence was optimized according to the codon preference of Saccharomyces cerevisiae (the optimized sequence is shown in SEQ ID NO: 2 in Table 1), and synthesized by BGI. The IDI sequence after the restriction site was introduced was obtained by PCR (primer sequences are shown in Table 3). In order to express these three genes, first select three inducible strong promoter plasmids GAL1p-HcKan, GAL7p-HcKan and GAL10p-HcKan (i.e., the plasmids obtained by connecting the GAL1p, GAL7p and GAL10p promoters known in the prior art to the vector HcKan_P, respectively, and the construction method of the plasmid can be found in Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, Wang L, Shen Y, Luo Y, Zheng Y, Lin J, Chen GQ, Wu Q, Cai Y, Dai J. Yeast Fab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res. 2015 Jul. 27; 43(13):e88.), and terminator plasmids CIT1t-HcKan, FUM1t-HcKan, and ERP2t-HcKan (for the construction methods of the three terminator plasmids, please refer to Zhang Y, Chiu TY, Zhang JT, Wang SJ, Wang SW, Liu LY, Ping Z, WangY, Chen A, Zhang WW, Chen T, Wang Y, Shen Y. Systematical Engineering of SyntheticYeast for Enhanced Production of Lycopene. Bioengineering (Basel). 2021Jan 19; 8(1):14.), and then the three promoters, genes, and terminators were digested with BsmBI according to the YeastFab method and ligated into POT2, POT4, and POT5 vectors (for the specific information on the construction method or map of the vector, please refer to reference

[21] ) respectively to obtain the transcription unit plasmids ERG20 of the three genes. N96W-N127W-POT2, IDI-POT4, tHMG1-POT5 (Table 2). The target fragments on the POT2, POT4, and POT5 vectors were digested with BsaI and then ligated in sequence using T4 DNA ligase. The three transcription units were then connected in series and assembled into the pRS415 vector (for detailed information on the construction method or map of the vector, see reference

[22] ) to obtain the MVA pathway optimized plasmid MVA-pRS415 (Table 2). The plasmid was digested with BsmBI (NEB, R0739L) to obtain tHMG1, IDI, and ERG20. N96W-N127W The fragments of the three gene transcription units were amplified by PCR with 500 bp of the upstream homology arm of the GAL80 gene, the LEU2 nutritional tag, and 500 bp of the downstream homology arm of the GAL80 gene (primer sequences are shown in Table 2), and the above fragments were transformed into the yeast syn2369R_Cr obtained in step 1) to achieve the three known transformation targets of the geraniol biosynthesis pathway (i.e., tHMG1, IDI and ERG20 N96W-N127W ) genome integration. -Ura-Leu The screening plate was cultured at 30°C for 3 days to obtain a geraniol-producing strain (syn2369R_Cr MVA) as the starting strain for genome rearrangement.

[0041] Example 2: Synthetic yeast genome rearrangement and high-yield strain screening

[0042] (1) Synthetic yeast genome rearrangement

[0043] The synthetic geraniol-producing Saccharomyces cerevisiae starting strain syn2369R_Cr MVA obtained in Example 1 was subjected to SCRaMbLE evolution to obtain a genome rearranged strain, as follows:

[0044] First, the Cre recombinase plasmid (pRS413-Cre / EBD) was transformed into the starting strain syn2369R_Cr MVA.

[20] (For the specific construction method or map information of this plasmid, please refer to reference

[20] ) The inducible rearrangement strain syn2369R_Cr MVA+Cre was obtained, and 90 positive clones after transformation were selected and inoculated into 500 μL SC -Ura-Leu-His The strain was revived in a 96-well deep-well plate containing liquid culture medium (see Table 6 for culture medium formula) at 30°C and 200 rpm shaking overnight. The overnight revived bacterial solution was measured at OD 600 = 0.1 transferred to 1 mL SC containing 1 μM estradiol (Toronto Research Chemicals, E888000) -Ura-Leu-HisIn liquid culture medium, SCRaMbLE was induced at 30°C and 220 rpm for 24 h. 1 μL of SCRaMbLE bacterial solution was applied to SC -Ura-Leu Solid plates (culture medium formula see Table 6), cultured at 30°C for 3 days, and a large number of genome rearrangement strains were obtained.

[0045] (2) Screening of high-yield strains

[0046] This experiment uses a rapid detection method for geraniol combined with a coupled enzyme (for specific principles, see Figure 3 ), quickly and efficiently screen out high-geraniol-producing strains from the large number of SCRaMbLE post-genome rearrangement strains obtained in step (1). The specific principle is as follows Figure 3 Geranial dehydrogenase (GeDH, GeneBank: CCF55024.1) can catalyze the oxidation of geraniol in vitro, accompanied by the cofactor NAD + Reduced to NADH, the NADH produced by reduction can be detected by the microplate reader at a wavelength of 340nm, so according to OD 340 The change of the value can determine the content of the substrate geraniol. This method can be used to quickly determine the geraniol in the fermentation broth of the strain.

[0047] To screen the rearrangement strains, the in vitro catalytic enzyme GeDH must first be obtained as follows: CdGeDH (GeneBank: CCF55024.1) from Castellaniella defragrans was synthesized (synthesized by BGI) and connected to the pET-28a(+) plasmid (Solarbio, catalog number: P3110) to construct the protein expression plasmid GeDH-pET-28a (Table 2). The cells were transformed into Escherichia coli BL21 (DE3) cells, induced by 0.1 mM IPTG (BBI, A600168) at 20°C for 20 h, and after cell lysis, the protein was purified by a protein fast chromatography system (GE Healthcare, HisTrapTM FF chromatography column) was used to successfully purify the GeDH protein, which was dissolved in 50mM Tris (BBI, A600194), 50% glycerol (Diamond, A100854-0500), and 300mM KCl (Tianjin Damao Chemical Reagent Factory) solution. The concentration of the purified GeDH protein was 1.81mg / mL. The enzyme activity was defined as the amount of enzyme required to convert 1μM substrate geraniol within 1min at 25°C. The specific enzyme activity of the GeDH obtained in this example was 2988U / mg, which can be used for the catalytic reaction with geraniol in the fermentation broth of the strain.

[0048] The rearranged strains obtained in step (1) of this example were screened to obtain high-geraniol-producing strains, as follows. Single colonies of the genome rearranged strains obtained in step (1) were randomly selected and inoculated into a deep-well plate containing 1 mL of liquid culture medium, and cultured at 30°C, 220 rpm, and shaken for 24 h to recover. The OD 600 =0.1 was transferred to a deep-well plate containing 1 mL of liquid culture medium and fermented at 30°C and 220 rpm for 48 h. The culture was centrifuged at 2000 rpm for 20 min to separate the bacteria and the fermentation supernatant. 50 μL of the fermentation supernatant was mixed with 2×GeDH reaction solution in an ELISA plate at a ratio of 1:1, wherein the 2×GeDH reaction solution contained 200 mM Tris-HCl (Invitrogen, AM9855G), 4 mM NAD + (NEB, B9007S), 0.054U GeDH (prepared as above), and the absorbance change at 340nm was detected by an ELISA reader (BioTek, Synergy H1) within 30min. The fermentation supernatant of each deep-well plate was detected 3 times. A total of 2160 strains after genome rearrangement were screened using this scheme, and 49 strains with relative OD 340 Strains with increased variation values ​​(such as Figure 4 shown).

[0049] 49 candidate strains were fermented to verify whether the actual geraniol production of these candidate strains was improved. A single yeast colony was inoculated into 5 mL SC -Ura-Leu In liquid culture medium, shake and culture at 30°C and 220 rpm overnight. 600 =0.1 was transferred to 10 mL of liquid culture medium and fermented in a 50 mL centrifuge tube covered with a breathable membrane at 30°C and 220 rpm for 5 days. The fermentation broth was centrifuged at 4000 rpm for 10 min, 700 μL of the supernatant was added to 300 μL of the organic phase (25% acetone and 75% ethyl acetate), and the mixture was extracted by shaking for 2 min. The geraniol content was detected by gas chromatography-mass spectrometry (GC-MS) (ThermoFisher, gas phase: TRACE1300; mass spectrometry: TSQ9000). The test results showed that 22 strains with improved geraniol production were obtained. Compared with the starting strain syn2369R_Cr MVA obtained in Example 1, the improvement ratio ranged from 12.5% ​​to 69.04% (eg Figure 5 shown).

[0050] Example 3: Gene target analysis and verification of high-geraniol-producing strains

[0051] (1) Sequencing and target analysis

[0052] The 22 rearranged strains with high geraniol production obtained in Example 2 were subjected to whole genome sequencing by a sequencer (Shenzhen HuaDa, DIPSEQ), and then sequence variation was analyzed to analyze gene targets related to high geraniol production. According to the genome sequence data, some high-yield strains underwent simple structural variation (as shown in Table 4). For example, two deletions were found in the sequencing results of the rearranged strain SCR (4) P4-B4, one of which was 902 bp, and the function of the related gene YFR010W-A involved was unknown, and the other gene was MIC19 (systematic name YFR011C, NCBI Gene ID: 850563), the function of the MIC19 gene is to express a component of the mitochondrial inner membrane complex MICOS. The genome variation of the rearranged strain SCR (4) P5-B9 was a tandem duplication of 893 bp, and the gene involved in the variation was ECM15 (systematic name YBL001C, NCBI Gene ID: 852285), which is speculated to play a role in yeast cell wall biogenesis.

[0053] (2) Gene target verification

[0054] The inventors further explored the correlation between genotype and phenotype, and added the upstream 60bp and downstream 60bp homology arms of the MIC19 gene to both ends of the HIS3 tag gene transcription unit by PCR amplification (primer sequences are shown in Table 2), and obtained the knockout fragment of the MIC19 gene (or the MIC19 gene can be inactivated or attenuated by point mutation or CRISPR method to obtain similar effects). After transforming the starting strain syn2369R_Cr MVA obtained in Example 1, the SC -Ura-Leu-His The screening plate was cultured at 30°C for 3 days to obtain the reconstructed strain delMIC19 with the MIC19 gene deleted. In addition, the ECM15 gene and its promoter and terminator were amplified using the genome of the starting strain syn2369R_Cr MVA as a template (primer sequences are shown in Table 2), and the ECM15 gene transcription unit obtained by PCR was ligated to the pRS413 vector

[22] The plasmid ECM15-pRS413 was obtained. The plasmid was transformed into the starting strain syn2369R_Cr MVA obtained in Example 1 to achieve low-copy overexpression of ECM15, and the corresponding reconstructed strain dupECM15 was obtained. The two reconstructed strains were fermented at 30°C for 5 days, and the geraniol production in the fermentation broth was detected by GC-MS. The geraniol production of the reconstructed strains delMIC19 and dupECM15 was measured to be 1.75 mg / L and 1.87 mg / L, respectively, which were 1.4 and 1.5 times higher than that of the starting strain syn2369R_CrMVA, respectively (as shown in Figure 2). Figure 6As shown), these two mutation sites are important regulatory targets for the adaptive evolution of geraniol synthesis and high geraniol production of the rearranged strain.

[0055] The two newly discovered metabolic targets were combined to further explore the effects of these two metabolic target combinations on geraniol synthesis. By PCR amplifying the ECM15 gene transcription unit and HIS nutritional label, adding the upstream 60bp and downstream 60bp homologous arms of the MIC19 gene to both ends of the primers, and transforming the DNA fragments into the starting strain syn2369R_Cr MVA, the MIC19 gene knockout and the ECM15 gene duplication can be achieved simultaneously to obtain the target combination strain mic19::ECM15. The target combination strain was fermented in 10mL culture medium at 30°C and 220rpm for 5 days with glucose as the carbon source. The fermentation products were detected by GC-MS, and it was found that the geraniol production of the target combination strain was further improved to 2.14mg / L, which is 1.71 times that of the starting strain (such as Figure 7 ). This proves that knocking out the MIC19 gene and / or duplication of the ECM15 gene can enhance the geraniol synthesis of yeast strains.

[0056] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0057] Table 1 CrGES gene codon optimized sequence

[0058]

[0059]

[0060] Table 2 Plasmids used in the examples

[0061]

[0062]

[0063] Table 3 Primer sequences used in the examples

[0064]

[0065]

[0066] Table 4 Strain information in the examples

[0067]

[0068]

[0069] Table 5 Structural variations observed in the synthetic chromosomes of the rearranged strains with high geraniol production

[0070]

[0071] Table 6 Culture medium formula

[0072]

[0073] Table 7 Yeast promoter sequence

[0074]

[0075]

[0076] References:

[0077] [1]CHEN W,VILJOEN A M.Geraniol—a review of a commercially important fragmentation material[J].South African Journal of Botany,2010,76(4):643-51.

[0078] [2]PERALTA-YAHYA PP,KEASLING J D.Advanced biofuel production inmicrobes[J].Biotechnology journal,2010,5(2):147-62.

[0079] [3]ZHANG J,HANSEN LG,GUDICH O,et al.A microbial supply chain for production of the anti-cancer drug vinblastine[J].Nature,2022,609(7926):341-7.

[0080] [4]JIANG GZ,YAO MD,WANG Y,et al.Manipulation of GES and ERG20 forgeraniol overproduction in Saccharomyces cerevisiae[J].Metabolic engineering,2017,41:57-66.

[0081] [5]ZHAO J,BAO X,LI C,et al.Improving monoterpene geraniol productionthrough geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae[J].Applied microbiology and biotechnology,2016,100(10):4561-71.

[0082] [6]BLANCHARD L,KARST F.Characterization of a lysine-to-glutamic acidmutation in a conservative sequence of farnesyl diphosphate synthase fromSaccharomyces cerevisiae[J].Gene,1993,125(2):185-9.

[0083] [7]FISCHER M J,MEYER S,CLAUDEL P,et al.Metabolic engineering ofmonoterpene synthesis in yeast[J].Biotechnology and bioengineering,2011,108(8):1883-92.

[0084] [8]IGNEA C,PONTINI M,MAFFEI M E,et al.Engineering monoterpeneproductionin yeast using a synthetic dominant negative geranyl diphosphatesynthase[J].ACSsynthetic biology,2014,3(5):298-306.

[0085] [9]RO D K,PARADISE E M,OUELLET M,et al.Production of the antimalarialdrugprecursor artemisinic acid in engineered yeast[J].Nature,2006,440(7086):940-3.

[0086]

[10] LIU J,ZHANG W,DU G,et al.Overproduction of geraniol byenhancedprecursor supply in Saccharomyces cerevisiae[J].Journal ofbiotechnology,2013,168(4):446-51.

[0087]

[11] LIN J-L,EKAS H,MARKHAM K,et al.An enzyme-coupled assayenablesrapid protein engineering for geraniol production in yeast[J].Biochemical EngineeringJournal,2018,139:95-100.

[0088]

[12] YEE D A,DENICOLA AB,BILLINGSLEY J M,et al.Engineeredmitochondrialproduction of monoterpenes in Saccharomyces cerevisiae[J].Metabolicengineering,2019,55:76-84.

[0089]

[13] GERKE J,FRAUENDORF H,SCHNEIDER D,et al.Production of thefragrancegeraniol in peroxisomes of a product-tolerant baker’s yeast[J].Frontiersinbioengineering and biotechnology,2020:1097.

[0090]

[14] LUO B,JIN MM,LI X,et al.Yeast surface display for in vitrobiosyntheticpathway reconstruction[J].ACS Synthetic Biology,2021,10(11):2938-46.

[0091]

[15] SARAH M.RICHARDSON L AM,GIOVANNI STRACQUADANIO,KUNYANG,JESSICAS.DYMOND,JAMES E.DICARLO,DONGWON LEE,CHENG LAIVICTOR HUANG,SRINIVASANCHANDRASEGARAN,YIZHI CAI,JEF D.BOEKE,JOEL S.BADER.Design of synthetic yeastgenome[J].science,2017.

[0092]

[16] SHEN Y, STRACQUADANIO G, WANG Y, et al.

[0093]

[17] SHEN Y,WANG Y,CHEN T,et al.Deep functional analysis of synII,a770-kilobase synthetic yeast chromosome[J].Science,2017,355(6329).

[0094]

[18] ANNALURU N,MULLER H,MITCHELL LA,et al.Total synthesis ofafunctional designer eukaryotic chromosome[J].Science,2014,344(6179):55-8.

[0095]

[19] MITCHELL L A,WANG A,STRACQUADANIO G,et al.Synthesis,debugging,andeffects of synthetic chromosome consolidation:synVI and beyond[J].Science,2017,355(6329).

[0096]

[20] DYMOND J S,RICHARDSON S M,COOMBES C E,et al.Syntheticchromosomearms function in yeast and generate phenotypic diversity by design[J].Nature,2011,477(7365):471-6.

[0097]

[21] GUO Y,DONG J,ZHOU T,et al.YeastFab:the design and constructionofstandard biological parts for metabolic engineering in Saccharomycescerevisiae[J].Nucleicacids research,2015,43(13):e88-e.

[0098]

[22] SIKORSKI R S,HIETER P.Asystem of shuttle vectors and yeast hoststrainsdesigned for efficient manipulation of DNAin Saccharomyces cerevisiae[J].Genetics,1989,122(1):19-27。

Claims

1. A method for producing a high-geraniol-producing yeast or a method for increasing the yield of geraniol in a yeast, comprising reducing the expression of the MIC19 gene and / or increasing the expression of the ECM15 gene in the yeast; wherein: The yeast has a geraniol synthase gene.

2. The method according to claim 1, wherein: The reducing the expression of the MIC19 gene includes deleting the MIC19 gene, or adding or deleting one or more bases in the nucleotide sequence of the MIC19 gene, or causing nucleotide substitutions that lead to missense mutations so that the expression of the MIC19 gene is reduced or absent, or using gene silencing technology to achieve reduced expression of the MIC19 gene; wherein, the increasing the expression of the ECM15 gene includes tandemly repeating the ECM15 gene or introducing a recombinant plasmid containing the ECM15 gene.

3. The method according to claim 1, wherein: The geraniol synthase gene is introduced from an exogenous source.

4. The method according to claim 1, wherein: The geraniol synthase gene is a codon-optimized sequence (preferably as shown in SEQ ID NO: 1) and the PTS region sequence is removed; Preferably, the HMG1, ERG20 and / or IDI genes in the yeast are also optimized (for example, the HMG1 gene is truncated, the ERG20 gene is mutated to ERG20 N96W-N127W , and / or codon optimization of the IDI gene).

5. The method according to claim 1, wherein: The yeast is haploid yeast or diploid yeast, preferably haploid yeast; Wherein, the haploid yeast includes Saccharomyces cerevisiae and Saccharomyces pastorianus.

6. The method according to any one of claims 1 to 5, wherein: The yeast is a yeast containing synthetic chromosomes; preferably, it is a cerevisiae yeast containing synthetic chromosomes; more preferably, it is a haploid cerevisiae yeast containing artificially synthesized chromosomes II, III, VI and IX, the right arms of chromosomes, a total of 3.5 synthetic chromosomes.

7. Geraniol high-yielding yeast, among which: The MIC19 gene in the yeast is deleted or silenced, or the nucleotide sequence of the MIC19 gene contains the addition or deletion of one or more bases in the wild-type MIC19 gene or nucleotide substitutions resulting in missense mutations, so that the expression of the MIC19 gene is reduced or absent; and / or, the ECM15 gene in the yeast is repeated in tandem, or the yeast contains a recombinant plasmid containing the ECM15 gene.

8. A kit comprising compounds or reagents for reducing MIC19 gene expression and / or increasing ECM15 gene expression in yeast; preferably, the kit is used for producing geraniol high-yielding yeast or increasing geraniol production in yeast. 9 . The kit according to claim 8 , wherein the compound or agent for reducing the expression of the MIC19 gene in yeast comprises a compound or agent for yeast homologous recombination, or siRNA against the MIC19 gene.

10. The kit according to claim 8, wherein The compound or reagent for increasing the expression of the ECM15 gene in yeast includes a recombinant plasmid containing the ECM15 gene.

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