CN115786154A - High-yield squalene recombinant saccharomyces cerevisiae engineering strain by improving ethanol tolerance and application thereof - Google Patents

Abstract

The invention discloses a high-yield squalene recombination saccharomyces cerevisiae engineering strain by improving ethanol tolerance and application thereof, belonging to the technical field of genetic engineering and biological engineering. The invention enlarges the metabolic flow of MVA path, removes the metabolic obstruction of the path, leads the whole metabolic path to be smooth and ensures the maximum limit of sufficient acetyl coenzyme A for product accumulation. Through the synthesis of trehalose and the regulation of heat shock protein, the strain tolerance under a stress environment is enhanced, so that the engineering strain still has excellent metabolism and growth production capacity under high-concentration ethanol. The activity of the modified saccharomyces cerevisiae strain is improved by 80.21%, the ethanol tolerance is improved to 30g/L, and the saccharomyces cerevisiae strain still has excellent activity under the stress of 50g/L ethanol. Through the combination of metabolic engineering modification and modification of stress environment tolerance capacity, the yield of squalene fermented by saccharomyces cerevisiae reaches 27.33g/L, and the dry weight of cells is 650.13mg/g DCW.

Description

High-yield squalene recombinant saccharomyces cerevisiae engineering strain by improving ethanol tolerance and application thereof

Technical Field

The invention relates to a high-yield squalene recombination saccharomyces cerevisiae engineering strain by improving ethanol tolerance and application thereof, belonging to the technical field of genetic engineering and biological engineering.

Background

Squalene is a linear triterpene that is widely distributed in plants, animals, fungi, and humans and is a key precursor in the biosynthesis of various triterpenes and steroids. The squalene has different antioxidation effects, and has advantages in anticancer, antitumor, dermatosis treating, and drug delivery. Squalene can stimulate and enhance immune response to antigens, and is used as a vaccine additive to improve vaccine efficacy including pandemic influenza, malaria and COVID-19 vaccines. Squalene is mainly extracted from animal and vegetable oils and fats such as shark liver oil, olive oil, amaranth oil, etc., however, separation of squalene from animals or plants may cause over-fishing and other environmental problems. The method for synthesizing the squalene by using the microorganisms has the advantages of short production period, simplicity in operation, high economic benefit, no limitation of raw materials and environment and the like, and the development of a synthetic biological technology provides an opportunity for improving the efficiency of synthesizing the squalene by using the microorganisms in metabolic engineering.

In recent years, the research on producing squalene and terpenoids by metabolically engineered microorganisms is not limited to the modification of the inherent MVA pathway in cytoplasm, but also various organelles in cells are widely researched and applied to the production of terpenoids. Mitochondria, peroxisomes, lipid droplets, etc. have all been used as reservoirs to enhance the accumulation of terpenoids in microorganisms. However, excessive organelle engineering can result in excess intermediates being retained in the organelle, increasing the metabolic stress of the organelle, hindering its normal metabolic function, and thus causing significant cellular metabolic stress. Stress on the fermentation environment, as compared to compartmentalization, is also an important factor limiting the production of terpenes and other substances by microorganisms. Therefore, the improvement of the activity of the strain and the stress environment tolerance of the strain cannot be ignored while modifying the terpenoid synthesis path. In the research process, the target product is produced by fully utilizing the advantages of microbial synthesis by combining the joint regulation and control of multiple aspects such as growth and production coupling, metabolic path and the like.

Disclosure of Invention

The invention provides a genetically engineered bacterium for high yield of squalene, which is a bacterium obtained by genetically modifying a saccharomyces cerevisiae serving as an initial strain; the genetically engineered bacterium is modified to knock out transcription factors YPL062W, ROX, YJL064W, DOS and DPP1 on a genome, integrate expression genes tHMG1, IDI1, ERG19, ERG20, ERG10, ERG13, ERG12, ERG8, ERG9, ADH2, ALD6, ACS, HSP104 and TPS1, and complement genes URA.

In one embodiment, genes tHMG1 and IDI1 are integrated at Ty1 and Ty4 sites in multiple copies, and genes ERG19 and ERG20 are integrated at YPL062W site after knockout of YPL062W site; after the ROX1 site is knocked out, integrating genes ERG10, ERG13, ERG12, ERG8 and ERG9 at the ROX1 site; after knocking out YPL062W site, genes ADH2, ALD6 and ACS are integrated at YPL062W site, and after knocking out DPP1 site, genes HSP104 and TPS1 are integrated at DPP1 site.

In one embodiment, saccharomyces cerevisiae C800 is the starting strain, said saccharomyces cerevisiae C800 being disclosed in, e.g., gao, s.; zhou, h.; zhou, J.et al, promoter-library-based dpathwayoptimization for efficacy (2S) -naringenin production from coriicacid Saccharomyces cerevisiae, 2020.

In one embodiment, the nucleotide sequence of gene tmg 1 is shown in SEQ ID No.1 and the nucleotide sequence of gene IDI1 is shown in SEQ ID No. 2.

In one embodiment, the nucleotide sequence of YPL062W is shown as SEQ ID NO.4, the nucleotide sequence of ROX1 is shown as SEQ ID NO.5, and the nucleotide sequence of YJL064W is shown as SEQ ID NO. 18.

In one embodiment, the nucleotide sequence of the gene DOS2 is shown as SEQ ID No.23, and the nucleotide sequence of the gene DPP1 is shown as SEQ ID No. 24.

In one embodiment, the nucleotide sequence of ERG20 is shown as SEQ ID NO.7, the nucleotide sequence of ERG19 is shown as SEQ ID NO.9, the nucleotide sequence of ERG9 is shown as SEQ ID NO.10, the nucleotide sequence of ERG10 is shown as SEQ ID NO.12, the nucleotide sequence of ERG13 is shown as SEQ ID NO.13, the nucleotide sequence of ERG12 is shown as SEQ ID NO.14, and the nucleotide sequence of ERG8 is shown as SEQ ID NO. 16.

In one embodiment, the nucleotide sequence of the gene ADH2 is shown in SEQ ID No.20, the nucleotide sequence of the gene ALD6 is shown in SEQ ID No.21, and the nucleotide sequence of the gene ACS is shown in SEQ ID No. 22.

In one embodiment, the nucleotide sequence of the gene HSP104 is shown in SEQ ID NO.25, and the nucleotide sequence of the gene TPS1 is shown in SEQ ID NO. 26.

In one embodiment, the nucleotide sequence of the gene URA is shown in SEQ ID NO. 27.

In one embodiment, a promoter P is utilized _GAL10/1 、P _TDH1 、P _MET6 Or P _TEF The expression of the gene is initiated.

In one embodiment, a bidirectional promoter P is utilized _GAL10/1 The expression of gene tHMG1 and gene IDI1 is initiated.

In one embodiment, a bidirectional promoter P is utilized _GAL10/1 The expression of the gene ERG19 and the gene ERG9 is started.

In one embodiment, a bidirectional promoter P is utilized _GAL10/1 The expression of the gene ERG13 and the gene ERG12 is started.

In one embodiment, a bidirectional promoter P is utilized _GAL10/1 The expression of the gene ALD6 and the gene ACS is initiated.

In one embodiment, a promoter P is utilized _TDH1 The expression of genes ERG20, ERG10 and HSP104 is respectively promoted.

In one embodiment, the promoter P is utilized _MET6 The expression of the genes ERG8 and TPS1 is respectively promoted.

In one embodiment, a promoter P is utilized _TEF Respectively openExpression of the genes ADH2 and URA.

In one embodiment, the bidirectional promoter P _GAL10/1 The nucleotide sequence of (A) is shown as SEQ ID NO.3, and the promoter P _TDH1 The nucleotide sequence of (A) is shown as SEQ ID NO.6, and the promoter P _MET6 The nucleotide sequence of (A) is shown as SEQ ID NO.17, and the promoter P _TEF The nucleotide sequence of (A) is shown in SEQ ID NO.19,

in one embodiment, the bidirectional terminator T _ter22 The nucleotide sequence of (A) is shown as SEQ ID NO.8, and the terminator T _TAT1 The nucleotide sequence of (A) is shown as SEQ ID NO.11, and the bidirectional terminator T _TET The nucleotide sequence of (A) is shown in SEQ ID NO. 15.

The invention also provides a method for producing squalene, which is to inoculate the genetically engineered bacteria in a culture medium for fermentation culture.

In one embodiment, the medium is a YPD medium.

In one embodiment, the method is to produce squalene by shake flask fermentation, and the seed solution of the genetically engineered bacteria is inoculated into YPD culture medium for fermentation culture for 80-100 h.

In one embodiment, the method is a fermentation tank for producing squalene by fermentation, the seed solution of the genetically engineered bacteria is inoculated into a fermentation medium containing 30-50 g/L glucose for fermentation production, and after the glucose is exhausted, ethanol is fed into the medium, so that the final concentration of the ethanol in the medium is maintained below 20g/L.

In one embodiment, the fermentation medium contains 15-25 g/L of peptone, 5-15 g/L of yeast powder and 30-50 g/L of glucose.

In one embodiment, the seed solution is prepared by selecting single colony of the above genetically engineered bacteria, culturing in 5mLYPD medium for 20-24h, 28-32 deg.C, 200-220 rpm as primary seed solution; inoculating the first-class seed solution into 50mL YPD culture medium with the inoculation amount of 2% (v/v) for culturing for 20-24h, and taking the seed solution at the temperature of 28-32 ℃ and the rpm of 200-220.

The invention also provides application of the genetic engineering bacteria in producing a product containing squalene.

Has the advantages that:

the invention takes saccharomyces cerevisiae engineering bacteria C800 (CEN. PK2-1D: delta gal80: G418) as a host, and aims to solve the problems that saccharomyces cerevisiae is stressed by high-concentration ethanol and the activity of an intermediate product is accumulated to inhibit a strain in the fermentation process. Firstly, the invention knocks out transcription factors YPL062W, ROX, YJL064W and DOS2 by overexpressing all non-rate-limiting step enzyme genes of key rate-limiting enzyme genes tHMG1, IDI1, ERG10, ERG13, ERG12, ERG8, ERG9, ERG19 and ERG20 of an MVA pathway, improves the flux of the MVA pathway, synthesizes key genes TSP1 and heat shock protein gene HSP104 by overexpressing trehalose, enhances the trehalose generated by saccharomyces cerevisiae to resist ethanol stress and supplements URA genes. Compared with the initial strain SQ1, the activity of the engineered saccharomyces cerevisiae strain SQ7 is improved by 80.21%, the ethanol tolerance is improved to 30g/L, and the strain still has excellent activity under the stress of 50g/L of ethanol.

The invention further optimizes fermentation conditions of the fermentation tank, optimizes the carbon source and the feeding time of the carbon source, realizes that the yield of the fermented squalene on a 5L fermentation tank reaches 27.33g/L, and the dry weight of cell production is 650mg/g DCW, which is the highest yield and the highest production capacity of squalene produced by microorganisms reported at present. The invention makes it possible to synthesize squalene from acetyl-CoA by completely enhancing the MVA pathway (FIG. 1). Based on the characteristic of ethanol accumulation in saccharomyces cerevisiae fermentation, the stress resistance of the strain ethanol is improved by strengthening the synthesis way of trehalose of the yeast and assisting regulation and control of heat shock protein.

Drawings

FIG. 1 is a schematic representation of the metabolism of squalene synthesis in Saccharomyces cerevisiae.

FIG. 2 is a diagram of the synthesis of the enhanced squalene by the MVA pathway; a, SQ1 strain, B: SQ2 strain, C: SQ2, SQ3 and SQ4 strains.

FIG. 3 is a control diagram of the overall enhancement of ethanol metabolic pathways.

FIG. 4 is a diagram of the regulation of ethanol tolerance of engineering strains; a, squalene production of SQ5 strain under different concentrations of ethanol feed, B: squalene yield of SQ6 strain under ethanol feed of different concentrations, C: trehalose content of SQ5 and SQ6 strains.

FIG. 5 is a graph showing the growth of cells and the production of squalene in a 5L bioreactor; a, optimization of molasses feeding time, B: optimization of molasses feed time, C: optimization of glucose and ethanol feed time, D: optimization of glucose and ethanol feed time, E: optimization of glucose and ethanol feed times, F: and (4) optimizing the ethanol feeding time.

Detailed Description

(I) culture Medium

LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. 20g/L agar powder was added to prepare an LB solid medium.

YNB medium: yeastNutritionBase67.4g/L, glucose 20g/L, amino acids (5 g/L uracil, 10g/L tryptophan, 10g/L leucine, 10g/L histidine, appropriate deletion of corresponding amino acids as required).

YPD medium: peptone 20g/L, yeast powder 10g/L, and glucose 20g/L.

Fermentation medium: peptone 20g/L, yeast powder 10g/L, glucose 40g/L.

(II) preparation and transformation of Saccharomyces cerevisiae competence

(1) Selecting a single colony of the saccharomyces cerevisiae, inoculating the single colony to 5mL of YPD culture medium, and culturing the single colony of the saccharomyces cerevisiae in a 50mL shake flask at 30 ℃ and 220rpm for about 17 hours to obtain saccharomyces cerevisiae seed solution.

(2) Measuring OD value, sucking appropriate amount of the above seed solution into 50mL fresh YPD medium to make OD ₆₀₀ Approximately equal to 0.3.

(3) Culturing at 30 deg.C and 220rpm to OD ₆₀₀ Between 1.2 and 1.5.

(4) The cells were transferred to a 50mL centrifuge tube and placed on ice for 5min.

(5) 3500rpm, 4 deg.C, centrifuging for 5min, and discarding the supernatant.

(6) Washed with 25mL of pre-cooled sterile water, centrifuged at 3500rpm at 4 ℃ for 5min, and the supernatant was discarded.

(7) 1mL of 0.1M lithium acetate was added to the suspension, and the cells were resuspended in a 1.5mL EP tube.

(8) Centrifuging at 4 deg.C and 3500rpm for 2.5min, and discarding the supernatant.

(9) The cells were resuspended in pre-cooled 100-400. Mu.L of 0.1M lithium acetate.

(10) 3500rpm, 2min, and the supernatant was discarded, and 24. Mu.L of 50% PEG3350 (M/V), 36. Mu.L of 1M lithium acetate, 25. Mu.L of ssDNA (concentration: 2 mg/mL), and 50. Mu.L of DNA fragment were added in this order.

(11) Shake vigorously for 10s.

(12) Incubate at 30 ℃ for 50min.

(13) In a water bath at 42 ℃ for 20min.

(14) Centrifuging at 3500rpm for 2.5min, and discarding the supernatant.

(15) 500-600. Mu.L of ddH2O was used to resuspend the cells.

(16) 50-80 μ L of the bacterial liquid was spread on corresponding YNB medium and cultured at 30 ℃ for 3-5 days.

(III) Shake flask fermentation of squalene

The shake flask fermentation conditions are as follows: individual colonies were removed and cultured in 50mL 5mLYPD medium in shake flasks for 22h,30 ℃ at 220rpm as seed solutions. The fermentation test adopts 1 percent of seed solution to be cultured with 25mLYPD culture medium in a 250mL shake flask for 96h;

in example 3, the ethanol tolerance of the strain was examined, and the seed solution was inoculated and fermented for 24 hours, and then added to the fermentation broth (10 g/L, 20g/L, 30g/L, 40g/L, and 50g/L, respectively, absolute ethanol, and used after filtration with a sterile filter);

when the attenuation expression of 10mg/L of terbinafine on ERG1 is considered, 10mg/L of terbinafine is added into the fermentation liquor after the seed solution is inoculated and fermented for 24 h.

Extraction and detection of squalene

Aspirate 500. Mu.L of broth into a 2mL disruption tube (MP), centrifuge, and discard the supernatant. The precipitated cells were retained, disrupted beads (0.5 mm) of equal cell volume were added to the disruption tube, 1.5mL of acetone was added, and disrupted for 4-6 cycles using the Saccharomyces cerevisiae program of FastPrep apparatus. And (4) centrifuging to separate the thalli from an acetone organic layer, and extracting squalene from an upper organic phase for detection. The liquid phase detector used was Waters UPLC, UV detector. Using a ThermoFisher (250X 4.6mm,5 μm) C18 column, chromatographic conditions: isocratic elution, phase B with acetonitrile (0.1% trifluoroacetic acid); the detection wavelength is 195nm; the column temperature is 40 ℃; and 15min.

(V) Strain information is shown in Table 1.

TABLE 1 strains involved in the invention

Example 1 Overall enhancement of the Saccharomyces cerevisiae MVA pathway

(1) Construction of SQ1 Strain

tHMG1 (nucleotide sequence is shown as SEQ ID NO. 1) and IDI1 (nucleotide sequence is shown as SEQ ID NO. 2) are integrated into the Ty1 site of the chassis strain C800 in multiple copies. The gene tHMG1 is amplified by using a primer Ty1-tHMG1-F1 and Ty1-tHMG1-R1 and a bidirectional promoter P is amplified by using a primer Gal10-F1 and Gal10-R1 by using a saccharomyces cerevisiae genome as a template _GAL10/1 (the nucleotide sequence is shown as SEQ ID NO. 3), and amplifying a gene IDI1 by using primers Ty1-IDI1-F1 and Ty1-IDI 1-R1; using Pct125 (disclosed in patent publication No. CN 113403334A) as a template, PCR primers Ty1-TMTD1-R1 and Ty1-TLEU2-F1 were used to amplify an integrated fragment of the Pct125 vector, and a bidirectional promoter P was obtained _GAL10/1 Plasmid TY1-11 was constructed by Gibson assembly of the vector integration fragments of gene tHMG1, gene IDI1 and Pct 125.

Then, using plasmid TY1-11 as a template, and using primers Ty1-11-up and Ty1-11-down to amplify a Ty1armup-tHMG1-PGAL10/1-IDI 1-HISneg-Ty 1armdown integration fragment. 10. Mu.g of the integration fragment Ty1armup-tHMG1-PGAL10/1-IDI 1-HISneg-Ty 1armdow was transformed into the engineered Saccharomyces cerevisiae strain C800 by the efficient Saccharomyces cerevisiae transformation method, spread on a screening YNB solid medium, and cultured at 30 ℃ for 3-5 days until colonies appeared, and the correct clone was verified and named SQ1.

A series of single colonies were picked and subjected to YPD shake flask fermentation for 96h, and finally the SQ1-1 strain with the squalene yield of 593.3mg/L was obtained (FIG. 2A).

(2) Construction of SQ2 Strain

tHMG1 and IDI1 are integrated in multiple copies at the Ty4 site of SQ 1-1. Amplification with the plasmid TY1-11 as template and primers TY4-tHMG1-F1 and Ty4-IDI1-R1tHMG1-P is produced _GAL10/1 -an IDI1 expression cassette; using Pct44 (disclosed in patent publication No. CN 113403334A) as a template, an integrated fragment of Pct44 vector was amplified using primers Ty4-TCYC1-F1 and Ty4-TADH1-R1, and tHMG1-P was amplified _GAL10/1 the-IDI 1 expression cassette and the integration fragment of the Pct44 vector were assembled by Gibbson to construct plasmid TY22-1.

Ty44armup-tHMG1-PGAL10/1-IDI 1-TRPD-Ty 44armdown was amplified using plasmid TY22-1 as a template and primers Ty44-11-up and Ty44-11-down, and the integrated fragment Ty44armup-tHMG1-PGAL10/1-IDI 1-TRPD-Ty 44armdown was transformed into the engineered Saccharomyces cerevisiae strain SQ1-1 in step (1) by the Saccharomyces cerevisiae high efficiency transformation method using 10. Mu.g, spread on a screening YNB solid medium, cultured at 30 ℃ for 3-5 days until colonies appeared, and the correct clone was verified and named SQ2.

A series of single colonies are picked to carry out YPD shake flask fermentation for 96h, and after the shake flask fermentation is carried out for 96h, a high-yield strain SQ2-7 is obtained, the yield of the strain is 919.1mg/L, and the yield of the strain is improved by 54.9% compared with that of an SQ1-1 strain copied at a Ty1 locus (figure 2B).

(3) Construction of SQ3 Strain

YPL062W (nucleotide sequence shown as SEQ ID NO. 4) and ROX1 (nucleotide sequence shown as SEQ ID NO. 5) on the SQ2-7 strain genome are knocked out by utilizing a multi-gene editing technology. Constructing a gene knockout plasmid R-Y-1 by using primers of 3-1-ROX1-PAM-1-F, 3-1-ROX1-PAM-1-R, 3-2-ROX1-PAM-2-F, 3-2-ROX1-PAM-2-R, 3-3-YP-PAM-1-F, 3-3-YP-PAM-1-R, 3-4-YP-PAM-2-F and 1-4-R, and referring to a CRISPR-Cas9 system which is applied to a one-step multi-target gene editing technology of saccharomyces cerevisiae, wherein 20nt for knocking out ROX1 is CACGACCCTTCAACGAGACA, CGGTGTCAAGCTCGAACAGC; the 20nt used for knock-out of YPL062W was AAGCAACCAGCACGTCGCCG, CACGGGAATAAGGCAGCCGA.

A saccharomyces cerevisiae genome is taken as a template, homologous arms of about 500bp upstream and downstream of an YPL062W site are amplified by using a primer YPL-UP-F, YPL-UP-R, YPL-down-F, YPL-down-R, homologous arms of about 500bp upstream and downstream of an ROX1 site are amplified by using a primer ROX1-UP-F, ROX-UP-R, ROX-down-F, ROX-down-R, the homologous arms of about 500bp upstream and downstream of the ROX1 site are respectively fused by using a fusion PCR method, and fused segments are named as ROX1-UP/down and YPL062W-UP/down. About 500-700ng plasmid R-Y-1 with ROX1 and YPL062W knocked out and 10 mu g fragments ROX1-up/down and YPL062W-up/down are transformed into a saccharomyces cerevisiae engineering strain SQ2-7 by a saccharomyces cerevisiae high-efficiency transformation method, the engineered strain is coated on a screening YNB solid culture medium and cultured for 3-5 days at 30 ℃ until colonies appear, and the correct clone is verified and named as SQ3-1.

Knocking out YPL062W and ROX1 on the genome of the SQ2-7 strain to obtain the SQ3-1 strain, and the squalene yield is reduced compared with that of the SQ2-7 strain serving as a control strain. After the expression of ERG1 is weakened by 10mg/L of terbinafine, the squalene yield of the SQ3-1 strain is 1080.3mg/L. Knockout of ROX1 increased the expression of not only ERGs genes before squalene, but also ERGs genes downstream of squalene (fig. 2C).

(4) Construction of SQ4 Strain

YPL062W and ROX1 on the genome of the saccharomyces cerevisiae SQ2-7 strain are knocked out by utilizing a multi-gene editing technology, ERG19 and ERG20 are overexpressed at YPL062W sites, and ERG10, ERG13, ERG12, ERG8 and ERG9 are overexpressed at ROX1 sites. Using Saccharomyces cerevisiae genome as template, primer YPL-UP-F, YPL-UP-R, YPL-down-F, YPL-down-R is used to amplify homologous arm about 500bp upstream and downstream of YPL062W, and primer YPL-PTDH1-F1 and YPL-PTDH1-R1 are used to amplify P _TDH1 Promoter (nucleotide sequence shown as SEQ ID NO. 6), ERG20 fragment (nucleotide sequence shown as SEQ ID NO. 7) amplified by using primers YPL-ERG20-F1 and YPL-ERG20-R1, and bidirectional terminator T amplified by using primers YPL-ter22T-F1 and YPL-ter22T-R1 _ter22 (nucleotide sequence is shown as SEQ ID NO. 8), ERG19 fragment (nucleotide sequence is shown as SEQ ID NO. 9) is amplified by using primers YPL-ERG19-F1 and YPL-ERG19-R1, and bidirectional promoter P is amplified by using primers YPL-GAL10-F1 and YPL-GAL10-R1 _GAL10/1 Amplifying ERG9 segment (nucleotide sequence is shown as SEQ ID NO. 10) by using primers YPL-ERG9-F1 and YPL-ERG9-R1, and amplifying terminator T by using primers YPL-TAT1T-F1 and YPL-TAT1T-R1 _TAT1 (the nucleotide sequence is shown as SEQ ID NO. 11), a pY26 expression vector is used as a template, and primers YPL-MVA-F1 and YPL-MVA-R1 are used for amplifying a pY26 vector fragment. The homologous arm and P of about 500bp upstream and downstream of YPL062W _TDH1 Promoter, ERG20 fragment, bidirectional terminator T _ter22 ERG19 fragment, bidirectional promoter P _GAL10/1 ERG9 fragment, terminator T _TAT1 And the pY26 vector fragment is assembled by Gibbson to construct plasmid 5. The plasmid 5 is used as a template, and an expression frame segment YPL-1 is amplified by using a primer YPL-UP-F, YPL-down-R.

Using Saccharomyces cerevisiae genome as template, amplifying homologous arms of about 500bp upstream and downstream of ROX1 by using primers ROX1-UP-F, ROX-UP-R, ROX1-down-F, ROX-down-R, and amplifying P by using primers ROX1-PTDH1-F1 and ROX1-PTDH1-R1 _TDH1 Promoter, ERG10 fragment (nucleotide sequence shown as SEQ ID NO. 12) amplified by primers ROX1-ERG10-F1 and ROX1-ERG10-R1, and bidirectional terminator T amplified by primers ROX1-ter22T-F1 and ROX1-ter22T-R1 _ter22 The ERG13 fragment (the nucleotide sequence is shown as SEQ ID NO. 13) is amplified by using the primers ROX1-ERG13-F1 and ROX1-ERG13-R1, and the bidirectional promoter P is amplified by using the primers ROX1-GAL10-F1 and ROX1-GAL10-R1 _GAL10/1 Amplifying ERG12 segment (nucleotide sequence is shown as SEQ ID NO. 14) by using primers ROX1-ERG12-F1 and ROX1-ERG12-R1, and amplifying T by using primers ROX1-TEFt-F1 and ROX1-TEFt-R1 _TET A bidirectional terminator (nucleotide sequence is shown as SEQ ID NO. 15), an ERG8 fragment (nucleotide sequence is shown as SEQ ID NO. 16) is amplified by using primers ROX1-ERG8-F1 and ROX1-ERG8-R1, and a promoter P is amplified by using primers ROX1-PMET6-F1 and ROX1-PMET6-R1 _MET6 (the nucleotide sequence is shown as SEQ ID NO. 17); and (3) taking the pY26 expression vector as a template, and amplifying a pY26 vector fragment by using primers ROX1-MVA-F1 and ROX 1-MVA-R1. The homology arm P of about 500bp at the upstream and downstream of ROX1 _TDH1 Promoter, ERG10 fragment, bidirectional terminator T _ter22 ERG13 fragment, bidirectional Start P _GAL10/1 ERG12 fragment, T _TET Bidirectional terminator, ERG8 fragment, promoter P _MET6 And pY26 vector fragment was assembled with Gibbson to construct plasmid 6. An expression frame segment ROX1-1 is amplified by using a primer ROX1UP-F, ROX-down-R by taking the plasmid 6 as a template.

And (3) converting the plasmid R-Y-1 with the ROX1 and YPL062W knocked out by about 500-700ng in the step (3) and the expression frame fragments ROX1-1 and YPL-1 by a saccharomyces cerevisiae high-efficiency conversion method into a saccharomyces cerevisiae engineering strain SQ2-7, coating the saccharomyces cerevisiae engineering strain SQ2-7 on a screening YNB solid culture medium, culturing for 3-5 days at 30 ℃ until colonies appear, and verifying that the correct clone is named as SQ4-2.

The yield of squalene produced by the strain SQ4-2 in the shake flask fermentation reaches 1405.3mg/L (FIG. 2C). After the expression of ERG1 is weakened by 10mg/L of terbinafine, the squalene yield of the SQ4-2 strain reaches 1856.4mg/L.

TABLE 2 primer sequences

Example 2 modification of ethanol metabolic pathway

Since the self promoter of ADH2 can be inhibited when the concentration of ethanol is more than 2.3g/L, the constitutive strong promoter PTEF is used for regulating the expression of ADH2 in the experiment, and the metabolic inhibition effect under high-concentration ethanol is relieved to promote the accumulation of acetyl coenzyme A. YJL064W and DOS2 on the genome of the saccharomyces cerevisiae SQ4-2 strain are knocked out by utilizing a multi-gene editing technology, and ADH2, ALD6 and ACS are overexpressed at a YPL062W locus.

Using Saccharomyces cerevisiae genome as template, amplifying YJL064W (nucleotide sequence shown as SEQ ID NO. 18) upstream and downstream 500bp homologous arm segments by using primers YD 01-YJUp-F1, YD 01-YJUp-R1, YD01-YJLdown-F1 and YD01-YJLdown-R1, and amplifying P by using primers YD01-TEF-F1 and YD01-TEF-R1 _TEF A promoter (nucleotide sequence is shown as SEQ ID NO. 19), primers YD01-ADH2-F1 and YD01-ADH2-R1 are used for amplifying ADH2 fragments (nucleotide sequence is shown as SEQ ID NO. 20), and primers YD01-TER-F1 and YD01-TER-R1 are used for amplifying bidirectional terminator T _ter22 An ALD6 fragment (the nucleotide sequence is shown as SEQ ID NO. 21) is amplified by using the primers YD01-ALD-F1 and YD01-ALD-R1, and a bidirectional promoter P is amplified by using the primers YD01-GAL-F1 and YD01-GAL-R1 _GAL10/1 An ACS fragment (nucleotide sequence is shown as SEQ ID NO. 22) is amplified by using primers YD01-ACS-F1 and YD01-ACS-R1, and a terminator T is amplified by using primers YD01-TAT-F1 and YD01-TAT-R1 _TAT1 (ii) a And (3) amplifying a pY26 vector fragment by using primers YD01-KJ-F1 and YD01-KJ-R1 by using the pY26 expression vector as a template. Will be provided withYJL064W upstream and downstream 500bp homology arm fragments, P _TEF Promoter, ADH2 fragment, bidirectional terminator T _ter22 ALD6 fragment, bidirectional promoter P _GAL10/1 ACS fragment, terminator T _TAT1 And pY26 vector fragment was assembled with Gibbson to construct plasmid 7.

A saccharomyces cerevisiae genome is taken as a template, primers d-DOSup-F, d-DOSup-R1, d-DOSdawn-F1 and d-DOSdawn-R are used for amplifying segments of DOS2 (nucleotide sequence is shown in SEQ ID NO. 23) with 500bp of upstream and downstream homologous arms, the upstream and downstream homologous arms are respectively fused by a fusion PCR method, and the obtained segment is DOS2-1.

The method comprises the steps of utilizing primers 6-1-DOS2-PAM-1-F, 6-1-DOS2-PAM-1-R, 6-2-DOS2-PAM-2-F, 6-2-DOS2-PAM-2-R, 6-3-YJL064W-PAM-1-F, 6-3-YJL064W-PAM-1-R, 6-4-YJL064W-PAM-2-F and 6-4-YJL064W-PAM-2-R to construct a gene knockout plasmid D-Y-1 by a goldengate method, wherein 20nt for knocking out DOS2 is 3238 zft 3238, and 20nt for knocking out YJL064W is 3262 zft 3262. The plasmid 7 is used as a template, and an expression frame segment YJL064W-1 is amplified by using a primer d-YJUp-F, d-YJLdown-R.

About 500-700ng of plasmid D-Y-1 with DOS2 and YJL064W knocked out and 10 mu g of segment DOS2-1 and YJL064W-1 are transformed into a saccharomyces cerevisiae engineering strain SQ4-2 by a saccharomyces cerevisiae high-efficiency transformation method, coated on a screening YNB solid culture medium, cultured for 3-5 days at 30 ℃ until colonies appear, and the correct clone is verified and named as SQ5.

Knocking out a transcription factor YJL064W, DOS on the basis of the SQ4-2 strain, promoting acetyl coenzyme A to flow to an MVA pathway and strengthening the synthesis of squalene. On this basis, the squalene yield of the overexpressed strain SQ5 integrated with ADH2, ALD6, ACS2 was increased from 1405.3mg/L to 1469.3mg/L (FIG. 3).

TABLE 3 primer sequences

Example 3: saccharomyces cerevisiae ethanol tolerance regulation

The strain SQ5 obtained by strengthening the ethanol pathway gene in the example 2 is fermented under the feeding of ethanol with different concentrations (10 g/L, 20g/L, 30g/L, 40g/L and 50 g/L), and the strain shows the best squalene yield and growth activity when 20g/L of ethanol is added, wherein the squalene yield is 1701.1mg/L, OD ₆₀₀ Is 22.54. When the ethanol concentration is higher than 30g/L, the activity of the strain begins to decline, and the yield is reduced along with the activity.

The DPP1 on the genome of a saccharomyces cerevisiae SQ5 strain is knocked out by utilizing a multigene editing technology, and HSP104 (the nucleotide sequence is shown in SEQ ID NO. 25) and TPS1 (the nucleotide sequence is shown in SEQ ID NO. 26) are overexpressed at the DPP1 (the nucleotide sequence is shown in SEQ ID NO. 24) site.

The saccharomyces cerevisiae genome is taken as a template, primers TT-DPPUP-F1, TT-DPPUP-R1, TT-DPPIDN-F1 and TT-DPP1DN-R1 are used for amplifying upstream and downstream homologous arms of DPP1, and primers TT-PTDH1-F1 and TT-PTDH1-R1 are used for amplifying promoter P _TDH1 The HSP104 fragment is amplified by primers TT-HSP-F1 and TT-HSP-R1, and the T fragment is amplified by primers TT-ter-F1 and TT-ter-R1 _ter22 Bidirectional terminator, using primers TT-tsp1-F1 and TT-tsp1-R1 to amplify a TPS1 fragment, and using primers TT-PMET6-F1 and TT-PMET6-R1 to amplify a promoter P _MET6 (ii) a And (3) taking the pY26 expression vector as a template, and amplifying a pY26 vector fragment by using primers TT-KJ-F1 and TT-KJ-R1. DPP1 upstream and downstream homology arms, promoter P _TDH1 HSP104 fragment, T _ter22 Bidirectional terminator, TPS1 fragment, promoter P _MET6 And the pY26 vector fragment is assembled by Gibbson to construct the plasmid TT-1. The plasmid TT-1 is taken as a template, and the primer TT-UP-F, TT-DN-R is used for amplifying an expression frame segment TT-01.

The primers D-D-PAM-F3, D-D-PAM-R3, D-D-PAM-F4 and D-D-PAM-R4 are used for constructing a gene knockout plasmid DTT-1 by a goldengate method, and 20nt for knocking out DPP1 is ATTAGTGCAGCTCCTAACAG, ACCAAGCGAGTGATAAACCA. About 500-700ng of plasmid DTT-1 with DPP1 knocked out and 10 mu g of expression frame fragment TT-01 are transformed into a saccharomyces cerevisiae engineering strain SQ5 by a saccharomyces cerevisiae high-efficiency transformation method, the saccharomyces cerevisiae engineering strain SQ5 is coated on a screening YNB solid culture medium, the culture is carried out for 3-5 days at 30 ℃ until colonies appear, and the clone with correct verification is named as SQ6.

Shake flask fermentation culture of squalene was performed using SQ5 in example 2 as a control strain and SQ6 as a modified strain, and feeding concentrations of ethanol were set to 10g/L, 20g/L, 40g/L, and 50g/L. The results show that the modified strain SQ6 overexpressing HSP104 (Saccharomyces cerevisiae heat shock protein), TPS1 (key gene for trehalose synthesis) in SQ5 shake flask culture OD 96h ₆₀₀ The growth activity is obviously improved from 20 of the control strain SQ5 to 40, and the optimal ethanol feeding addition amount of the strain is improved to 30g/L. Under the condition, the yield of squalene is 5649.8mg/L and OD is obtained when the shake flask is fermented for 96 hours ₆₀₀ Is 40.62. Compared with the control strain SQ5, the trehalose content of the modified strain SQ6 is also obviously improved. By improving the stress resistance, the efficiency and the accumulation amount of the SQ6 strain for accumulating squalene are obviously improved. When the shake flask ethanol feeding concentration of the SQ6 modified strain is higher than 30g/L, the activity of the strain begins to decline, which indicates that high-concentration ethanol still has an influence on the SQ6 engineering strain. However, the activity of the SQ6 strain at 50g/L ethanol feed was still higher than that of the control strain SQ5 at 10g/L ethanol feed, indicating that the modification of stress resistance greatly enhances the ethanol tolerance of Saccharomyces cerevisiae (FIG. 4).

TABLE 4 primer sequences

Example 4: replement of URA Gene

In order to further improve the activity of the strain, the defect of the engineering strain SQ6 is complemented back. The promoter P is amplified by using a saccharomyces cerevisiae genome as a template and using primers TY2-PTEF-F1 and TY2-PTEF-R1 _TEF Amplifying a URA (nucleotide sequence is shown as SEQ ID NO. 27) fragment by using primers TY2-URA-F1 and TY2-URA-R1, and amplifying a terminator T by using primers TY2-TAT1-F1 and TY2-TAT1-R1 _TAT1 Fragments, PCR of Pct22 (disclosed in patent publication No. CN 113403334A) amplified using primers TY2-KJ-R1 and TY2-KJ-F1Synthesizing the fragment, and transforming the promoter P _TEF URA fragment, terminator T _TAT1 And Pct22 integration fragment was assembled using Gibbson to synthesize plasmid TY2-U. Using plasmid TY2-U as template, using TY2-L-U-F1, TY2-L-U-R1, obtaining homologous recombination fragment Ty2armup-P by PCR _TEF -URA-LEUdeg-Ty2armdown (TY 2-1). About 10 mu g of fragment TY2-1 is transformed into a saccharomyces cerevisiae engineering strain SQ6 by a saccharomyces cerevisiae high-efficiency transformation method, the strain is coated on a screening YNB solid culture medium, the strain is cultured for 3-5 days at 30 ℃ until colonies appear, and the clone with correct verification is named as SQ7.

TABLE 5 primer sequences

Example 5 fermentation Condition optimization

(1) Optimization of glucose and molasses feeding time

A single colony of SQ7 strain of example 4 was picked, cultured in 50mL of 5mLYPD medium in a shake flask for 22h,30 ℃ and 220rpm as a primary seed solution. Inoculating the primary seed solution into a 250mL shaking flask 50mLYPD culture medium with the inoculation amount of 2% (v/v) for culturing 22h, culturing at 30 ℃ at 220rpm as a secondary seed solution, inoculating the secondary seed solution into a 5L bioreactor with the liquid loading amount of 2.5LYPD culture medium (the initial concentration of glucose is 40 g/L) for fermentation culture with the inoculation amount of 2% (v/v), starting to supplement the glucose with the final concentration of 500g/L (the residual sugar in the reaction system is controlled to be 0) about 11h of the fermentation, stopping glucose feeding about 24h and 36h respectively, starting to supplement molasses (the residual sugar in the reaction system is controlled to be 0, and the ethanol concentration of the metabolite is not more than 20 g/L), and carrying out co-fermentation culture for 120h.

The results show that the feed of molasses was started at 24h and the squalene yield reached 7.72g/L at 120h of fermentation as shown in FIG. 5A. To avoid premature molasses replenishment, growth is not favored, thus avoiding the growth vigor phase. As shown in FIG. 5B, the molasses feeding is started at 36h, and the squalene yield reaches 9.06g/L after 120h of fermentation. The scheme provides reference for producing high-value terpenoid from industrial waste molasses.

(2) Optimization of glucose and ethanol feeding time

A single colony of the SQ7 strain of example 4 was picked and cultured in 50mL of shake flask 5mLYPD medium for 22h,30 ℃ and 220rpm as a primary seed solution. The primary seed solution was inoculated at an inoculum size of 2% (v/v) into a 250mL shake flask of 50mLYPD medium for 22h,30 ℃ and 220rpm as a secondary seed solution. Inoculating the secondary seed solution into a 5L bioreactor containing 2.5LYPD culture medium (initial concentration of glucose is 40 g/L) at an inoculation amount of 2% (v/v), performing fermentation culture, beginning to supplement glucose with a final concentration of 500g/L in about 11h of fermentation, stopping glucose feeding in about 60h, 48h, 24h and 12h, and beginning to supplement ethanol (controlling final concentration of ethanol in the reaction system not to exceed 20 g/L) for about 120-156h of co-fermentation culture.

The results show that as shown in FIG. 5C, glucose feeding is started at 11h, glucose feeding is stopped at 60h, ethanol is fed, the yield of YPD in a 5L fermentation tank reaches 9.91g/L after 130h of fermentation, and the growth condition and the product accumulation of the strain are poor during the whole fermentation process.

As shown in FIG. 5D, glucose starts to be supplemented in 11h, glucose feeding is stopped in 48h, ethanol is supplemented, the yield of the fermentation reaches 15.11g/L in 156h, and the whole fermentation process is analyzed, compared with the common saccharomyces cerevisiae, the saccharomyces cerevisiae strain with the regulated tolerance has better growth capacity, still has activity in the later growth period, and the product accumulation can be maintained at 156h from 24 h. The growth condition of the strain and the product accumulation in the fermentation process are analyzed, the growth condition of the strain reaches a certain level after 36 hours, but the product accumulation is lacked, and the accumulation of the product is better compared with the accumulation of ethanol supplemented 24 hours after the glucose in the culture medium in the shake flask is exhausted. Therefore, the fermentation strategy is adjusted, and the accumulation condition of the product is inspected by adding ethanol in the early stage.

The yield of the fermentation is 22.55g/L for 156h as shown in FIG. 5E. Analyzing the whole fermentation process, and when glucose is supplemented and ethanol is directly supplemented in the growth rapid stage (24 h) of the fermentation process, the growth of the thalli is premature. When the thalli grow to 84h, obvious reduction of growth occurs, so that carbon source replacement is not advisable during the vigorous growth period of 24h logarithmic growth phase.

The yield of the fermentation reaches 27.33g/L in 156h as shown in FIG. 5F. In combination with the shake flask fermentation, in the first stage, 40g/L glucose is used to prepare a fermentation medium for growing the cells during the 5L fermentation tank expansion fermentation. In the second stage, after glucose in the culture medium is exhausted, the whole fermentation process is regulated and controlled after 16 hours, and the thalli are grown and produced by using proper ethanol. Through a series of fermentation strategy adjustment, the yield of squalene produced by saccharomyces cerevisiae SQ7 fermentation reaches 27.33g/L, and the dry weight produced by cells is 650.13mg/g DCW.

(3) 5L bioreactor fermentation production of squalene

A single colony of SQ7 strain of example 4 was picked and cultured in 50mL of 5mLYPD medium in a shake flask for 22h,30 ℃ and 220rpm as the primary seed solution. The primary seed solution was inoculated at an inoculum size of 2% (v/v) into a 250mL shake flask 50mLYPD medium for 22h,30 ℃ and 220rpm as a secondary seed solution. The secondary seed liquid was inoculated in a 5L bioreactor with 2.5L fermentation medium at an inoculum size of 2% (v/v) for the fermentative production of squalene. In the first stage (0 h-16 h), a YPD medium containing 40g/L glucose is used for the growth of the thalli, and in the second stage (16 h-156 h), after the glucose is exhausted, ethanol is supplemented (the final concentration of the ethanol in the reaction system is maintained below 20 g/L) for the growth of the thalli and the accumulation of squalene. Through a series of fermentation optimization, the yield of squalene produced by saccharomyces cerevisiae and the dry weight of cell production respectively reach 27.33g/L and 650.13mg/g DCW.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. A genetically engineered bacterium for high yield of squalene is characterized in that the genetically engineered bacterium is obtained by genetically modifying a starting strain saccharomyces cerevisiae; the genetically engineered bacterium is transformed into a knockout genome transcription factor YPL062W, ROX, YJL064W, DOS and DPP1, integrates and expresses genes tHMG1, IDI1, ERG19, ERG20, ERG10, ERG13, ERG12, ERG8, ERG9, ADH2, ALD6, ACS, HSP104 and TPS1, and supplements the gene URA.

2. The genetically engineered bacterium of claim 1, wherein genes ERG19 and ERG20 are integrated at the site of YPL062W after multiple copies of genes tHMG1 and IDI1 are integrated at the site of Ty1 and Ty4 and YPL062W is knocked out; after the ROX1 site is knocked out, integrating genes ERG10, ERG13, ERG12, ERG8 and ERG9 at the ROX1 site; after knocking out YPL062W site, genes ADH2, ALD6 and ACS are integrated at YPL062W site, and after knocking out DPP1 site, genes HSP104 and TPS1 are integrated at DPP1 site.

3. The genetically engineered bacterium of claim 1 or 2, wherein the nucleotide sequence of gene tmg 1 is shown as SEQ ID No.1, the nucleotide sequence of gene IDI1 is shown as SEQ ID No.2, the nucleotide sequence of gene YPL062W is shown as SEQ ID No.4, the nucleotide sequence of gene ROX1 is shown as SEQ ID No.5, the nucleotide sequence of gene YJL064W is shown as SEQ ID No.18, the nucleotide sequence of gene DOS2 is shown as SEQ ID No.23, and the nucleotide sequence of gene DPP1 is shown as SEQ ID No. 24.

4. The genetically engineered bacterium of claim 1 or 2, wherein the nucleotide sequence of ERG20 is shown as SEQ ID No.7, the nucleotide sequence of ERG19 is shown as SEQ ID No.9, the nucleotide sequence of ERG9 is shown as SEQ ID No.10, the nucleotide sequence of ERG10 is shown as SEQ ID No.12, the nucleotide sequence of ERG13 is shown as SEQ ID No.13, the nucleotide sequence of ERG12 is shown as SEQ ID No.14, the nucleotide sequence of ERG8 is shown as SEQ ID No.16, the nucleotide sequence of gene ADH2 is shown as SEQ ID No.20, the nucleotide sequence of gene ALD6 is shown as SEQ ID No.21, the nucleotide sequence of gene ACS is shown as SEQ ID No.22, the nucleotide sequence of gene HSP104 is shown as SEQ ID No.25, the nucleotide sequence of gene TPS1 is shown as SEQ ID No.26, and the nucleotide sequence of gene URA is shown as SEQ ID No. 27.

5. The genetically engineered bacterium of any one of claims 1 to 4, wherein the promoter P is used _GAL10/1 、P _TDH1 、P _MET6 Or P _TEF The expression of the gene is initiated.

6. The genetically engineered bacterium of any one of claims 1 to 5, wherein the bidirectional promoter P is _GAL10/1 The nucleotide sequence of (A) is shown as SEQ ID NO.3, and the promoter P _TDH1 The nucleotide sequence of (A) is shown as SEQ ID NO.6, and the promoter P _MET6 The nucleotide sequence of (A) is shown as SEQ ID NO.17, and the promoter P _TEF The nucleotide sequence of (A) is shown in SEQ ID NO. 19.

7. The genetically engineered bacterium of any one of claims 1 to 6, wherein Saccharomyces cerevisiae C800 is used as an initial strain.

8. A method for producing squalene, which comprises inoculating the genetically engineered bacterium of any one of claims 1 to 6 into a culture medium for fermentation culture.

9. The method according to claim 8, wherein the seed solution of the genetically engineered bacterium according to any one of claims 1 to 6 is inoculated into a fermentation medium containing 30 to 50g/L glucose for fermentation production, and after the glucose is exhausted, ethanol is fed into the medium to maintain the final concentration of ethanol in the medium to be less than 20g/L.

10. Use of the genetically engineered bacterium of any one of claims 1 to 6 in the production of a squalene-containing product.

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