A method for producing ethanol by fermentation of saccharomyces cerevisiae based on a dual antibacterial strategy
By overexpressing cyanamide hydratase in Saccharomyces cerevisiae and combining it with the antimicrobial peptide DptB and endosomalin LysKB317, a dual antimicrobial strategy was formed, which solved the problem of bacterial inhibition during Saccharomyces cerevisiae fermentation and improved ethanol yield and production stability.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING TECH UNIV
- Filing Date
- 2026-04-08
- Publication Date
- 2026-06-02
AI Technical Summary
Existing technologies are insufficient to effectively inhibit contaminating microorganisms during the fermentation of brewer's yeast, leading to reduced ethanol production or fermentation failure. Existing antimicrobial strategies suffer from insufficient broad-spectrum, targeted, and stable properties.
By overexpressing cyanamide hydratase in Saccharomyces cerevisiae and combining it with the antimicrobial peptide DptB and endosomalin LysKB317, a dual antimicrobial strategy is formed. This strategy utilizes the chemical antimicrobial effect of cyanamide and the synergistic biological antimicrobial effect of the antimicrobial peptide-endosomalin to target and inhibit Acetobacter and Lactobacillus.
It achieves an organic combination of broad-spectrum antibacterial and targeted antibacterial effects, improving the stability and economy of ethanol production, and increasing ethanol yield by 10.1% to 23.7%.
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Figure CN122128121A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for producing ethanol by fermentation of Saccharomyces cerevisiae based on a dual antimicrobial strategy. Background Technology
[0002] Bioethanol, a key renewable energy source in the global carbon neutrality strategy, is widely used in energy, industry, and other fields. Its industrial production mainly relies on the efficient fermentation capacity of Saccharomyces cerevisiae. However, contamination during large-scale fermentation severely restricts production efficiency. Acetic acid bacteria, lactic acid bacteria, and other contaminating microorganisms can reduce ethanol yield by 1% to 30% by competing for carbon sources and secreting inhibitory metabolites such as lactic acid and acetic acid. In extreme cases, this can even render batch fermentation economically worthless. Existing contamination control methods have significant limitations: physical sterilization is energy-intensive and difficult to handle dynamic contamination in continuous fermentation; chemical methods easily lead to drug resistance and environmental residues, failing to meet sustainable production requirements; single biological antimicrobial strategies (such as antimicrobial peptides and bacteriophages), while highly targeted, are limited by narrow antimicrobial spectra or insufficient stability, making them difficult to address complex contamination scenarios. While cyanamide, as a broad-spectrum chemical antimicrobial agent, inhibits contaminating microorganisms by blocking the respiratory chain, it also inhibits the growth of Saccharomyces cerevisiae; antimicrobial peptides and endosomalins, although capable of targeting and killing Gram-negative and Gram-positive bacteria, are easily affected by pH and protease in the fermentation system, leading to activity attenuation. The dual challenges of initial and continuous contamination in industrial fermentation necessitate antimicrobial strategies that possess both the ability to rapidly inhibit initial contamination and the long-term control of subsequent pollution. Existing single strategies struggle to balance broad-spectrum activity, targeted targeting, and long-term stability; therefore, there is an urgent need to develop synergistic and efficient composite antimicrobial solutions. Summary of the Invention
[0003] The technical problem to be solved by the present invention is to provide a genetically engineered brewing yeast strain that addresses the shortcomings of the prior art.
[0004] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned genetically engineered brewer's yeast.
[0005] The final technical problem to be solved by this invention is to provide a method for producing ethanol by fermentation using the above-mentioned genetically engineered Saccharomyces cerevisiae.
[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:
[0007] The first aspect of this invention provides a genetically engineered *Saccharomyces cerevisiae* strain in which the gene encoding cyanamide hydratase is overexpressed in the starting strain of *Saccharomyces cerevisiae*. CAH The amino acid sequence of the monocyanamide hydratase is shown in SEQ ID NO:6.
[0008] Preferably, the gene encoding the monocyanamide hydratase... CAHThe nucleotide sequence is shown in SEQ ID NO:1.
[0009] The brewing yeast is brewing yeast CICC 1308.
[0010] A second aspect of this invention provides a method for constructing the genetically engineered *Saccharomyces cerevisiae* strain, wherein the gene encoding the cyanamide hydratase is... CAH It is obtained by inserting it into the genome of the brewer's yeast.
[0011] In some embodiments, the method for constructing the genetically engineered Saccharomyces cerevisiae includes the following steps: constructing a gene encoding cyanamide hydratase. CAH The expression cassette was used to insert the gene encoding cyanamide hydratase into the CRISPR / Cas9 gene editing technology. CAH The expression cassette is inserted into the genome of the brewer's yeast to obtain the product.
[0012] In some embodiments, the gene encoding cyanamide hydratase CAH The insertion site of the expression cassette is the 1622b site of the Saccharomyces cerevisiae genome.
[0013] The monocyanamide hydratase overexpressed in the genetically engineered Saccharomyces cerevisiae can degrade monocyanamide, thereby reducing the inhibitory effect of monocyanamide on Saccharomyces cerevisiae. The genetically engineered Saccharomyces cerevisiae can grow normally in a system containing 0.5 g / L monocyanamide.
[0014] The third aspect of the present invention provides a method for producing ethanol by fermentation using the aforementioned Saccharomyces cerevisiae genetically engineered bacteria, wherein the Saccharomyces cerevisiae genetically engineered bacteria are inoculated into a fermentation medium for fermentation culture.
[0015] The fermentation medium contains the antimicrobial peptide DptB, the endosomalin LysKB317, and cyanamide.
[0016] The amino acid sequence of the antimicrobial peptide DptB is shown in SEQ ID NO:2; the amino acid sequence of the endolysin LysKB317 is shown in SEQ ID NO:3.
[0017] In some embodiments, the preparation method of the antimicrobial peptide DptB and endolysin LysKB317 includes the following steps:
[0018] The genes encoding the antimicrobial peptide DptB and the endolysin LysKB317 were ligated into plasmid pET28a to construct recombinant expression vectors p-DptB and p-LysKB317, respectively. These vectors were then introduced into *E. coli* BL21(DE3) to construct recombinant *E. coli* BL21(DE3)-pET28a-DptB and BL21(DE3)-pET28a-LysKB317, respectively. Overexpression of the antimicrobial peptide DptB and endolysin LysKB317 genes was induced in these recombinant *E. coli* BL21(DE3)-pET28a-DptB and BL21(DE3)-pET28a-LysKB317 genes. Bacterial cells were collected, lysed, and centrifuged. The supernatants were collected and treated with Ni-NTA. Purification using a 6FF pre-packed gravity column yielded the antimicrobial peptide DptB and endolysin LysKB317.
[0019] The nucleotide sequences of the antimicrobial peptide DptB encoding gene and the endolysin LysKB317 encoding gene are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively; the inducing agent used to induce the expression of recombinant Escherichia coli BL21(DE3)-pET28a-DptB and BL21(DE3)-pET28a-LysKB317 is IPTG.
[0020] The initial concentrations of the antimicrobial peptide DptB, endolysin LysKB317, and cyanamide in the fermentation medium were 8-12 g / L, 28-32 g / L, and 0.4-0.6 g / L, respectively.
[0021] Preferably, the initial concentrations of the antimicrobial peptide DptB, endolysin LysKB317, and cyanamide in the fermentation medium are 10 g / L, 30 g / L, and 0.5 g / L, respectively.
[0022] Antimicrobial peptide DptB and endosomalin LysKB317 can respectively target and inhibit Acetic Acid Bacillus ( Acetobacter spp.) and lactobacillus ( Lactobacillus (spp.), when the two are combined, the actual inhibitory concentration of the compound against Lactobacillus is lower than the minimum inhibitory concentration of endolysin LysKB317 alone due to the synergistic effect. The minimum inhibitory concentration of the combined antibacterial agent against Acetobacter is 0.8 mM (protein concentration), while the minimum inhibitory concentration against Lactobacillus is reduced to below 1.0 mM (protein concentration) due to the synergistic effect.
[0023] The engineered Saccharomyces cerevisiae is inoculated into the fermentation medium in the form of a seed culture; the OD of the seed culture... 600The inoculation amount of the seed solution is 4 to 6; the inoculation amount of the seed solution is 8 to 12% v / v.
[0024] The fermentation culture is carried out at a temperature of 30-37℃, a pH of 3.0-4.5, a rotation speed of 150-250 rpm, and the fermentation time is adjusted according to the initial glucose concentration. Fermentation ends when the remaining glucose concentration in the fermentation medium is less than 10 g / L.
[0025] The fermentation medium comprises the following components: 50-200 g / L glucose, 8-12 g / L yeast extract, 18-22 g / L peptone, 0.8-1.2 g / L potassium dihydrogen phosphate, and 0.4-0.6 g / L magnesium sulfate.
[0026] Beneficial effects:
[0027] This invention combines the advantages of the chemical antibacterial properties of monocyanate with the biological antibacterial properties of the antimicrobial peptide-endolysin, forming a dual antimicrobial strategy in the fermentation process of Saccharomyces cerevisiae to produce ethanol. The synergistic effect of chemical and biological antimicrobial agents achieves an organic combination of broad-spectrum and targeted antimicrobial activity, enhancing the antimicrobial effect. It effectively inhibits contaminating bacteria while reducing the interference of chemical antimicrobial agents on the metabolism of Saccharomyces cerevisiae, reducing the risk of contamination caused by Saccharomyces cerevisiae in industrial ethanol production, and ultimately improving the stability and economy of ethanol production. The ethanol yield of the 50-200 g / L glucose fermentation system is increased by 10.1% to 23.7% compared with the control group. Attached Figure Description
[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.
[0029] Figure 1 This is the plasmid map of the recombinant plasmid pCas-1622b in Example 1.
[0030] Figure 2 The Saccharomyces cerevisiae genetically engineered strain 1308-P constructed in Example 1 DDI2 -CAH colony PCR verification results.
[0031] Figure 3 The Saccharomyces cerevisiae 1308 and the genetically engineered Saccharomyces cerevisiae 1308-P in Example 1 are examples. DDI2 -Image of CAH plate growth verification.
[0032] Figure 4 The results of the plate inhibition experiment of antimicrobial peptide DptB and endolysin LysKB317 in Example 2 are shown.
[0033] Figure 5The results of the plate inhibition experiment of the mixture of 10 mg / mL antimicrobial peptide DptB and 30 mg / mL endolysin LysKB317 in Example 2 against Acetobacter and Lactobacillus.
[0034] Figure 6 This is a statistical chart showing the biomass, residual sugar content, and ethanol production of different fermentation systems during the fermentation verification experiment of the antimicrobial peptide-endolysin antimicrobial strategy in Example 2.
[0035] Figure 7 Figures 1-2 show the biomass and residual sugar content of different fermentation systems during the fermentation process in Example 3. Figures a-c show the biomass of different fermentation systems during the fermentation process under the conditions of initial glucose concentrations of 50 g / L, 100 g / L, and 200 g / L in the fermentation medium, respectively. Figures d-f show the residual sugar content of different fermentation systems during the fermentation process under the conditions of initial glucose concentrations of 50 g / L, 100 g / L, and 200 g / L in the fermentation medium, respectively.
[0036] Figure 8 Figures 1-2 show the ethanol yield and production rate of different fermentation systems in Example 3. Figures a-c show the ethanol yield of different fermentation systems under the conditions of initial glucose concentrations of 50 g / L, 100 g / L, and 200 g / L in the fermentation medium, respectively. Figures d-f show the ethanol titer and production rate of different fermentation systems under the conditions of initial glucose concentrations of 50 g / L, 100 g / L, and 200 g / L in the fermentation medium, respectively. Detailed Implementation
[0037] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.
[0038] For any specific techniques or conditions not specified in the examples, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.
[0039] Example 1: Genetically engineered Saccharomyces cerevisiae strain 1308-P DDI2- CAH Construction and Functional Verification
[0040] Using CRISPR / Cas9 gene editing technology to edit the gene encoding cyanamide hydratase CAH Expression Box (P) DDI2 -CAH-T CYC1 It was integrated into the 1622b site of the Saccharomyces cerevisiae 1308 genome.
[0041] 1. Recombinant plasmid pFA6a-P DDI2 -CAH Construction
[0042] 1.1 Amplification of fragments expressing cassettes
[0043] Using the Saccharomyces cerevisiae 1308 genome as a template, primer P was used. DDI2 -F / P DDI2 -DDI2-R was used for PCR amplification to obtain the DDI2 promoter (P DDI2 The fragment (theoretical size 925 bp) was synthesized using the pUC57-CAH recombinant plasmid (codon-optimized encoding gene of Aspergillus niger cyanamide hydratase) synthesized by General Biotechnology (Anhui) Co., Ltd. CAH Using ) as a template, primer pair P DDI2- CAH-F / CAH-T CYC1 -R is used for PCR amplification to obtain... CAH Gene fragment (theoretical size 735 bp). Using the genome of Saccharomyces cerevisiae 1308 as a template, primer pair CAH-T was used. CYC1 -F / T CYC1 -R was used for PCR amplification to obtain the terminator T. CYC1 Fragment (theoretical size 284 bp).
[0044] 1.2 Construction of Recombinant Plasmids
[0045] Using pFA6a plasmid as a vector, the plasmid was double-digested with Bgl II and BamHI. The digestion products were verified by electrophoresis and then recovered by gel extraction. The recovered P... DDI2 promoter, CAH Genes, T CYC1 The terminator fragment was cloned in one step with the linearized pFA6a vector, transformed into E. coli DH5α, plated on LB agar plates containing 50 μg / mL Kan, and cultured for 12–16 h. Single colonies were picked for PCR verification (primers pFA6a-Check-F / R) and sequencing to obtain the recombinant plasmid pFA6a-P. DDI2 -CAH.
[0046] 2. Construction of Donor DNA
[0047] 2.1 Homologous Arms and CAH Expression cassette amplification
[0048] Using the Saccharomyces cerevisiae 1308 genome as a template, the upper and lower homologous arms (both 1020 bp) of the 1622b site in the Saccharomyces cerevisiae 1308 genome were amplified using primer pairs 1622b-up-F / R and 1622b-down-F / R, respectively. pFA6a-PDDI2 -CAH is used as a template, with primer pair 1622b-P DDI2 -F / T CYC1 -1622b-R amplification of the CAH expression cassette (P DDI2 -CAH-T CYC1 ,1914 bp), glue recovery of each segment.
[0049] 2.2 Overlap PCR Ligation
[0050] The upper homologous arm, CAH expression cassette, and lower homologous arm were ligated by overlap PCR, and the product was recovered by gel electrophoresis to obtain Donor DNA (3874 bp). The primer pair used for overlap PCR was 1622b-up-F and 1622b-down-R.
[0051] 3. Construction of recombinant plasmid pCas-1622b
[0052] Using the pCas plasmid as a template, upstream primer loop p1622b-F and downstream primer loop p1622b-R were added for reverse PCR amplification. Verification was performed by 1% agarose gel electrophoresis. The linearized vector was recovered from the gel and cloned in one step, transformed into *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL Kan, and cultured for 12–16 h. Single colonies were picked and sequenced to obtain the recombinant plasmid pCas-1622b, the plasmid map of which is shown below. Figure 1 As shown, its nucleotide sequence is shown in SEQ ID NO:7.
[0053] 4. Construction and validation of genetically engineered Saccharomyces cerevisiae
[0054] To prepare competent *Saccharomyces cerevisiae* 1308 cells, 100 μL of competent cells were added to pCas-1622b plasmid and Donor DNA, and electroporated. Immediately after electroporation, 1 mL of YPD medium was added, and the cells were incubated at 30°C for 1 h. After centrifugation, the cells were resuspended in 100 μL of YPD medium and plated onto YPD plates containing 500 μg / mL G418. The plates were then incubated at 30°C for 48 h. Single colonies were picked from the plates, treated with 0.02 M NaOH aqueous solution, and PCR verification was performed using Check-1622b-F / R primers. The gel electrophoresis image of the colony PCR amplification products is shown below. Figure 2 As shown, positive clones were sent for sequencing confirmation. The correctly sequenced strains were passaged three times on YPD plates (without G418), and single colonies were streaked onto YPD and YPD+G418 plates, respectively, and incubated at 30°C for 24 h. Strains that grew exclusively on YPD plates were screened, and pCas plasmid loss was verified by PCR, yielding the *Saccharomyces cerevisiae* genetically engineered strain 1308-P with the CAH expression cassette knocked in. DDI2-CAH.
[0055] 5. Functional verification
[0056] Saccharomyces cerevisiae 1308 and Saccharomyces cerevisiae genetically engineered strain 1308-P were used. DDI2 -CAH bacterial suspensions were serially diluted to 1, 10, and so on. -1 10 -2 10 -3 The solutions were added dropwise to YPD plates containing 0.5 g / L cyanamide (CY) and incubated at 30°C for 60 h. The results are as follows: Figure 3 As shown, 1308-P DDI2 -CAH colony density was significantly higher than that of Saccharomyces cerevisiae 1308, indicating CAH The expression box is stably integrated and functions normally.
[0057] The primers used in this embodiment are shown in Table 1.
[0058] Table 1 Primers and their sequences used in Example 1
[0059]
[0060] Example 2 Implementation of the antimicrobial peptide-endolysin antimicrobial strategy
[0061] 1. Construction of recombinant strains BL21-DptB and BL21-LysKB317
[0062] 1.1 Amplification of the genes encoding the antimicrobial peptide DptB and the endolysin LysKB317
[0063] Based on the gene sequences of the antimicrobial peptide DptB and the endolysin LysKB317 encoding genes provided by NCBI, an 18 bp 6×His tag sequence was added before the stop codon of each gene. The final sequences were synthesized by General Biotechnology (Anhui) Co., Ltd. (the nucleotide sequences of the antimicrobial peptide DptB and endolysin LysKB317 encoding genes are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively), and existed in the form of plasmids pUC57-DptB and pUC57-LysKB317, respectively. Using plasmids pUC57-DptB and pUC57-LysKB317 as templates, and using DptB-F / R and LysKB317-F / R as primers, respectively, the antimicrobial peptide DptB and endolysin LysKB317 encoding genes were amplified by PCR. The double restriction enzyme sites were Nco I and Hind III, respectively.
[0064] 1.2 Construction of recombinant strains BL21-DptB and BL21-LysKB317
[0065] The pET28a plasmid was digested with restriction endonucleases Nco I and Hind III, and the reaction was carried out at 37°C for 2 h. The linearized vector was then recovered by gel electrophoresis. Using a one-step cloning technique, the genes encoding the antimicrobial peptide DptB and the endolysin LysKB317 were ligated into the linearized pET28a vector, respectively. The ligation was then performed in *E. coli* DH5α, plated on LB agar plates containing 50 μg / mL Kan, and incubated at 37°C for 12–16 h. Colony PCR verification (primers Check-F / R) was performed, and after confirmation by sequencing of positive clones, recombinant plasmids pET28a-DptB and pET28a-LysKB317 were obtained. These plasmids were then transformed into *E. coli* BL21(DE3) to obtain recombinant expression strains BL21-DptB and BL21-LysKB317.
[0066] 2. Induced expression and purification of antimicrobial peptide DptB and endolysin LysKB317
[0067] BL21-DptB and BL21-LysKB317 were inoculated into LB liquid medium containing 50 μg / mL Kan and cultured at 37°C and 200 rpm until OD500. 600 The initial concentration of IPTG in the culture medium was 0.6-0.8 mM, and the medium was induced at 25°C and 180 rpm for 12 h. The culture medium was centrifuged at 6000 rpm for 5 min, and the bacterial sludge was collected. After washing with 1×PBS, the cells were resuspended and disrupted using an ultrasonic cell disruptor. The intracellular protein supernatant was collected by centrifugation. Purification was performed using a Ni-NTA pre-packed gravity column (purchased from Sangon Biotech (Shanghai) Co., Ltd.; catalog number: C600791). The elution buffer was an imidazole aqueous solution. First, 10 mM imidazole was used to wash away non-specifically bound proteins, and then 250 mM imidazole was used to elute the target protein. The DptB elution buffer and LysKB317 elution buffer were collected separately. The DptB eluent was ultrafiltered using an ultrafiltration tube with an MWCO of 3 kDa (Millipore; catalog number: UFC8003) to remove imidazole, and the LysKB317 eluent was ultrafiltered using an ultrafiltration tube with an MWCO of 10 kDa (Millipore; catalog number: UFC8010) to remove imidazole, thus obtaining purified antimicrobial peptide DptB solution and endolysin LysKB317 solution.
[0068] 3. Validation of antibacterial activity and determination of concentration of antimicrobial peptide-endolysin.
[0069] 3.1 Validation of antibacterial activity
[0070] The antibacterial effect was verified using a perforated plate test. OD 600Acetobacter and Lactobacillus bacterial suspensions (with a concentration of 1) were spread onto LB and MRS plates, respectively. Several wells (4 mm inner diameter) were evenly punched in each plate using a sterile punch. Then, 10 mg / mL of the antimicrobial peptide DptB and 30 mg / mL of the endolysin LysKB317 were added to the wells of the LB and MRS plates, respectively. The plates were incubated at 30°C for 12 h, and the diameter of the inhibition zone was measured (formula: inhibition zone diameter = total measured diameter - 4 mm). BL21-DptB / BL21-LysKB317 cell lysate and PBS were used as controls. Results are as follows: Figure 4 As shown, the inhibition zone diameter of DptB against Acetobacter was 10.3 mm, and the inhibition zone diameter of LysKB317 against Lactobacillus was 5.7 mm.
[0071] 3.2 Determination of Antimicrobial Peptide-Endolysin Addition Concentration
[0072] A mixed solution of the antimicrobial peptide DptB and the endolysin LysKB317 was prepared, with DptB at a concentration of 10 mg / mL and LysKB317 at a concentration of 30 mg / mL. The inhibitory effect of this mixed solution on Acetobacter and Lactobacillus was verified using the plating method. The results are as follows: Figure 5 As shown, a mixed solution of the antimicrobial peptide DptB and the endosomalin LysKB317 strongly inhibits the growth of other microorganisms.
[0073] 4. Fermentation validation of the antimicrobial peptide-endolysin antimicrobial strategy
[0074] Four fermentation media were set up and named as follows: Group 1308, Group 1308-mixed bacteria (1308+CM), Group 1308-antimicrobial peptide-endolysin (1308+DL), and Group 1308-antimicrobial peptide-endolysin-mixed bacteria (1308+DL+CM). The fermentation medium of Group 1308 was inoculated with *Saccharomyces cerevisiae* 1308 seed culture (seed culture OD). 600 The inoculum size was 5, and the inoculum size was 10% v / v; the Saccharomyces cerevisiae 1308 seed culture was inoculated into a fermentation medium of 1308+CM (seed culture OD). 600 The inoculum size was 5, with an inoculum size of 10% v / v; acetic acid bacteria seed culture (OD) 600 The inoculum size was 8 (1% v / v) and the seed culture of Lactobacillus (OD). 600 The inoculum size was 8, and the inoculum size was 1% v / v; the fermentation medium of 1308+DL was inoculated with Saccharomyces cerevisiae 1308 seed culture (seed culture OD). 600 The inoculum size was 5, with an inoculum size of 10% v / v, and antimicrobial peptide DptB and endolysin LysKB317 were added to a final concentration of 10 mg / mL and 30 mg / mL, respectively; the fermentation medium of 1308+DL+CM was inoculated with Saccharomyces cerevisiae 1308 seed culture (seed culture OD). 600The inoculum size was 5, with an inoculum size of 10% v / v; acetic acid bacteria seed culture (OD) 600 The inoculum size was 8 (1% v / v) and the seed culture of Lactobacillus (OD). 600 The inoculum was 8 (1% v / v), and antimicrobial peptide DptB and endolysin LysKB317 were added to final concentrations of 10 mg / mL and 30 mg / mL, respectively.
[0075] The above four fermentation systems were cultured at 35℃ and 200 rpm. The initial glucose concentration in the fermentation medium was 50 g / L (the fermentation medium consisted of the following components: 50 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and deionized water). Samples were taken every 3 hours to measure the OD. 600 Residual sugar (biosensor analyzer) and ethanol yield (gas chromatograph). Experimental results are as follows: Figure 6 As shown, the results indicate that the ethanol yield of the 1308+DL+CM group was 11.9% higher than that of the 1308+CM group, verifying that the combined use of antimicrobial peptides and endosomalin can effectively inhibit miscellaneous bacteria.
[0076] The primers used in this embodiment are shown in Table 2.
[0077] Table 2 Primers and their sequences used in Example 2
[0078]
[0079] Example 3: Strain 1308-P DDI2 Validation of the efficacy of a dual antimicrobial strategy combining CAH and antimicrobial peptide-endolysin
[0080] The fermentation medium was divided into four main groups: a control group (1308 group), a contaminated control group (1308+CM), a dual-antimicrobial group (1308-CAH+CDL), and a combined validation group (1308-CAH+CDL+CM). Each main group's fermentation medium was further divided into three subgroups, with initial glucose concentrations of 50 g / L, 100 g / L, and 200 g / L for the three subgroups within each main group. The fermentation medium for the control group (1308 group) was inoculated with *Saccharomyces cerevisiae* 1308; the fermentation medium for the contaminated control group (1308+CM) was inoculated with *Saccharomyces cerevisiae* 1308, *Acetobacter*, and *Lactobacillus*; and the fermentation medium for the dual-antimicrobial group (1308-CAH+CDL) was inoculated with the genetically engineered strain 1308-P. DDI2-CAH was added, along with cyanamide, antimicrobial peptide DptB, and endolysin LysKB317 at final concentrations of 0.5 g / L, 10 mg / mL, and 30 mg / mL, respectively; the fermentation medium of the validation group (1308-CAH+CDL+CM) was inoculated with genetically engineered strain 1308-P. DDI2 -CAH, Acetobacter, and Lactobacillus were added, along with cyanamide, antimicrobial peptide DptB, and endolysin LysKB317 at final concentrations of 0.5 g / L, 10 mg / mL, and 30 mg / mL, respectively.
[0081] The inoculation methods and inoculation amounts for the above-mentioned strains are the same as those in "4. Fermentation verification of the antimicrobial peptide-endolysin antimicrobial strategy" in Example 2; genetically engineered strain 1308-P DDI2 The inoculation method and inoculation amount for CAH were the same as those for Saccharomyces cerevisiae 1308. The above four fermentation systems were cultured at 35℃ and 200 rpm. Samples were taken every 3 hours during fermentation to determine the OD (oxidative stress). 600 ( Figure 7 ), residual sugar ( Figure 7 ) and ethanol production ( Figure 8 ).
[0082] The results showed that, regarding the improvement in ethanol production: when the initial glucose concentration was 50 g / L, the ethanol production of the 1308 group was 18.78 g / L, the 1308+CM group was 14.75 g / L, and the 1308-CAH+CDL+CM group was 18.25 g / L, with the 1308-CAH+CDL+CM group showing a 23.7% increase in ethanol production compared to the 1308+CM group; when the initial glucose concentration was 100 g / L, the ethanol production of the 1308 group was 38.66 g / L, the 1308-CM group was 32.32 g / L, and the 1308-CAH+CDL+CM group was 36.37 g / L, with the 1308-CAH+CDL+CM group showing a 12.5% increase in ethanol production compared to the 1308+CM group; when the initial glucose concentration was 200 g / L, the ethanol production of the 1308 group was 72.72 g / L, and the 1308-CM group was 61.42 g / L. The ethanol yield of the 1308-CAH+CDL+CM group was 68.31 g / L, and the ethanol yield of the 1308-CAH+CDL+CM group was 10.1% higher than that of the 1308+CM group.
[0083] Ethanol in the fermentation broth was separated and purified from the fermentation broth through a distillation process at a temperature of 78.5℃, which is far below the boiling point of cyanamide (260℃). Therefore, no cyanamide residue was found in the obtained ethanol product. Simultaneously, the antimicrobial peptide endolysin added to the fermentation medium remained in the bottom residue and did not evaporate with the ethanol vapor. Furthermore, due to the low distillation temperature, no antimicrobial peptide endolysin was detected in the distilled ethanol.
[0084] This invention provides a concept and method for producing ethanol by fermentation of Saccharomyces cerevisiae based on a dual antibacterial strategy. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. A genetically engineered brewer's yeast, characterized in that, Overexpression of the gene encoding cyanamide hydratase in the starting strain *Saccharomyces cerevisiae* CAH The amino acid sequence of the monocyanamide hydratase is shown in SEQ ID NO:
6.
2. The genetically engineered Saccharomyces cerevisiae according to claim 1, characterized in that, The gene encoding the cyanamide hydratase CAH The nucleotide sequence is shown in SEQ ID NO:
1.
3. The Saccharomyces cerevisiae genetically engineered strain according to claim 1, characterized in that, The brewing yeast is brewing yeast CICC 1308.
4. The method for constructing the *Saccharomyces cerevisiae* genetically engineered strain according to any one of claims 1 to 3, characterized in that, The steps include: encoding the cyanamide hydratase gene... CAH It is obtained by inserting it into the genome of the brewer's yeast.
5. The construction method according to claim 4, characterized in that, The gene encoding the monocyanamide hydratase is described. CAH The method for inserting the enzyme into the genome of the brewer's yeast includes the following steps: constructing the gene encoding the monocyanamide hydratase. CAH The expression cassette was used to insert the gene encoding the monocyanamide hydratase using CRISPR / Cas9 gene editing technology. CAH The expression cassette is inserted into the genome of the brewer's yeast.
6. A method for producing ethanol by fermentation using the genetically engineered *Saccharomyces cerevisiae* strain according to any one of claims 1 to 3, characterized in that, The process includes the following steps: inoculating the genetically engineered Saccharomyces cerevisiae into a fermentation medium for fermentation culture; The fermentation medium contains the antimicrobial peptide DptB, the endolysin LysKB317, and cyanamide. The amino acid sequence of the antimicrobial peptide DptB is shown in SEQ ID NO:2; the amino acid sequence of the endolysin LysKB317 is shown in SEQ ID NO:
3.
7. The method according to claim 6, characterized in that, The initial concentrations of the antimicrobial peptide DptB, endolysin LysKB317, and cyanamide in the fermentation medium were 8-12 g / L, 28-32 g / L, and 0.4-0.6 g / L, respectively.
8. The method according to claim 6, characterized in that, The genetically engineered Saccharomyces cerevisiae was inoculated into the fermentation medium in the form of a seed culture. The OD of the seed liquid 600 The inoculation amount of the seed solution is 4 to 6; the inoculation amount of the seed solution is 8 to 12% v / v.
9. The method according to claim 6, characterized in that, The fermentation culture was carried out at a temperature of 30-37℃, a pH of 3.0-4.5, and a stirring speed of 150-250 rpm.
10. The method according to claim 6, characterized in that, The fermentation medium comprises the following components: 50-200 g / L glucose, 8-12 g / L yeast extract, 18-22 g / L peptone, 0.8-1.2 g / L potassium dihydrogen phosphate, and 0.4-0.6 g / L magnesium sulfate.