WO2023134104A1 - Method for improving efficiency of integrated bioprocessing by means of using artificial microorganisms - Google Patents
Method for improving efficiency of integrated bioprocessing by means of using artificial microorganisms Download PDFInfo
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- WO2023134104A1 WO2023134104A1 PCT/CN2022/095508 CN2022095508W WO2023134104A1 WO 2023134104 A1 WO2023134104 A1 WO 2023134104A1 CN 2022095508 W CN2022095508 W CN 2022095508W WO 2023134104 A1 WO2023134104 A1 WO 2023134104A1
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- trichoderma reesei
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
Definitions
- the invention relates to the technical field of microorganisms, in particular to a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms.
- CBP is a highly integrated biorefinery process with the least number of unit operations, that is, one-step process.
- SECS steam exploded corn stalks
- Glucaric acid is produced by microbial fermentation, and the current mainstream is recombinant Escherichia coli (Escherichia coli) and recombinant Saccharomyces cerevisiae (Saccharomyces cerevisiae) for the production of glucose and inositol.
- the purpose of the present invention is to provide a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which can solve the current technical problems of low output, yield and yield of integrated bioprocessing.
- the present invention solves its technical problems by adopting the following technical solutions.
- the application provides a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which includes the following steps: mixing various artificial microorganisms to obtain a mixed flora, mixing the mixed flora with raw materials and then fermenting to complete the processing.
- the artificial microorganisms include: Trichoderma reesei and yeast. This method can greatly promote the efficiency of artificial microbial flora CBP.
- this method can not only be used to improve the efficiency of artificial microbial flora to produce glucaric acid in one step, but also has generalizability, and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, For example, distiller's grains produce single cell protein (SCP).
- SCP single cell protein
- the production of SCP is significantly improved, which proves that the push-pull strategy of division of labor and cooperation is an effective and generally applicable (generalizable) method to improve the efficiency of artificial microbial flora integration bioprocessing, for example, by engineering Trichoderma reesei to make It can degrade lignocellulose more efficiently and produce more fermentable sugar for Saccharomyces cerevisiae.
- the process can be described as pull. Therefore, we call it division of labor and collaborative push and pull.
- the present invention has at least the following advantages or beneficial effects:
- the invention provides a method for improving the efficiency of integrated biological processing by using artificial microorganisms, which can greatly promote the efficiency of artificial microbial flora CBP. Moreover, this method can not only be used to improve the efficiency of one-step production of glucaric acid by artificial microbial flora, but also has universality and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, such as distiller's grains Single-cell protein production.
- the production of SCP was significantly improved, which proved that the push-pull strategy of division of labor and cooperation is an effective and generally applicable method to improve the efficiency of artificial microbial flora integration bioprocessing.
- Figure 1 is a schematic diagram of the plasmid construction of Saccharomyces cerevisiae in the present invention, pY26-CDT1- ⁇ G (A), pY26-CDT2- ⁇ G (B) and pY26-CDT1-CDT2- ⁇ G (C) plasmid construction diagram;
- Fig. 2 is the output of the glucaric acid in effect example 1 of the present invention, the schematic diagram of the contrast result of productive rate and yield, (A) division of labor and cooperative push-pull strategy promotes the mechanism diagram of synthetic microbial flora one-step method to produce glucaric acid; Filter paper enzyme activity (B) and glucaric acid (C) during CBP 30g/L steam-exploded corn stover; filter paper enzyme activity (D) and glucaric acid (E) during CBP 50g/L steam-exploded corn stover ); filter paper enzyme activity (F) and glucaric acid (G) in the course of CBP 80g/L steam-exploded corn stover;
- Fig. 3 is the schematic diagram of the comparison result of SCP output, yield and yield in effect example 2 of the present invention
- A take 33.3g/L distiller's grains as the course of substrate CBP to produce SCP
- B use 50g/L distiller's grains The process of producing SCP with distiller's grains as substrate CBP
- C the process of producing SCP with 80g/L distiller's grains as substrate CBP
- D the process of producing SCP with 100g/L distiller's grains as substrate CBP;
- Fig. 4 is a schematic flow chart of the method for improving the efficiency of integrated bioprocessing by using artificial microorganisms provided by the present invention.
- the application provides a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which includes the following steps: mixing various artificial microorganisms to obtain a mixed flora, mixing the mixed flora with raw materials and then fermenting to complete the processing.
- the artificial microorganisms include: Trichoderma reesei and yeast. This method can greatly promote the efficiency of artificial microbial flora CBP.
- this method can not only be used to improve the efficiency of one-step production of glucaric acid by artificial microbial flora, but also has universality and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, such as distiller's grains Single-cell protein production.
- the production of SCP was significantly improved, which proved that the push-pull strategy of division of labor and cooperation is an effective and universally applicable method to improve the efficiency of artificial microbial flora integration bioprocessing.
- the aforementioned raw materials include agricultural waste and industrial waste. Waste utilization is achieved by using waste as the main raw material to save energy.
- Trichoderma reesei C10 is constructed by placing the Trichoderma reesei cbh2 gene between the promoter and terminator of the cbh1 gene, constructing a strong expression cassette P cbh1 -cbh2-T cbh1 , and introducing it into Reesei by the Agrobacterium tumefaciens transformation method Trichoderma, transformants of Trichoderma reesei were screened to obtain cellulase high-yield strain C10; the construction of Saccharomyces cerevisiae PC3 included the following steps: Engineering Saccharomyces cerevisiae to have the ability to metabolize cellodextrin method reference, plasmid pRS426-PGK- References for construction methods of CDT2-CYC1, pRS426-PGK
- the cdt-1, cdt-2 and gh1-1 genes were amplified on the plasmids pRS426-PGK-CDT2-CYC1, pRS426-PGK-CDT2-CYC1 and pRS425-PGK-gh1-1-cyc1, respectively. Since the promoters and terminators of the three are the same, in order to prevent the mismatch of primers, the gh1-1 gene was reversely expressed in this paper.
- the fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-cdt2-Kan-gh1-1 were also constructed by overlapping extension PCR technique.
- connection of CDT1 and CDT2 in cdt1-cdt2-Kan-gh1-1 is realized by means of AatII restriction site. Both ends of the three fragments have NTS homology arms, and the three target fragments are connected with the PCR linearized pY26 plasmid using the Sosoo seamless cloning kit of Qingke to construct plasmids pY26-CDT1- ⁇ G, pY26-CDT2- ⁇ G and pY26-CDT1-CDT2- ⁇ G, as shown in Figure 1.
- CDT1 and CDT2 in the fragment CDT1-CDT2-Kan-gh1-1 is achieved by means of the AatII restriction site, or directly from the plasmid pY26-CDT1- ⁇ G, pY26-CDT2- ⁇ G and pY26-CDT1-CDT2 -Amplify the target fragment on ⁇ G for subsequent transformation of Saccharomyces cerevisiae.
- the A1 fragment (pGPD-ZIgA-linker3-INO1-tCYC1—pTEF-ZHER2-linker2-INM1-tADH1-Kan1) and the A2 fragment (pGPD-ZIgA-linker3-INO1-linker5) were constructed by overlapping extension PCR technology -ZIgA-tCYC1—pTEF-ZHER2-linker2-INM1-linker1-ZHER2-tADH1-Kan1) and fragment B (Kan2-pGPM1-ZWt-linker4--miox4-linker1-udh-tRPL3-pENO2-Scaffold-tDIT1) fragment,
- the A1 and A2 fragments constructed above were ligated with the pY26 plasmid linearized with primer pY26-1F/R using the Qingke Seamless Cloning Kit, and the B fragment was ligated with the pY26 plasmid
- scaffold protein integrated gene fragments For the construction of scaffold protein integrated gene fragments: firstly, four key enzyme genes miox4, udh, INO1, INM; three kinds of affibody and scaffold; promoters TEF, GPD, GPM1, ENO2; terminators ADH, CYC1, RPL3, DIT1; the first 830bp and the last 858bp of the screening marker Kan gene were amplified with corresponding primers, and A1, A2, and B fragments were constructed by overlapping extension PCR technology, or directly from pY26-A1 plasmid, pY26-A2 plasmid and pY26-B The corresponding fragments were amplified on the plasmid for subsequent transformation of Saccharomyces cerevisiae.
- Fragment details are as follows: Fragment A1 (5920bp): delta1-pGPD-ZIgA-linker3-INO1-tCYC1-pTEF-ZHER2-linker2-INM1-tADH1-Kan (first 830bp) Fragment A2 (6400bp): delta1-pGPD- ZIgA-linker3-INO1-linker5-ZIgA-tCYC1-pTEF-ZHER2-linker2-INM1-linker1-ZHER2-tADH1-Kan (before 830bp) Fragment B (5862bp):Kan (back 858bp)-pGPM1-ZWt-linker4--- After MIOX4-linker1-UDH-tRPL3-ENO2p-scaffolds-DIT1t obtained fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-CDT2-
- the inoculation ratio of Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 is (2.5-3.5):1.
- the inoculum ratio can ensure a higher yield of the obtained SCP and a higher fermentation rate, thereby improving the rate and yield of integrated bioprocessing.
- the above-mentioned raw materials include distiller's grains, and the inoculation rate of mixed flora of Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 and distiller's grains is 2-6%.
- the inoculum rate is the ratio of the mass of the bacterial solution of the mixed flora to the mass of distiller's grains.
- the inoculum rate range can ensure that the mixed flora can make full use of the substrate raw materials, so as to ensure that the yield of the produced SCP is sufficient without causing waste of raw materials and interference to its own flora.
- the aforementioned artificial microorganisms include Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2.
- the construction of Saccharomyces cerevisiae LGA-1C3S2 includes the following steps: starting from LGA-1. References for the method of engineering Saccharomyces cerevisiae to have the ability to metabolize cellodextrin, and references for the construction methods of plasmids pRS426-PGK-CDT2-CYC1, pRS426-PGK-CDT2-CYC1, and pRS425-PGK-gh1-1-cyc1.
- the cdt-1, cdt-2 and gh1-1 genes were amplified on the plasmids pRS426-PGK-CDT2-CYC1, pRS426-PGK-CDT2-CYC1 and pRS425-PGK-gh1-1-cyc1, respectively. Since the promoters and terminators of the three are the same, in order to prevent the mismatch of primers, the gh1-1 gene was reversely expressed in this paper.
- the fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-cdt2-Kan-gh1-1 were also constructed by overlapping extension PCR technique.
- connection of CDT1 and CDT2 in cdt1-cdt2-Kan-gh1-1 is realized by means of AatII restriction site. Both ends of the three fragments have NTS homology arms, and the three target fragments are connected with the PCR linearized pY26 plasmid using the Sosoo seamless cloning kit of Qingke to construct plasmids pY26-CDT1- ⁇ G, pY26-CDT2- ⁇ G and pY26-CDT1-CDT2- ⁇ G, as shown in Figure 1.
- CDT1 and CDT2 in the fragment CDT1-CDT2-Kan-gh1-1 is achieved by means of the AatII restriction site, or directly from the plasmid pY26-CDT1- ⁇ G, pY26-CDT2- ⁇ G and pY26-CDT1-CDT2 -Amplify the target fragment on ⁇ G for the subsequent transformation of Saccharomyces cerevisiae.
- the A1 fragment (pGPD-ZIgA-linker3-INO1-tCYC1—pTEF-ZHER2-linker2-INM1-tADH1-Kan1) and the A2 fragment (pGPD-ZIgA-linker3-INO1-linker5) were constructed by overlapping extension PCR technology -ZIgA-tCYC1—pTEF-ZHER2-linker2-INM1-linker1-ZHER2-tADH1-Kan1) and fragment B (Kan2-pGPM1-ZWt-linker4--miox4-linker1-udh-tRPL3-pENO2-Scaffold-tDIT1) fragment,
- the A1 and A2 fragments constructed above were ligated with the pY26 plasmid linearized with primer pY26-1F/R using the Qingke Seamless Cloning Kit, and the B fragment was ligated with the pY26 plasmid
- scaffold protein integrated gene fragments For the construction of scaffold protein integrated gene fragments: firstly, four key enzyme genes miox4, udh, INO1, INM; three kinds of affibody and scaffold; promoters TEF, GPD, GPM1, ENO2; terminators ADH, CYC1, RPL3, DIT1; the first 830bp and the last 858bp of the screening marker Kan gene were amplified with corresponding primers, and A1, A2, and B fragments were constructed by overlapping extension PCR technology, or directly from pY26-A1 plasmid, pY26-A2 plasmid and pY26-B The corresponding fragments were amplified on the plasmid for subsequent transformation of Saccharomyces cerevisiae.
- Fragment details are as follows: Fragment A1 (5920bp): delta1-pGPD-ZIgA-linker3-INO1-tCYC1-pTEF-ZHER2-linker2-INM1-tADH1-Kan (first 830bp) Fragment A2 (6400bp): delta1-pGPD- ZIgA-linker3-INO1-linker5-ZIgA-tCYC1-pTEF-ZHER2-linker2-INM1-linker1-ZHER2-tADH1-Kan (before 830bp) Fragment B(5862bp):Kan(back 858bp)-pGPM1-ZWt-linker4- -MIOX4-linker1-UDH-tRPL3-ENO2p-scaffolds-DIT1t obtained fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-CDT2-K
- A1 and B After obtaining the target gene fragments A1 and B, A2 and B, use kan as a screening marker (subsequently use the Cre-LoxP system to achieve knockout), transform it into the Saccharomyces cerevisiae LGA-1C3 strain with lithium acetate high-efficiency transformation method, and spread G418 resistant plates.
- the basic expression (A1+B) recombinant strain and the network expression (A2+B) recombinant strain were obtained, and the strain with the highest glucaric acid production was selected after fermentation verification, named LGA-1C3S2.
- the modified source strains used in the present invention are Trichoderma reesei Rut-C30 and Saccharomyces cerevisiae INVSc1, both of which were purchased from ACCT.
- the inoculum ratio of the above Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 is 1:1.
- the inoculation ratio can ensure that Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 can fully exert the push-pull function, thereby increasing the yield of the product.
- the above-mentioned raw materials include straw, and the inoculation rate of the mixed flora of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 on the straw is 8-12%.
- the inoculation rate is the ratio of the mass of the bacterial solution of the mixed flora to the mass of the straw. This range of inoculation rate can ensure that the mixed flora can make full use of straw and other materials, so as to ensure the production of sufficient glucaric acid. When the inoculation rate is too high or too low, the mixed flora or raw materials will be wasted.
- the temperature of above-mentioned fermentation is 28-32 °C. This temperature range can ensure that the above-mentioned mixed flora is in a highly active state, thereby improving the efficiency of integrated bioprocessing.
- the time of above-mentioned fermentation is at least 7 days. Only after seven days can it be ensured that most of the raw materials have been reacted, thereby ensuring the highest content of the product.
- This embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: culturing Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 hours, and culturing Saccharomyces cerevisiae LGA-1C3S2 in YPD medium Cultivate to an OD 600 value of 5, mix Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 at an inoculation ratio of 1:1 to obtain a mixed flora, and inoculate the mixed flora at the same time at a total inoculum of 8% (v/v) In the CBP glucaric acid production medium, ferment at 28°C and 180rpm, and the processing is completed.
- the Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
- the CBP glucaric acid production medium includes: 50g/L steam-exploded corn stover, 1g/L peptone, 1g/L yeast extract, 6g/L (NH 4 ) 2 SO 4 , 2.0g/L KH 2 PO 4 , 0.3g/LMgSO 4 , 0.3g/L CaCl 2 , 0.005g/L FeSO 4 , 0.0016g/L MnSO4, 0.0014g/LZnSO 4 , 0.0037g/L CoCl 2 , citric acid buffer 0.05mol/L (final Concentration), 0.1g/L Tween 80. Sterilization conditions: 121°C, 30min.
- This embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: culturing Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 hours, and culturing Saccharomyces cerevisiae LGA-1C3S2 in YPD medium Cultivate to an OD 600 value of 5, mix Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 at an inoculation ratio of 1:1 to obtain a mixed flora, and insert the mixed flora at the same time at a total inoculum of 12% (v/v) In the CBP glucaric acid production medium, ferment at 32°C to complete the processing.
- the Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
- the CBP glucaric acid production medium includes: 50g/L steam-exploded corn stover, 1g/L peptone, 1g/L yeast extract, 6g/L (NH 4 ) 2 SO 4 , 2.0g/L KH 2 PO 4 , 0.3g/LMgSO 4 , 0.3g/L CaCl 2 , 0.005g/L FeSO 4 , 0.0016g/L MnSO4, 0.0014g/LZnSO 4 , 0.0037g/L CoCl 2 , citric acid buffer 0.05mol/L (final Concentration), 0.1g/L Tween 80. Sterilization conditions: 121°C, 30min.
- This embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: culturing Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 hours, and culturing Saccharomyces cerevisiae LGA-1C3S2 in YPD medium Cultivate to an OD 600 value of 5, mix Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 at an inoculation ratio of 1:1 to obtain a mixed flora, and insert the mixed flora at the same time at a total inoculum of 10% (v/v) In the CBP glucaric acid production medium, ferment at 30°C and 180rpm, and the processing is completed.
- the Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
- the CBP glucaric acid production medium includes: 50g/L steam-exploded corn stover, 1g/L peptone, 1g/L yeast extract, 6g/L (NH 4 ) 2 SO 4 , 2.0g/L KH 2 PO 4 , 0.3g/LMgSO 4 , 0.3g/L CaCl 2 , 0.005g/L FeSO 4 , 0.0016g/L MnSO4, 0.0014g/LZnSO 4 , 0.0037g/L CoCl 2 , citric acid buffer 0.05mol/L (final Concentration), 0.1g/L Tween 80. Sterilization conditions: 121°C, 30min.
- the present embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: cultivating Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 h, cultivating Saccharomyces cerevisiae PC3 in YPD medium to The OD 600 value is 5, Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 are mixed at an inoculum ratio of 2.5:1 to obtain a mixed flora, and the mixed flora is simultaneously inserted into CBP to produce SCP at a total inoculum of 2% (v/v) for culture Fermentation at 28°C and 180rpm in the base, the processing is completed.
- the Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
- composition of the CBP SCP production medium is as follows: 50g/L distiller's grains, 1g/L glucose, 5g/L (NH 4 ) 2 SO 4 , 5g/L NH 4 NO 3 , 4g/L KH 2 PO 4 , 0.6g/L MgSO 4 , 0.6g/L CaCl 2 , 0.17mL/L Mandels trace element nutrient salt (Mandels et al., 1981), citric acid buffer solution 0.05mol/L (final concentration), sterilization conditions: 121°C, 30min.
- the present embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: cultivating Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 h, cultivating Saccharomyces cerevisiae PC3 in YPD medium to The OD 600 value is 5, Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 are mixed at an inoculum ratio of 3.5:1 to obtain a mixed flora, and the mixed flora is simultaneously inserted into CBP to produce SCP at a total inoculum of 2% (v/v) for culture Fermentation at 32°C and 180rpm in the base, the processing is completed.
- the Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
- composition of the CBP SCP production medium is as follows: 50g/L distiller's grains, 1g/L glucose, 5g/L (NH 4 ) 2 SO 4 , 5g/L NH 4 NO 3 , 4g/L KH 2 PO 4 , 0.6g/L MgSO 4 , 0.6g/L CaCl 2 , 0.17mL/L Mandels trace element nutrient salt (Mandels et al., 1981), citric acid buffer solution 0.05mol/L (final concentration), sterilization conditions: 121°C, 30min.
- the present embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: cultivating Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 h, cultivating Saccharomyces cerevisiae PC3 in YPD medium to The OD 600 value is 5, Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 are mixed at an inoculum ratio of 3:1 to obtain a mixed flora, and the mixed flora is simultaneously inserted into CBP to produce SCP at a total inoculum of 2% (v/v) for culture In the base, ferment at 30°C to complete the processing.
- the Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
- composition of the CBP SCP production medium is as follows: 50g/L distiller's grains, 1g/L glucose, 5g/L (NH 4 ) 2 SO 4 , 5g/L NH 4 NO 3 , 4g/L KH 2 PO 4 , 0.6g/L MgSO 4 , 0.6g/L CaCl 2 , 0.17mL/L Mandels trace element nutrient salt (Mandels et al., 1981), citric acid buffer solution 0.05mol/L (final concentration), sterilization conditions: 121°C, 30min.
- Trichoderma reesei C10+Saccharomyces cerevisiae LGA-1C3 and Trichoderma reesei Rut-C30+Saccharomyces cerevisiae LGA-1 (Trichoderma reesei Rut-C30 purchased from ACCT) were used to ferment under the same conditions as comparative example 1 and comparative example 2, and detect the output, productive rate and yield of the glucaric acid that embodiment 1-3, comparative example 1-2 obtain, wherein the detection result of embodiment 3, comparative example 1 and comparative example 2 is as Fig. 2 Shown, according to the output of Trichoderma reesei Rut-C30+Saccharomyces cerevisiae LGA-1 and embodiment 3 shown in Fig.
- Yeast LGA-1 is higher, and the output, yield and yield of glucaric acid of embodiment 3 are higher than Trichoderma reesei Rut-C30+Saccharomyces cerevisiae LGA-1, therefore, the method provided by the invention can effectively Improve integrated bioprocessing efficiency.
- Trichoderma reesei Rut-C30+Saccharomyces cerevisiae INVSc1 (Trichoderma reesei Rut-C30 and Saccharomyces cerevisiae INVSc1 are all purchased from ACCT) to ferment under the same conditions as comparative example 3, and detect examples 4-6 and comparative examples 3
- the output, yield and yield of the obtained SCP wherein the detection results of the yield, yield and yield of SCP in Example 6 and Comparative Example 3 are as shown in Figure 3, according to the results shown in Figure 3, the embodiment The yield, yield and yield of SCP in 6 are higher than that of Comparative Example 3, therefore, the method provided by the present invention can effectively improve the efficiency of integrated bioprocessing.
- the present invention provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing:
- This method can greatly promote the efficiency of artificial microbial flora CBP. Moreover, this method can not only be used to improve the efficiency of one-step production of glucaric acid by artificial microbial flora, but also has universality and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, such as distiller's grains Single-cell protein production. After adopting the push-pull strategy of division of labor and cooperation, the production of SCP was significantly improved, which proved that the push-pull strategy of division of labor and cooperation is an effective and universally applicable method to improve the efficiency of artificial microbial flora integration bioprocessing.
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Abstract
Provided is a method for improving the efficiency of integrated bioprocessing by means of using artificial microorganisms, which relates to the technical field of microorganisms. The method comprises the following steps: mixing a plurality of artificial microorganisms to obtain a mixed micropopulation; and mixing the mixed flora with raw materials, and performing fermentation to complete the processing. The technical problems of low output, low production and low yield of integrated bioprocessing at present can be solved.
Description
本发明涉及微生物技术领域，具体而言，涉及一种利用人工微生物提高整合生物加工效率的方法。The invention relates to the technical field of microorganisms, in particular to a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms.
CBP是一种高度整合的生物炼制工艺，拥有最少的单元操作数，即一步法。研究中发现CBP蒸汽爆破玉米秸秆(steam exploded corn store，SECS)有较高的得率，优于SSF，虽然在产量上不如SHF，但得率较为接近，因此CBP拥有较大的潜力。微生物发酵法产葡糖二酸，目前的主流是重组大肠杆菌(Escherichia coli)和重组酿酒酵母(Saccharomyces cerevisiae)发酵葡萄糖和肌醇生产，前者的最大产量为接近5g/L，由麻省理工学院的Prather教授课题组报道，酿酒酵母的最大产量为6g/L，由江南大学的Deng教授课题组报道，将酿酒酵母的产量提高到了11.21g/L。但这些高产的成果都是建立在高投入的基础上，这些工艺都需要添加10.8g/L肌醇(myo-inositol)，肌醇本身就是高附加值产品，CBP秸秆产葡糖二酸虽然产量低，15g/L SECS产0.45g/L葡糖二酸，但底物是农业废弃物，价格低廉，环保而又可再生，因此具有良好的应用前景。但现有的CBP秸秆产葡糖二酸的加工方法，其葡糖二酸的产量、产率和得率都较低，难以进行商业化应用。CBP is a highly integrated biorefinery process with the least number of unit operations, that is, one-step process. The study found that CBP steam exploded corn stalks (steam exploded corn store, SECS) has a higher yield, better than SSF, although the yield is not as good as SHF, but the yield is relatively close, so CBP has great potential. Glucaric acid is produced by microbial fermentation, and the current mainstream is recombinant Escherichia coli (Escherichia coli) and recombinant Saccharomyces cerevisiae (Saccharomyces cerevisiae) for the production of glucose and inositol. Professor Prather's research group reported that the maximum yield of Saccharomyces cerevisiae was 6g/L, and Professor Deng's research group of Jiangnan University reported that the yield of Saccharomyces cerevisiae was increased to 11.21g/L. However, these high-yield results are all based on high investment. These processes require the addition of 10.8g/L myo-inositol. Myo-inositol itself is a high-value-added product. Although the output of glucaric acid produced by CBP straw Low, 15g/L SECS produces 0.45g/L glucaric acid, but the substrate is agricultural waste, which is cheap, environmentally friendly and renewable, so it has a good application prospect. However, the existing processing methods for producing glucaric acid from CBP straw have low glucaric acid yield, yield and yield, making it difficult to apply commercially.
发明内容Contents of the invention
本发明的目的在于提供一种利用人工微生物提高整合生物加工效率的方法，其能够解决现目前整合生物加工产量、产率和得率低的技术问题。The purpose of the present invention is to provide a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which can solve the current technical problems of low output, yield and yield of integrated bioprocessing.
本发明解决其技术问题是采用以下技术方案来实现的。The present invention solves its technical problems by adopting the following technical solutions.
本申请提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤，将多种人工微生物混合后得到混合菌群，将混合菌群与原材料混合后发酵，即完成加工，人工微生物包括里氏木霉菌和酵母菌。该方法可以极大地促进人工微生物菌群CBP的效率。而且该法不仅可以用于提高人工微生物菌群一步法产葡糖二酸的效率，还具有普遍性(generalizability)，可用于其它由里氏木霉与酿酒酵母组成的微生物菌群的CBP效率，比如白酒糟产单细胞蛋白(single cell protein，SCP)。采用分工协作推拉策略以后SCP产量明显提高，证明了分工协作推拉策略是一种有效的、普遍适用的(generalizable)提高人工微生物菌群整合生物加工效率的方法，例如通过工程改造里氏木霉使其能够更高效地降解木质纤维素产更多的可发酵糖供给酿酒酵母，整个过程可形象地描述为推；酿酒酵母可摄入更多的可发酵糖，产更多葡糖二酸，这个过程可描述为拉。因此，我们称之为分工协作推拉。The application provides a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which includes the following steps: mixing various artificial microorganisms to obtain a mixed flora, mixing the mixed flora with raw materials and then fermenting to complete the processing. The artificial microorganisms include: Trichoderma reesei and yeast. This method can greatly promote the efficiency of artificial microbial flora CBP. Moreover, this method can not only be used to improve the efficiency of artificial microbial flora to produce glucaric acid in one step, but also has generalizability, and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, For example, distiller's grains produce single cell protein (SCP). After adopting the push-pull strategy of division of labor and cooperation, the production of SCP is significantly improved, which proves that the push-pull strategy of division of labor and cooperation is an effective and generally applicable (generalizable) method to improve the efficiency of artificial microbial flora integration bioprocessing, for example, by engineering Trichoderma reesei to make It can degrade lignocellulose more efficiently and produce more fermentable sugar for Saccharomyces cerevisiae. The process can be described as pull. Therefore, we call it division of labor and collaborative push and pull.
相对于现有技术，本发明至少具有如下优点或有益效果：Compared with the prior art, the present invention has at least the following advantages or beneficial effects:
本发明提供了一种利用人工微生物提高整合生物加工效率的方法，该方法可以极大地促进人工微生物菌群CBP的效率。而且该法不仅可以用于提高人工微生物菌群一步法产葡糖二酸的效率，还具有普遍性，可用于其它由里氏木霉与酿酒酵母组成的微生物菌群的CBP效率， 比如白酒糟产单细胞蛋白。采用分工协作推拉策略以后SCP产量明显提高，证明了分工协作推拉策略是一种有效的、普遍适用的提高人工微生物菌群整合生物加工效率的方法。The invention provides a method for improving the efficiency of integrated biological processing by using artificial microorganisms, which can greatly promote the efficiency of artificial microbial flora CBP. Moreover, this method can not only be used to improve the efficiency of one-step production of glucaric acid by artificial microbial flora, but also has universality and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, such as distiller's grains Single-cell protein production. After adopting the push-pull strategy of division of labor and cooperation, the production of SCP was significantly improved, which proved that the push-pull strategy of division of labor and cooperation is an effective and generally applicable method to improve the efficiency of artificial microbial flora integration bioprocessing.
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.
图1为本发明中酿酒酵母的质粒构建示意图，pY26-CDT1-βG(A)、pY26-CDT2-βG(B)和pY26-CDT1-CDT2-βG(C)质粒构建图；Figure 1 is a schematic diagram of the plasmid construction of Saccharomyces cerevisiae in the present invention, pY26-CDT1-βG (A), pY26-CDT2-βG (B) and pY26-CDT1-CDT2-βG (C) plasmid construction diagram;
图2为本发明效果例1中的葡糖二酸的产量、产率和得率的对照结果示意图，(A)分工协作推拉策略促进合成微生物菌群一步法产葡糖二酸的机理图；CBP 30g/L蒸汽爆破玉米秸秆历程中的滤纸酶活(B)和葡糖二酸(C)；CBP 50g/L蒸汽爆破玉米秸秆历程中的滤纸酶活(D)和葡糖二酸(E)；CBP 80g/L蒸汽爆破玉米秸秆历程中的滤纸酶活(F)和葡糖二酸(G)；Fig. 2 is the output of the glucaric acid in effect example 1 of the present invention, the schematic diagram of the contrast result of productive rate and yield, (A) division of labor and cooperative push-pull strategy promotes the mechanism diagram of synthetic microbial flora one-step method to produce glucaric acid; Filter paper enzyme activity (B) and glucaric acid (C) during CBP 30g/L steam-exploded corn stover; filter paper enzyme activity (D) and glucaric acid (E) during CBP 50g/L steam-exploded corn stover ); filter paper enzyme activity (F) and glucaric acid (G) in the course of CBP 80g/L steam-exploded corn stover;
图3为本发明效果例2中的SCP产量、产率和得率的对照结果示意图，(A)以33.3g/L白酒糟为底物CBP产SCP的历程；(B)以50g/L白酒糟为底物CBP产SCP的历程；(C)以80g/L白酒糟为底物CBP产SCP的历程；(D)以100g/L白酒糟为底物CBP产SCP的历程；Fig. 3 is the schematic diagram of the comparison result of SCP output, yield and yield in effect example 2 of the present invention, (A) take 33.3g/L distiller's grains as the course of substrate CBP to produce SCP; (B) use 50g/L distiller's grains The process of producing SCP with distiller's grains as substrate CBP; (C) the process of producing SCP with 80g/L distiller's grains as substrate CBP; (D) the process of producing SCP with 100g/L distiller's grains as substrate CBP;
图4为本发明提供的该利用人工微生物提高整合生物加工效率的方法的流程示意图。Fig. 4 is a schematic flow chart of the method for improving the efficiency of integrated bioprocessing by using artificial microorganisms provided by the present invention.
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考具体实施例来详细说明本发明。It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. Hereinafter, the present invention will be described in detail with reference to specific examples.
本申请提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤，将多种人工微生物混合后得到混合菌群，将混合菌群与原材料混合后发酵，即完成加工，人工微生物包括里氏木霉菌和酵母菌。该方法可以极大地促进人工微生物菌群CBP的效率。而且该法不仅可以用于提高人工微生物菌群一步法产葡糖二酸的效率，还具有普遍性，可用于其它由里氏木霉与酿酒酵母组成的微生物菌群的CBP效率，比如白酒糟产单细胞蛋白。采用分工协作推拉策略以后SCP产量明显提高，证明了分工协作推拉策略是一种有效的、普遍适用的提高人工微生物菌群整合生物加工效率的方法。The application provides a method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which includes the following steps: mixing various artificial microorganisms to obtain a mixed flora, mixing the mixed flora with raw materials and then fermenting to complete the processing. The artificial microorganisms include: Trichoderma reesei and yeast. This method can greatly promote the efficiency of artificial microbial flora CBP. Moreover, this method can not only be used to improve the efficiency of one-step production of glucaric acid by artificial microbial flora, but also has universality and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, such as distiller's grains Single-cell protein production. After adopting the push-pull strategy of division of labor and cooperation, the production of SCP was significantly improved, which proved that the push-pull strategy of division of labor and cooperation is an effective and universally applicable method to improve the efficiency of artificial microbial flora integration bioprocessing.
上述原材料包括农业废料和工业废料。通过将废料作为主要原材料使用以节约能源，实现废物利用。The aforementioned raw materials include agricultural waste and industrial waste. Waste utilization is achieved by using waste as the main raw material to save energy.
上述人工微生物包括里氏木霉C10和酿酒酵母PC3。其中里氏木霉C10的构建为将里氏木霉cbh2基因置于cbh1基因的启动子和终止子之间，构建强表达盒P
cbh1-cbh2-T
cbh1，通过根瘤农杆菌转化法导入里氏木霉，筛选里氏木霉转化子，得纤维素酶高产菌株C10；酿酒酵母PC3的构建包括如下步骤：工程改造酿酒酵母使其具有代谢纤维糊精能力的方法参考文献，质粒pRS426-PGK-CDT2-CYC1、pRS426-PGK-CDT2-CYC1、pRS425-PGK-gh1-1-cyc1的构建方法参考文献。分别在质粒pRS426-PGK-CDT2-CYC1、pRS426-PGK-CDT2-CYC1、pRS425-PGK-gh1-1-cyc1上扩增cdt-1、cdt-2和gh1-1基因。由于三者的启动子与终止子一样，后续为防止引物的错配，本文将gh1-1基因反向表达。同样利用利用重叠延伸PCR技术构建片段CDT1-Kan-gh1-1、CDT2-Kan-gh1-1和CDT1-cdt2-Kan-gh1-1。cdt1-cdt2-Kan-gh1-1中CDT1和CDT2的连接借助于AatII酶切位点实现。三个片段两端均带有NTS同源臂，利用擎科的Sosoo无缝克隆试剂盒将三个目的片段与PCR线性化的pY26质粒进行连接，构建质粒pY26-CDT1-βG、pY26-CDT2-βG和pY26-CDT1-CDT2-βG，如图1所示。对于纤维糊精转途径整合基因片段的构建：分别在质粒pRS426-PGK-CDT1-CYC1、pRS426-PGK-CDT2-CYC1、pRS425-PGK-gh1-1-cyc1上扩增cdt-1、cdt-2和gh1-1基因，筛选标记Kan基因的前830bp和后858bp用相应引物进行扩增，之后利用重叠延伸PCR技术将CDT1与Kan1相连，Kan2与gh1-1相连，二轮连接构建CDT1-Kan-gh1-1片段，同理可得CDT2-Kan-gh1-1，CDT1-CDT2-Kan-gh1-1。需要注意的是片段CDT1-CDT2-Kan-gh1-1中CDT1和CDT2的连接借助于AatII酶切位点实现，或直接从质粒pY26-CDT1-βG、pY26-CDT2-βG和pY26-CDT1-CDT2-βG上扩增目的片段用于后续酿酒酵母的转化。
The aforementioned artificial microorganisms include Trichoderma reesei C10 and Saccharomyces cerevisiae PC3. Among them, Trichoderma reesei C10 is constructed by placing the Trichoderma reesei cbh2 gene between the promoter and terminator of the cbh1 gene, constructing a strong expression cassette P cbh1 -cbh2-T cbh1 , and introducing it into Reesei by the Agrobacterium tumefaciens transformation method Trichoderma, transformants of Trichoderma reesei were screened to obtain cellulase high-yield strain C10; the construction of Saccharomyces cerevisiae PC3 included the following steps: Engineering Saccharomyces cerevisiae to have the ability to metabolize cellodextrin method reference, plasmid pRS426-PGK- References for construction methods of CDT2-CYC1, pRS426-PGK-CDT2-CYC1, pRS425-PGK-gh1-1-cyc1. The cdt-1, cdt-2 and gh1-1 genes were amplified on the plasmids pRS426-PGK-CDT2-CYC1, pRS426-PGK-CDT2-CYC1 and pRS425-PGK-gh1-1-cyc1, respectively. Since the promoters and terminators of the three are the same, in order to prevent the mismatch of primers, the gh1-1 gene was reversely expressed in this paper. The fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-cdt2-Kan-gh1-1 were also constructed by overlapping extension PCR technique. The connection of CDT1 and CDT2 in cdt1-cdt2-Kan-gh1-1 is realized by means of AatII restriction site. Both ends of the three fragments have NTS homology arms, and the three target fragments are connected with the PCR linearized pY26 plasmid using the Sosoo seamless cloning kit of Qingke to construct plasmids pY26-CDT1-βG, pY26-CDT2- βG and pY26-CDT1-CDT2-βG, as shown in Figure 1. For the construction of integrated gene fragments of the cellodextrin transfer pathway: amplify cdt-1 and cdt-2 on plasmids pRS426-PGK-CDT1-CYC1, pRS426-PGK-CDT2-CYC1, and pRS425-PGK-gh1-1-cyc1, respectively and gh1-1 gene, the first 830bp and the last 858bp of the screening marker Kan gene were amplified with corresponding primers, and then CDT1 was connected to Kan1, Kan2 was connected to gh1-1 by overlapping extension PCR technology, and the second round of connection was used to construct CDT1-Kan- gh1-1 fragment, CDT2-Kan-gh1-1, CDT1-CDT2-Kan-gh1-1 can be obtained in the same way. It should be noted that the connection of CDT1 and CDT2 in the fragment CDT1-CDT2-Kan-gh1-1 is achieved by means of the AatII restriction site, or directly from the plasmid pY26-CDT1-βG, pY26-CDT2-βG and pY26-CDT1-CDT2 -Amplify the target fragment on βG for subsequent transformation of Saccharomyces cerevisiae.
生工生物工程股份有限公司合成Anti-ZWT-anti-ZHER2-anti-ZIgA scaffold with(SSSSG)
4linkers、ZWT、ZHER2、ZIgA后，分别连接在通用载体PUC19上。从相应模板上将支架蛋白、4种关键酶基因miox4、udh、INO1、INM；启动子TEF、GPD、GPM1、ENO2；终止子ADH、CYC1、RPL3、DIT1；筛选标记kan1和kan2一起利用相应引物对进行扩增(见表2-10)。目的片段准备结束后，利用重叠延伸PCR技术构建A1片段(pGPD-ZIgA-linker③-INO1-tCYC1—pTEF-ZHER2-linker②-INM1-tADH1-Kan1)，A2片段(pGPD-ZIgA-linker③-INO1-linker⑤-ZIgA-tCYC1—pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan1)及B片段(Kan2-pGPM1-ZWt-linker④--miox4-linker①-udh-tRPL3-pENO2-Scaffold-tDIT1)片段，将上述构建好的A1，A2片段利用擎科无缝克隆试剂盒与用引物pY26-1F/R线性化的pY26质粒连接，B片段与用引物pY26-2F/R线性化的pY26质粒连接，最终获得pY26-A1质粒、pY26-A2质粒及pY26-B质粒。
Sangon Bioengineering Co., Ltd. synthesized Anti-ZWT-anti-ZHER2-anti-ZIgA scaffold with (SSSSG) 4 linkers, ZWT, ZHER2, and ZIgA, and connected them to the universal carrier PUC19 respectively. From the corresponding templates, the scaffold protein, four key enzyme genes miox4, udh, INO1, INM; promoters TEF, GPD, GPM1, ENO2; terminators ADH, CYC1, RPL3, DIT1; screening markers kan1 and kan2 were used together with the corresponding primers Amplify (see Table 2-10). After the preparation of the target fragment, the A1 fragment (pGPD-ZIgA-linker③-INO1-tCYC1—pTEF-ZHER2-linker②-INM1-tADH1-Kan1) and the A2 fragment (pGPD-ZIgA-linker③-INO1-linker⑤) were constructed by overlapping extension PCR technology -ZIgA-tCYC1—pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan1) and fragment B (Kan2-pGPM1-ZWt-linker④--miox4-linker①-udh-tRPL3-pENO2-Scaffold-tDIT1) fragment, The A1 and A2 fragments constructed above were ligated with the pY26 plasmid linearized with primer pY26-1F/R using the Qingke Seamless Cloning Kit, and the B fragment was ligated with the pY26 plasmid linearized with primer pY26-2F/R, and finally The pY26-A1 plasmid, pY26-A2 plasmid and pY26-B plasmid were obtained.
对于支架蛋白整合基因片段的构建：首先从相应模板上将4种关键酶基因miox4，udh，INO1，INM；3种affibody及scaffold；启动子TEF，GPD，GPM1，ENO2；终止子ADH，CYC1，RPL3，DIT1；筛选标记Kan基因的前830bp和后858bp用相应引物进行扩增，利用重叠延伸PCR技术构建A1，A2，B片段，或者直接从pY26-A1质粒、pY26-A2质粒及pY26-B质粒上扩增相应片段用于后续酿酒酵母的转化。片段详细信息如下所示：片段A1(5920bp)：delta1-pGPD-ZIgA-linker③-INO1-tCYC1-pTEF-ZHER2-linker②-INM1-tADH1-Kan(前830bp)片段A2(6400bp)：delta1-pGPD-ZIgA-linker③-INO1-linker⑤-ZIgA-tCYC1-pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan(前830bp)片段B(5862bp):Kan(后858bp)-pGPM1-ZWt-linker④--MIOX4-linker①-UDH-tRPL3-ENO2p-scaffolds-DIT1t得到片段CDT1-Kan-gh1-1、CDT2-Kan-gh1-1和CDT1-CDT2-Kan-gh1-1后，同以kan作为筛选标记(后续利用Cre-LoxP系统实现敲除)，用醋酸锂高效转化法转化至酿酒酵母PC3，涂布G418抗性平板，得到酿酒酵母重组菌株，经发酵验证选择里面SCP产量最高的菌株，命名为PC3。For the construction of scaffold protein integrated gene fragments: firstly, four key enzyme genes miox4, udh, INO1, INM; three kinds of affibody and scaffold; promoters TEF, GPD, GPM1, ENO2; terminators ADH, CYC1, RPL3, DIT1; the first 830bp and the last 858bp of the screening marker Kan gene were amplified with corresponding primers, and A1, A2, and B fragments were constructed by overlapping extension PCR technology, or directly from pY26-A1 plasmid, pY26-A2 plasmid and pY26-B The corresponding fragments were amplified on the plasmid for subsequent transformation of Saccharomyces cerevisiae. Fragment details are as follows: Fragment A1 (5920bp): delta1-pGPD-ZIgA-linker③-INO1-tCYC1-pTEF-ZHER2-linker②-INM1-tADH1-Kan (first 830bp) Fragment A2 (6400bp): delta1-pGPD- ZIgA-linker③-INO1-linker⑤-ZIgA-tCYC1-pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan (before 830bp) Fragment B (5862bp):Kan (back 858bp)-pGPM1-ZWt-linker④-- After MIOX4-linker①-UDH-tRPL3-ENO2p-scaffolds-DIT1t obtained fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-CDT2-Kan-gh1-1, kan was used as a screening marker (subsequent Knockout was achieved by Cre-LoxP system), transformed into Saccharomyces cerevisiae PC3 by high-efficiency lithium acetate transformation method, and coated with G418 resistance plate to obtain the recombinant strain of Saccharomyces cerevisiae. After fermentation verification, the strain with the highest SCP production was selected and named PC3.
上述里氏木霉C10和酿酒酵母PC3的接种比为(2.5-3.5):1。该接种比例能够保证所得到的SCP产量较高，且保证较高的发酵速率，从而提高整合生物加工的速率和得率。The inoculation ratio of Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 is (2.5-3.5):1. The inoculum ratio can ensure a higher yield of the obtained SCP and a higher fermentation rate, thereby improving the rate and yield of integrated bioprocessing.
上述原材料包括酒糟，里氏木霉C10和酿酒酵母PC3的混合菌群与酒糟的接种率为2-6％。接种率为混合菌群的菌液的质量与酒糟的质量比。该接种率区间能够保证混合菌群可以充分利用底物原料，从而保证所制得的SCP的产量充足的同时又不会造成原料的浪费和对本身菌群的干扰。The above-mentioned raw materials include distiller's grains, and the inoculation rate of mixed flora of Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 and distiller's grains is 2-6%. The inoculum rate is the ratio of the mass of the bacterial solution of the mixed flora to the mass of distiller's grains. The inoculum rate range can ensure that the mixed flora can make full use of the substrate raw materials, so as to ensure that the yield of the produced SCP is sufficient without causing waste of raw materials and interference to its own flora.
上述人工微生物包括里氏木霉C10和酿酒酵母LGA-1C3S2。其中酿酒酵母LGA-1C3S2的构建包括如下步骤：由LGA-1出发。工程改造酿酒酵母使其具有代谢纤维糊精能力的方法参考文献，质粒pRS426-PGK-CDT2-CYC1、pRS426-PGK-CDT2-CYC1、pRS425-PGK-gh1-1-cyc1的构建方法参考文献。分别在质粒pRS426-PGK-CDT2-CYC1、pRS426-PGK-CDT2-CYC1、pRS425-PGK-gh1-1-cyc1上扩增cdt-1、cdt-2和gh1-1基因。由于三者的启动子与终止子一样，后续为防止引物的错配，本文将gh1-1基因反向表达。同样利用利用重叠延伸PCR技术构建片段CDT1-Kan-gh1-1、CDT2-Kan-gh1-1和CDT1-cdt2-Kan-gh1-1。cdt1-cdt2-Kan-gh1-1中CDT1和CDT2的连接借助于AatII酶切位点实现。三个片段两端均带有NTS同源臂，利用擎科的Sosoo无缝克隆试剂盒将三个目的片段与PCR线性化的pY26质粒进行连接，构建质粒pY26-CDT1-βG、pY26-CDT2-βG和pY26-CDT1-CDT2-βG，如图1所示。对于纤维糊精转途径整合基因片段的构建：分别在质粒pRS426-PGK-CDT1-CYC1、pRS426-PGK-CDT2-CYC1、pRS425-PGK-gh1-1-cyc1上扩增cdt-1、cdt-2和gh1-1基因，筛选标记Kan基因的前830bp和后858bp用相应引物进行扩增，之后利用重叠延伸PCR技术将CDT1与Kan1相连，Kan2与gh1-1相连，二轮连接构建CDT1-Kan-gh1-1片段，同理可得CDT2-Kan-gh1-1，CDT1-CDT2-Kan-gh1-1。需要注意的是片段CDT1-CDT2-Kan-gh1-1中CDT1和CDT2的连接借助于AatII酶切位点实现，或直接从质粒pY26-CDT1-βG、pY26-CDT2-βG和pY26-CDT1-CDT2-βG上扩增目的片段用于后续酿酒酵母的转化。The aforementioned artificial microorganisms include Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2. The construction of Saccharomyces cerevisiae LGA-1C3S2 includes the following steps: starting from LGA-1. References for the method of engineering Saccharomyces cerevisiae to have the ability to metabolize cellodextrin, and references for the construction methods of plasmids pRS426-PGK-CDT2-CYC1, pRS426-PGK-CDT2-CYC1, and pRS425-PGK-gh1-1-cyc1. The cdt-1, cdt-2 and gh1-1 genes were amplified on the plasmids pRS426-PGK-CDT2-CYC1, pRS426-PGK-CDT2-CYC1 and pRS425-PGK-gh1-1-cyc1, respectively. Since the promoters and terminators of the three are the same, in order to prevent the mismatch of primers, the gh1-1 gene was reversely expressed in this paper. The fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-cdt2-Kan-gh1-1 were also constructed by overlapping extension PCR technique. The connection of CDT1 and CDT2 in cdt1-cdt2-Kan-gh1-1 is realized by means of AatII restriction site. Both ends of the three fragments have NTS homology arms, and the three target fragments are connected with the PCR linearized pY26 plasmid using the Sosoo seamless cloning kit of Qingke to construct plasmids pY26-CDT1-βG, pY26-CDT2- βG and pY26-CDT1-CDT2-βG, as shown in Figure 1. For the construction of integrated gene fragments of the cellodextrin transfer pathway: amplify cdt-1 and cdt-2 on plasmids pRS426-PGK-CDT1-CYC1, pRS426-PGK-CDT2-CYC1, and pRS425-PGK-gh1-1-cyc1, respectively and gh1-1 gene, the first 830bp and the last 858bp of the screening marker Kan gene were amplified with corresponding primers, and then CDT1 was connected to Kan1, Kan2 was connected to gh1-1 by overlapping extension PCR technology, and the second round of connection was used to construct CDT1-Kan- gh1-1 fragment, CDT2-Kan-gh1-1, CDT1-CDT2-Kan-gh1-1 can be obtained in the same way. It should be noted that the connection of CDT1 and CDT2 in the fragment CDT1-CDT2-Kan-gh1-1 is achieved by means of the AatII restriction site, or directly from the plasmid pY26-CDT1-βG, pY26-CDT2-βG and pY26-CDT1-CDT2 -Amplify the target fragment on βG for the subsequent transformation of Saccharomyces cerevisiae.
生工生物工程股份有限公司合成Anti-ZWT-anti-ZHER2-anti-ZIgA scaffold with(SSSSG)
4linkers、ZWT、ZHER2、ZIgA后，分别连接在通用载体PUC19上。从相应模板上将支架蛋白、4种关键酶基因miox4、udh、INO1、INM；启动子TEF、GPD、GPM1、ENO2；终止子ADH、CYC1、RPL3、DIT1；筛选标记kan1和kan2一起利用相应引物对进行扩增(见表2-10)。目的片段准备结束后，利用重叠延伸PCR技术构建A1片段(pGPD-ZIgA-linker③-INO1-tCYC1—pTEF-ZHER2-linker②-INM1-tADH1-Kan1)，A2片段(pGPD-ZIgA-linker③-INO1-linker⑤-ZIgA-tCYC1—pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan1)及B片段(Kan2-pGPM1-ZWt-linker④--miox4-linker①-udh-tRPL3-pENO2-Scaffold-tDIT1)片段，将上述构建好的A1，A2片段利用擎科无缝克隆试剂盒与用引物pY26-1F/R线性化的pY26质粒连接，B片段与用引物pY26-2F/R线性化的pY26质粒连接，最终获得pY26-A1质粒、pY26-A2质粒及pY26-B质粒。
Sangon Bioengineering Co., Ltd. synthesized Anti-ZWT-anti-ZHER2-anti-ZIgA scaffold with (SSSSG) 4 linkers, ZWT, ZHER2, and ZIgA, and connected them to the universal carrier PUC19 respectively. From the corresponding templates, the scaffold protein, four key enzyme genes miox4, udh, INO1, INM; promoters TEF, GPD, GPM1, ENO2; terminators ADH, CYC1, RPL3, DIT1; screening markers kan1 and kan2 were used together with the corresponding primers Amplify (see Table 2-10). After the preparation of the target fragment, the A1 fragment (pGPD-ZIgA-linker③-INO1-tCYC1—pTEF-ZHER2-linker②-INM1-tADH1-Kan1) and the A2 fragment (pGPD-ZIgA-linker③-INO1-linker⑤) were constructed by overlapping extension PCR technology -ZIgA-tCYC1—pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan1) and fragment B (Kan2-pGPM1-ZWt-linker④--miox4-linker①-udh-tRPL3-pENO2-Scaffold-tDIT1) fragment, The A1 and A2 fragments constructed above were ligated with the pY26 plasmid linearized with primer pY26-1F/R using the Qingke Seamless Cloning Kit, and the B fragment was ligated with the pY26 plasmid linearized with primer pY26-2F/R, and finally The pY26-A1 plasmid, pY26-A2 plasmid and pY26-B plasmid were obtained.
对于支架蛋白整合基因片段的构建：首先从相应模板上将4种关键酶基因miox4，udh，INO1，INM；3种affibody及scaffold；启动子TEF，GPD，GPM1，ENO2；终止子ADH，CYC1，RPL3，DIT1；筛选标记Kan基因的前830bp和后858bp用相应引物进行扩增，利用重叠延伸PCR技术构建A1，A2，B片段，或者直接从pY26-A1质粒、pY26-A2质粒及pY26-B质粒上扩增相应片段用于后续酿酒酵母的转化。片段详细信息如下所示：片段A1(5920bp)：delta1-pGPD-ZIgA-linker③-INO1-tCYC1-pTEF-ZHER2-linker②-INM1-tADH1-Kan(前830bp)片段A2(6400bp)：delta1-pGPD-ZIgA-linker③-INO1-linker⑤-ZIgA-tCYC1-pTEF-ZHER2-linker ②-INM1-linker①-ZHER2-tADH1-Kan(前830bp)片段B(5862bp):Kan(后858bp)-pGPM1-ZWt-linker④--MIOX4-linker①-UDH-tRPL3-ENO2p-scaffolds-DIT1t得到片段CDT1-Kan-gh1-1、CDT2-Kan-gh1-1和CDT1-CDT2-Kan-gh1-1后，同以kan作为筛选标记(后续利用Cre-LoxP系统实现敲除)，用醋酸锂高效转化法转化至酿酒酵母LGA-1菌株，涂布G418抗性平板，得到酿酒酵母重组菌株，经发酵验证选择里面葡糖二酸产量最高的菌株，命名为LGA-1C3。For the construction of scaffold protein integrated gene fragments: firstly, four key enzyme genes miox4, udh, INO1, INM; three kinds of affibody and scaffold; promoters TEF, GPD, GPM1, ENO2; terminators ADH, CYC1, RPL3, DIT1; the first 830bp and the last 858bp of the screening marker Kan gene were amplified with corresponding primers, and A1, A2, and B fragments were constructed by overlapping extension PCR technology, or directly from pY26-A1 plasmid, pY26-A2 plasmid and pY26-B The corresponding fragments were amplified on the plasmid for subsequent transformation of Saccharomyces cerevisiae. Fragment details are as follows: Fragment A1 (5920bp): delta1-pGPD-ZIgA-linker③-INO1-tCYC1-pTEF-ZHER2-linker②-INM1-tADH1-Kan (first 830bp) Fragment A2 (6400bp): delta1-pGPD- ZIgA-linker③-INO1-linker⑤-ZIgA-tCYC1-pTEF-ZHER2-linker②-INM1-linker①-ZHER2-tADH1-Kan (before 830bp) Fragment B(5862bp):Kan(back 858bp)-pGPM1-ZWt-linker④- -MIOX4-linker①-UDH-tRPL3-ENO2p-scaffolds-DIT1t obtained fragments CDT1-Kan-gh1-1, CDT2-Kan-gh1-1 and CDT1-CDT2-Kan-gh1-1, and used kan as a screening marker ( Subsequent use of the Cre-LoxP system to achieve knockout) was transformed into the Saccharomyces cerevisiae LGA-1 strain with lithium acetate high-efficiency transformation method, and the G418 resistance plate was coated to obtain the recombinant strain of Saccharomyces cerevisiae, which had the highest glucaric acid production after fermentation verification The strain named LGA-1C3.
得到目的基因片段A1与B片段，A2与B片段后，以kan作为筛选标记(后续利用Cre-LoxP系统实现敲除)，用醋酸锂高效转化法转化至酿酒酵母LGA-1C3菌株中，涂布G418抗性平板。获得基础表达(A1+B)重组菌株和网状表达(A2+B)重组菌株，经发酵验证选择里面葡糖二酸产量最高的菌株，命名为LGA-1C3S2。After obtaining the target gene fragments A1 and B, A2 and B, use kan as a screening marker (subsequently use the Cre-LoxP system to achieve knockout), transform it into the Saccharomyces cerevisiae LGA-1C3 strain with lithium acetate high-efficiency transformation method, and spread G418 resistant plates. The basic expression (A1+B) recombinant strain and the network expression (A2+B) recombinant strain were obtained, and the strain with the highest glucaric acid production was selected after fermentation verification, named LGA-1C3S2.
本发明中所使用的改造来源菌株为里氏木霉Rut-C30和酿酒酵母INVSc1，二者均购自ACCT。The modified source strains used in the present invention are Trichoderma reesei Rut-C30 and Saccharomyces cerevisiae INVSc1, both of which were purchased from ACCT.
上述里氏木霉C10和酿酒酵母LGA-1C3S2的接种比为1:1。该接种的比例能够保证里氏木霉C10和酿酒酵母LGA-1C3S2能够充分发挥推拉功效，从而提高产物的得率。The inoculum ratio of the above Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 is 1:1. The inoculation ratio can ensure that Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 can fully exert the push-pull function, thereby increasing the yield of the product.
上述原材料包括秸秆，里氏木霉C10和酿酒酵母LGA-1C3S2混合菌群与秸秆的接种率为8-12％。接种率为混合菌群的菌液的质量与秸秆的质量比。该接种率区间能够保证混合菌群能够充分利用秸秆等材料，从而保证产足够的葡糖二酸，当接种率过高或过低会导致混合菌群或原料的浪费。The above-mentioned raw materials include straw, and the inoculation rate of the mixed flora of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 on the straw is 8-12%. The inoculation rate is the ratio of the mass of the bacterial solution of the mixed flora to the mass of the straw. This range of inoculation rate can ensure that the mixed flora can make full use of straw and other materials, so as to ensure the production of sufficient glucaric acid. When the inoculation rate is too high or too low, the mixed flora or raw materials will be wasted.
上述发酵的温度为28-32℃。该温度区间能保证上述混合菌群处于高活性状态，从而提高整合生物加工的效率。The temperature of above-mentioned fermentation is 28-32 ℃. This temperature range can ensure that the above-mentioned mixed flora is in a highly active state, thereby improving the efficiency of integrated bioprocessing.
上述发酵的时间至少为7天。七天后才能保证大部分的原料已反应完，从而保证产物的含量最高。The time of above-mentioned fermentation is at least 7 days. Only after seven days can it be ensured that most of the raw materials have been reacted, thereby ensuring the highest content of the product.
以下结合实施例对本发明的特征和性能作进一步的详细描述。The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.
实施例1Example 1
本实施例提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤：将里氏木霉C10在里氏木霉种子培养基中培养36h，酿酒酵母LGA-1C3S2在YPD培养基中培养到OD
600值5，将里氏木霉C10与酿酒酵母LGA-1C3S2以接种比1:1混合，得到混合菌群，将混合菌群按总接种量8％(v/v)同时接入CBP产葡糖二酸培养基中，于28℃、180rpm发酵，即完成加工。
This embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: culturing Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 hours, and culturing Saccharomyces cerevisiae LGA-1C3S2 in YPD medium Cultivate to an OD 600 value of 5, mix Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 at an inoculation ratio of 1:1 to obtain a mixed flora, and inoculate the mixed flora at the same time at a total inoculum of 8% (v/v) In the CBP glucaric acid production medium, ferment at 28°C and 180rpm, and the processing is completed.
其中里氏木霉培养基包括：葡萄糖10g/L，蛋白胨1g/L，Mandels营养盐浓液5mL/瓶，Mandels微量元素浓液0.05mL/瓶，1mol/L柠檬酸缓冲液2.5mL/瓶，Tween-802滴/瓶，以上溶液定容至50mL，倒入250mL的三角瓶，塞上棉花塞，盖上牛皮纸，用皮筋扎紧，于121℃，灭菌30min。The Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
其中CBP产葡糖二酸培养基包括：50g/L蒸汽爆破玉米秸秆，1g/L蛋白胨，1g/L酵母浸膏，6g/L(NH
4)
2SO
4，2.0g/L KH
2PO
4，0.3g/LMgSO
4，0.3g/L CaCl
2，0.005g/L FeSO
4，0.0016g/L MnSO4，0.0014g/LZnSO
4，0.0037g/L CoCl
2，柠檬酸缓冲液0.05mol/L(终浓度)，0.1g/L吐温80。灭菌条件：121℃、30min。
Among them, the CBP glucaric acid production medium includes: 50g/L steam-exploded corn stover, 1g/L peptone, 1g/L yeast extract, 6g/L (NH 4 ) 2 SO 4 , 2.0g/L KH 2 PO 4 , 0.3g/LMgSO 4 , 0.3g/L CaCl 2 , 0.005g/L FeSO 4 , 0.0016g/L MnSO4, 0.0014g/LZnSO 4 , 0.0037g/L CoCl 2 , citric acid buffer 0.05mol/L (final Concentration), 0.1g/L Tween 80. Sterilization conditions: 121°C, 30min.
实施例2Example 2
本实施例提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤：将里氏木霉C10在里氏木霉种子培养基中培养36h，酿酒酵母LGA-1C3S2在YPD培养基中培养到OD
600值5，将里氏木霉C10与酿酒酵母LGA-1C3S2以接种比1:1混合，得到混合菌群，将混合菌群按总接种量12％(v/v)同时接入CBP产葡糖二酸培养基中，于32℃发酵，即完成加工。
This embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: culturing Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 hours, and culturing Saccharomyces cerevisiae LGA-1C3S2 in YPD medium Cultivate to an OD 600 value of 5, mix Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 at an inoculation ratio of 1:1 to obtain a mixed flora, and insert the mixed flora at the same time at a total inoculum of 12% (v/v) In the CBP glucaric acid production medium, ferment at 32°C to complete the processing.
其中里氏木霉培养基包括：葡萄糖10g/L，蛋白胨1g/L，Mandels营养盐浓液5mL/瓶，Mandels微量元素浓液0.05mL/瓶，1mol/L柠檬酸缓冲液2.5mL/瓶，Tween-802滴/瓶，以上溶液定容至50mL，倒入250mL的三角瓶，塞上棉花塞，盖上牛皮纸，用皮筋扎紧，于121℃，灭菌30min。The Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
其中CBP产葡糖二酸培养基包括：50g/L蒸汽爆破玉米秸秆，1g/L蛋白胨，1g/L酵母浸膏，6g/L(NH
4)
2SO
4，2.0g/L KH
2PO
4，0.3g/LMgSO
4，0.3g/L CaCl
2，0.005g/L FeSO
4，0.0016g/L MnSO4，0.0014g/LZnSO
4，0.0037g/L CoCl
2，柠檬酸缓冲液0.05mol/L(终浓度)，0.1g/L吐温80。灭菌条件：121℃、30min。
Among them, the CBP glucaric acid production medium includes: 50g/L steam-exploded corn stover, 1g/L peptone, 1g/L yeast extract, 6g/L (NH 4 ) 2 SO 4 , 2.0g/L KH 2 PO 4 , 0.3g/LMgSO 4 , 0.3g/L CaCl 2 , 0.005g/L FeSO 4 , 0.0016g/L MnSO4, 0.0014g/LZnSO 4 , 0.0037g/L CoCl 2 , citric acid buffer 0.05mol/L (final Concentration), 0.1g/L Tween 80. Sterilization conditions: 121°C, 30min.
实施例3Example 3
本实施例提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤：将里氏木霉C10在里氏木霉种子培养基中培养36h，酿酒酵母LGA-1C3S2在YPD培养基中培养到OD
600值5，将里氏木霉C10与酿酒酵母LGA-1C3S2以接种比1:1混合，得到混合菌群，将混合菌群按总接种量10％(v/v)同时接入CBP产葡糖二酸培养基中，于30℃、180rpm发酵，即完成加工。
This embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: culturing Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 hours, and culturing Saccharomyces cerevisiae LGA-1C3S2 in YPD medium Cultivate to an OD 600 value of 5, mix Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 at an inoculation ratio of 1:1 to obtain a mixed flora, and insert the mixed flora at the same time at a total inoculum of 10% (v/v) In the CBP glucaric acid production medium, ferment at 30°C and 180rpm, and the processing is completed.
其中里氏木霉培养基包括：葡萄糖10g/L，蛋白胨1g/L，Mandels营养盐浓液5mL/瓶，Mandels微量元素浓液0.05mL/瓶，1mol/L柠檬酸缓冲液2.5mL/瓶，Tween-802滴/瓶，以上溶液定容至50mL，倒入250mL的三角瓶，塞上棉花塞，盖上牛皮纸，用皮筋扎紧，于121℃，灭菌30min。The Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
其中CBP产葡糖二酸培养基包括：50g/L蒸汽爆破玉米秸秆，1g/L蛋白胨，1g/L酵母浸膏，6g/L(NH
4)
2SO
4，2.0g/L KH
2PO
4，0.3g/LMgSO
4，0.3g/L CaCl
2，0.005g/L FeSO
4，0.0016g/L MnSO4，0.0014g/LZnSO
4，0.0037g/L CoCl
2，柠檬酸缓冲液0.05mol/L(终浓度)，0.1g/L吐温80。灭菌条件：121℃、30min。
Among them, the CBP glucaric acid production medium includes: 50g/L steam-exploded corn stover, 1g/L peptone, 1g/L yeast extract, 6g/L (NH 4 ) 2 SO 4 , 2.0g/L KH 2 PO 4 , 0.3g/LMgSO 4 , 0.3g/L CaCl 2 , 0.005g/L FeSO 4 , 0.0016g/L MnSO4, 0.0014g/LZnSO 4 , 0.0037g/L CoCl 2 , citric acid buffer 0.05mol/L (final Concentration), 0.1g/L Tween 80. Sterilization conditions: 121°C, 30min.
实施例4Example 4
本实施例提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤：将里氏木霉C10在里氏木霉种子培养基中培养36h，酿酒酵母PC3在YPD培养基中培养到OD
600值5，将里氏木霉C10与酿酒酵母PC3以接种比2.5:1混合，得到混合菌群，将混合菌群按总接种量2％(v/v)同时接入CBP产SCP培养基中，于28℃、180rpm发酵，即完成加工。
The present embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: cultivating Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 h, cultivating Saccharomyces cerevisiae PC3 in YPD medium to The OD 600 value is 5, Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 are mixed at an inoculum ratio of 2.5:1 to obtain a mixed flora, and the mixed flora is simultaneously inserted into CBP to produce SCP at a total inoculum of 2% (v/v) for culture Fermentation at 28°C and 180rpm in the base, the processing is completed.
其中里氏木霉培养基包括：葡萄糖10g/L，蛋白胨1g/L，Mandels营养盐浓液5mL/瓶，Mandels微量元素浓液0.05mL/瓶，1mol/L柠檬酸缓冲液2.5mL/瓶，Tween-802滴/瓶，以 上溶液定容至50mL，倒入250mL的三角瓶，塞上棉花塞，盖上牛皮纸，用皮筋扎紧，于121℃，灭菌30min。The Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
其中CBP产SCP培养基组成如下：50g/L白酒糟，1g/L葡萄糖，5g/L(NH
4)
2SO
4，5g/L NH
4NO
3，4g/LKH
2PO
4，0.6g/L MgSO
4，0.6g/L CaCl
2，0.17mL/LMandels微量元素营养盐(Mandels等，1981)，柠檬酸缓冲液0.05mol/L(终浓度)，灭菌条件：121℃、30min。
The composition of the CBP SCP production medium is as follows: 50g/L distiller's grains, 1g/L glucose, 5g/L (NH 4 ) 2 SO 4 , 5g/L NH 4 NO 3 , 4g/L KH 2 PO 4 , 0.6g/L MgSO 4 , 0.6g/L CaCl 2 , 0.17mL/L Mandels trace element nutrient salt (Mandels et al., 1981), citric acid buffer solution 0.05mol/L (final concentration), sterilization conditions: 121°C, 30min.
实施例5Example 5
本实施例提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤：将里氏木霉C10在里氏木霉种子培养基中培养36h，酿酒酵母PC3在YPD培养基中培养到OD
600值5，将里氏木霉C10与酿酒酵母PC3以接种比3.5:1混合，得到混合菌群，将混合菌群按总接种量2％(v/v)同时接入CBP产SCP培养基中，于32℃、180rpm发酵，即完成加工。
The present embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: cultivating Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 h, cultivating Saccharomyces cerevisiae PC3 in YPD medium to The OD 600 value is 5, Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 are mixed at an inoculum ratio of 3.5:1 to obtain a mixed flora, and the mixed flora is simultaneously inserted into CBP to produce SCP at a total inoculum of 2% (v/v) for culture Fermentation at 32°C and 180rpm in the base, the processing is completed.
其中里氏木霉培养基包括：葡萄糖10g/L，蛋白胨1g/L，Mandels营养盐浓液5mL/瓶，Mandels微量元素浓液0.05mL/瓶，1mol/L柠檬酸缓冲液2.5mL/瓶，Tween-802滴/瓶，以上溶液定容至50mL，倒入250mL的三角瓶，塞上棉花塞，盖上牛皮纸，用皮筋扎紧，于121℃，灭菌30min。The Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
其中CBP产SCP培养基组成如下：50g/L白酒糟，1g/L葡萄糖，5g/L(NH
4)
2SO
4，5g/L NH
4NO
3，4g/LKH
2PO
4，0.6g/L MgSO
4，0.6g/L CaCl
2，0.17mL/LMandels微量元素营养盐(Mandels等，1981)，柠檬酸缓冲液0.05mol/L(终浓度)，灭菌条件：121℃、30min。
The composition of the CBP SCP production medium is as follows: 50g/L distiller's grains, 1g/L glucose, 5g/L (NH 4 ) 2 SO 4 , 5g/L NH 4 NO 3 , 4g/L KH 2 PO 4 , 0.6g/L MgSO 4 , 0.6g/L CaCl 2 , 0.17mL/L Mandels trace element nutrient salt (Mandels et al., 1981), citric acid buffer solution 0.05mol/L (final concentration), sterilization conditions: 121°C, 30min.
实施例6Example 6
本实施例提供了一种利用人工微生物提高整合生物加工效率的方法，包括如下步骤：将里氏木霉C10在里氏木霉种子培养基中培养36h，酿酒酵母PC3在YPD培养基中培养到OD
600值5，将里氏木霉C10与酿酒酵母PC3以接种比3:1混合，得到混合菌群，将混合菌群按总接种量2％(v/v)同时接入CBP产SCP培养基中，于30℃发酵，即完成加工。
The present embodiment provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing, comprising the steps of: cultivating Trichoderma reesei C10 in Trichoderma reesei seed medium for 36 h, cultivating Saccharomyces cerevisiae PC3 in YPD medium to The OD 600 value is 5, Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 are mixed at an inoculum ratio of 3:1 to obtain a mixed flora, and the mixed flora is simultaneously inserted into CBP to produce SCP at a total inoculum of 2% (v/v) for culture In the base, ferment at 30°C to complete the processing.
其中里氏木霉培养基包括：葡萄糖10g/L，蛋白胨1g/L，Mandels营养盐浓液5mL/瓶，Mandels微量元素浓液0.05mL/瓶，1mol/L柠檬酸缓冲液2.5mL/瓶，Tween-802滴/瓶，以上溶液定容至50mL，倒入250mL的三角瓶，塞上棉花塞，盖上牛皮纸，用皮筋扎紧，于121℃，灭菌30min。The Trichoderma reesei medium includes: glucose 10g/L, peptone 1g/L, Mandels nutrient salt concentrate 5mL/bottle, Mandels trace element concentrate 0.05mL/bottle, 1mol/L citric acid buffer 2.5mL/bottle, Tween-802 drops/bottle, the above solution was adjusted to 50mL, poured into a 250mL Erlenmeyer flask, plugged with a cotton plug, covered with kraft paper, tied tightly with a rubber band, and sterilized at 121°C for 30min.
其中CBP产SCP培养基组成如下：50g/L白酒糟，1g/L葡萄糖，5g/L(NH
4)
2SO
4，5g/L NH
4NO
3，4g/LKH
2PO
4，0.6g/L MgSO
4，0.6g/L CaCl
2，0.17mL/LMandels微量元素营养盐(Mandels等，1981)，柠檬酸缓冲液0.05mol/L(终浓度)，灭菌条件：121℃、30min。
The composition of the CBP SCP production medium is as follows: 50g/L distiller's grains, 1g/L glucose, 5g/L (NH 4 ) 2 SO 4 , 5g/L NH 4 NO 3 , 4g/L KH 2 PO 4 , 0.6g/L MgSO 4 , 0.6g/L CaCl 2 , 0.17mL/L Mandels trace element nutrient salt (Mandels et al., 1981), citric acid buffer solution 0.05mol/L (final concentration), sterilization conditions: 121°C, 30min.
效果例1Effect Example 1
使用里氏木霉C10+酿酒酵母LGA-1C3和里氏木霉Rut-C30+酿酒酵母LGA-1(里氏木霉Rut-C30购自ACCT)在相同条件下进行发酵，作为对比例1和对比例2，并检测实施例1-3、对比例1-2所得到的葡糖二酸的产量、产率和得率，其中实施例3的、对比例1和对比例2的检测结果如图2所示，根据图2结果所示里氏木霉Rut-C30+酿酒酵母LGA-1和实施例3中的葡糖二酸的产量、产率和得率均明显较里氏木霉Rut-C30+酿酒酵母LGA-1更高，且实施 例3的葡糖二酸的产量、产率和得率较里氏木霉Rut-C30+酿酒酵母LGA-1更高，因此，本发明提供的该方法能够有效提高整合生物加工效率。Trichoderma reesei C10+Saccharomyces cerevisiae LGA-1C3 and Trichoderma reesei Rut-C30+Saccharomyces cerevisiae LGA-1 (Trichoderma reesei Rut-C30 purchased from ACCT) were used to ferment under the same conditions as comparative example 1 and comparative example 2, and detect the output, productive rate and yield of the glucaric acid that embodiment 1-3, comparative example 1-2 obtain, wherein the detection result of embodiment 3, comparative example 1 and comparative example 2 is as Fig. 2 Shown, according to the output of Trichoderma reesei Rut-C30+Saccharomyces cerevisiae LGA-1 and embodiment 3 shown in Fig. Yeast LGA-1 is higher, and the output, yield and yield of glucaric acid of embodiment 3 are higher than Trichoderma reesei Rut-C30+Saccharomyces cerevisiae LGA-1, therefore, the method provided by the invention can effectively Improve integrated bioprocessing efficiency.
效果例2Effect example 2
使用里氏木霉Rut-C30+酿酒酵母INVSc1(里氏木霉Rut-C30和酿酒酵母INVSc1均购自ACCT)在相同条件下进行发酵，作为对比例3，并检测实施例4-6和对比例3所得到的SCP的产量、产率和得率，其中实施例6和对比例3中SCP的产量、产率和得率的检测结果如图3所示，根据图3结果所示，实施例6中SCP的产量、产率和得率均高于对比例3，因此，本发明提供的该方法能够有效提高整合生物加工效率。Use Trichoderma reesei Rut-C30+Saccharomyces cerevisiae INVSc1 (Trichoderma reesei Rut-C30 and Saccharomyces cerevisiae INVSc1 are all purchased from ACCT) to ferment under the same conditions as comparative example 3, and detect examples 4-6 and comparative examples 3 The output, yield and yield of the obtained SCP, wherein the detection results of the yield, yield and yield of SCP in Example 6 and Comparative Example 3 are as shown in Figure 3, according to the results shown in Figure 3, the embodiment The yield, yield and yield of SCP in 6 are higher than that of Comparative Example 3, therefore, the method provided by the present invention can effectively improve the efficiency of integrated bioprocessing.
综上所述，本发明提供了一种利用人工微生物提高整合生物加工效率的方法：In summary, the present invention provides a method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing:
该方法可以极大地促进人工微生物菌群CBP的效率。而且该法不仅可以用于提高人工微生物菌群一步法产葡糖二酸的效率，还具有普遍性，可用于其它由里氏木霉与酿酒酵母组成的微生物菌群的CBP效率，比如白酒糟产单细胞蛋白。采用分工协作推拉策略以后SCP产量明显提高，证明了分工协作推拉策略是一种有效的、普遍适用的提高人工微生物菌群整合生物加工效率的方法。This method can greatly promote the efficiency of artificial microbial flora CBP. Moreover, this method can not only be used to improve the efficiency of one-step production of glucaric acid by artificial microbial flora, but also has universality and can be used for the CBP efficiency of other microbial flora composed of Trichoderma reesei and Saccharomyces cerevisiae, such as distiller's grains Single-cell protein production. After adopting the push-pull strategy of division of labor and cooperation, the production of SCP was significantly improved, which proved that the push-pull strategy of division of labor and cooperation is an effective and universally applicable method to improve the efficiency of artificial microbial flora integration bioprocessing.
以上所描述的实施例是本发明一部分实施例，而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The embodiments described above are some, not all, embodiments of the present invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the claimed invention but to represent only selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.
Claims (10)
- 一种利用人工微生物提高整合生物加工效率的方法，其特征在于，包括如下步骤，将多种人工微生物混合后得到混合菌群，将所述混合菌群与原材料混合后发酵，即完成加工，所述人工微生物包括里氏木霉菌和酵母菌。A method for improving the efficiency of integrated bioprocessing by using artificial microorganisms, which is characterized in that it comprises the steps of mixing a variety of artificial microorganisms to obtain a mixed flora, mixing the mixed flora with raw materials and then fermenting to complete the processing. The artificial microorganisms include Trichoderma reesei and yeast.
- 根据权利要求1所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述原材料包括农业废料和工业废料。The method for improving integrated bioprocessing efficiency by using artificial microorganisms according to claim 1, wherein the raw materials include agricultural waste and industrial waste.
- 根据权利要求2所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述人工微生物包括里氏木霉C10和酿酒酵母PC3。The method for improving integrated bioprocessing efficiency by using artificial microorganisms according to claim 2, wherein the artificial microorganisms comprise Trichoderma reesei C10 and Saccharomyces cerevisiae PC3.
- 根据权利要求3所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述里氏木霉C10和酿酒酵母PC3的接种比为(2.5-3.5):1。The method for utilizing artificial microorganisms to improve integrated bioprocessing efficiency according to claim 3, wherein the inoculation ratio of Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 is (2.5-3.5):1.
- 根据权利要求4所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述原材料包括酒糟，所述里氏木霉C10和酿酒酵母PC3的混合菌群与所述酒糟的接种率为2-6％。The method for utilizing artificial microorganisms to improve integrated bioprocessing efficiency according to claim 4, wherein the raw materials include distiller's grains, the inoculum rate of the mixed flora of Trichoderma reesei C10 and Saccharomyces cerevisiae PC3 and the distiller's grains 2-6%.
- 根据权利要求2所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述人工微生物包括里氏木霉C10和酿酒酵母LGA-1C3S2。The method for improving integrated bioprocessing efficiency by using artificial microorganisms according to claim 2, wherein the artificial microorganisms include Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2.
- 根据权利要求6所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述里氏木霉C10和酿酒酵母LGA-1C3S2的接种比为1:1。The method for utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing according to claim 6, wherein the inoculation ratio of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 is 1:1.
- 根据权利要求6所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述原材料包括秸秆，所述里氏木霉C10和酿酒酵母LGA-1C3S2混合菌群与所述秸秆的接种率为8-12％。The method of utilizing artificial microorganisms to improve the efficiency of integrated bioprocessing according to claim 6, wherein the raw materials include stalks, and the inoculation of the mixed flora of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 with the stalks The rate is 8-12%.
- 根据权利要求1-8任意一项所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述发酵的温度为28-32℃。The method for improving integrated bioprocessing efficiency by using artificial microorganisms according to any one of claims 1-8, characterized in that the fermentation temperature is 28-32°C.
- 根据权利要求9所述的利用人工微生物提高整合生物加工效率的方法，其特征在于，所述发酵的时间至少为7天。The method for improving integrated bioprocessing efficiency by using artificial microorganisms according to claim 9, characterized in that the fermentation time is at least 7 days.
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