WO2023134104A1

WO2023134104A1 - Method for improving efficiency of integrated bioprocessing by means of using artificial microorganisms

Info

Publication number: WO2023134104A1
Application number: PCT/CN2022/095508
Authority: WO
Inventors: 方浩; 李朝风; 邓云涛
Original assignee: 浙江大学杭州国际科创中心
Priority date: 2022-01-14
Filing date: 2022-05-27
Publication date: 2023-07-20
Also published as: CN114410728A

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

A method for improving the efficiency of integrated bioprocessing using artificial microorganisms

technical field

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.

Background technique

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.

Contents of the invention

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. 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.

Compared with the prior art, 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. 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.

Description of drawings

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.

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;

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.

Detailed ways

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.

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.

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.

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.

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.

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.

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.

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. 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.

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.

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 ℃. 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.

The characteristics and performance of the present invention will be described in further detail below in conjunction with the examples.

Example 1

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.

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.

Example 2

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.

Example 3

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.

Example 4

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 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.

Example 5

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.

Example 6

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.

Effect Example 1

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.

Effect example 2

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.

In summary, 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.

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

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.
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.
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.
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.
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%.
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.
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.
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%.
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.
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.