A tobacco leaf culture medium and its preparation method and a method for directly fermenting and producing bio-based chemicals

Abstract

A tobacco leaf culture medium and its preparation method as well as a method for directly fermenting and producing bio-based chemicals belong to the technical field of biomass resource utilization. The biomass tobacco leaves of the present invention can be directly utilized, mainly using tobacco leaves and water as raw materials, without adding or adding no more than 10% of other biomass, and sterilizing the culture medium to be directly fermented and utilized. The raw materials of the present invention may include only tobacco leaves and water, or may include adding no more than 10% of other biomass, tobacco leaves and water, and directly obtaining the fermentation culture medium after sterilization. The culture medium has low production cost and simple production process. The present invention not only simplifies the steps of using traditional biomass as fermentation culture medium to produce bio-based chemicals, but also greatly reduces the production cost and dependence on petroleum resources. The method of the present invention provides a brand-new way to utilize tobacco leaves, which not only has broad application prospects in the field of biosynthesis, but also has great development potential in replacing animal-derived proteins.

Description

Technical Field

[0001] The invention belongs to the technical field of biomass energy utilization, and specifically relates to a tobacco leaf culture medium and its preparation, as well as a method for directly fermenting and producing bio-based chemicals. Background Art

[0002] Microbial synthesis of bio-based chemicals and biofuels has the advantages of being natural, environmentally friendly, and having a short cycle. However, due to the high price of carbon and nitrogen sources in the culture medium, the cost remains high, and the industrialization process is hindered. Although biomass resources provide a large amount of fermentable sugars for microbial synthesis of bio-based chemicals, alleviating dependence on petrochemical resources and reducing environmental pollution, the process is inefficient and costly due to the complex structure of lignocellulose in biomass, the high cost of enzymes used in the process, and the cumbersome application steps, which limits its large-scale production and application process. The current research focus is mainly on reducing the overall production cost. On the one hand, a large number of studies focus on understanding the structure of different lignocelluloses and designing appropriate pretreatment methods, so as to improve the efficiency of lignocellulose use and reduce costs; on the other hand, different technologies are used to reduce the cost of enzymes; in addition, there are also studies focusing on genetically modified energy plants and breeding low-lignin or high-cellulose plants. Although these methods can reduce the utilization cost of lignocellulose to a certain extent, the industrial application of this process is still not possible and needs further improvement. Summary of the invention

[0003] The purpose of the present invention is to provide a more economical and environmentally friendly new method and new material for promoting industrial fermentation to produce biofuels and bio-based chemicals. The method of the present invention can directly use the solution of tobacco leaves after water sterilization as a fermentation medium to produce bio-based chemicals, providing a new idea for the utilization of biomass and the transformation and utilization of tobacco resources. It also plays a positive role in maximizing resource utilization efficiency, reducing environmental pollution and alleviating environmental pressure. The method of the present invention is simple to operate, energy-saving and emission-reducing, and has great development potential. The method of the present invention uses tobacco leaves as raw materials, and the treated liquid obtained after one-step treatment is directly used as a microbial fermentation medium, which can not only improve the shortcomings of the application process of lignocellulosic biomass due to its complex lignocellulosic structure, the high cost of the enzyme used in the process, and the cumbersome process steps, but also has important significance for the development and utilization of biomass resources, and has guiding significance for the transformation and utilization of tobacco resources. In addition, the plant-derived nitrogen source in tobacco leaves is used to replace animal-derived protein, thereby avoiding the potential risks of viral contamination, unclear composition and disadvantageous product purification brought by animal-derived protein found in recent years during use, and ensuring the safety of subsequent products.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The tobacco leaf culture medium is mainly made of tobacco leaves and water as raw materials, and the supernatant after sterilization, wherein the mass ratio of solid to water is 1:(12.5-5); specifically, it is completed by the following steps:

[0006] Step 1: Put the tobacco leaves into an oven and dry them at a temperature not higher than 40°C until they can be crushed with fingers. Take out the dried tobacco from the oven and grind it immediately. The grinding time should not exceed 2 minutes. Put the ground tobacco into a clean, dry, light-proof container and seal it. Shake it thoroughly and mix it to obtain tobacco leaf powder.

[0007] Step 2: Add pure water to the tobacco leaf powder obtained in step 1 at a mass ratio of solid to water of 1: (12.5-5), sterilize directly, and after centrifugation, take the supernatant to obtain the tobacco leaf culture medium.

[0008] The tobacco leaf culture medium can also be a whole solution of tobacco leaves and water as raw materials, sterilized, and then treated with enzymes; specifically, it is completed by the following steps:

[0009] Step 1: Put the tobacco leaves into an oven and dry them at a temperature not higher than 40°C until they can be crushed with fingers. Take out the dried tobacco from the oven and grind it immediately. The grinding time should not exceed 2 minutes. Put the ground tobacco into a clean, dry, light-proof container and seal it. Shake it thoroughly and mix it to obtain tobacco leaf powder.

[0010] Step 2: Add pure water to the tobacco powder obtained in step 1 at a solid to water mass ratio of 1: (12.5-5), sterilize directly, then add cellulase, xylanase and β-glucose enzyme for treatment, collect the solution, and obtain tobacco culture medium.

[0011] It is further defined that the raw material includes one or a combination of several of straw, corn cobs, corn stalks, rice straw, switchgrass, miscanthus, and sawdust, but is not limited to these; the preparation step is different from the above method in that: one or a combination of several of straw, corn cobs, corn stalks, rice straw, switchgrass, miscanthus, and sawdust is processed according to the operation of step one to obtain powder A; in step two, pure water is first added to the tobacco powder A obtained in step one

[0012] It is further defined that the tobacco leaves are flue-cured tobacco leaves and / or air-dried tobacco leaves.

[0013] It is further defined that the raw material also includes acid water, alkaline water, organic solvent or ionic liquid.

[0014] It is further defined that the acid water is one of hydrochloric acid, acetic acid, sulfuric acid, nitric acid, and phosphoric acid, but is not limited thereto.

[0015] It is further defined that the alkaline water is one of sodium hydroxide, calcium hydroxide, ammonia water, and potassium hydroxide, but is not limited thereto.

[0016] In a further aspect, the organic solvent is ethanol or methanol, but is not limited thereto.

[0017] The method for producing bio-based chemicals by fermenting tobacco leaf culture medium is characterized in that the method for producing bio-based chemicals is as follows: microorganisms are added to the above-mentioned tobacco leaf culture medium or the tobacco leaf culture medium prepared by the above-mentioned method to carry out liquid fermentation.

[0018] Tobacco leaves are the raw materials of the tobacco industry and are widely planted. Compared with traditional biomass, tobacco leaves have the advantages of high soluble component content, high sugar content, high nitrogen content, and low lignin content, so tobacco leaves are feasible for direct use. The treated liquid obtained after a one-step treatment of tobacco leaves can be directly used as a microbial fermentation medium, which can not only improve the shortcomings of the lignocellulosic biomass application process due to its complex lignocellulosic structure, high enzyme cost used in the process, and cumbersome process steps, but also has important significance for the development and utilization of biomass resources, and has guiding significance for the transformation and utilization of tobacco resources. In addition, tobacco leaves can be used as a plant-derived nitrogen source due to its high nitrogen content to replace animal-derived proteins, thereby avoiding the potential risks of viral contamination, unclear composition, and inconvenience for product purification brought by animal-derived proteins found in recent years, and ensuring the safety of subsequent products.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The present invention provides new materials for industrial fermentation production of biofuels and bio-based chemicals. The bio-based chemicals of the present invention can be directly fermented in liquid form to produce bio-based chemicals and biofuels. The bio-based chemicals of the present invention have high water solubility, high soluble sugar content, high nitrogen content, and high utilization efficiency.

[0021] 2) The raw materials of the present invention only include tobacco leaves and water, which are directly obtained after sterilization treatment, with low cost and simple process. The present invention not only simplifies the steps of using traditional biomass as fermentation medium to produce bio-based chemicals, but also greatly reduces production costs and dependence on petroleum resources.

[0022] 3) The present invention provides new materials for industrial fermentation production of biofuels and bio-based chemicals. The bio-based chemicals of the present invention have a wide planting range, rapid growth, large biomass, simple field management and low acquisition cost.

[0023] 4) The present invention provides new materials for industrial fermentation production of biofuels and bio-based chemicals. The bio-based chemicals of the present invention can be used to replace animal-derived nitrogen sources, thereby reducing the risks of potential viral contamination, unclear ingredients, and disadvantages in product purification during the use of animal-derived proteins, and ensuring the safety of subsequent products. The method of the present invention provides a new way to utilize tobacco leaves, which not only has broad application prospects in the field of biosynthesis, but also has great development potential in replacing animal-derived proteins.

[0024] 5) The present invention provides a new method for industrial fermentation to produce biofuels and bio-based chemicals that is more economical and environmentally friendly. The processing method is simple to operate, saves energy and reduces emissions, and has great development potential.

[0025] 6) The present invention provides a method for producing bio-based chemicals by direct liquid fermentation of tobacco biomass, which can produce bio-based fuels and bio-based chemicals in a green manner and has good economy and sustainability. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Mass spectrum of farnesene obtained by fermentation in Example 3;

[0027] Figure 2 Mass spectrum of 2,3-butanediol obtained for Example 7 fermentation. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with embodiments.

[0029] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or the product instructions are used. If the manufacturer of the materials or instruments used is not specified, they are all conventional products that can be purchased.

[0030] Example 1: The bio-based chemicals described in this example are made from tobacco leaves and water as raw materials, without adding other biomass, and the supernatant after sterilization treatment; the specific preparation method is completed by the following steps:

[0031] Step 1: Prepare tobacco powder

[0032] 1. Sample Collection

[0033] The tobacco leaf samples were collected by random sampling, and mainly the tobacco leaf products after drying were sampled.

[0034] 2. Sample Preparation

[0035] Put the collected and dried tobacco leaves into an oven and dry them at a temperature not higher than 40°C until they can be crushed with fingers. Then take out the dried tobacco from the oven and grind it immediately. The grinding time should not exceed 2 minutes. Then put the ground tobacco powder into a clean and dry brown wide-mouth bottle and seal it. Shake it thoroughly and mix it evenly. Then get the tobacco powder. This is the prepared sample.

[0036] Step 2: Take 4.00 g of the prepared sample, add 50 mL of pure water, sterilize directly at 115°C for 15 min, centrifuge, and store the supernatant in a refrigerator for later use.

[0037] The following test is used to verify the effect of the invention:

[0038] 1. Solubility characterization:

[0039] Weigh 4.00 g of tobacco powder obtained in step 1, add 50 mL of pure water and sterilize in a high-temperature and high-pressure sterilizer. Filter the treated sample while hot, store the filtrate at -20°C for later use, wash the filter residue three times with 100 mL of pure water, and dry it at 42°C to constant weight. The results show that the soluble matter in tobacco leaves can reach 62.23±0.34%.

[0040] 2. Determination of total sugar content

[0041] 0.1g of tobacco powder obtained in step 1 was added to 20mL of pure water, and after ultrasonic treatment for 30min, the supernatant was centrifuged and the total sugar was measured by sulfuric acid phenol method. The final results showed that the total sugar content in the tobacco sample could reach 24.04±0.16%.

[0042] 3. Determination of total nitrogen content

[0043] Accurately weigh 0.2500g of the bio-based chemical obtained in step 2, add it to the digestion tube, and add copper sulfate, potassium sulfate and concentrated sulfuric acid to carry out digestion reaction, digestion at 420℃ for 1h. After digestion and cooling, nitrogen determination is carried out using a Kjeldahl nitrogen analyzer, and finally the liquid in the receiving bottle is titrated with 0.5M hydrochloric acid, and the total nitrogen content in the tobacco leaf is finally calculated based on the consumption of hydrochloric acid. The results show that the total nitrogen content in the tobacco leaf sample is 3.01±0.43%.

[0044] Example 2: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0045] 50 mL of tobacco leaf culture medium obtained by the method of Example 1 was directly used as a fermentation medium, and the specific steps for producing farnesene were as follows:

[0046] The farnesene-producing genetically engineered Escherichia coli was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0047] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0048] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 209.78±1.24 mg / L.

[0049] The construction method of the farnesene-producing genetically engineered Escherichia coli used in Example 2 (CN111607546A, publication date 20200901).

[0050] 1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: After the gene sequence of β-farnesene synthase AaFS from Artemisia annua was optimized for codon preference in Escherichia coli, restriction endonuclease BglII and XhoI were added at both ends, and the sequence was synthesized by BGI and cloned into the pUC57-simple vector to obtain plasmid pUC57-AaFS. The construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS was carried out by restriction endonuclease-ligation method. First, the plasmid pACYC-mvaE-mvaS-ispA-Sab1 and pUC57-AaFS were double-digested with restriction endonucleases BglII and XhoI, respectively, and the enzyme digestion system was as follows:

[0051]

[0052]

[0053] The products after enzyme digestion were subjected to agarose gel electrophoresis and the target bands were cut and recovered. Among them, pACYC-mvaE-mvaS-ispA-Sab1 was double-digested with BglII and XhoI to recover an 8260bp fragment as a vector; pUC57-AaFS was double-digested with BglII and XhoI to recover a 1725bp fragment as an insert fragment. The recovered products were subjected to ligation reaction:

[0054]

[0055] 10 μL of the ligation product was heat-shocked and transformed into DH5α competent cells and coated on LBCm plates, and cultured overnight at 37°C. The next day, the colonies on the plate were observed, and a single colony was picked into liquid culture medium, cultured at 37°C until it was relatively dense, and then the colony was identified by PCR or restriction digestion of the extracted plasmid, and sent for sequencing to obtain the plasmid pACYC-mvaE-mvaS-ispA-AaFS.

[0056] 2) Construction of plasmid pTrcLower-ΔIDI: To investigate the catalytic effect of heterologous IDI when all gene copies are 1, single-copy gene recombinant bacteria were prepared, and the yeast ScIDI originally contained in the plasmid pTrcLower was removed. The construction of plasmid pTrcLower-ΔIDI adopted the Gibson Assembly method, and the pTrcLower plasmid was used as a template to amplify the vector part without ScIDI, namely pTrc-ERG12-ERG8-ERG19. The PCR amplification system is as follows:

[0057]

[0058] The PCR product was subjected to agarose gel electrophoresis, and the pTrc-ERG12-ERG8-ERG19 vector fragment of about 8352 bp was recovered by gel cutting. The NEBuilder kit was used for self-ligation reaction. The ratio of the fragments and the amount of each component were calculated according to the instructions. The ligation reaction was carried out at 50°C for 60 minutes. The product was diluted with an equal volume of sterile water, and 5 μL was taken to heat-shock transform DH5α competent cells and coated on LBAmp plates, and cultured at 37°C overnight. The colonies on the plate were observed the next day, and a single colony was picked into a liquid culture medium, cultured at 37°C until it was relatively concentrated, and the colony PCR identification or the extracted plasmid enzyme digestion identification was performed, and it was sent for sequencing to obtain the plasmid pTrcLower-ΔIDI.

[0059] 3) Construction of plasmid pTrcLower-AaIDI: The gene sequence of isopentenyl diphosphate isomerase AaIDI from Artemisia annua was optimized for codon preference in E. coli, synthesized by BGI, and cloned into pUC57-simple vector to obtain plasmid pUC57-AaIDI. The construction of plasmid pTrcLower-AaIDI was carried out by restriction digestion-ligation method. First, using plasmid pUC57-AaIDI and pTrcLower as templates, primers with SacI and PstI were used to amplify the AaIDI gene fragment and pTrc-ERG12-ERG8-ERG19 vector fragment respectively:

[0060]

[0061] The PCR products were subjected to agarose gel electrophoresis, and the AaIDI gene fragment of about 708 bp and the pTrc-ERG12-ERG8-ERG19 vector fragment of 8412 bp were recovered by gel electrophoresis, and double-digested with SacI and PstI:

[0062]

[0063] The products after enzyme digestion were subjected to agarose gel electrophoresis, and the AaIDI gene fragment of about 708 bp and the pTrc-ERG12-ERG8-ERG19 vector fragment of 8412 bp were recovered from the gel, and the recovered products were subjected to ligation reaction:

[0064]

[0065] 10 μL of the ligation product was heat-shocked and transformed into DH5α competent cells and coated on LB Amp plates, and cultured overnight at 37°C. The next day, the colonies on the plate were observed, and a single colony was picked into liquid culture medium, cultured at 37°C until it was relatively dense, and then the colony was identified by PCR or restriction digestion of the extracted plasmid, and sent for sequencing to obtain the plasmid pTrc-ERG12-ERG8-ERG19-AaIDI, i.e., pTrcLower-AaIDI.

[0066] 4) Construction of plasmid pET28a-AaFS-ispA-IDI:

[0067] The isopentenyl diphosphate isomerase AaIDI gene from Artemisia annua was optimized for Escherichia coli codon preference, sequenced by BGI, and cloned into the pUC57-simple vector to obtain the plasmid pUC57-AaIDI; the isopentenyl diphosphate isomerase SlIDI gene from tomato was optimized for Escherichia coli codon preference, sequenced by BGI, and cloned into the pUC57-simple vector to obtain the plasmid pUC57-SlIDI.

[0068] The construction of plasmid pET28a-AaFS-ispA-IDI adopts Gibson Assembly method. First, pET28a vector and three gene fragments of AaFS, ispA and IDI (from Artemisia annua and tomato, respectively) are amplified, and pET28a plasmid, pUC57-AaFS plasmid, Escherichia coli BL21 (DE3) bacterial solution, pUC57-AaIDI plasmid and pUC57-SlIDI plasmid are used as templates respectively:

[0069]

[0070] The PCR products were subjected to agarose gel electrophoresis and the target bands were cut and recovered. The concentration of the recovered products was determined. The NEBuilder kit was used for Gibson Assembly. The ratio of the fragments and the amount of each component were calculated according to the instructions. The ligation reaction was carried out at 50°C for 60 minutes. The product was diluted with an equal volume of sterile water. 5 μL was heat-shocked to transform DH5α competent cells and coated on LBKan plates, and cultured at 37°C overnight. The colonies on the plate were observed the next day, and a single colony was picked into a liquid culture medium, cultured at 37°C until it was relatively concentrated, and the colony PCR identification or the extracted plasmid enzyme digestion identification was performed, and it was sent for sequencing to obtain plasmids pET28a-AaFS-ispA-AaIDI and plasmids pET28a-AaFS-ispA-SlIDI, respectively.

[0071] 5) Plasmid transformation: The correctly sequenced plasmid pACYC-mvaE-mvaS, the pTrcLower-ΔIDI prepared in step 2), and the pET28a-AaFS-ispA-SlIDI / AaIDI prepared in step 4 (single copy), or the pACYC-mvaE-mvaS-ispA-AaFS prepared in step 1), the pTrcLower-AaIDI prepared in step 3), and the pET28a-AaFS-ispA-AaIDI prepared in step 4 (double copy) were combined and transformed into E. coli BL21 (DE3) competent cells were coated with corresponding three-antibody (Cm, Amp and Kan) LB medium plates, wherein the final concentration of Cm in the LB medium was 34 mg / L, the final concentration of Amp in the LB medium was 100 mg / L, and the final concentration of Kan in the LB medium was 50 mg / L, and cultured at 37°C until a single colony grew out, thereby obtaining a genetically engineered bacterium that synthesizes farnesene.

[0072] Genetically engineered bacteria containing the AaIDI gene from Artemisia annua (single copy): a recombinant bacteria overexpressing the acetyl-CoA acyltransferase / HMG-CoA reductase mvaE gene, the HMG-CoA synthetase mvaS gene, the mevalonate-5-phosphate kinase ERG8 gene, the mevalonate kinase ERG12 gene, the mevalonate-5-diphosphate decarboxylase ERG19 gene, the isopentenyl diphosphate isomerase AaIDI gene from Artemisia annua, the farnesyl diphosphate synthase ispA gene and the β-farnesene synthase AaFS gene, wherein the copy number of the IDI gene, ispA gene and AaFS gene on the plasmid is 1.

[0073] In this example, the farnesene image is obtained as shown in Figure 1 As shown, it can be seen from the figure that the fermentation product is farnesene.

[0074] Example 3: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0075] The fermentation medium of this example has a total volume of 50 mL: 9.8 g / L K2HPO4; 1 mL / L trace elements; 0.06 g / L MgSO4; and the supernatant obtained by the method of Example 1 is used to make up to 50 mL. The specific steps for producing farnesene are as follows:

[0076] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0077] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0078] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 234.00±0.17 mg / L.

[0079] Example 4: Production of farnesene by direct liquid fermentation of tobacco leaves using Saccharomyces cerevisiae

[0080] The fermentation medium of this example has a total volume of 50 mL: 15 g / L urea; 8 g / L KH2PO4; 6 g / LMgSO4, and the supernatant obtained by the method of Example 1 is used to make up to 50 mL. The specific steps for producing farnesene are as follows:

[0081] The farnesene-producing yeast was inoculated into 5 mL of YPD liquid medium to prepare the primary seed solution, and 5 mL of it was inoculated into 50 mL of YPD fermentation medium and cultured at 30 °C until OD 600 Equal to 5 as the secondary seed liquid, inoculated into the fermentation medium, and cultured at 30°C for 48 hours. After the fermentation is completed, 20% n-dodecane is added, and after extraction, 1 mL of the solvent layer is filtered and subjected to gas chromatography detection. The detection conditions are:

[0082] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0083] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 94.55±2.62 mg / L.

[0084] Construction of genetically engineered bacteria for farnesene-producing yeast in this example (application publication number CN111378588A, publication date 20200707):

[0085] The starting strain of the target genetically engineered bacteria is yeast, which integrates the farnesene synthase gene AaFS, the phosphate acetyltransferase gene CkPTA, the phosphoketolase gene LmPK, the acetaldehyde dehydrogenase gene DzeutE, and the hydroxymethylglutaryl-CoA reductase gene RpHMGR.

[0086] The engineering strain uses yeast strain CEN.PK2-1C (MATa; ura3-52; trp1-289; leu2-3_112; his3Δ1; MAL2-8c; SUC2) as the starting strain, and the simple construction process is as follows:

[0087] (1) The genes described above were obtained by PCR, wherein the farnesene synthase gene AaFS was derived from Artemisia annua L., with a sequence accession number of GenBank: AY835398.1; the phosphate acetyltransferase gene CkPTA was derived from Clostridium kluyveri, with a sequence accession number of GenBank: CP018335.1; the phosphoketolase gene LmPK was derived from Leuconostoc mesenteroides, with a sequence accession number of GenBank: AY804190.1; the acetaldehyde dehydrogenase gene DzeutE was derived from Dickeyazeae, with a sequence accession number of GenBank: CP006929.1; and the hydroxymethylglutaryl-CoA reductase gene RpHMGR was derived from Ruegeria pomeroyi, with a sequence accession number of NCBI Reference Sequence: NC_006569.1.

[0088] (2) Construction of integration fragments: PGAL1-AaFS-TCYC1, PGAL1-LmPK-TCYC1_PGAL10-CkPTA-TADH1_HIS,

[0089] PGAL1-AaFS-TCYC1_PGAL10-DzeutE-TADH1_URA3, PGAL1-AaFS-TCYC1_PGAL10-RpHMGR-TADH1_LEU2

[0090] P(OC)GAL4-GAL4_PGAL1-tHMG1_KanMX. The above fragments were integrated into the GAL80, ALD4, ALD6, ADH5, and RHR2 sites, respectively. PGAL1 and _PGAL10 are GAL1 and GAL10 promoters, respectively. TCYC1 and TADH1 are terminators.

[0091] (3) The obtained integration vector is transformed into the starting strain to obtain the target transgenic engineering strain.

[0092] The engineering strain uses yeast strain CEN.PK2-1C (MATa; ura3-52; trp1-289; leu2-3_112; his3Δ1; MAL2-8c; SUC2) as the starting strain, and the construction process is as follows:

[0093] Specific process of constructing yeast engineering strain containing farnesene synthase gene AaFS:

[0094] (1) The farnesene synthase gene AaFS was inserted into the GAL80 site of the yeast genome. The farnesene synthase gene was derived from Artemisia annua L., the sequence accession number of which was GenBank: AY835398.1, and the gene was codon optimized. AaFS was cut from the synthesized plasmid by BamHI and SalI, and connected to the pESC-HIS plasmid to obtain the recombinant plasmid pESC-HIS-AaFS. The PGAL1-AaFS-T CYC1 fragment was obtained by PCR, where PGAL1 was the GAL1 promoter and TCYC1 was the CYC1 terminator, and it was connected to the pUC19 plasmid using restriction endonucleases NdeI and PstI. Then, the upstream (GAL80US) 511bp of GAL80 and the downstream (GAL80DS) 501bp of GAL80 were sequentially connected to the two ends of P GAL1-AaFS-T CYC1 through NdeI and PstI to obtain the recombinant plasmid pUC19-GAL80US-P GAL1-AaFS-T CYC1-GAL80DS. The donor fragment was obtained by NdeI and PstI digestion: GAL80US-P GAL1-AaFS-T CYC1-GAL80DS.

[0095] (2) The sequence TAAGGCTGCTGCTGAACGT was used as the target sequence to construct the gRNA expression fragment. The 19bp target sequence was introduced when designing the primers, and the linearized pML104 was used as the template to PCR amplify the 500bp DNA sequences at both ends. Both upstream and downstream fragments contained the 19bp GAL80 target sequence. The two fragments were then fused together by overlapping PCR to obtain a complete gRNA expression fragment, and its 19bp target sequence was located at the center of the gRNA expression fragment. The plasmid pML104 was double-digested with restriction endonucleases SwaI and BclI to obtain a linearized plasmid; the above donor DNA fragment, linearized pML104 plasmid, and gRNA expression fragment were simultaneously transferred into the yeast competent cell CEN.PK2-1C by chemical transformation to obtain the yeast genetic engineering bacteria FS1.

[0096] Construction of yeast engineered strain containing phosphoketolase gene LmPK and phosphate acetyltransferase gene CkPTA.

[0097] (1) Based on the FS1 strain, gene integration was performed by homologous recombination. The phosphate acetyltransferase gene CkPTA and the phosphoketolase gene LmPK were inserted into the ALD6 site of the yeast genome. The phosphate acetyltransferase gene CkPTA was derived from Clostridium kluyveri, and the sequence accession number was GenBank: CP018335.1; the phosphoketolase gene LmPK was derived from Leuconostoc mesenteroides, and the sequence accession number was GenBank: AY804190.1. The CkPTA gene was linked to the pESC-URA plasmid through EcoRI and SacI to obtain the recombinant plasmid pESC-URA-CkPTA. The LmPK gene was linked to the pESC-URA-CkPTA plasmid through BamHI and SalI to obtain the recombinant plasmid pESC-URA-LmPK-CkPTA. The P GAL1-LmPK-T CYC1_P GAL10-CkPTA-T ADH1 fragment was obtained by PCR.

[0098] (2) The upstream and downstream fragments of ALD6 gene, ALD6US and ALD6DS, were obtained by PCR, and the HIS expression fragment was obtained by PCR using the pESC-HIS plasmid as a template. The pUC19 plasmid was linearized by restriction endonucleases Sma I and Pst I, and then P GAL1 -LmPK-TCYC1_P GAL10 -CkPTA-TADH1, ALD6US, ALD6DS, and HIS were connected into the pUC19 plasmid using the Hieff Plus Multi OneStep Cloning Kit (10912ES10) to obtain the recombinant plasmid pUC19-ALD6US-P GAL1 -LmPK-TCYC1_P GAL10 -CkPTA-TADH1 -HIS-ALD6DS. The donor fragment was obtained by digesting the recombinant plasmid with SmaI and SphI: ALD6US-P GAL1 -LmPK-T CYC1_P GAL10 -CkPTA-T ADH1 -HIS-ALD6DS. The donor fragment was transferred into yeast strain FS1 by chemical transformation to obtain yeast strain FS2.

[0099] Construction of yeast engineered strain containing acetaldehyde dehydrogenase gene DzeutE.

[0100] (1) Gene integration was performed on the basis of FS2 strain by homologous recombination. The acetaldehyde dehydrogenase gene DzeutE and the farnesene synthase gene AaFS were inserted into the ALD4 site of the yeast genome. The acetaldehyde dehydrogenase gene DzeutE was derived from Dickeya zeae, and the sequence accession number was GenBank: CP006929.1.

[0101] The DzeutE gene was connected into the pESC-HIS-AaFS plasmid via NotI and PacI to obtain the recombinant plasmid pESC-HIS-AaFS-DzeutE. The PGAL1-AaFS-T CYC1_P GAL10-DzeutE-T ADH1 fragment was obtained by PCR.

[0102] (2) The upstream and downstream fragments of ALD4 gene, ALD4US and ALD4DS, were obtained by PCR. The URA expression fragment was obtained by PCR using the pESC-URA plasmid as a template. The four fragments, P GAL1-AaFS-T CYC1_P GAL10-DzeutE-T ADH1, ALD4US, ALD4DS, and URA, were transferred into the yeast strain FS2 by chemical transformation to obtain the yeast strain FS3.

[0103] Construction of an engineered yeast strain containing the hydroxymethylglutaryl-CoA reductase gene RpHMGR.

[0104] (1) Based on the FS3 strain, gene integration was performed by homologous recombination. The hydroxymethylglutaryl-CoA reductase gene RpHMGR and the farnesene synthase gene AaFS were inserted into the ADH5 site of the yeast genome. The hydroxymethylglutaryl-CoA reductase gene RpHMGR is derived from Ruegeria pomeroyi, and the sequence registration number is NCBI Reference Sequence: NC_006569.1. The RpHMGR described in the present invention is an optimized sequence.

[0105] The RpHMGR gene was obtained by PCR and linked into the pESC-HIS-AaFS plasmid via SpeI and SacI to obtain the recombinant plasmid pESC-HIS-AaFS-RpHMGR. The P GAL1-AaFS-T CYC1_P GAL10-RpHMGR-T ADH1 fragment was obtained by PCR.

[0106] (2) The upstream and downstream fragments of the ADH5 gene, ADH5US and ADH5DS, were obtained by PCR. The LEU expression fragment was obtained by PCR using the pESC-LEU plasmid as a template. The four fragments, P GAL1-AaFS-T CYC1_P GAL10-RpHMGR-T ADH1, ADH5US, ADH5DS, and LEU, were transferred into the yeast strain FS3 by chemical transformation to obtain the yeast strain FS4.

[0107] Construction of yeast engineering bacteria overexpressing yeast GAL4 gene and tHMG1 gene.

[0108] (1) Based on the FS4 strain, gene integration was performed by homologous recombination. The GAL4 gene is derived from Saccharomyces cerevisiae, and the sequence registration number is NCBI Reference Sequence: NC_001148.4. The GAL4 gene contains a 251 bp downstream sequence after the stop codon. The promoter of the GAL4 gene is an optimized GAL4 promoter. The tHMG1 gene is derived from Saccharomyces cerevisiae, and the sequence registration number is NCBI Reference Sequence: NC_001145.3. The tHMG1 gene is a truncation lacking 529 amino acids at the N-terminus, and contains a 225 bp sequence after the stop codon, and the promoter is the PGAL1 promoter. The GAL4 gene and tHMG1 are inserted into the RHR2 site of the yeast genome.

[0109] (2) The GAL4 expression fragment (including the promoter, coding gene and terminator sequence) was divided into three fragments A, B and C, which were amplified by PCR. The tHMG1 expression fragment (including the promoter, coding gene and terminator sequence) was divided into two fragments D and E, which were amplified by PCR. The selection marker gene KanMX with loxP site was obtained by PCR using plasmid pUC6 as a template. The upstream and downstream fragments RHR2US and RHR2DS of the RHR2 gene were obtained by PCR.

[0110] (3) Overlap extension PCR was used to fuse RHR2US and A, C and D, E and KanMX, and KanMX and RHR2-DS into one fragment, respectively. Then, the fused RHR2US+A and B were fused into one fragment. These four fragments were simultaneously transferred into yeast strain FS4 by chemical transformation to obtain yeast strain FS5.

[0111] Construction of yeast engineered bacteria that downregulate the expression of yeast ERG9 gene.

[0112] (1) Based on the FS5 strain, gene integration was performed by homologous recombination. The ERG9 gene is derived from Saccharomyces cerevisiae, and the sequence registration number is NCBI Reference Sequence: NC_001140.6. The promoter of the down-regulating ERG9 gene expression fragment (including the promoter, the coding gene and the downstream 537bp sequence) is the MET3 promoter.

[0113] (2) Using the yeast genome as a template, the upstream and downstream fragments of the ERG9 gene, ERG9US and ERG9+ERG9DS, were obtained by PCR. Using the yeast genome as a template, the MET3 promoter P MET3 was obtained by PCR. Using the plasmid pESC-TRP as a template, the TRP expression fragment was obtained by PCR.

[0114] (3) The fragments ERG9US and TRP were fused into one fragment by Overlap extension PCR, and the three fragments ERG9US+TRP, P MET3 and ERG9 were simultaneously transferred into the yeast strain FS5 by chemical transformation to obtain the yeast strain FS6, which is the genetically engineered bacteria used in the present invention for converting cellulose hydrolyzate to synthesize farnesene.

[0115] Example 5: Production of farnesene by direct liquid fermentation of tobacco leaves using Saccharomyces cerevisiae

[0116] 50 mL of the supernatant obtained by the method of Example 2 was directly used as a fermentation medium. The specific steps for producing farnesene are as follows:

[0117] Farnesene-producing yeast (same as in Example 4) was inoculated into 5 mL YPD liquid medium to prepare primary seed solution, and 5 mL was inoculated into 50 mL YPD fermentation medium and cultured at 30°C until OD 600 Equal to 5 as the secondary seed liquid, inoculated into the fermentation medium, and cultured at 30°C for 48 hours. After the fermentation is completed, 20% n-dodecane is added, and after extraction, 1 mL of the solvent layer is filtered and subjected to gas chromatography detection. The detection conditions are:

[0118] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0119] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 95.67±0.87 mg / L.

[0120] Example 6: Production of 2,3-butanediol by direct liquid fermentation of tobacco leaves using Bacillus amyloliquefaciens

[0121] 50 mL of the supernatant obtained by the method of Example 2 was directly used as a fermentation medium. The specific steps for producing 2,3-butanediol are as follows:

[0122] Bacillus amyloliquefaciens was inoculated in 5mL LB liquid medium to prepare the primary seed liquid, and then inoculated in a 250mL conical flask containing 100mL seed medium with a 5% inoculation amount for secondary subculture, and then inoculated in a fermentation medium (250mL conical flask containing 100mL fermentation medium) with a 5% inoculation amount at 37°C and 200rpm for 9h, and cultured at 37°C and 200rpm for 84h to obtain the fermentation liquid for gas phase detection. The above fermentation liquid was centrifuged at 12000r / min for 10min, the precipitate was discarded, and the supernatant of the fermentation liquid was extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract, which was filtered with a 0.22μm organic phase membrane and quantitatively detected by GC. Capillary gas chromatography was used, with a gas flow rate of 1mL / min, a split ratio of 30:1, and an injection volume of 1μL. The initial column temperature was 100°C, retained for 1min, then heated to 180°C at a rate of 5°C / min, retained for 3min, and the detector and injection port temperatures were both 250°C.

[0123] The 2,3-butanediol yield was calculated based on the 2,3-butanediol gas phase detection standard curve, and the final yield was 2.27±0.87 g / L.

[0124] In this example, 2,3-butanediol was obtained. Figure 2 As shown in the figure, it can be seen that the produced compound is 2,3-butanediol.

[0125] Example 7: Production of 2,3-butanediol by direct liquid fermentation of tobacco leaves using Bacillus amyloliquefaciens

[0126] The fermentation medium composition of this example is: KH2PO4 6g / L, K2HPO4·3H2O 14g / L, sodium citrate 8g / L, total volume 50mL, and the supernatant obtained by the method of Example 1 is used to make up 50mL. The specific steps for producing 2,3-butanediol are as follows:

[0127] Bacillus amyloliquefaciens was inoculated into 5 mL LB liquid medium to prepare a primary seed liquid, and then inoculated into a 250 mL conical flask containing 100 mL seed medium with a 5% inoculum for secondary subculture, and then inoculated into a fermentation medium (250 mL conical flask containing 100 mL fermentation medium) with a 5% inoculum after culturing at 37 ° C and 200 rpm for 9 hours, and cultured at 37 ° C and 200 rpm for 84 hours to obtain the fermentation liquid for gas phase detection. The above fermentation liquid was centrifuged at 12000 r / min for 10 minutes, the precipitate was discarded, and the supernatant of the fermentation liquid was extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract, which was filtered with a 0.22 μm organic phase membrane and quantitatively detected by GC. Capillary gas chromatography was used, with a gas flow rate of 1 mL / min, a split ratio of 30:1, and an injection volume of 1 μL. The initial column temperature was 100 ° C, retained for 1 minute, and then heated to 180 ° C at a rate of 5 ° C / min, retained for 3 minutes, and the detector and injection port temperatures were both 250 ° C.

[0128] The 2,3-butanediol yield was calculated based on the 2,3-butanediol gas phase detection standard curve, and the final yield was 2.89±0.15 g / L.

[0129] Example 8: Production of phenylethanol by direct liquid fermentation of tobacco leaves using Saccharomyces cerevisiae S288c

[0130] 50 mL of the supernatant obtained by the method of Example 1 was directly used as a fermentation medium. The specific steps for producing phenylethanol are as follows:

[0131] Saccharomyces cerevisiae S288c was inoculated into 5 mL LB liquid medium to prepare a primary seed solution, and then inoculated into a 250 mL conical flask containing 50 mL seed medium at a 5% inoculation amount for secondary subculture, and then inoculated into a fermentation medium (250 mL conical flask containing 50 mL fermentation medium) at a 2% inoculation amount at 30°C and 200 rpm for 16 h, and cultured at 37°C and 200 rpm for 84 h. After the fermentation was completed, 2% TRPO was added, and after extraction, 1 mL of the solvent layer was filtered and subjected to gas chromatography detection. The detection conditions were:

[0132] Chromatographic column: Agilent DB-WAX; injection volume: 1 μL; column temperature: initially 50 °C for 1 min, then heated to 180 °C at a rate of 25 °C / min, then held for 1 min, then heated to 240 °C at a rate of 20 °C / min, then held for 15 min; flow rate 0.5 mL / min, detector and injection port temperatures were both 250 °C.

[0133] The phenylethanol yield was calculated based on the phenylethanol gas phase detection standard curve, and the final yield was 0.57±0.22 g / L.

[0134] Example 9: The tobacco leaf culture medium described in this example is made of tobacco leaves and water as raw materials, 10% switchgrass is added, and the supernatant after sterilization is obtained; the specific preparation method is completed by the following steps:

[0135] Step 1: Preparation of tobacco leaf and switchgrass powder

[0136] 1. Sample Collection

[0137] The tobacco leaf samples were collected by random sampling, mainly sun-dried tobacco leaf products and switchgrass samples.

[0138] 2. Sample Preparation

[0139] Put the collected tobacco leaves and switchgrass samples after drying in an oven and dry them at a temperature not higher than 40°C until they can be crushed with fingers; then take out the dried samples from the oven and grind them immediately. The grinding time should not exceed 2 minutes; then immediately put the ground samples into a clean and dry brown wide-mouth bottle and seal it. Shake thoroughly and mix well; obtain sample powder. This is the prepared sample.

[0140] Step 2: Take 4.00 g of the prepared sample, add 0.4 g of switchgrass sample powder, add 50 mL of pure water, sterilize directly at 115° C. for 15 min, centrifuge, and store the supernatant in a refrigerator for later use.

[0141] Example 10: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0142] 50 mL of the supernatant obtained by the method of Example 9 was directly used as a fermentation medium. The specific steps for producing farnesene were as follows:

[0143] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0144] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0145] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 189.78±0.29 mg / L.

[0146] Example 11: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0147] The fermentation medium of this example has a total volume of 50 mL: 9.8 g / L K2HPO4; 1 mL / L trace elements; 0.06 g / L MgSO4; and the supernatant obtained by the method of Example 9 is used to make up to 50 mL. The specific steps for producing farnesene are as follows:

[0148] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0149] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0150] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 214.00±0.47 mg / L.

[0151] Example 12: The tobacco leaf culture medium described in this example is made of tobacco leaves and water as raw materials, without adding other biomass, sterilized, and added with enzymes. The specific preparation method is completed by the following steps:

[0152] Step 1: Prepare tobacco leaves

[0153] 1. Sample Collection

[0154] The tobacco leaf samples were collected based on the principle of random sampling, and mainly the samples were sun-dried tobacco leaves.

[0155] 2. Sample Preparation

[0156] Put the collected tobacco leaf samples after drying in an oven and dry them at a temperature not higher than 40°C until they can be crushed with fingers; then take out the dried samples from the oven and grind them immediately. The grinding time should not exceed 2 minutes; then immediately put the ground samples into a clean and dry brown wide-mouth bottle and seal it. Shake thoroughly and mix well; obtain sample powder. This is the prepared sample.

[0157] Step 2: Take 4.00 g of the prepared sample, add 50 mL of pure water, sterilize directly at 115°C for 15 min, then add 12 mg of 1.44 U / mg cellulase, 5 mg of 7.7 U / mg xylanase, and 10 mg of 60 U / mg β-glucosidase to obtain an enzymatic solution, and store it in a refrigerator for use.

[0158] Example 13: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0159] 50 mL of the enzymatic hydrolyzate obtained by the method of Example 12 was directly used as a fermentation medium. The specific steps for producing farnesene were as follows:

[0160] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0161] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0162] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 225.78±0.76 mg / L.

[0163] Example 14: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0164] The fermentation medium composition of this embodiment is: 9.8 g / L K2HPO4; 1 mL / L trace elements; 0.06 g / L MgSO4 in a total volume of 50 mL; and the enzymatic hydrolysate obtained by the method of Example 12 is used to make up to 50 mL. The specific steps for producing farnesene are as follows:

[0165] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0166] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0167] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 309.00±1.48 mg / L.

[0168] Example 15: The tobacco leaf culture medium described in this example is made of tobacco leaves and water as raw materials, other biomass is added, sterilized, and enzymes are added. The specific preparation method is completed by the following steps:

[0169] Step 1: Preparation of tobacco leaf and switchgrass powder

[0170] 1. Sample Collection

[0171] The tobacco leaf samples were collected by random sampling, mainly sun-dried tobacco leaf products and switchgrass samples.

[0172] 2. Sample Preparation

[0173] Put the collected tobacco leaves and switchgrass samples after drying in an oven and dry them at a temperature not higher than 40°C until they can be crushed with fingers; then take out the dried samples from the oven and grind them immediately. The grinding time should not exceed 2 minutes; then immediately put the ground samples into a clean and dry brown wide-mouth bottle and seal it. Shake thoroughly and mix well; obtain sample powder. This is the prepared sample.

[0174] Step 2: Take 4.00 g of the prepared sample, add 0.4 g of switchgrass sample powder, add 50 mL of pure water, sterilize directly at 115 ° C for 15 min, then add 16 mg of 1.44 U / mg cellulase, 10 mg of 7.7 U / mg xylanase, and 10 mg of 60 U / mg β-glucosidase to obtain enzymatic solution, and store it in the refrigerator for use.

[0175] Example 16: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0176] 50 mL of the enzymatic hydrolyzate obtained by the method of Example 15 was directly used as a fermentation medium. The specific steps for producing farnesene were as follows:

[0177] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0178] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0179] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 389.78±2.19 mg / L.

[0180] Example 17: Direct liquid fermentation of Escherichia coli using tobacco leaf culture medium to produce farnesene

[0181] The fermentation medium of this embodiment has a total volume of 50 mL: 9.8 g / L K2HPO4; 1 mL / L trace elements; 0.06 g / L MgSO4; and the enzymatic hydrolysate obtained by the method of Example 15 is used to make up to 50 mL. The specific steps for producing farnesene are as follows:

[0182] Farnesene-producing genetically engineered Escherichia coli (same as in Example 2) was inoculated into LB liquid medium to prepare seed solution, and then inoculated into fermentation medium at an inoculum of 1%, and cultured at 37°C until OD 600 Equal to 0.6, add IPTG, and add a stopper. Culture at 30℃ for 24h. After the fermentation is completed, add 20% n-dodecane, extract, take 1mL of the solvent layer, filter it, and perform gas chromatography detection. The detection conditions are:

[0183] Chromatographic column: Agilent DB-5MS; injection volume: 1 μL; column temperature: initially 60°C, maintained for 0.75 min, heated to 180°C at a rate of 10°C / min, and then dropped to the initial temperature; running time: 13 min.

[0184] The farnesene yield was calculated based on the farnesene gas phase detection standard curve, and the final yield was 414.00±1.07 mg / L.

Claims

1. Application of tobacco leaf culture medium in the production of bio-based chemicals or biofuels, characterized in that: The tobacco leaf culture medium is the supernatant after the raw material is sterilized or the solution obtained after the raw material is sterilized and then treated with an enzyme; wherein the raw material is a combination of solid and water, the solid is tobacco leaves or a combination of tobacco leaves and biomass, when the solid is a combination of tobacco leaves and biomass, the tobacco leaves account for more than 90% of the solid mass, and the biomass is any one of corn cobs, corn stalks, rice straw, switchgrass, Miscanthus, and sawdust, or a combination of several thereof; the mass ratio of solid to water is 1:(12.5-5); The application is to use the tobacco leaf culture medium to ferment Escherichia coli or Saccharomyces cerevisiae to produce farnesene, or to use the tobacco leaf culture medium to ferment Bacillus amyloliquefaciens to produce 2,3-butanediol, or to use the tobacco leaf culture medium to ferment Saccharomyces cerevisiae to produce phenylethanol.

2. The use according to claim 1, characterized in that: The tobacco leaves are flue-cured tobacco leaves and / or air-dried tobacco leaves.

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