CN115678919A - Tobacco culture medium, preparation thereof and method for producing bio-based chemicals through direct fermentation - Google Patents
Tobacco culture medium, preparation thereof and method for producing bio-based chemicals through direct fermentation Download PDFInfo
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- CN115678919A CN115678919A CN202110845667.1A CN202110845667A CN115678919A CN 115678919 A CN115678919 A CN 115678919A CN 202110845667 A CN202110845667 A CN 202110845667A CN 115678919 A CN115678919 A CN 115678919A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A tobacco culture medium and a preparation method thereof as well as a method for producing bio-based chemicals by direct fermentation belong to the technical field of biomass resource utilization. The biomass tobacco leaf can be directly utilized, and is mainly prepared by taking tobacco leaf and water as raw materials, adding no other biomass or adding no more than 10 percent of other biomass, sterilizing to obtain a culture medium, and directly fermenting and utilizing the culture medium. The raw materials of the invention can only comprise tobacco leaves and water, and can also comprise other biomasses, tobacco leaves and water which are not more than 10 percent, and the culture medium for fermentation is directly obtained after sterilization treatment. The invention not only simplifies the steps of producing the bio-based chemicals by taking the traditional biomass as the fermentation culture medium, but also greatly reduces the production cost and the dependence on petroleum resources. The method provides a brand-new tobacco leaf utilization way, has wide application prospect in the field of biosynthesis, and also has great development potential in replacing animal-derived proteins.
Description
Technical Field
The invention belongs to the technical field of biomass energy utilization; in particular to a tobacco culture medium and a preparation method thereof as well as a method for producing bio-based chemicals by direct fermentation.
Background
The microbial synthesis of bio-based chemicals and biofuels has the advantages of natural and environment-friendly properties, short period and the like. However, the carbon source and the nitrogen source of the culture medium are expensive, so that the cost is high, and the industrialization process is hindered. Although biomass resources provide a large amount of fermentation sugar for microorganisms to synthesize bio-based chemicals, the dependence on petrochemical resources is relieved, and environmental pollution is reduced, due to the reasons that the structure of lignocellulose in biomass is complex, the cost of enzyme used in the process is high, the application steps are complicated and the like, the efficiency of the process is low, the cost is high, and the process of large-scale production and application is limited. The key point of the current research is mainly to reduce the overall production cost, on one hand, a great deal of research focuses on understanding the structure of different lignocelluloses and designing proper pretreatment methods, so that the use efficiency of the lignocelluloses can be improved and the cost can be reduced; on the other hand, different technologies are applied to reduce the cost of the enzyme; in addition, there have been studies focused on genetically modifying energy plants to breed low-lignin or high-cellulose plants. Although the methods can reduce the utilization cost of lignocellulose to a certain extent, the industrial application of the process cannot be realized at present, and further improvement is needed.
Disclosure of Invention
The invention aims to provide a new method and a new material which are more economical and environment-friendly for promoting industrial fermentation to produce biofuel and bio-based chemicals. The method can directly use the solution of tobacco leaves after water sterilization treatment as a fermentation medium to produce bio-based chemicals, and provides a new idea for biomass utilization and transformation utilization of tobacco resources. The method also has positive effects of maximizing the resource utilization efficiency, reducing the environmental pollution and relieving the environmental pressure. The method disclosed by the invention is simple to operate, energy-saving and emission-reducing, and has great development potential. The method takes the tobacco leaves as the raw material, and the treatment liquid obtained after one-step treatment is directly used as the microbial fermentation culture medium, so that the defects of complex lignocellulose structure, higher enzyme cost used in the process, complicated process steps and the like in the lignocellulose biomass application process can be improved, and the method 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 the tobacco leaves is used for replacing animal-derived protein, so that the risks of potential virus pollution, undefined components, inconvenience for product purification and the like caused by the discovery of the animal-derived protein in the use process in recent years are avoided, and the safety of subsequent products is guaranteed.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the tobacco leaf culture medium mainly takes tobacco leaves and water as raw materials, and supernatant liquid is obtained after sterilization treatment, wherein the mass ratio of solids to water is 1 (12.5-5); the method is specifically completed by the following steps:
step one, putting tobacco leaves into an oven, drying the tobacco leaves at the temperature of not higher than 40 ℃ until the tobacco leaves can be twisted by fingers, taking the dried tobacco leaves out of the oven, and immediately grinding the tobacco leaves for a continuous grinding time of not more than 2min; immediately putting the ground tobacco leaves into a clean and dry lightproof container, sealing, fully shaking, and uniformly mixing to obtain tobacco leaf powder;
and step two, adding pure water into the tobacco powder obtained in the step one according to the mass ratio of the solid to the water of 1 (12.5-5), directly sterilizing, centrifuging, and taking supernatant fluid to obtain the tobacco culture medium.
The tobacco leaf culture medium can also be a whole solution which is prepared by taking tobacco leaves and water as raw materials, sterilizing and then carrying out enzyme treatment; the method is specifically completed by the following steps:
step one, putting tobacco leaves into an oven, drying the tobacco leaves at the temperature of not higher than 40 ℃ until the tobacco leaves can be twisted by fingers, taking the dried tobacco leaves out of the oven, and immediately grinding the tobacco leaves for a continuous grinding time of not more than 2min; immediately putting the ground tobacco leaves into a clean and dry light-proof container, sealing, fully shaking, and uniformly mixing to obtain tobacco leaf powder;
and step two, adding pure water into the tobacco powder obtained in the step one according to the mass ratio of the solid to the water of 1 (12.5-5), directly sterilizing, adding cellulase, xylanase and beta-glucosidase for treatment, and collecting the solution to obtain the tobacco culture medium.
Further defined, the raw material comprises one or any ratio combination of straw, corncob, cornstalk, straw, switchgrass, miscanthus and wood chip, but is not limited to the above; the preparation steps are different from the method in that: treating one or a combination of several of straws, corncobs, cornstalks, straw, switchgrass, miscanthus and wood chips according to the operation of the step one to obtain powder A; in the second step, pure water is added into the tobacco powder A obtained in the first step
The tobacco leaves are further limited to flue-cured tobacco leaves and/or sun-cured tobacco leaves.
Further limited, the raw material also comprises acid water, alkaline water, an organic solvent or ionic liquid.
The acid water is one of hydrochloric acid, acetic acid, sulfuric acid, nitric acid and phosphoric acid, but is not limited to these.
The alkaline water is further limited to one of sodium hydroxide, calcium hydroxide, ammonia water and potassium hydroxide, but is not limited to the above.
Further, the organic solvent is, but not limited to, ethanol or methanol.
The method for producing the bio-based chemicals by fermenting the tobacco culture medium is characterized in that the method for producing the bio-based chemicals comprises the following steps: and (3) adding microorganisms into the tobacco culture medium or the tobacco culture medium prepared by the method, and performing liquid fermentation.
The tobacco leaves are used as raw materials of the tobacco industry, and the planting range is wide. Compared with the traditional biomass, the tobacco leaves have the advantages of high soluble component content, high sugar content, high nitrogen content, low lignin content and the like, so the tobacco leaves have the feasibility of direct utilization. The tobacco leaves can be directly used as a microbial fermentation culture medium through the treatment liquid obtained after one-step treatment, so that the defects of complex structure of lignocellulose, higher cost of enzyme used in the process, complex process steps and the like in the application process of the lignocellulose biomass can be improved, and the method has important significance for development and utilization of biomass resources and has guiding significance for transformation and utilization of tobacco resources. In addition, the tobacco leaves can be used as a plant-derived nitrogen source due to high nitrogen content to replace animal-derived proteins, so that the risks of potential virus pollution, undefined components, unfavorable product purification and the like caused by the discovery of the animal-derived proteins in the use process in recent years are avoided, and the safety of subsequent products is guaranteed.
Compared with the prior art, the invention has the beneficial effects that:
1) The present invention provides a new material for producing biofuel and bio-based chemicals by industrial fermentation. The bio-based chemical can be directly fermented in a liquid state to produce the bio-based chemical and biofuel, and has the advantages of high water solubility, high content of soluble sugar, high nitrogen content and high utilization efficiency.
2) The raw materials of the invention only comprise tobacco leaves and water, and the tobacco leaves are directly obtained after sterilization treatment, so the cost is low and the process is simple. The invention not only simplifies the steps of producing the bio-based chemicals by taking the traditional biomass as the fermentation culture medium, but also greatly reduces the production cost and the dependence on petroleum resources.
3) The present invention provides a new material for producing biofuel and bio-based chemicals by industrial fermentation. The bio-based chemical variety has the advantages of wide planting range, rapid growth, large biomass, simple and convenient field management and low acquisition cost.
4) The present invention provides a new material for producing biofuel and bio-based chemicals by industrial fermentation. The bio-based chemical can be used for replacing an animal-derived nitrogen source, so that the risks of potential virus pollution, undefined components, unfavorable product purification and the like in the use process of animal-derived protein are reduced, and the safety of subsequent products is guaranteed. The method provides a brand-new tobacco leaf utilization way, has wide application prospect in the field of biosynthesis, and has huge development potential in replacing animal-derived proteins.
5) The invention provides a new method for producing biofuel and bio-based chemicals by industrial fermentation, which is more economical and environment-friendly. The treatment method is simple to operate, energy-saving and emission-reducing, and has great development potential.
6) The invention provides a method for producing bio-based chemicals through direct liquid fermentation of tobacco biomass.
Drawings
FIG. 1 is a mass spectrum of farnesene obtained by fermentation in example 3;
FIG. 2 is a mass spectrum of 2,3-butanediol obtained from the fermentation of example 7.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. Those skilled in the art will recognize that the specific techniques or conditions, not specified in the examples, are according to the techniques or conditions described in the literature of the art or according to the product specification. The materials or instruments used are not indicated by manufacturers, and are all conventional products available by purchase.
Example 1: the bio-based chemicals in the embodiment are supernatants obtained by sterilizing tobacco leaves and water as raw materials without adding other biomass; the preparation method is completed by the following steps:
step one, preparing tobacco powder
1. Sample collection
The tobacco leaf sample is collected by adopting a random sampling principle. Mainly sampling the aired tobacco leaf product.
2. Sample preparation
Putting the collected and aired tobacco leaves into an oven, and drying the tobacco leaves at the temperature of not higher than 40 ℃ until the tobacco leaves can be twisted into pieces by fingers; then taking out the baked tobacco from the oven, and immediately grinding the tobacco for a time not exceeding 2min; and immediately filling the ground tobacco powder into a clean and dry brown wide-mouth bottle and sealing. Fully shaking and mixing; obtaining the tobacco powder. This is the prepared sample.
And step two, taking 4.00g of prepared sample, adding 50mL of pure water, directly sterilizing at 115 ℃ for 15min, centrifuging, and storing the supernatant into a refrigerator for later use.
The following tests are adopted to verify the effect of the invention:
1. and (3) solubility characterization:
weighing 4.00g of the tobacco powder obtained in the step one, adding 50mL of pure water into a high-temperature high-pressure sterilization pot, and performing sterilization treatment. The treated sample is filtered while hot, the filtrate is stored at-20 ℃ for standby, the filter residue is washed three times by 100mL pure water and then is dried to constant weight at 42 ℃, and the result shows that the soluble substance of the tobacco leaves can reach 62.23 +/-0.34%.
2. Determination of Total sugar content
And (3) adding 0.1g of the tobacco powder obtained in the first step into 20mL of pure water, carrying out ultrasonic treatment for 30min, centrifuging to obtain a supernatant, and measuring total sugar by adopting a sulfuric acid phenol method. The final result shows that the total sugar content in the tobacco leaf sample can reach 24.04 +/-0.16%.
3. Determination of total nitrogen content
And (3) accurately weighing 0.2500g of the bio-based chemical obtained in the step two, adding the bio-based chemical into a digestion tube, adding copper sulfate, potassium sulfate and concentrated sulfuric acid, performing digestion reaction, and digesting for 1 hour at 420 ℃. And after digestion is finished and cooling is carried out, carrying out nitrogen determination by using a Kjeldahl azotometer, finally titrating the liquid in the receiving bottle by using 0.5M hydrochloric acid, and finally calculating the total nitrogen content in the tobacco leaves according to the consumption of the hydrochloric acid. The results show that the total nitrogen content in the tobacco leaf samples is 3.01 +/-0.43%.
Example 2: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
Taking 50mL of tobacco culture medium obtained by the method in the embodiment 1 as a fermentation culture medium directly, and producing farnesene by the method comprises the following specific steps:
inoculating the farnesene-producing genetic engineering escherichia coli into an LB liquid culture medium to prepare a seed solution, inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 1 percent, and culturing the seed solution to OD at 37 DEG C 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After fermentation, 20% of n-dodecane is added, after extraction, 1mL of solvent layer is filtered, and gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 209.78 +/-1.24 mg/L.
The method for constructing farnesene-producing genetically engineered Escherichia coli used in example 2 (CN 111607546A, published Japanese 20200901).
1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: after the gene sequence of beta-farnesene synthetase AaFS from southernwood is optimized by the codon preference of escherichia coli, restriction enzymes BglII and XhoI are respectively added at two ends, the sequence synthesis is carried out by Huada gene, and the plasmid pUC57-AaFS is obtained by cloning the gene sequence to pUC57-simple vector. The construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS adopts an enzyme digestion-connection method. Firstly, carrying out double enzyme digestion on plasmids pACYC-mvaE-mvaS-ispA-Sab1 and pUC57-AaFS by using restriction enzymes BglII and XhoI respectively, wherein the enzyme digestion system is as follows:
performing agarose gel electrophoresis and target strip gel cutting recovery on the product after enzyme digestion, wherein the 8260bp fragment is recovered from pACYC-mvaE-mvaS-ispA-Sab1 after BglII and XhoI double enzyme digestion and is used as a carrier; after BglII and XhoI double digestion, pUC57-AaFS is recovered into 1725bp fragment as an insert fragment, and the product is recovered for ligation reaction:
mu.L of the ligation product was heat-shock transformed into DH 5. Alpha. Competent cells and plated with LBCm plates and incubated overnight at 37 ℃. Observing the colony condition on the plate the next day, selecting single bacteria, dropping into liquid culture medium, culturing at 37 deg.C to be concentrated, carrying out colony PCR identification or plasmid enzyme digestion identification, and delivering to sequence to obtain plasmid pACYC-mvaE-mvaS-ispA-AaFS.
2) Plasmid pTrcLower- Δ IDI construction: in order to examine the catalytic effect of heterologous IDI when the copy number of all genes was 1, a single-copy gene recombinant strain was prepared, and the yeast ScIDI originally contained in the plasmid pTrcLower was deleted. Construction of plasmid pTrcLower- Δ IDI A Gibson Assembly method was used to amplify the ScIDI-free vector fraction, pTrc-ERG12-ERG8-ERG19, using pTrcLower plasmid as template. The PCR amplification system is as follows:
carrying out agarose gel electrophoresis on the PCR product, tapping, recovering about 8352bp pTrc-ERG12-ERG8-ERG19 vector fragment, carrying out self-ligation reaction by using a NEBuilder kit, calculating the proportion of the fragment and the amount of each component according to the instruction, carrying out ligation reaction at 50 ℃ for 60min, diluting the product with equal volume of sterile water, taking 5 mu L of heat shock transformed DH5 alpha competent cells, coating an LBAmp plate, and culturing at 37 ℃ overnight. Observing colony conditions on the plate the next day, selecting single bacteria to fall into a liquid culture medium, culturing at 37 ℃ until the single bacteria are concentrated, carrying out colony PCR identification or extraction plasmid enzyme digestion identification, and carrying out transfer sequencing to obtain the plasmid pTrcLower-delta IDI.
3) Construction of plasmid pTrcLower-AaIDI: after the isopentenyl diphosphate isomerase AaIDI gene sequence from the sweet wormwood is optimized by the codon preference of escherichia coli, a Huada gene is subjected to sequence synthesis and is cloned to a pUC57-simple vector to obtain a plasmid pUC57-AaIDI, and the construction of the plasmid pTrcLower-AaIDI adopts an enzyme digestion-connection method. Firstly, using plasmids pUC57-AaIDI and pTrcLower as templates, respectively amplifying AaIDI gene fragment and pTrc-ERG12-ERG8-ERG19 vector fragment by using primers with SacI and PstI:
the PCR product was subjected to agarose gel electrophoresis, and the AaIDI gene fragment of about 708bp and the pTrc-ERG12-ERG8-ERG19 vector fragment of 8412bp were recovered by tapping, respectively, and double digestion was carried out with SacI and PstI:
performing agarose gel electrophoresis on the product after enzyme digestion, respectively tapping and recovering an about 708bp AaIDI gene fragment and an 8412bp pTrc-ERG12-ERG8-ERG19 vector fragment, and recovering the products for connection reaction:
10 μ L of the ligation product was heat-shocked to transform DH 5. Alpha. Competent cells and plated on LB Amp plates and incubated overnight at 37 ℃. Observing colony condition on the plate the next day, selecting single bacteria, dropping into liquid culture medium, culturing at 37 deg.C to relatively thick, performing colony PCR identification or extraction plasmid restriction enzyme identification, and subjecting to sequencing to obtain plasmid pTrc-ERG12-ERG8-ERG19-AaIDI (pTrcLower-AaIDI).
4) Construction of plasmid pET28 a-AaFS-ispA-IDI:
isopentenyl diphosphate isomerase AaIDI gene from sweet wormwood is optimized through codon preference of escherichia coli, sequence synthesis is carried out on Huada gene, and the Huada gene is cloned to pUC57-simple vector to obtain plasmid pUC57-AaIDI; isopentenyl diphosphate isomerase SlIDI gene from tomato is optimized by codon preference of escherichia coli, sequence synthesis is carried out on Huada gene, and the Huada gene is cloned on pUC57-simple vector to obtain plasmid pUC57-SlIDI.
Construction of plasmid pET28a-AaFS-ispA-IDI A Gibson Assembly method is adopted, firstly pET28a vector and three gene fragments of AaFS, ispA and IDI (from sweet wormwood and tomato respectively) are amplified, and pET28a plasmid, pUC57-AaFS plasmid, escherichia coli BL21 (DE 3) bacterial liquid, pUC57-AaIDI plasmid and pUC57-SlIDI plasmid are taken as templates respectively:
performing agarose gel electrophoresis and gel cutting recovery of target bands on PCR products, determining the concentration of gel recovery products, performing Gibson Assembly by using a NEBuilder kit, calculating the proportion of fragments and the amount of each component according to the instruction, performing ligation reaction at 50 ℃ for 60min, diluting the products by adding sterile water with the same volume, taking 5 mu L of heat shock transformed DH5 alpha competent cells, coating LBKan plates, and culturing at 37 ℃ overnight. Observing colony conditions on the plate the next day, selecting single bacteria to fall into a liquid culture medium, culturing at 37 ℃ until the single bacteria are concentrated, carrying out colony PCR identification or extraction plasmid restriction enzyme identification, and carrying out transfer sequencing to obtain a plasmid pET28a-AaFS-ispA-AaIDI and a plasmid pET28a-AaFS-ispA-SlIDI respectively.
5) And (3) plasmid transformation: e.coli BL21 (DE 3) competent cells were transformed by combining pACYC-mvaE-mvaS, plasmid pACYC-mvaE-mvaS with correct sequencing, pTrcLower- Δ IDI prepared in step 2), pET28a-AaFS-ispA-SlIDI/AaIDI (single copy) prepared in step 4), pACYC-mvaE-mvaS-ispA-AaFS prepared in step 1), pTrcLower-AaIDI prepared in step 3), pET28a-AaFS-ispA-AaIDI (double copy) prepared in step 4), and E.coli BL21 (DE 3) competent cells, plated with corresponding triple antibody (Cm, amp and Kan) medium plates, wherein the final concentration of Cm in LB medium is 34mg/L, the final concentration of Amp in LB medium is 100mg/L, and the final concentration of Kan in LB medium is 50mg/L, cultured at 37 ℃ to obtain a single-length gene engineering bacterium colony.
A genetically engineered bacterium (single copy) containing AaIDI gene from sweet wormwood herb: the recombinant bacterium overexpressing acetyl CoA acyltransferase/HMG-CoA reductase mvaE gene, HMG-CoA synthetase mvaS gene, mevalonate-5-phosphokinase ERG8 gene, mevalonate kinase ERG12 gene, mevalonate-5-diphosphate decarboxylase ERG19 gene, isopentenyl diphosphate isomerase AaIDI gene from artemisia apiacea, farnesyl diphosphate synthetase ispA gene and beta-farnesene synthetase AaFS gene, wherein the copy number of the IDI gene, the ispA gene and the AaFS gene on the plasmid is 1.
The farnesene profile obtained in this example is shown in FIG. 1, which shows that the fermentation product is farnesene.
Example 3: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
The total volume of the fermentation medium components in this example was 50mL:9.8g/L K 2 HPO 4 (ii) a 1mL/L of trace elements; 0.06g/L MgSO 4 (ii) a The volume of the supernatant obtained in example 1 was 50mL. The specific steps for producing farnesene are as follows:
the farnesene-producing genetically engineered escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, 1% of the seed solution was inoculated into a fermentation medium, and the mixture was cultured to OD at 37 ℃ 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 234.00 +/-0.17 mg/L.
Example 4: method for producing farnesene by direct liquid state fermentation of tobacco leaves by saccharomyces cerevisiae
The total volume of the components of the fermentation medium in this example was 50mL:15g/L urea; 8g/L KH 2 PO 4 ；6g/L MgSO 4 The volume of the supernatant obtained in example 1 was 50mL. The specific steps for producing farnesene are as follows:
inoculating farnesene-producing yeast into 5mL YPD liquid culture medium to prepare first-stage seed solution, inoculating 5mL yeast into 50mL YPD fermentation culture medium, and culturing at 30 deg.C to OD 600 Equal to 5 as a secondary seed solution, inoculating into a fermentation medium, and culturing at 30 ℃ for 48h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 94.55 +/-2.62 mg/L.
In this example, a genetically engineered bacterium producing farnesene yeast was constructed (application publication No. CN111378588a, publication No. 20200707):
the starting strain of the target genetic engineering bacteria is saccharomycetes, and the saccharomycetes integrates farnesene synthetase gene AaFS, phosphotransacetylase gene CkPTA, phosphoketolase gene LmpK, acetaldehyde dehydrogenase gene Dzeute and hydroxymethyl glutaryl coenzyme A reductase gene RpHMGR.
The engineering strain takes a yeast strain CEN.PK2-1C (MATa; ura3-52, trp1-289, leu 3 delta 1, MAL2-8c SUC 2) as a starting strain, and has the following simple construction process:
(1) The genes are obtained by a PCR method, wherein a farnesene synthetase gene AaFS is derived from southernwood (Artemisia annua L.) with a sequence registration number of GenBank: AY835398.1; the phosphotransacetylase gene CkPTA is derived from Clostridium kluyveri (Clostridium kluyveri), and the sequence registration number is GenBank: CP018335.1; the phosphoketolase gene LmpK is derived from Leuconostoc (Leuconostoc cmesenteroides) and has the sequence registration number of GenBank: AY804190.1; the acetaldehyde dehydrogenase gene Dzeute is derived from rice-based rotting fungi (Dickeyaeae), and has a sequence registration number of GenBank: CP006929.1; the hydroxymethyl glutaryl coenzyme A reductase gene RpHGR is derived from Ruegeriapomeroyi and has the sequence registration number NCBIReferenceSequence of NC-006569.1.
(2) Construction of integration fragment: PGAL1-AaFS-TCYC1, PGAL1-LmpK-TCYC1_ PGAL10-CkPTA-TADH1_ HIS,
PGAL1-AaFS-TCYC1_PGAL10-DzeutE-TADH1_URA3、PGAL1-AaFS-TCYC1_PGAL10-RpHMGR-TADH1_LEU2
P (OC) GAL4-GAL4_ PGAL1-tHMG1_ KanMX. The above fragments were integrated into GAL80, ALD4, ALD6, ADH5, RHR2 sites, respectively. PGAL1 and _ PGAL10 are GAL1 and GAL10 promoters, respectively. TCYC1 and TADH1 are terminators.
(3) And transforming the obtained integration vector into an original strain to obtain a target transgenic engineering strain.
The engineering strain takes a yeast strain CEN.PK2-1C (MATa; ura3-52, trp1-289, leu 3 delta 1, MAL2-8c SUC 2) as a starting strain, and the construction process is as follows:
the specific process of constructing the yeast engineering strain containing the farnesene synthetase gene AaFS comprises the following steps:
(1) The farnesene synthetase gene AaFS is inserted into GAL80 site of yeast genome. The farnesene synthetase gene is derived from Artemisia annua L sequence registration number GenBank: AY835398.1, and codon optimization is carried out on the gene. AaFS was cleaved from the synthesized plasmid with BamHI and SalI, and ligated into pESC-HIS plasmid to obtain recombinant plasmid pESC-HIS-AaFS. A PGAL1-AaFS-T CYC1 fragment was obtained by PCR, P GAL1 was GAL1 promoter, T CYC1 was CYC1 terminator, and it was ligated into pUC19 plasmid using restriction enzymes NdeI and PstI. Then, upstream (GAL 80 US) 511bp of GAL80 and downstream (GAL 80 DS) 501bp of GAL80 are sequentially ligated to both ends of P GAL1-AaFS-T CYC1 by NdeI and PstI to obtain recombinant plasmid pUC19-GAL80US-P GAL1-AaFS-T CYC1-GAL80DS. The donor fragment was obtained by NdeI and PstI cleavage: GAL80US-P GAL1-AaFS-T CYC1-GAL80DS.
(2) And constructing an expression fragment of the gRNA by taking the sequence TAAGGCTGCTGCTGAACGT as a target sequence. A19 bp target sequence is introduced during primer design, a linearized pML104 is used as a template, 500bpDNA sequences at two ends of the primer are amplified by PCR, two upstream and downstream fragments both contain a 19bp GAL80 target sequence, the two fragments are fused together by overlap PCR to obtain a complete gRNA expression fragment, and the 19bp target sequence is positioned in the center of the gRNA expression fragment. Carrying out double enzyme digestion on the plasmid pML104 by using restriction enzymes SwaI and BclI to obtain a linearized plasmid; and (3) simultaneously transferring the donor DNA fragment, the linearized pML104 plasmid and the gRNA expression fragment into a yeast competent cell CEN.PK2-1C by a chemical conversion method to obtain the yeast genetic engineering bacteria FS1.
Constructing a yeast engineering strain containing a phosphoketolase gene LmpK and a phosphotransacetylase gene CkPTA.
(1) On the basis of the FS1 strain, gene integration is carried out by means of homologous recombination. Phosphotransacetylase gene CkPTA and phosphoketolase gene LmPK are inserted into ALD6 site of yeast genome. The phosphotransacetylase gene CkPTA is derived from Clostridium kluyveri (Clostridium kluyveri), and the sequence registration number is GenBank: CP018335.1; the phosphoketolase gene LmpK is derived from Leuconostoc mesenteroides (Leuconostoc mesenteroides) and has the sequence registration number of GenBank: AY804190.1. The CkPTA gene is connected into pESC-URA plasmid through EcoRI and SacI to obtain recombinant plasmid pESC-URA-CkPTA. The LmPK gene is ligated into the pESC-URA-CkPTA plasmid through BamHI and SalI to obtain a recombinant plasmid pESC-URA-LmPK-CkPTA. P GAL1-LmpK-T CYC1_ P GAL10-CkPTA-T ADH1 fragment was obtained by PCR.
(2) The upstream and downstream fragments ALD6US and ALD6DS of the ALD6 gene were obtained by PCR, and HIS-expressing fragments were obtained by PCR using pESC-HIS plasmid as a template. The pUC19 plasmid was linearized with restriction enzymes Sma I and PstI, and then P GAL1-LmpK-TCYC1_ P GAL10-CkPTA-T ADH1, ALD6US, ALD6DS, HIS were ligated into the pUC19 plasmid by means of a Multi-fragment one-step rapid Cloning Kit (Hieff Plus Multi OneStep Cloning Kit,10912ES 10) to obtain a recombinant plasmid pUC19-ALD6US-P GAL1-LmpK-T CYC1_ P GAL10-CkPTA-T ADH 1-HIS-ALD 6DS. The donor fragment was obtained by cleavage of the recombinant plasmid with SmaI and SphI: ALD6US-P GAL1-LmpK-T CYC1_ P GAL10-CkPTA-T ADH 1-HIS-ALD 6DS. The donor fragment was transferred into yeast strain FS1 by chemical transformation to obtain yeast strain FS2.
Constructing a yeast engineering strain containing acetaldehyde dehydrogenase gene Dzeute.
(1) On the basis of the FS2 strain, gene integration is carried out by means of homologous recombination. The acetaldehyde dehydrogenase gene Dzeute and the farnesene synthetase gene AaFS are inserted into the ALD4 site of the yeast genome. The acetaldehyde dehydrogenase gene Dzeute is derived from rice-based rotting fungi (Dickeya zeae), and has the sequence registration number of GenBank: CP006929.1.
The Dzeute gene was ligated to pESC-HIS-AaFS plasmid via NotI and PacI to obtain recombinant plasmid pESC-HIS-AaFS-Dzeute. The PGAL1-AaFS-T CYC1_ P GAL10-Dzeute-T ADH1 fragment was obtained by PCR.
(2) The upstream and downstream fragments ALD4US and ALD4DS of the ALD4 gene were obtained by PCR. The URA expression fragment was obtained by PCR using pESC-URA plasmid as a template. The yeast strain FS3 is obtained by transferring 4 fragments of P GAL1-AaFS-T CYC1_ P GAL10-Dzeute-T ADH1, ALD4US, ALD4DS and URA into yeast strain FS2 by chemical conversion method.
Constructing the yeast engineering strain containing the hydroxymethyl glutaryl coenzyme A reductase gene RHMGR.
(1) On the basis of FS3 strain, gene integration is carried out by means of homologous recombination. The hydroxymethylglutaryl-CoA reductase gene RpHGR and the farnesene synthase gene AaFS are inserted into the ADH5 site of the yeast genome. The hydroxymethyl glutaryl coenzyme A reductase gene RpHGR is derived from Ruegeriapomeroyi and has the sequence registration number of NCBIReferenceSequence: NC-006569.1. The RpHGR is an optimized sequence.
The RpHMGR gene is obtained through PCR, and the pESC-HIS-AaFS plasmid is connected through SpeI and SacI to obtain a recombinant plasmid pESC-HIS-AaFS-RpHMGR. A P GAL1-AaFS-T CYC1_ P GAL10-RpHMGR-T ADH1 fragment was obtained by PCR.
(2) The upstream and downstream fragments ADH5US and ADH5DS of ADH5 gene were obtained by PCR. The LEU expression fragment was obtained by PCR using the pESC-LEU plasmid as a template. The 4 fragments P GAL1-AaFS-T CYC1_ P GAL10-RpHMGR-T ADH1, ADH5US, ADH5DS and LEU were transformed into yeast strain FS3 by chemical transformation to obtain yeast strain FS4.
Constructing yeast engineering bacteria of over-expressing yeast GAL4 gene and tHMG1 gene.
(1) On the basis of the FS4 strain, gene integration is carried out by means of homologous recombination. The GAL4 gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) and has the Sequence accession number NCBI Reference Sequence NC-001148.4. The GAL4 gene contains a downstream sequence of 251bp after the stop codon. The GAL4 gene promoter is an optimized GAL4 promoter. the tHMG1 gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the Sequence registration number NCBI Reference Sequence: NC-001145.3. The tHMG1 gene is a truncation body with 529 amino acids of the N end deleted and contains a 225bp sequence behind a stop codon, and the promoter is a PGAL1 promoter. GAL4 gene and tHMG1 are inserted into the RHR2 site of the yeast genome.
(2) GAL4 expression fragment (including promoter, coding gene and terminator sequence) is divided into A, B, C fragments, and the three fragments are obtained by PCR amplification respectively. the tHMG1 expression fragment (including promoter, coding gene and terminator sequence) is divided into D, E fragments, which are respectively obtained by PCR amplification. The selection marker gene KanMX having a loxP site was obtained by PCR using plasmid pUC6 as a template. The upstream and downstream fragments RHR2US, RHR2DS of the RHR2 gene were obtained by PCR.
(3) The fragments RHR2US, A, C, D, E, kanMX and RHR2-DS are respectively fused into a fragment by using Overlap extension PCR, the fused RHR2US + A and B are fused into a fragment, and the four fragments are simultaneously transferred into a yeast strain FS4 by a chemical conversion method to obtain a yeast strain FS5.
Constructing the yeast engineering bacteria for down-regulating and expressing the yeast ERG9 gene.
(1) On the basis of FS5 strain, gene integration is carried out by means of homologous recombination. The ERG9 gene is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the Sequence registration number NCBI Reference Sequence: NC-001140.6. The promoter for down-regulating the ERG9 gene expression fragment (comprising a promoter, an encoding gene and a downstream 537bp sequence) is a MET3 promoter.
(2) The upstream and downstream fragments ERG9US and ERG9+ ERG9DS of ERG9 gene were obtained by PCR using yeast genome as template. MET3 promoter P MET3 was obtained by PCR using yeast genome as a template. Taking plasmid pESC-TRP as a template, and obtaining a TRP expression fragment through PCR.
(3) The fragments ERG9US and TRP are fused into one fragment by Overlap extensionPCR, and the three fragments ERG9US + TRP, P MET3 and ERG9 are simultaneously transferred into a yeast strain FS5 by a chemical conversion method to obtain a yeast strain FS6, namely the genetically engineered bacterium for converting cellulose hydrolysate to synthesize farnesene.
Example 5: method for producing farnesene by direct liquid state fermentation of tobacco leaves by saccharomyces cerevisiae
50mL of the supernatant obtained in example 2 was used as the fermentation medium. The method for producing farnesene comprises the following specific steps:
a farnesene-producing yeast (same as in example 4) was inoculated into 5mL of YPD liquid medium to prepare a primary seed solution, 5mL of the primary seed solution was inoculated into 50mL of YPD fermentation medium, and the primary seed solution was cultured at 30 ℃ to OD 600 Equal to 5 as a secondary seed solution, inoculating into a fermentation medium, and culturing at 30 ℃ for 48h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 95.67 +/-0.87 mg/L.
Example 6: bacillus amyloliquefaciens for producing 2,3-butanediol by direct liquid fermentation of tobacco leaves
50mL of the supernatant obtained in example 2 was used as the fermentation medium. The specific steps for producing 2,3-butanediol are as follows:
inoculating bacillus amyloliquefaciens into 5mL of LB liquid culture medium to prepare a primary seed solution, then inoculating the primary seed solution into a 250mL conical flask containing 100mL of seed culture medium with an inoculum size of 5% for secondary subculture, inoculating the primary seed solution into a fermentation culture medium (containing 100mL of fermentation culture medium in the 250mL conical flask) with an inoculum size of 5% after culturing at 37 ℃ and 200rpm for 9h, and culturing at 37 ℃ and 200rpm for 84h to take the fermentation liquid for gas phase detection. Centrifuging the fermentation liquor at 12000r/min for 10min, removing precipitate, extracting the supernatant with equal volume of ethyl acetate to obtain ethyl acetate extract, filtering with 0.22 μm organic phase membrane, and quantitatively detecting by GC. Capillary gas chromatography is adopted, the gas flow rate is set to be 1mL/min, the split ratio is set to be 30. The initial column temperature was 100 ℃ and held for 1min, then the temperature was raised to 180 ℃ at a rate of 5 ℃/min and held for 3min, both the detector and injection port temperatures were 250 ℃.
The 2,3-butanediol yield is calculated according to 2,3-butanediol gas phase detection standard curve, and the final yield is 2.27 +/-0.87 g/L.
The 2,3-butanediol obtained in this example is shown in FIG. 2, from which it can be seen that the compound produced is 2,3-butanediol.
Example 7: bacillus amyloliquefaciens for producing 2,3-butanediol by direct liquid fermentation of tobacco leaves
The total volume of the components of the fermentation medium in this example was 50mL: KH (Perkin Elmer) 2 PO 4 6g/L，K 2 HPO 4 ·3H 2 O14 g/L, sodium citrate 8g/L, and 50mL of the supernatant obtained in example 1 was replenished. The specific steps for producing 2,3-butanediol are as follows:
inoculating bacillus amyloliquefaciens into a 5mLLB liquid culture medium to prepare a primary seed solution, then inoculating the primary seed solution into a 250mL conical flask containing 100mL seed culture medium with an inoculum size of 5 percent for secondary subculture, inoculating the primary seed solution into a fermentation culture medium (containing 100mL fermentation culture medium in the 250mL conical flask) with an inoculum size of 5 percent after culturing at 37 ℃ and 200rpm for 9h, and culturing at 37 ℃ and 200rpm for 84h to take the fermentation liquid for gas phase detection. Centrifuging the fermentation liquor at 12000r/min for 10min, removing precipitate, extracting the supernatant with ethyl acetate of equal volume to obtain ethyl acetate extract, filtering with 0.22 μm organic phase membrane, and quantitatively detecting by GC. Capillary gas chromatography is adopted, the gas flow rate is set to be 1mL/min, the split ratio is set to be 30. The initial column temperature was 100 ℃ and held for 1min, then the temperature was raised to 180 ℃ at a rate of 5 ℃/min and held for 3min, both detector and injection port temperatures were 250 ℃.
The 2,3-butanediol yield is calculated according to 2,3-butanediol gas phase detection standard curve, and the final yield is 2.89 +/-0.15 g/L.
Example 8: saccharomyces cerevisiae S288c for producing phenethyl alcohol by direct liquid fermentation of tobacco leaves
50mL of the supernatant obtained in example 1 was used as the fermentation medium. The specific steps for producing the phenethyl alcohol are as follows:
saccharomyces cerevisiae S288c was inoculated into 5mLLB liquid medium to prepare a primary seed solution, which was inoculated into 50mL seed medium in a 250mL Erlenmeyer flask at an inoculum size of 5% for secondary subculture, and then inoculated into fermentation medium at an inoculum size of 2% after culturing at 30 ℃ and 200rpm for 16 hours (50 mL fermentation medium in a 250mL Erlenmeyer flask), and cultured at 37 ℃ and 200rpm for 84 hours. After the fermentation was completed, 2% TRPO was added, and after extraction, 1mL of the solvent layer was filtered and subjected to gas chromatography under the following conditions:
and (3) chromatographic column: agilent DB-WAX; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 50 ℃ for 1min, heating to 180 ℃ at the speed of 25 ℃/min, keeping the temperature for 1min, heating to 240 ℃ at the speed of 20 ℃/min, and keeping the temperature for 15min; the volume flow rate was 0.5mL/min, and the detector and injection port temperatures were 250 ℃.
According to the gas phase detection standard curve of the phenethyl alcohol, the yield of the phenethyl alcohol is calculated and obtained, and the final yield is 0.57 +/-0.22 g/L.
Example 9: in the embodiment, the tobacco leaf culture medium is prepared by taking tobacco leaves and water as raw materials, adding 10% of switchgrass, and sterilizing the supernatant; the preparation method is completed by the following steps:
step one, preparing tobacco leaf and switchgrass powder
1. Sample collection
The tobacco leaf samples are collected by adopting a random sampling principle. Mainly sampled air-cured tobacco products and switchgrass samples.
2. Sample preparation
Putting the collected and aired tobacco leaves and switchgrass samples into an oven, and drying at the temperature of not higher than 40 ℃ until the tobacco leaves and the switchgrass samples can be twisted into pieces by fingers; then taking out the baked sample from the oven, and immediately grinding the sample, wherein the continuous grinding time is not more than 2min; and putting the ground mixture into a clean and dry brown wide-mouth bottle immediately and sealing the bottle. Fully shaking and mixing; a sample powder was obtained. This is the prepared sample.
And step two, taking 4.00g of prepared sample, adding 0.4g of switchgrass sample powder, adding 50mL of pure water, directly sterilizing at 115 ℃ for 15min, centrifuging, and storing supernatant in a refrigerator for later use.
Example 10: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
Taking 50mL of the supernatant obtained in the method of example 9 as a fermentation medium directly, the specific steps for producing farnesene are as follows:
the farnesene-producing genetically engineered escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, 1% of the seed solution was inoculated into a fermentation medium, and the mixture was cultured to OD at 37 ℃ 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After fermentation, 20% of n-dodecane is added, after extraction, 1mL of solvent layer is filtered, and gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 189.78 +/-0.29 mg/L.
Example 11: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
The total volume of the components of the fermentation medium in this example was 50mL:9.8g/L K 2 HPO 4 (ii) a 1mL/L of trace elements; 0.06g/L MgSO 4 (ii) a The volume of the supernatant obtained in example 9 was 50mL. The specific steps for producing farnesene are as follows:
the farnesene-producing genetically engineered escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, 1% of the seed solution was inoculated into a fermentation medium, and the mixture was cultured to OD at 37 ℃ 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After the fermentation is finished, addingExtracting 20% n-dodecane, filtering 1mL of solvent layer, and performing gas chromatography detection under the following detection conditions:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 214.00 +/-0.47 mg/L.
Example 12: in the embodiment, the tobacco leaf culture medium is prepared by taking tobacco leaves and water as raw materials, adding no other biomass, sterilizing, and adding enzyme, and the specific preparation method comprises the following steps:
step one, preparing tobacco leaves
1. Sample collection
The tobacco leaf samples are collected by adopting a random sampling principle. Mainly sampling the aired tobacco leaf sample.
2. Sample preparation
Putting the collected and aired tobacco leaf sample into an oven, and drying at the temperature of not higher than 40 ℃ until the tobacco leaf sample can be twisted into pieces by fingers; then taking out the baked sample from the oven, and immediately grinding the sample, wherein the continuous grinding time is not more than 2min; and putting the ground mixture into a clean and dry brown wide-mouth bottle immediately and sealing the bottle. Fully shaking and mixing; a sample powder was obtained. This is the prepared sample.
And step two, taking 4.00g of prepared sample, adding 50mL of pure water, directly sterilizing at 115 ℃ for 15min, adding 12mg of 1.44U/mg cellulase, 5mg of 7.7U/mg xylanase and 10mg of 60U/mg beta-glucosidase to obtain enzymatic hydrolysate, and storing the enzymatic hydrolysate in a refrigerator for later use.
Example 13: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
Taking 50mL of the enzymolysis solution obtained by the method in the embodiment 12 as a fermentation medium directly, and the specific steps for producing farnesene are as follows:
a farnesene-producing genetically engineered Escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, and the amount of the seed solution was determined1% of the total amount of the active ingredients, inoculating the active ingredients into a fermentation medium, and culturing the active ingredients at 37 ℃ to OD 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And (4) calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 225.78 +/-0.76 mg/L.
Example 14: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
The total volume of the components of the fermentation medium in this example was 50mL:9.8g/L K 2 HPO 4 (ii) a 1mL/L of trace elements; 0.06g/L MgSO 4 (ii) a 50mL of the enzymatic hydrolysate obtained in example 12 was used as a supplement. The specific steps for producing farnesene are as follows:
the farnesene-producing genetically engineered escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, 1% of the seed solution was inoculated into a fermentation medium, and the mixture was cultured to OD at 37 ℃ 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 309.00 +/-1.48 mg/L.
Example 15: in the embodiment, the tobacco leaf culture medium is prepared by taking tobacco leaves and water as raw materials, adding other biomass, sterilizing, and adding enzyme, and the specific preparation method comprises the following steps:
step one, preparing tobacco leaves and switchgrass powder
1. Sample collection
The tobacco leaf sample is collected by adopting a random sampling principle. Mainly sampled after-air-cured tobacco products and switchgrass samples.
2. Sample preparation
Putting the collected and aired tobacco leaves and switchgrass samples into an oven, and drying at the temperature of not higher than 40 ℃ until the tobacco leaves and the switchgrass samples can be twisted into pieces by fingers; then taking out the baked sample from the oven, and immediately grinding the sample, wherein the continuous grinding time is not more than 2min; and putting the ground mixture into a clean and dry brown wide-mouth bottle immediately and sealing the bottle. Fully shaking and mixing; a sample powder was obtained. This is the prepared sample.
And step two, taking 4.00g of prepared sample, adding 0.4g of switchgrass sample powder, adding 50mL of pure water, directly sterilizing at 115 ℃ for 15min, adding 16mg of 1.44U/mg of cellulase, 10mg of 7.7U/mg of xylanase and 10mg of 60U/mg of beta-glucosidase to obtain enzymatic hydrolysate, and storing the enzymatic hydrolysate in a refrigerator for later use.
Example 16: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
Taking 50mL of the enzymolysis solution obtained by the method in the embodiment 15 as a fermentation medium directly, and the specific steps for producing farnesene are as follows:
the farnesene-producing genetically engineered escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, 1% of the seed solution was inoculated into a fermentation medium, and the mixture was cultured to OD at 37 ℃ 600 Equal to 0.6, IPTG was added and the stopper was added. Culturing at 30 deg.C for 24h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample injection amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 389.78 +/-2.19 mg/L.
Example 17: method for producing farnesene by directly fermenting escherichia coli in liquid state by using tobacco leaf culture medium
The total volume of the components of the fermentation medium in this example was 50mL:9.8g/L K 2 HPO 4 (ii) a 1mL/L of trace elements; 0.06g/L MgSO 4 (ii) a 50mL of the enzymatic hydrolysate obtained in example 15 was used as a supplement. The specific steps for producing farnesene are as follows:
the farnesene-producing genetically engineered escherichia coli (same as in example 2) was inoculated into an LB liquid medium to prepare a seed solution, 1% of the seed solution was inoculated into a fermentation medium, and the mixture was cultured to OD at 37 ℃ 600 Equal to 0.6, IPTG is added and the stopper is plugged. Culturing at 30 deg.C for 24h. After fermentation, 20% n-dodecane is added, after extraction, 1mL of solvent layer is taken out and filtered, and then gas chromatography detection is carried out, wherein the detection conditions are as follows:
a chromatographic column: agilent DB-5MS; sample introduction amount: 1 mu L of the solution; column temperature: keeping the temperature at 60 ℃ for 0.75min initially, heating to 180 ℃ at the speed of 10 ℃/min, and then cooling to the initial temperature; operating time: and 13min.
And calculating to obtain the farnesene yield according to a farnesene gas phase detection standard curve, wherein the final yield is 414.00 +/-1.07 mg/L.
Claims (9)
1. The tobacco leaf culture medium is characterized in that the tobacco leaf culture medium mainly takes tobacco leaves and water as raw materials, and the supernatant after sterilization treatment or the supernatant after sterilization treatment and enzyme treatment are carried out to obtain all solutions; wherein the mass ratio of the solid to the water is 1 (12.5-5).
2. The tobacco leaf culture medium of claim 1, wherein the raw material further comprises one or a combination of straw, corn cob, corn stalk, rice straw, switchgrass, miscanthus, wood chips, and the tobacco leaves comprise more than 90% of the solid mass.
3. The tobacco leaf culture medium according to claim 1 or 2, characterized in that the tobacco leaf is a cured tobacco leaf and/or an air cured tobacco leaf.
4. The tobacco leaf culture medium according to claim 1 or 2, characterized in that the raw material further comprises acid water, alkaline water, organic solvent or ionic liquid.
5. The tobacco leaf culture medium according to claim 4, wherein the acid water is one of hydrochloric acid water solution, acetic acid water solution, sulfuric acid water solution, nitric acid water solution and phosphoric acid water solution; the alkaline water is one of sodium hydroxide aqueous solution, calcium hydroxide aqueous solution, ammonia water and potassium hydroxide aqueous solution; the organic solvent is one of ethanol and methanol.
6. The method for preparing a tobacco leaf culture medium according to claim 1, wherein the preparation method is performed by the following steps:
step one, putting tobacco leaves into an oven, drying the tobacco leaves at the temperature of not higher than 40 ℃ until the tobacco leaves can be twisted by fingers, taking the dried material out of the oven, and immediately grinding the material to respectively obtain tobacco leaf powder;
and step two, adding pure water into the tobacco powder obtained in the step one, directly sterilizing, centrifuging, and taking supernatant to obtain the tobacco culture medium.
7. The method for preparing the tobacco leaf culture medium according to claim 6, wherein one or a combination of several of straw, corncob, cornstalk, straw, switchgrass, miscanthus and wood chips is treated according to the operation of the step one to obtain powder A; and step two, adding pure water into the tobacco powder A obtained in the step one.
8. The preparation method of the tobacco culture medium according to claim 6 or 7, characterized in that after the direct sterilization treatment in the step two, cellulase, xylanase and beta-glucosidase are added for treatment, and the solution is collected to obtain the tobacco culture medium.
9. The method for producing the bio-based chemicals by fermenting the tobacco culture medium is characterized by comprising the following steps of: adding microorganism into the tobacco leaf culture medium according to any one of claims 1 to 5 or the tobacco leaf culture medium prepared by the method according to claim 6, 7 or 8, and performing liquid fermentation.
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