Construction method and application of engineering escherichia coli for producing humulone
阅读说明：本技术 一种生产葎草酮用工程大肠杆菌的构建方法及其应用 (Construction method and application of engineering escherichia coli for producing humulone ) 是由 邹慧斌 黄静玲 张楠 王亚群 梁秀红 于 2020-06-04 设计创作，主要内容包括：本发明提出了一种生产葎草酮用工程大肠杆菌的构建方法及其应用。本发明的构建方法包括：以工程大肠杆菌为出发菌株,将源于工程大肠杆菌的异戊二烯焦磷酸异构酶进化后的基因和来源于啤酒花的异戊二烯基转移酶进化后的基因以及单加氧酶基因构建到载体质粒中,得第一表达载体；将来源于啤酒花的戊二酮合酶进化后的基因、胞质辅酶A连接酶进化后的基因和异戊二烯基转移酶进化后的基因片段构建到另外一个载体质粒中,得第二表达载体；将表达载体转入工程大肠杆菌,得生产葎草酮用工程大肠杆菌；本发明还给出利用上述工程大肠杆菌生物法合成葎草酮的方法。本发明的合成方法步骤少,纯度高,质量稳定,摆脱了对啤酒花原料的依赖,避免了溶剂的大量消耗。(The invention provides a construction method and application of engineering escherichia coli for producing humulone. The construction method comprises the following steps: constructing an evolved gene of isoprene pyrophosphate isomerase derived from the engineering escherichia coli, an evolved gene of prenyl transferase derived from hops and a monooxygenase gene into a vector plasmid by taking the engineering escherichia coli as an initial strain to obtain a first expression vector; constructing genes derived from the hop after the evolution of the pentanedione synthase, genes derived from the cytoplasmic coenzyme A ligase and gene segments derived from the prenyltransferase into another vector plasmid to obtain a second expression vector; transferring the expression vector into engineering escherichia coli to obtain the engineering escherichia coli for producing the humulone; the invention also provides a method for synthesizing humulone by using the engineering escherichia coli biological method. The synthesis method has the advantages of few steps, high purity and stable quality, gets rid of dependence on hop raw materials and avoids large consumption of solvents.)
1. A construction method of engineering escherichia coli for producing humulone is characterized by comprising the following steps:
1) the method comprises the steps of taking pTrcHis2B as a starting plasmid, constructing an evolved gene of isoprene pyrophosphate isomerase originated from engineering escherichia coli, an evolved gene of prenyltransferase originated from hops and a monooxygenase gene into a vector plasmid pTrcHis2B to obtain an expression vector pTrcHis2B-idi-pt 1-monooxyenase, wherein the nucleotide sequence of the evolved gene of the prenyl pyrophosphate isomerase is shown as SEQ ID NO:1, the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO:2, and the nucleotide sequence of the monooxygenase gene is shown as SEQ ID NO: 3;
2) constructing a gene derived from hop after the evolution of pentanedione synthase, a gene derived from hop after the evolution of cytoplasmic coenzyme A ligase and a gene fragment derived from isoprene transferase into a vector plasmid pACYCDuet-1 by taking pACYCDuet-1 as a starting plasmid to obtain an expression vector pACYCDuet-1-vps-ccl2-pt2, wherein the nucleotide sequence of the gene derived from the pentanedione synthase is shown as SEQ ID NO:4, the nucleotide sequence of the gene derived from the cytoplasmic coenzyme A ligase is shown as SEQ ID NO:5, and the nucleotide sequence of the gene derived from isoprene transferase is shown as SEQ ID NO: 6;
3) the expression vector pTrcHis2B-idi-pt 1-monooxgene obtained in the step 1) and the expression vector pACYCDuet-1-vps-ccl2-pt2 obtained in the step 2) are transferred into engineering escherichia coli BL21(DE3) -Trc-low, and the engineering escherichia coli for producing the humulone is obtained.
2. An engineering escherichia coli for producing humulone, which is characterized in that:
the engineered Escherichia coli for humulone production is the engineered Escherichia coli obtained by the method for constructing the engineered Escherichia coli for humulone production according to claim 1.
3. The application of engineering escherichia coli in biological method synthesis of humulone is characterized by comprising the following steps:
taking the engineering escherichia coli obtained by the construction method of the engineering escherichia coli for producing the humulone according to claim 1, fermenting, centrifugally filtering, freeze-drying, extracting and purifying to obtain the humulone.
4. The use of engineered escherichia coli for the biosynthesis of humulone as claimed in claim 3, wherein:
the fermentation is shake flask fermentation, and the shake flask fermentation comprises the following specific steps:
activating a strain of engineering escherichia coli, preparing a seed solution, inoculating according to an inoculation amount of 3-5%, carrying out shake culture at 37 ℃ at 220r/min, and culturing for 12 h; adding an inducer isopropyl thiogalactoside, wherein the addition amount of the inducer is 0.2-0.6 mL/L, and continuing to shake and culture; sampling every 12h, measuring OD value and pH value, and adjusting the pH value of the fermentation liquor to 7.0 by using 10% sodium hydroxide when the fermentation liquor is acidic; and when the OD value does not rise any more and the pH value is alkaline, ending the fermentation to obtain the fermentation liquor.
5. The use of engineered escherichia coli for the biosynthesis of humulone as claimed in claim 4, wherein:
in the fermentation process, firstly, adding 2mL/L magnesium sulfate mother liquor and 1mL/L microelement mother liquor into a fermentation culture medium; then, 0.7mL/L ampicillin mother liquor and 0.7mL/L chloramphenicol mother liquor were added; finally, 7.5mL/L of mevalonic acid and 15mL/L of leucine mother liquor are added;
the concentration of the magnesium sulfate mother liquor is 0.24g/L, and the microelement mother liquor comprises the following components: 3.7g/L of ammonium molybdate tetrahydrate, 2.9g/L of zinc sulfate heptahydrate, 24.7g/L of boric acid, 2.5g/L of copper sulfate pentahydrate and 15.8g/L of manganese chloride tetrahydrate;
the concentration of the leucine mother liquor is 17.5g/L, the concentration of the chloramphenicol mother liquor is 34mg/L, and the concentration of the ampicillin mother liquor is 100 mg/L.
6. The use of engineered escherichia coli in the biosynthesis of humulone according to claim 4 or 5, wherein:
in the process of shake flask fermentation, the fermentation medium is as follows: (NH)2)4SO41g/L，K2HPO4·3H2O3 g/L, KCl 1.7g/L, sodium citrate 1g/L, betaine 1g/L, glucose 20g/L, citric acid 1g/L and yeast extract 5-10 g/L; sterilizing with high pressure steam at 115 deg.C for 30 min;
the mevalonic acid and the leucine mother liquor are added into the fermentation liquor after the addition of the inducer, and the mevalonic acid is added into the fermentation liquor in 3 batches.
7. The use of engineered escherichia coli for the biosynthesis of humulone as claimed in claim 3, wherein:
and during centrifugal filtration, the centrifugal rotating speed is 5000-10000 r/min, the centrifugal temperature is below 20 ℃, and the centrifugal time is 15-30 min.
8. The use of engineered escherichia coli for the biosynthesis of humulone as claimed in claim 3, wherein:
during freeze drying, the vacuum degree is 10Pa, the drying temperature is-20 to-60 ℃, and the drying time is 12 to 36 hours.
9. The use of engineered escherichia coli for the biosynthesis of humulone as claimed in claim 3, wherein:
the extraction and purification method comprises any one or more of ultrasonic extraction, reagent extraction, thermal extraction and magnetic stirring extraction.
10. A humulone, comprising:
the humulone is obtained by applying the engineering escherichia coli in biological synthesis of the humulone according to any one of claims 3 to 9.
Technical Field
The invention relates to the technical field of humulone, in particular to a construction method of engineering escherichia coli for producing humulone and application of the engineering escherichia coli in producing humulone.
Background
Humulone is a phloroglucinol derivative separated from hop glands and named humulone in English, has a structure with natural product commonality, is relatively complex and consists of more carbon molecules, and C is C21H30O5Is the chemical molecular formula of the humulone, the specific molecular weight is 362.466, and the CAS number of the humulone which can be found on the network is 26472-41-3.
Humulone is a yellowish low-melting crystalline substance that is easily soluble in alkali and organic solvents, but hardly soluble in water. Humulone and other picric acids and other allylated polyketides are found primarily in the hop glands, which are glandular trichomes found on the cones and leaves of hops. Glandular trichomes generally have little or no photosynthetic activity and, typically, contain only some highly active biosynthetic pathways, however, humulone production in hops proceeds in the glands.
Currently, the production status of humulone is to extract from hop plants, the extraction method needs many steps and consumes a large amount of organic reagents, and the purity of the humulone obtained by the extraction method is not high, the humulone is often mixed with cohumulone and cohumulone, and even a small amount of beta-picric acid is contained; in addition, the raw materials of the method for extracting the humulone greatly depend on the supply of hops, and the large-scale production of the humulone is limited due to slow growth of beer peanuts; secondly, the humulone concentration can fluctuate greatly due to climate and geographical differences in the beer peanut growth process.
Disclosure of Invention
The invention provides a construction method of engineering escherichia coli for producing humulone and application of the engineering escherichia coli in producing humulone, and aims to solve the problems that in the prior art, a method for extracting humulone from hops has multiple operation steps, large organic solvent consumption, low purity, strong dependence on raw materials and unstable quality of products in different batches.
In order to solve the technical problem, the technical scheme of the invention is realized as follows:
in one aspect, the invention provides a method for constructing engineering escherichia coli for producing humulone, which comprises the following steps: 1) the method comprises the steps of taking pTrcHis2B as a starting plasmid, constructing an evolved gene of isoprene pyrophosphate isomerase originated from engineering escherichia coli, an evolved gene of prenyltransferase originated from hops and a monooxygenase gene into a vector plasmid pTrcHis2B to obtain an expression vector pTrcHis2B-idi-pt 1-monooxyenase, wherein the nucleotide sequence of the evolved gene of the prenyl pyrophosphate isomerase is shown as SEQ ID NO:1, the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO:2, and the nucleotide sequence of the monooxygenase gene is shown as SEQ ID NO: 3; 2) constructing a gene derived from hop after the evolution of pentanedione synthase, a gene derived from hop after the evolution of cytoplasmic coenzyme A ligase and a gene fragment derived from isoprene transferase into a vector plasmid pACYCDuet-1 by taking pACYCDuet-1 as a starting plasmid to obtain an expression vector pACYCDuet-1-vps-ccl2-pt2, wherein the nucleotide sequence of the gene derived from the pentanedione synthase is shown as SEQ ID NO:4, the nucleotide sequence of the gene derived from the cytoplasmic coenzyme A ligase is shown as SEQ ID NO:5, and the nucleotide sequence of the gene derived from isoprene transferase is shown as SEQ ID NO: 6; 3) the expression vector pTrcHis2B-idi-pt 1-monooxgene obtained in the step 1) and the expression vector pACYCDuet-1-vps-ccl2-pt2 obtained in the step 2) are transferred into engineering escherichia coli BL21(DE3) -Trc-low, and the engineering escherichia coli for producing the humulone is obtained.
The construction method of the engineering escherichia coli for producing the humulone is based on the existing engineering escherichia coli BL21(DE3) -Trc-low strain in a laboratory, and constructs the gene (idi) derived from the evolution of isoprene pyrophosphate isomerase of the escherichia coli, the gene (pt1) derived from the evolution of prenyltransferase of hop hairy glands and the monooxygenase gene (monooxygenase) into a vector plasmid pTrcHis2B by using a gene recombination technology; constructing the evolved gene (vps) of the pentanedione synthase derived from the hairy gland of the hop, the evolved gene (ccl2) of the cytoplasmic coenzyme A ligase and the evolved gene (pt2) of another prenyltransferase into a vector plasmid pACYCDuet-1; then, the two plasmids are transferred into the original engineering bacteria of the escherichia coli, so that a complete humulone total biosynthesis pathway is constructed in a target strain. Coli has many advantages over other expression systems (e.g., yeast, protozoan, etc. cells), such as: the method has the advantages of clear genetic background, simple culture method, high propagation speed, strong anti-pollution capability and high expression level of target genes, and the method has few operation steps, short flow and simple and easy operation no matter culture preparation or separation and purification.
In another aspect, the engineered escherichia coli for humulone production of the present invention is an engineered escherichia coli obtained according to the above-described construction method for engineered escherichia coli for humulone production.
The engineering escherichia coli can be directly used for synthesizing the humulone, so that a brand new synthesis method is provided for the synthesis technology of the humulone, and the engineering escherichia coli is a biosynthesis method; the method for synthesizing the humulone can avoid the limitation of hop raw materials, realizes the retention of biological activity which is difficult to realize by chemical synthesis, has higher selectivity for preparing the humulone by utilizing engineering strains than a natural product method, only contains the humulone and does not contain humulone derivatives, has high purity and stable product quality, and does not have the phenomenon that the concentration of the humulone fluctuates greatly due to climate and geographical differences in the process of beer peanut growth.
In still another aspect, the invention provides a use of engineered escherichia coli in the biological synthesis of humulone, comprising the following steps: taking the engineering escherichia coli obtained by the construction method for producing the engineering escherichia coli for humulone, fermenting, centrifuging, filtering, freeze-drying, extracting and purifying to obtain the humulone.
The invention starts from the existing engineering strains in a laboratory, utilizes the metabolic engineering technology, introduces key enzyme genes in the humulone synthesis way into engineering escherichia coli strains through a vector, and constructs fermentation strains for synthesizing the humulone in a full-biological way; the obtained engineering escherichia coli is subjected to fermentation, centrifugal filtration, freeze drying and extraction and purification to obtain the humulone, which is a brand-new humulone synthesis method; the humulone synthesis method disclosed by the invention has the advantages of fewer operation steps, short flow, simplicity, easiness in control and low cost, gets rid of dependence on hop raw materials, can realize large-scale production, avoids large consumption of solvents, fully retains the biological activity of humulone, has high selectivity, ensures that the obtained product only contains humulone and does not contain humulone derivatives, is good in purity and stable in product quality, and does not have the phenomenon of large fluctuation of the concentration of humulone due to climate and geographical differences in the beer peanut growing process.
As a preferred embodiment, the fermentation is a shake flask fermentation, and the specific steps of the shake flask fermentation are as follows: activating a strain of engineering escherichia coli, preparing a seed solution, inoculating according to an inoculation amount of 3-5%, carrying out shake culture at 37 ℃ at 220r/min, and culturing for 12 h; adding an inducer isopropyl thiogalactoside, wherein the addition amount of the inducer is 0.2-0.6 mL/L, and continuing to shake and culture; sampling every 12h, measuring OD value and pH value, and adjusting the pH value of the fermentation liquor to 7.0 by using 10% sodium hydroxide when the fermentation liquor is acidic; and when the OD value does not rise any more and the pH value is alkaline, ending the fermentation to obtain the fermentation liquor. The fermentation process of the invention is shake flask fermentation, which is usually carried out on a constant temperature oscillator, and an inducer is also added in the shake flask fermentation process, and the inducer IPTG is used as an important fermentation additive for inducing enzyme protein expression in the strain fermentation process, provides an enzyme for catalyzing product synthesis, and also influences the fermentation level of the strain to a certain extent; the inducer is usually added to the fermentation liquor 12-20 hours after the beginning of the fermentation.
As a preferred embodiment, in the fermentation process, firstly, 2mL/L magnesium sulfate mother liquor and 1mL/L microelement mother liquor are added into a fermentation medium; then, 0.7mL/L ampicillin mother liquor and 0.7mL/L chloramphenicol mother liquor were added; finally, 7.5mL/L of mevalonic acid and 15mL/L of leucine mother liquor are added; the concentration of the magnesium sulfate mother liquor is 0.24g/L, and the microelement mother liquor comprises the following components: 3.7g/L of ammonium molybdate tetrahydrate, 2.9g/L of zinc sulfate heptahydrate, 24.7g/L of boric acid, 2.5g/L of copper sulfate pentahydrate and 15.8g/L of manganese chloride tetrahydrate; the concentration of the leucine mother liquor is 17.5g/L, the concentration of the chloramphenicol mother liquor is 34mg/L, and the concentration of the ampicillin mother liquor is 100 mg/L. In the invention, magnesium sulfate, trace elements, ampicillin and chloramphenicol are also added into the fermentation medium, and leucine and mevalonic acid are added in the fermentation process to increase the yield of humulone.
In the invention, the mevalonic acid is selected as a pure substance, and is prepared by adjusting the pH of mevalonolactone to 7.0 by using 1M NaOH and sterilizing the mevalonic lactone for 25min by using high-pressure steam at 121 ℃ without dilution; the leucine mother liquor is prepared by dissolving 0.525g leucine in 30mL sterilized UP water, slightly stirring with sterilized gun head to dissolve leucine, filtering with 0.22 μm sterile filter membrane, storing at 4 deg.C for a short time, and storing at-20 deg.C for a long time; the magnesium sulfate mother liquor is prepared by dissolving magnesium sulfate in RO water, sterilizing with high pressure steam at 121 deg.C for 25min, or sterilizing at 115 deg.C for 30min, and storing at 4 deg.C; dissolving the components in RO water, filtering with 0.22 μm sterile filter membrane, and storing at 4 deg.C; the mother liquid of chloramphenicol is prepared by filtering with sterile filter membrane with pore size of 0.22 μm after the preparation of chloramphenicol solution, and storing at-20 deg.C; the ampicillin mother liquor is prepared after ampicillin preparation, filtered by a sterile filter membrane with the aperture size of 0.22 mu m and stored at the temperature of minus 20 ℃.
In a preferred embodiment, the fermentation medium in the shake flask fermentation process is: (NH)2)4SO41g/L，K2HPO4·3H2O3 g/L, KCl 1.7g/L, sodium citrate 1g/L, betaine 1g/L, glucose 20g/L, citric acid 1g/L and yeast extract 5-10 g/L; sterilizing with high pressure steam at 115 deg.C for 30 min; the mevalonic acid and the leucine mother liquor are added into the fermentation liquor after the addition of the inducer, and the mevalonic acid is added into the fermentation liquor in 3 batches. The yeast extract in the fermentation medium is used as an important substrate in the fermentation process of the strain, can provide nitrogen resources for the growth and the propagation of the strain, and influences the fermentation of the strain to a certain extent; the strain of the invention has fast growth speed and good growth vigor.
In a preferred embodiment, the centrifugal rotation speed is 5000-10000 r/min, the centrifugal temperature is below 20 ℃, and the centrifugal time is 15-30 min during centrifugal filtration. The fermented thallus is centrifugally filtered in an ultracentrifuge, and pure water and alcohol are usually adopted to wash the thallus in the centrifugal process.
In a preferred embodiment, the freeze drying is carried out under the vacuum degree of 10Pa, the drying temperature of-20 to-60 ℃ and the drying time of 12 to 36 hours. In the invention, fermentation liquor after fermentation is centrifugally filtered, supernatant is poured off, and thallus precipitate is reserved; and transferring the thalli from the centrifugal bottle, drying in a freeze dryer, and drying the thalli in a low-temperature freeze drying manner, wherein the drying temperature is low, the drying efficiency is high, and the biological activity of the humulone is fully ensured.
As a preferred embodiment, the extraction and purification method comprises any one or more of ultrasonic extraction, reagent extraction, thermal extraction and magnetic stirring extraction. The extraction and purification method of the invention has a plurality of methods, the common methods are ultrasonic extraction method, reagent leaching method, thermal extraction and magnetic stirring extraction, the extracted solution is poured into a rotary steaming bottle, and rotary steaming is carried out at 40 ℃ under the action of dichloromethane and n-hexane solution to obtain the product; dissolving the product with methanol, and filtering with 0.45 μm filter membrane to obtain humulone.
In yet another aspect, a humulone of the present invention, which is a humulone obtained in the use of an engineered escherichia coli according to any one of the above in the biosynthesis of humulone. The humulone provided by the invention gets rid of the dependence on hop raw materials in the synthesis process, can realize large-scale production, avoids large consumption of solvents, fully retains the biological activity of the humulone, has high selectivity, good purity and stable quality, and does not generate the phenomenon of large fluctuation of the humulone concentration due to climate and geographical differences in the growth process of beer peanuts.
Compared with the prior art, the invention has the beneficial effects that: the invention starts from the existing engineering strains in a laboratory, utilizes the metabolic engineering technology, introduces key enzyme genes in the humulone synthesis way into engineering escherichia coli strains through a vector, and constructs fermentation strains for synthesizing the humulone in a full-biological way; the obtained strain is fermented, centrifugally filtered, freeze-dried and purified to obtain the humulone, which is a brand new humulone biosynthesis method; the humulone synthesis method disclosed by the invention has the advantages of few operation steps, short flow, simplicity, easiness in control and low cost, gets rid of the dependence on hop raw materials, can realize large-scale production, avoids large consumption of solvents, fully retains the biological activity of humulone, has high selectivity, only contains humulone in the obtained product, does not contain humulone derivatives, has good purity of humulone and stable product quality, and does not generate the phenomenon of large fluctuation of the concentration of humulone due to climate and geographical differences in the process of beer peanut growth.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph of the UV absorption spectrum of humulone obtained in example III of the present invention;
FIG. 2 is a high performance liquid phase diagram of humulone obtained in example III of the present invention;
FIG. 3 is the liquid phase mass spectrum of humulone obtained in the third embodiment of the present invention;
FIG. 4 is the hydrogen spectrum of humulone obtained in example III of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a construction method of engineering escherichia coli for producing humulone, which comprises the following steps:
1) the method comprises the steps of taking pTrcHis2B as a starting plasmid, constructing an evolved gene of isoprene pyrophosphate isomerase originated from engineering escherichia coli, an evolved gene of prenyltransferase originated from hops and a monooxygenase gene into a vector plasmid pTrcHis2B to obtain an expression vector pTrcHis2B-idi-pt 1-monooxyenase, wherein the nucleotide sequence of the evolved gene of the prenyl pyrophosphate isomerase is shown as SEQ ID NO:1, the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO:2, and the nucleotide sequence of the monooxygenase gene is shown as SEQ ID NO: 3;
2) constructing a gene derived from hop after the evolution of pentanedione synthase, a gene derived from hop after the evolution of cytoplasmic coenzyme A ligase and a gene fragment derived from isoprene transferase into a vector plasmid pACYCDuet-1 by taking pACYCDuet-1 as a starting plasmid to obtain an expression vector pACYCDuet-1-vps-ccl2-pt2, wherein the nucleotide sequence of the gene derived from the pentanedione synthase is shown as SEQ ID NO. 4, the nucleotide sequence of the gene derived from the cytoplasmic coenzyme A ligase is shown as SEQ ID NO. 5, and the nucleotide sequence of the gene derived from isoprene transferase is shown as SEQ ID NO. 6;
3) the expression vector pTrcHis2B-idi-pt 1-monooxgene obtained in the step 1) and the expression vector pACYCDuet-1-vps-ccl2-pt2 obtained in the step 2) are transferred into engineering escherichia coli BL21(DE3) -Trc-low, and the engineering escherichia coli for producing the humulone is obtained.
The engineered escherichia coli for producing humulone of the present invention is an engineered escherichia coli obtained according to the above-described construction method for an engineered escherichia coli for producing humulone.
The invention relates to an application of engineering escherichia coli in biological method synthesis of humulone, which comprises the following steps: taking the engineering escherichia coli obtained by the construction method for producing the engineering escherichia coli for humulone, fermenting, centrifuging, filtering, freeze-drying, extracting and purifying to obtain the humulone.
Preferably, the fermentation is shake flask fermentation, and the specific steps of the shake flask fermentation are as follows: activating a strain of engineering escherichia coli, preparing a seed solution, inoculating according to an inoculation amount of 3-5%, carrying out shake culture at 37 ℃ at 220r/min, and culturing for 12 h; adding an inducer isopropyl thiogalactoside, wherein the addition amount of the inducer is 0.2-0.6 mL/L, and continuing to shake and culture; sampling every 12h, measuring OD value and pH value, and adjusting the pH value of the fermentation liquor to 7.0 by using 10% sodium hydroxide when the fermentation liquor is acidic; and when the OD value does not rise any more and the pH value is alkaline, ending the fermentation to obtain the fermentation liquor.
Further, in the fermentation process, firstly, 2mL/L magnesium sulfate mother liquor and 1mL/L microelement mother liquor are added into a fermentation culture medium; then, 0.7mL/L ampicillin mother liquor and 0.7mL/L chloramphenicol mother liquor were added; finally, 7.5mL/L of mevalonic acid and 15mL/L of leucine mother liquor are added; the concentration of the magnesium sulfate mother liquor is 0.24g/L, and the microelement mother liquor comprises the following components: 3.7g/L of ammonium molybdate tetrahydrate, 2.9g/L of zinc sulfate heptahydrate, 24.7g/L of boric acid, 2.5g/L of copper sulfate pentahydrate and 15.8g/L of manganese chloride tetrahydrate; the concentration of the leucine mother liquor is 17.5g/L, the concentration of the chloramphenicol mother liquor is 34mg/L, and the concentration of the ampicillin mother liquor is 100 mg/L.
Specifically, in the shake flask fermentation process, the fermentation medium is: (NH)2)4SO41g/L，K2HPO4·3H2O3 g/L, KCl 1.7g/L, sodium citrate 1g/L, betaine 1g/L, glucose 20g/L, citric acid 1g/L and yeast extract 5-10 g/L; sterilizing with high pressure steam at 115 deg.C for 30 min; the mevalonic acid and the leucine mother liquor are added into the fermentation liquor after the addition of the inducer, and the mevalonic acid is added into the fermentation liquor in 3 batches.
More preferably, during centrifugal filtration, the centrifugal rotation speed is 5000-10000 r/min, the centrifugal temperature is below 20 ℃, and the centrifugal time is 15-30 min.
More preferably, the vacuum degree is 10Pa, the drying temperature is-20 to-60 ℃, and the drying time is 12 to 36 hours during freeze drying.
Further preferably, the extraction and purification method comprises any one or more of ultrasonic extraction, reagent extraction, thermal extraction and magnetic stirring extraction.
The humulone of the present invention is a humulone obtained by applying the engineered escherichia coli described in any one of the above aspects to biological synthesis of humulone.