阅读说明：本技术 一种生产蛇麻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 lupulone ) 是由 邹慧斌 张楠 黄静玲 梁秀红 王亚群 于 2020-06-04 设计创作，主要内容包括：本发明提出了一种生产蛇麻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下步骤：以大肠杆菌为出发菌株,将源于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构建到载体质粒中,得第一个表达载体；将来源于啤酒花的间苯三酚合酶进化后的基因、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,得第二个表达载体；将表达载体转入大肠杆菌,得生产蛇麻酮用工程大肠杆菌；本发明还给出利用上述工程大肠杆菌生物法合成蛇麻酮的方法。本发明的合成方法操作简单,流程短,成本低,摆脱了对啤酒花原料的依赖,实现清洁生产,避免了溶剂的大量消耗。(The invention provides a construction method and application of engineering escherichia coli for producing lupulone. The construction method comprises the following steps: constructing a gene derived from evolution of isoprene pyrophosphate isomerase of escherichia coli and a gene derived from evolution of prenyl transferase of hop into a vector plasmid by taking escherichia coli as an initial strain to obtain a first expression vector; constructing genes derived from the hop after the evolution of phloroglucinol synthase, genes derived from the evolution of cytoplasmic coenzyme A ligase and gene segments derived from the evolution of prenyltransferase into another vector plasmid to obtain a second expression vector; transferring the expression vector into escherichia coli to obtain engineering escherichia coli for producing lupulone; the invention also provides a method for synthesizing lupulone by using the engineering escherichia coli biological method. The synthesis method disclosed by the invention is simple to operate, short in flow and low in cost, gets rid of dependence on hop raw materials, realizes clean production and avoids large consumption of solvents.)

1. A construction method of engineering escherichia coli for producing lupulone is characterized by comprising the following steps:

1) constructing an evolved gene of isoprene pyrophosphate isomerase originated from engineering escherichia coli and an evolved gene of prenyltransferase originated from hops into a vector plasmid pTrcHis2B by taking pTrcHis2B as a starting plasmid to obtain an expression vector pTrcHis2B-idi-pt1, wherein the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO. 1, and the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO. 2;

2) constructing genes derived from hop after the evolution of phloroglucinol synthase, genes derived from the evolution of cytoplasmic coenzyme A ligase and gene segments derived from prenyltransferase into a vector plasmid pACYCDuet-1 by taking pACYCDuet-1 as a starting plasmid to obtain an expression vector pACYCDuet-1-ccl2-vps-pt2, wherein the nucleotide sequence of the genes derived from the phloroglucinol synthase is shown as SEQ ID NO. 3, the nucleotide sequence of the genes derived from the cytoplasmic coenzyme A ligase is shown as SEQ ID NO. 4, and the nucleotide sequence of the genes derived from the prenyltransferase is shown as SEQ ID NO. 5;

3) and (2) transferring the expression vector pTrcHis2B-idi-pt1 obtained in the step 1) and the expression vector pACYCDuet-1-ccl2-vps-pt2 obtained in the step 2) into engineering escherichia coli BL21(DE3) -Trc-low to obtain the engineering escherichia coli for producing lupulone.

2. An engineering escherichia coli for producing lupulone, which is characterized in that:

the engineered escherichia coli for producing lupulone is the engineered escherichia coli obtained by the construction method of the engineered escherichia coli for producing lupulone according to claim 1.

3. The application of engineering escherichia coli in the biological synthesis of lupulone is characterized by comprising the following steps:

taking the engineering escherichia coli obtained by the construction method of the engineering escherichia coli for producing lupulone according to claim 1, fermenting, centrifuging, filtering, freeze-drying, extracting and purifying to obtain the lupulone.

4. The use of the engineered escherichia coli according to claim 3 for the biosynthesis of lupulone, 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 10-15%, adjusting the pH value to be neutral by adopting 10% sodium hydroxide, performing shake culture at 37 ℃ and 220r/min for 12h, adding an inducer, namely isopropyl thiogalactoside, continuing shake culture, sampling every 12h, measuring the OD value, adjusting the pH value, and adjusting the culture period to be 90 h.

5. The use of the engineered escherichia coli of claim 4 for the biological synthesis of lupulone, wherein:

in the fermentation process, after inoculation, firstly, adding 7.5mL/L mevalonic acid and 5mL/L leucine, carrying out shake culture for 12h, adding an inducer, continuing to carry out shake culture for 12h, adding 5g/L yeast extract supplement liquid, 1g/L betaine supplement liquid and 5mL/L leucine, continuing to carry out shake culture for 12h, and then adding 5mL/L leucine;

the mevalonic acid is prepared by adjusting the pH of mevalonolactone to 7.0 with 1M NaOH, and sterilizing with high pressure steam at 121 deg.C for 25 min;

the leucine is prepared by dissolving 0.525g/L leucine in 30mL RO water, filtering with 0.22 μm sterile filter membrane, and storing at-20 deg.C;

the yeast extract supplementary liquid is prepared by dissolving 2g of yeast extract in 10mL of pure water, and sterilizing with high-pressure steam at 121 ℃ for 25 min;

the betaine supplementary liquid is prepared by dissolving 0.5g betaine in 5mL pure water, and sterilizing with high pressure steam at 121 deg.C for 25 min.

6. The use of the engineered escherichia coli according to claim 4 or 5 for the biosynthesis of lupulone, wherein:

in the process of shake flask fermentation, the fermentation medium is as follows: na (Na)₂HPO₄·12H₂O 30g/L，KH₂PO₄6g/L，NH₄Cl 2g/L, NaCl 1g/L, glucose 4g/L, citric acid 1g/L, yeast extract 2g/L, ampicillin 0.15g/L, chloramphenicol 0.1g/L, and water in balance, and sterilizing with 115 deg.C high pressure steam for 30 min.

7. The use of the engineered escherichia coli according to claim 3 for the biosynthesis of lupulone, wherein:

when in centrifugal filtration, the centrifugal rotation speed is 10000-50000r/min, the centrifugal temperature is below 5 ℃, and the centrifugal time is 15-30 min.

8. The use of the engineered escherichia coli according to claim 3 for the biosynthesis of lupulone, wherein:

during the freeze drying, the vacuum degree is 10Pa, the drying temperature is-80 ℃, and the drying time is 12 h.

9. The use of the engineered escherichia coli according to claim 3 for the biosynthesis of lupulone, wherein:

the extraction and purification comprises the following specific steps: adding n-hexane, refluxing at 80-90 deg.C for 6-9 hr, and drying.

10. Lupulone characterized by:

the lupulone is obtained by applying the engineering escherichia coli in the biological method for synthesizing the lupulone according to any one of claims 3 to 9.

Technical Field

The invention relates to the technical field of lupulone, in particular to a construction method of engineering escherichia coli for producing lupulone and application of the engineering escherichia coli in production of lupulone.

Background

Hops (Humulus lupulus), also known as hops, hops and yeast flowers, transliteration also known as "hops", and hermaphrodite strains, are the essential bittering agents in the beer brewing process, and are often used as medicinal plants in traditional medicine all the time. Lupulone is one of important secondary metabolites in hops, and has pharmacological effects of resisting bacteria, diminishing inflammation, tranquilizing, hypnotizing, resisting tumors and the like.

At present, the prior art mainly adopts a chemical method to extract lupulone from hops, such as: chinese patent CN104447264A discloses a method for extracting and separating lupulone from hops at 25/3/2015, and provides that the extraction method comprises the following steps: a. loading the hop compressed matter into a subcritical ultrasonic extractor, introducing the R134a mixed solvent into the extractor, dissolving the effective components in the extracted matter in the liquid or subcritical R134a mixed solvent, and pumping the filtrate into a separator; b. regulating temperature and pressure to convert R134a in the R134a mixed solvent from liquid state to gas state, separating from the extracting solution, concentrating the extracting solution containing methanol by a concentration evaporator to obtain hops extract, and entering a crystallization working section; c. putting a certain amount of hops extract into a reaction kettle, adding water, stirring, standing at room temperature, performing suction filtration, and collecting insoluble substances; d. adding water into the insoluble substance, stirring, standing at room temperature, and vacuum filtering; e. stirring and filtering, and collecting insoluble substances to obtain crude lupulone product; and recrystallizing the crude lupulone product by using normal hexane to obtain a white needle lupulone refined product.

The method for extracting lupulone has the advantages of long process flow, complex separation and purification steps, high cost and poor economy; moreover, the method for extracting lupulone also needs to consume a large amount of solvent, solvent residues are easy to cause, and the residual solvent can have harmful effects on the lupulone; in addition, the extraction method of lupulone also greatly depends on the supply of hops on raw materials, and the large-scale production of lupulone is limited due to the slow growth of beer peanuts; secondly, the hop peanut growth process can cause large fluctuation of lupulone concentration due to climate and geographical differences.

Disclosure of Invention

The invention aims to provide a construction method of engineering escherichia coli for producing lupulone and application of the construction method in production of lupulone, and aims to solve the problems of long process, complex operation, strong dependence on raw materials, high cost and solvent residue in a method for extracting lupulone from hops by a chemical method in the prior art.

In order to solve the technical problem, the main technical scheme of the invention is realized as follows:

in one aspect, the invention provides a construction method of engineering escherichia coli for producing lupulone, which comprises the following steps: 1) constructing an evolved gene of isoprene pyrophosphate isomerase originated from engineering escherichia coli and an evolved gene of prenyltransferase originated from hops into a vector plasmid pTrcHis2B by taking pTrcHis2B as a starting plasmid to obtain an expression vector pTrcHis2B-idi-pt1, wherein the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO:1, and the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO: 2; 2) constructing genes derived from hop after the evolution of phloroglucinol synthase, genes derived from the evolution of cytoplasmic coenzyme A ligase and gene segments derived from prenyltransferase into a vector plasmid pACYCDuet-1 by taking pACYCDuet-1 as a starting plasmid to obtain an expression vector pACYCDuet-1-ccl2-vps-pt2, wherein the nucleotide sequence of the genes derived from the phloroglucinol synthase is shown as SEQ ID NO. 3, the nucleotide sequence of the genes derived from the cytoplasmic coenzyme A ligase is shown as SEQ ID NO. 4, and the nucleotide sequence of the genes derived from the prenyltransferase is shown as SEQ ID NO. 5; 3) and (2) transferring the expression vector pTrcHis2B-idi-pt1 obtained in the step 1) and the expression vector pACYCDuet-1-ccl2-vps-pt2 obtained in the step 2) into engineering escherichia coli BL21(DE3) -Trc-low to obtain the engineering escherichia coli for producing lupulone.

The construction method of the engineering escherichia coli for producing lupulone provided by the invention starts from 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 and the gene (pt1) derived from prenyltransferase of hop hairy glands into a vector plasmid pTrcHis2B by using a gene recombination technology; constructing the segments of an evolved gene (vps) of phloroglucinol synthase derived from a hop hairy gland, an evolved gene (ccl2) of cytoplasmic coenzyme A ligase and another evolved gene (pt2) of prenyltransferase into a vector plasmid pACYCDuet-1; then, the two plasmids are transferred into the original escherichia coli engineering bacteria, so that a complete lupulone total biosynthesis way is constructed in the target bacterial 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 short flow, low cost, no limitation of raw materials and simple and easy operation no matter culture preparation or separation and purification.

In another aspect, the engineered escherichia coli for lupulone production according to the present invention is an engineered escherichia coli obtained according to the above-described method for constructing engineered escherichia coli for lupulone production.

The engineering escherichia coli can be directly used for synthesizing lupulone, so that a brand-new synthesis method is provided for a lupulone synthesis technology, the lupulone synthesis method is a biosynthesis method, the lupulone synthesis method can avoid the limitation of hop raw materials, the retention of biological activity which is difficult to realize by chemical synthesis is realized, the selectivity of lupulone prepared by utilizing engineering strains is higher than that of a natural product method, and the product only contains lupulone and does not contain lupulone derivatives.

In still another aspect, the invention provides an application of engineering escherichia coli in biological lupulone synthesis, which comprises the following steps: taking the engineering escherichia coli obtained by the construction method for producing the engineered escherichia coli for lupulone, fermenting, centrifuging, filtering, freeze-drying, extracting and purifying to obtain the lupulone.

The invention starts from the existing engineering strains in a laboratory, utilizes the metabolic engineering technology, introduces key enzyme genes in the lupulone synthesis way into engineering escherichia coli strains through a vector, and constructs fermentation strains for totally biologically synthesizing lupulone; the obtained engineering escherichia coli is subjected to fermentation, centrifugal filtration, freeze drying and extraction and purification to obtain lupulone, which is a brand new lupulone synthesis method; the lupulone synthesis method disclosed by the invention is simple to operate, short in flow and low in cost, gets rid of dependence on hop raw materials, can realize large-scale production, avoids large consumption of solvents, fully retains the bioactivity of lupulone, is high in selectivity, and the obtained product only contains lupulone and does not contain lupulone derivatives, so that the lupulone is good in purity and stable in quality.

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 10-15%, adjusting the pH value to be neutral by adopting 10% sodium hydroxide, carrying out shake culture at 37 ℃ and 220r/min for 12h, adding an inducer isopropyl thiogalactoside (IPTG), continuing shake culture, sampling every 12h, measuring the OD value, adjusting the pH value, and ensuring the culture period to be 90 h. 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 to promote the rapid progress of the fermentation reaction.

In a preferred embodiment, in the fermentation process, after inoculation, 7.5mL/L of mevalonic acid and 5mL/L of leucine are added firstly, shaking culture is carried out for 12h, an inducer is added, shaking culture is continued for 12h, 5g/L of yeast extract supplement, 1g/L of betaine supplement and 5mL/L of leucine are added, further shaking culture is continued for 12h, and then 5mL/L of leucine is added; the mevalonic acid is prepared by adjusting the pH of mevalonolactone to 7.0 with 1M NaOH, and sterilizing with high pressure steam at 121 deg.C for 25 min; the leucine is prepared by dissolving 0.525g/L of leucine in 30mL of vRO water, filtering with 0.22 μm sterile filter membrane, and storing at-20 deg.C; the yeast extract supplementary liquid is prepared by dissolving 2g of yeast extract in 10mL of pure water, and sterilizing with high-pressure steam at 121 ℃ for 25 min; the betaine supplementary liquid is prepared by dissolving 0.5g betaine in 5mL pure water, and sterilizing with high pressure steam at 121 deg.C for 25 min. According to the invention, the yield of the lupulone can be increased by additionally adding leucine and mevalonic acid in the process of shake flask fermentation, and the thallus density OD600 is increased from 4.70 to 14.00 by about 198%.

In a preferred embodiment, the fermentation medium in the shake flask fermentation process is: na (Na)₂HPO₄·12H₂O30g/L，KH₂PO₄6g/L，NH₄Cl 2g/L, NaCl 1g/L, glucose 4g/L, citric acid 1g/L, yeast extract 2g/L, ampicillin 0.15g/L, chloramphenicol 0.1g/L, and water in balance, and sterilizing with 115 deg.C high pressure steam for 30 min. The fermentation medium has good biocompatibility, convenient use and good cultivation effect.

In a preferred embodiment, the centrifugal filtration is performed at a centrifugal speed of 10000-50000r/min, at a centrifugal temperature of 5 ℃ or below, and for a centrifugal time of 15-30 min. 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 a vacuum degree of 10Pa, at a drying temperature of-80 ℃ and for a drying time of 12 hours. The invention adopts a low-temperature freeze drying mode to dry the thalli, has low drying temperature and high drying efficiency, and fully ensures the bioactivity of the lupulone.

As a preferred embodiment, the specific steps of the extraction and purification are as follows: adding n-hexane, refluxing at 80-90 deg.C for 6-9 hr, and drying. In the invention, the freeze-dried thallus is purified by normal hexane reflux, and a rotary evaporator is adopted to dry and remove the solvent, thereby obtaining the purified lupulone.

In a further aspect, the lupulone of the present invention is a lupulone obtained from the use of an engineered escherichia coli according to any one of the above in the biosynthesis of lupulone. The lupulone 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 bioactivity of lupulone, and has the advantages of high selectivity, good purity and stable quality.

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 lupulone synthesis way into engineering escherichia coli strains through a vector, and constructs fermentation strains for totally biologically synthesizing lupulone; the obtained strain is fermented, centrifugally filtered, freeze-dried, extracted and purified to obtain lupulone, which is a brand new lupulone biosynthesis method; the lupulone synthesis method disclosed by the invention is simple to operate, short in flow and low in cost, gets rid of dependence on hop raw materials, can realize large-scale production, avoids large consumption of solvents, fully retains the bioactivity of lupulone, is high in selectivity, and the obtained product only contains lupulone and does not contain lupulone derivatives, so that the lupulone is good in purity and stable in quality.

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 diagram showing the UV absorption spectrum of lupulone obtained in example III of the present invention;

FIG. 2 is a diagram showing the UV absorption spectrum of lupulone extracted from hops by a conventional chemical method;

FIG. 3 is a graph showing the UV absorption spectrum of a commercially available analytically pure lupulone;

FIG. 4 is a high performance liquid chromatography of lupulone obtained in example III of the present invention;

FIG. 5 is a high performance liquid chromatography of lupulone extracted from hops by prior art chemical methods;

FIG. 6 is a high performance liquid chromatography of commercially available analytical pure lupulone;

FIG. 7 is a hydrogen spectrum of lupulone obtained in example III of the present invention;

FIG. 8 is a hydrogen spectrum of lupulone extracted from hops by a prior chemical method;

FIG. 9 is a hydrogen spectrum of a commercially available analytically pure lupulone;

FIG. 10 is an LC-MS spectrum of lupulone obtained in example III of the present invention;

FIG. 11 is a LC-MS spectrum of commercially available analytically pure lupulone.

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 lupulone, which comprises the following steps:

1) constructing an evolved gene of isoprene pyrophosphate isomerase originated from engineering escherichia coli and an evolved gene of prenyltransferase originated from hops into a vector plasmid pTrcHis2B by taking pTrcHis2B as a starting plasmid to obtain an expression vector pTrcHis2B-idi-pt1, wherein the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO. 1, and the nucleotide sequence of the evolved gene of the prenyltransferase is shown as SEQ ID NO. 2;

2) constructing genes derived from hop after the evolution of phloroglucinol synthase, genes derived from the evolution of cytoplasmic coenzyme A ligase and gene segments derived from prenyltransferase into a vector plasmid pACYCDuet-1 by taking pACYCDuet-1 as a starting plasmid to obtain an expression vector pACYCDuet-1-ccl2-vps-pt2, wherein the nucleotide sequence of the genes derived from the phloroglucinol synthase is shown as SEQ ID NO. 3, the nucleotide sequence of the genes derived from the cytoplasmic coenzyme A ligase is shown as SEQ ID NO. 4, and the nucleotide sequence of the genes derived from the prenyltransferase is shown as SEQ ID NO. 5;

3) and (2) transferring the expression vector pTrcHis2B-idi-pt1 obtained in the step 1) and the expression vector pACYCDuet-1-ccl2-vps-pt2 obtained in the step 2) into engineering escherichia coli BL21(DE3) -Trc-low to obtain the engineering escherichia coli for producing lupulone.

The engineered escherichia coli for producing lupulone is obtained according to the construction method of the engineered escherichia coli for producing lupulone.

The application of the engineering escherichia coli in the biological synthesis of lupulone comprises the following steps: taking the engineering escherichia coli obtained by the construction method for producing the engineered escherichia coli for lupulone, fermenting, centrifuging, filtering, freeze-drying, extracting and purifying to obtain the lupulone.

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 10-15%, adjusting the pH value to be neutral by adopting 10% sodium hydroxide, carrying out shake culture at 37 ℃ and 220r/min for 12h, adding an inducer isopropyl thiogalactoside (IPTG), continuing shake culture, sampling every 12h, measuring the OD value, adjusting the pH value, and ensuring the culture period to be 90 h.

Further, in the fermentation process, after inoculation, 7.5mL/L mevalonic acid and 5mL/L leucine are added firstly, shaking culture is carried out for 12h, an inducer is added, shaking culture is continued for 12h, a yeast extract supplement liquid of 5g/L, a betaine supplement liquid of 1g/L and leucine of 5mL/L are added, further shaking culture is continued for 12h, and then 5mL/L leucine is added; the mevalonic acid is prepared by adjusting the pH of mevalonolactone to 7.0 with 1M NaOH, and sterilizing with high pressure steam at 121 deg.C for 25 min; the leucine is prepared by dissolving 0.525g/L leucine in 30mL RO water, filtering with 0.22 μm sterile filter membrane, and storing at-20 deg.C; the yeast extract supplementary liquid is prepared by dissolving 2g of yeast extract in 10mL of pure water, and sterilizing with high-pressure steam at 121 ℃ for 25 min; the betaine supplementary liquid is prepared by dissolving 0.5g betaine in 5mL pure water, and sterilizing with high pressure steam at 121 deg.C for 25 min.

Specifically, in the shake flask fermentation process, the fermentation medium is: na (Na)₂HPO₄·12H₂O 30g/L，KH₂PO₄6g/L，NH₄Cl 2g/L, NaCl 1g/L, glucose 4g/L, citric acid 1g/L, yeast extract 2g/L, ampicillin 0.15g/L, chloramphenicol 0.1g/L, and water in balance, and sterilizing with 115 deg.C high pressure steam for 30 min.

Preferably, during the centrifugal filtration, the centrifugal rotation speed is 10000-50000r/min, the centrifugal temperature is below 5 ℃, and the centrifugal time is 15-30 min.

Preferably, the vacuum degree is 10Pa, the drying temperature is-80 ℃ and the drying time is 12h during freeze drying.

Preferably, the specific steps of the extraction and purification are as follows: adding n-hexane, refluxing at 80-90 deg.C for 6-9 hr, and drying.

The lupulone is obtained by applying the engineering escherichia coli in biological synthesis of the lupulone.