Alpha-bisabolene synthetase mutant, genetically engineered bacterium for high yield of alpha-bisabolene and application of genetically engineered bacterium

Abstract

The invention discloses an alpha-bisabolene synthetase mutant, a genetically engineered bacterium for high yield of alpha-bisabolene and application of the genetically engineered bacterium, and belongs to the technical field of bioengineering. The purpose of the present invention is to produce alpha-bisabolene and to produce alpha-bisabolene with high yield. The invention discloses an alpha-bisabolene synthase mutant, which is characterized in that SEQ ID NO.2 is used as a starting sequence, and H at the site 806 is mutated into L. The invention further discloses a preparation method of the alpha-bisabolene synthase mutant. And a theoretical basis is provided for the production of alpha-bisabolene.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an α-bisabolene synthase mutant and a genetically engineered bacterium that produces high levels of α-bisabolene, and their applications. Background Technology

[0002] Bisabolene, also known as red bisabolene or sweet bisabolene, is a monocyclic sesquiterpene compound composed of three isopentenyl units, with the molecular formula C2. 15 H 24 Myrrhene has three isomers: α-myrrhene, β-myrrhene, and γ-myrrhene. Due to its unique fruity and balsam-like aroma, myrrhene is widely used as a high-value flavoring and fragrance compound, and is highly sought after in the food and cosmetics industries. Myrrhene also has significant applications in medicine, possessing anti-inflammatory, anti-allergic, sedative, and anticancer properties. Furthermore, because its properties are similar to traditional aviation fuels, myrrhene is considered a promising new type of biofuel.

[0003] The main production methods for commercial bisabolene are plant extraction and chemical synthesis. Due to the low bisabolene content in plants and the limitations imposed by arable land, climate, pests, and growth cycles on plant cultivation and growth, the cost of extracting bisabolene from plants is high, and the product is often a mixture of multiple isomers. Chemical synthesis of bisabolene is complex, inefficient, energy-intensive, and highly polluting. With the development of metabolic engineering and synthetic biology, the production of bisabolene using microorganisms as chassis cells has been achieved. However, the reported yields of bisabolene-synthesizing microorganisms are all low and cannot meet market demand. Currently, the highest bisabolene yield in *Saccharomyces cerevisiae* is 18.6 g / L, and the highest yield obtained using *Yarrowia lipolytica* is 15.5 g / L, which is also the highest yield achieved in the laboratory to date. In *Escherichia coli*, the highest yield is only 1150 mg / L. Since bisabolene itself is not toxic to common hosts, it is hoped that its yield can be further increased through genetic engineering or improvements in fermentation technology. Summary of the Invention

[0004] The purpose of this invention is to produce α-bimethene and to produce high-yield α-bimethene.

[0005] This invention provides an α-bisabolene synthase mutant, wherein the mutant is based on SEQ ID NO.2, and the H at position 806 is mutated to L.

[0006] The present invention provides a gene encoding the above-mentioned mutant.

[0007] This invention provides a recombinant vector containing the above-mentioned genes.

[0008] This invention provides a recombinant microbial cell containing the above-mentioned genes.

[0009] The present invention provides the application of the above-mentioned mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microorganism in the preparation of α-bisabolene.

[0010] This invention provides a genetically engineered bacterium that produces high levels of α-bimethanone. Using *Escherichia coli* as the starting strain, it overexpresses the following genes: acetyl-CoA acyltransferase / HMG-CoA reductase *mvaE*, HMG-CoA synthase *mvaS*, mevalonate kinase *ERG12*, mevalonate-5-phosphate kinase *ERG8*, mevalonate-5-bisphosphate decarboxylase *ERG19*, isopentenyl diphosphate isomerase *IDI*, farnesyl diphosphate synthase *ispA*, and the aforementioned genes.

[0011] This invention provides a genetically engineered bacterium that produces high levels of α-bimethene, using *Escherichia coli* as the starting strain, overexpressing the following genes: acetyl-CoA acyltransferase / HMG-CoA reductase *mvaE*, HMG-CoA synthase *mvaS*, mevalonate kinase *ERG12*, mevalonate-5-phosphate kinase *ERG8*, mevalonate-5-bisphosphate decarboxylase *ERG19*, isopentenyl diphosphate isomerase *IDI*, farnesyl diphosphate synthase *ispA*, the above-mentioned genes, and the *vgb* gene derived from *Vitreoscilla*. The nucleotide sequence of the *vgb* gene derived from *Vitreoscilla* is shown in SEQ ID NO. 5.

[0012] This invention provides a genetically engineered bacterium that produces high levels of α-bimethene. Using *Escherichia coli* as the starting strain, it overexpresses the following genes: acetyl-CoA acyltransferase / HMG-CoA reductase *mvaE*, HMG-CoA synthase *mvaS*, mevalonate kinase *ERG12*, mevalonate-5-phosphate kinase *ERG8*, mevalonate-5-bisphosphate decarboxylase *ERG19*, isopentenyl diphosphate isomerase *IDI*, farnesyl diphosphate synthase *ispA*, the aforementioned genes, the dCas9 gene, and the *vgb* gene derived from *Vitreoscilla*. It also knocks down the adenylate cyclase gene *cyaA*. The nucleotide sequence of the adenylate cyclase gene *cyaA* derived from *E. coli* is shown in SEQ ID NO. 6.

[0013] This invention provides the application of the above-mentioned genetically engineered bacteria in the preparation of α-bisabolene.

[0014] This invention provides a method for preparing α-bisabolene by adding the above-mentioned genetically engineered bacteria into a fermenter for fermentation.

[0015] Beneficial effects: The engineered Escherichia coli strain described in this invention yielded 29.49 g / L of α-bisabolene in a fermenter. Attached Figure Description

[0016] Figure 1 Myrrhene synthesis pathway; Figure 2 Plasmid maps: a) pET28a-AgBISH806L-ispA-IDI plasmid map; b) pACYC-mvaE-mvaS-vgb plasmid map; c) pACYC-mvaE-mvaS-vgb-dCas9 plasmid map; d) pET28a-AgBISH806L-ispA-IDI-gRNA plasmid map; Figure 3 Bisabolene yield of different strains at the shake-flask level; Figure 4 Myrrhene production at the fermenter level by strain F3. Detailed Implementation

[0017] The gene sequences of CoA acyltransferase / HMG-CoA reductase mvaE, HMG-CoA synthase mvaS, mevalonate kinase ERG12, mevalonate-5-phosphate kinase ERG8, mevalonate-5-bisphosphate decarboxylase ERG19, isopentenyl diphosphate isomerase IDI, and farnesyl pyrophosphate synthase ispA are described in the patent with publication number CN111607546 A.

[0018] SEQ ID NO.1, >AgBIS: SEQ ID NO.2,>AgBIS: MAGVSAVSKVSSLVCDLSSTSGLIRRTANPHPNVWGYDLVHSLKSPYIDSSYRERAEVLVSEIKAMLNPAITGDGESMITPSAYDTAWVARVPAIDGSARPQFPQTVDWILKNQLKDGSWGIQSHFLLSDRLLATLSCVLVLLKWNVGDLQVEQGIEFIKSNLELVKDETDQDSLVTDFE IIFPSLLREAQSLRLGLPYDLPYIHLLQTKRQERLAKLSREEIYAVPSPLLYSLEGIQDIVEWERIMEVQSQDGSFLSSPASTACVFMHTGDAKCLEFLNSVMIKFGNFVPCLYPVDLLERLLIVDNIVRLGIYRHFEKEIKEALDYVYRHWNERGIGWGRLNPIADLETTALGFRLLRLHRYNVSPAIFDNFKDANGKFICSTGQFNKDVASMLNLYRASQLAFPGENILDEAKSFATKYLREALEKSETSSAWNNKQNLSQEIKYALKTSWHASVPRVEAKRYCQVYRPDYARIAKCVYKLPYVNNEKFLELGKLDFNIIQSIHQEEMKNVTSWFRDSGLPLFTFARERPLEFYFLVAAGTYEPQYAKCRFLFTKVACLQTVLDDMYDTYGTLDELKLFTEAVRRWDLSFTENLPDYMKLCYQIYYDIVHEVAWEAEKEQGRELVSFFRKGWEDYLLGYYEEAEWLAAEYVPTLDEYIKNGITSIGQRILLLSGVLIMDGQLLSQEALEKVDYPGRRVLTELNSLISRLADDTKTYKAEKARGELASSIECYMKDHPECTEEEALDHIYSILEPAVKELTREFLKPDDVPFACKKMLFEETRVTMVIFKDGDGFGVSKLEVKDHIKECLIEPLPL* SEQ ID NO.3,>AgBIS(H806L): SEQ ID NO.4,>AgBIS(H806L): MAGVSAVSKVSSLVCDLSSTSGLIRRTANPHPNVWGYDLVHSLKSPYIDSSYRERAEVLVSEIKAMLNPAITGDGESMITPSAYDTAWVARVPAIDGSARPQFPQTVDWILKNQLKDGSWGIQSHFLLSDRLLATLSCVLVLLKWNVGDLQVEQGIEFIKSNLELVKDETDQDSLVTDFE IIFPSLLREAQSLRLGLPYDLPYIHLLQTKRQERLAKLSREEIYAVPSPLLYSLEGIQDIVEWERIMEVQSQDGSFLSSPASTACVFMHTGDAKCLEFLNSVMIKFGNFVPCLYPVDLLERLLIVDNIVRLGIYRHFEKEIKEALDYVYRHWNERGIGWGRLNPIADLETTALGFRLLRLHRYNVSPAIFDNFKDANGKFICSTGQFNKDVASMLNLYRASQLAFPGENILDEAKSFATKYLREALEKSETSSAWNNKQNLSQEIKYALKTSWHASVPRVEAKRYCQVYRPDYARIAKCVYKLPYVNNEKFLELGKLDFNIIQSIHQEEMKNVTSWFRDSGLPLFTFARERPLEFYFLVAAGTYEPQYAKCRFLFTKVACLQTVLDDMYDTYGTLDELKLFTEAVRRWDLSFTENLPDYMKLCYQIYYDIVHEVAWEAEKEQGRELVSFFRKGWEDYLLGYYEEAEWLAAEYVPTLDEYIKNGITSIGQRILLLSGVLIMDGQLLSQEALEKVDYPGRRVLTELNSLISRLADDTKTYKAEKARGELASSIECYMKDHPECTEEEALDHIYSILEPAVKELTREFLKPDDVPFACKKMLFEETRVTMVIFKDGDGFGVSKLEVKDLIKECLIEPLPL*; SEQ ID NO.5,>vgb: ATGCTGGATCAGCAGACCATTAATATCATTAAAGCGACCGTGCCGGTGCTGAAAGAACATGGCGTGACCATTACCACCACCTTTTATAAAAATCTGTTCGCGAAACACCCGGAAGTGCGCCCGTTGTTTGATATGGGCCGCCAGGAAAGCCTGGAACAGCCGAAAGCGCTGGCGATGACCGTGCTGGCAGCGGCGCAAAATATTGAAAATCTGCCGGCGATTCTGCCGGCGGTGAAAAAGATTGCGGTGAAACATTGCCAGGCGGGCGTGGCGGCGGCGCATTATCCAATTGTTGGCCAAGAACTGCTGGGCGCGATTAAAGAAGTGCTGGGCGATGCGGCGACCGATGATATTCTGGATGCGTGGGGCAAAGCGTATGGCGTGATTGCGGATGTGTTTATTCAGGTGGAAGCGGATCTGTATGCGCAGGCGGTGGAATAA; SEQ ID NO.6,>cyaA: SEQ ID NO.7,>dCas9:

[0019] The detection methods involved in the following embodiments are as follows: (1) Sample pretreatment: In order to analyze the yield of bisabolene during fermentation, samples were taken every 24 h. The samples were centrifuged at 14600 rpm for 15 min to collect the upper organic phase, which was then filtered through a 0.22 μm organic filter membrane.

[0020] (2) GC detection conditions: The organic phase of the fermentation sample was quantitatively analyzed by gas chromatography (GC, SHIMADZU GC-2014) equipped with a flame ionization detector (FID). The chromatographic column was a DB-5MS column (30 m × 0.25 mm × 0.25 μm). The injection volume was 1 μL, the injector temperature was 260℃, and the detector temperature was 300℃. The column oven temperature was initially 80℃ and held for 1 min, then increased to 250℃ at 10℃ / min and held for 1 min, then increased to 300℃ at 30℃ / min and held for 1 min. (3) Quantification of bisabolene: β-caryophyllene was added to all samples as an internal standard for quantification. β-caryophyllene was added to the filtered organic phase sample to make the final concentration 1 g / L.

[0021] The primer sequences involved in the following examples are shown in Table 1.

[0022] Table 1 Primer Sequences

[0023] Example 1: Construction method of genetically engineered bacteria for synthesizing bisabolene 1. Construction of expression vector for α-bisabolene synthase AgBIS and its mutant AgBISH806

[0024] Table 2 PCR amplification system

[0025] PCR reaction conditions: 98℃ for 2 min; 98℃ for 10 s, (Tm-5)℃ for 15 s, 72℃ for 15 s / kb, 35 cycles; 72℃ for 5 min.

[0026] (2) Construction of AgBIS mutant expression vector: Using plasmid pET28a-AgBIS-ispA-IDI as a template, the entire plasmid was amplified using complementary primers H806L-F and H806L-R containing the mutation site. The PCR amplification system and procedure are shown in Table 2, except that the amplification volume was reduced from 50 μL to 20 μL, and the annealing temperature was 60℃. After the reaction, restriction endonuclease DpnⅠ was added to the PCR product, and the mixture was digested at 37℃ for 30 min to remove the template plasmid. The digested product was transformed into E. coli competent cells trans1-T1, plated on LB agar plates containing Kan, and cultured at 37℃ until single colonies grew. Single colonies were picked and sent to the company for sequencing. If the sequencing was correct, the plasmid pET28a-AgBIS-ispA-IDI was obtained.

[0027] 2. Construction of plasmid pACYC-mvaE-mvaS-vgb The vgb gene sequence from Vitreoscilla was optimized according to the codon preference of E. coli, and the whole gene was synthesized by BGI Genomics and cloned into the pET28a vector to obtain the plasmid pET28a-vgb. The plasmid pACYC-mvaE-mvaS-vgb was constructed using an enzyme digestion-ligation method. Using plasmid pET28a-vgb as a template, the vgb gene was amplified using primers 1961-BglII-VHB-F and 1961-XhoI-VHB-R. The PCR product was subjected to agarose gel electrophoresis and gel extraction to obtain the vgb gene fragment. The plasmid pACYC-mvaE-mvaS and the vgb gene fragment were digested with BglII and XhoI. The enzyme digestion system is shown in Table 3. Table 3 Double enzyme digestion system

[0028] The enzyme digestion reaction was carried out at 37°C for 2 h. X: 10 μL of plasmid was added, and 40 μL of the gel-recovered fragment was added.

[0029] The enzyme digestion products were subjected to agarose gel electrophoresis, and the vgb gene fragment and pACYC-mvaE-mvaS vector fragment were recovered by gel excision. The recovered products were then subjected to ligation reaction, and the ligation system is shown in Table 4. Table 4 Connection System

[0030] The connection system was incubated overnight at 16°C.

[0031] The above ligation product was transformed into E. coli competent cells trans1-T1, plated on LB plates containing Cm, and cultured at 37°C until single colonies grew. Single colonies were picked for colony PCR identification or plasmid extraction and enzyme digestion identification. Positive clones were sent to the company for sequencing. If the sequencing was correct, the plasmid pACYC-mvaE-mvaS-vgb was obtained.

[0032] 3. Construction of the CRISPRi system The CRISPRi system in this invention consists of two CRISPRi plasmids: pACYC-mvaE-mvaS-vgb-dCas9 and pET28a-AgBISH806L-ispA-IDI-gRNA. pACYC-mvaE-mvaS-vgb-dCas9 expresses the dCas9 gene under the drive of the T7 promoter, and pET28a-AgBISH806L-ispA-IDI-gRNA expresses gRNA under the drive of the constitutive promoter J23119.

[0033] (1) Construction of pACYC-mvaE-mvaS-vgb-dCas9 plasmid: Using pRS416-dCas9-Mxi1+TetR+pRPR1(TetO)-NotI-gRNA plasmid (Addgene: 73796) as a template, the dCas9 gene was amplified using primers 1961-dCas9-F / 1961-dCas9-R according to the system in Table 2. Similarly, using plasmid pACYC-mvaE-mvaS-vgb as a template, primers 1961-vector-F / 1961-vec-R were used for amplification according to the system in Table 2. The PCR products were subjected to agarose gel electrophoresis and gel extraction to obtain the dCas9 gene fragment and the pACYC-mvaE-mvaS-vgb vector fragment. These fragments were then ligated using a seamless cloning kit; the ligation system and reaction conditions were as per the kit's instructions. The ligation product was transformed into E. coli competent cells trans1-T1, plated on LB agar plates containing Cm, and cultured at 37°C until single colonies grew. Single colonies were picked for colony PCR identification or plasmid extraction and enzyme digestion identification. Positive clones were sent to the company for sequencing. If the sequencing was correct, the plasmid pACYC-mvaE-mvaS-vgb-dCas9 was obtained.

[0034] (2) Construction of pGRB-cyaA-gRNA plasmid: Plasmid pGRB (Addgene: 71539) contains a complete gRNA expression system. In order to obtain a gRNA expression cassette that inhibits cyaA expression, the target sequence (N20) was first designed using the software "sgRNAcas9_V3.0_GUI". The sequence is as follows: cyaA-N20: ATGCAGCCTGCTGGAAAGCTGGG (SEQ ID NO. 27); Primers cyaA3-sgRNA-F / cyaA3-sgRNA-R were designed based on the N20 sequence. Using pGRB plasmid as a template, amplification was performed according to the system shown in Table 2. The PCR products were subjected to agarose gel electrophoresis, and the target band was purified using a gel extraction kit. The purified product was then ligated using the Golden Gate Assembly method. The ligation system is shown in Table 5. Table 5 Golden Gate Assembly System

[0035] Reaction conditions: 37℃ for 5 min, 25℃ for 5 min, for a total of 6 cycles; 37℃ for 30 min; 65℃ for 5 min.

[0036] The above ligation product was transformed into E. coli competent cells trans1-T1, plated on LB plates containing Amp, and cultured at 37°C until single colonies grew. Single colonies were picked for colony PCR identification. Positive clones were sent to the company for sequencing. If the sequencing was correct, the plasmid pGRB-cyaA-gRNA was obtained.

[0037] (3) Construction of pET28a-AgBISH806L-ispA-IDI-gRNA plasmid: Using pGRB-cyaA-gRNA as a template, the gRNA expression cassette sequence (including the promoter J23119 that drives gRNA expression, the N20 sequence that specifically binds to the target gene, and the gRNA scaffold sequence that binds to dCas9 protein) was amplified using primers 28a-gRNA-F / 28a-gRNA-R according to the system in Table 2. Using plasmid pET28a-AgBISH806L-ispA-IDI as a template, the system was amplified using primers GA-pET28a3FS-F / GA-IDItail-R according to the system in Table 2. The PCR products were subjected to agarose gel electrophoresis and gel extraction to obtain the gRNA fragment (ttgacagctagctcagtcctaggtataatactagtatgcagcctgctggaaagctgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcacc, SEQ ID NO. 28) and the pET28a-AgBISH806L-ispA-IDI vector fragment. These fragments were ligated using a seamless cloning kit, and the proportions and amounts of each component were calculated according to the manufacturer's instructions.

[0038] The above ligation product was transformed into E. coli competent cells trans1-T1, plated on LB agar containing Kan, and cultured at 37°C until single colonies grew. Single colonies were picked for colony PCR identification. Positive clones were sent to the company for sequencing. If the sequencing was correct, the plasmid pET28a-AgBISH806L-ispA-IDI-gRNA was obtained.

[0039] 4. Plasmid transformation: (1) The correctly sequenced plasmid pET28a-AgBISH806L-ispA-IDI, along with plasmid pACYC-mvaE-mvaS and plasmid pTrc-ERG12-ERG8-ERG19, were transformed into Escherichia coli BL21(DE3). The plasmids were then plated on LB agar plates containing the corresponding triple antibodies (Amp, Kan, and Cm) and cultured at 37°C until single colonies appeared, thus obtaining the bisabolene-synthesizing genetically engineered bacterium F1. (2) The correctly sequenced plasmids pET28a-AgBISH806L-ispA-IDI, pACYC-mvaE-mvaS-vgb and plasmid pTrc-ERG12-ERG8-ERG19 were transformed into Escherichia coli BL21(DE3), plated on LB medium plates with the corresponding triple antibodies (Amp, Kan and Cm), and cultured at 37°C until single colonies grew to obtain the genetically engineered bacterium F2 that synthesizes bisabolene; (3) The correctly sequenced plasmids pET28a-AgBISH806L-ispA-IDI-gRNA, pACYC-mvaE-mvaS-vgb-dCas9 and plasmid pTrc-ERG12-ERG8-ERG19 were transformed into Escherichia coli BL21(DE3), plated on LB medium plates with the corresponding triple antibodies (Amp, Kan and Cm), and cultured at 37°C until single colonies grew to obtain the genetically engineered bacterium F3 with high bisabolene production.

[0040] qRT-PCR results showed that cyaA expression was reduced by approximately 15% in F3 compared to F2. Comparative Example 1. The difference from Example 1 is that, except for step 4, the rest are the same. In this comparative example, the plasmid transformation in step 4 involves transforming plasmids pET28a-AgBIS-ispA-IDI, pACYC-mvaE-mvaS, and pTrc-ERG12-ERG8-ERG19 into Escherichia coli BL21(DE3), plasmids pET28a-AgBIS-ispA-IDI, pACYC-mvaE-mvaS, and pTrc-ERG12-ERG8-ERG19 together into Escherichia coli BL21(DE3), plating the corresponding triple-antibiotic (Amp, Kan, and Cm) LB agar plates, and culturing at 37°C until single colonies grow, thus obtaining the bisabolene-producing genetically engineered bacterium F0.

[0041] Example 2. Application of the constructed genetically engineered bacteria in the synthesis of bisabolene 1. The primary seed culture medium described in this embodiment is LB medium, the components of which are: 10 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract, and the remainder is water; The secondary seed culture medium and shake-flask fermentation culture medium in this embodiment consist of: 20 g / L glucose, 9.8 g / L K2HPO4, 5 g / L beef extract, 0.3 g / L ferric ammonium citrate, 2.1 g / L citric acid monohydrate, 0.06 g / L MgSO4, 1 mL / L trace element solution, and the remainder being water; the trace element solution contains (NH4)6Mo7O24·4H2O 0.37 g / L, ZnSO4·7H2O 0.29 g / L, H3BO3 2.47 g / L, CuSO4·5H2O 0.25 g / L and MnCl2·4H2O 1.58 g / L.

[0042] The fermenter culture medium is based on the shake flask culture medium, with the trace element solution adjusted to 1.5 mL / L, and the addition of betaine and ammonium sulfate at a final concentration of 1 g / L.

[0043] 2. Taking shake-flask fermentation as an example, the application of the genetically engineered bacteria constructed in Example 1 in the synthesis of bisabolene is described: A single colony of the genetically engineered bacteria obtained in Example 1 was picked and placed into 5 mL of LB medium containing the corresponding resistance (Cm / Amp / Kan), and cultured in a shaker at 37°C for 8–12 h to obtain a primary seed culture. Fermentation was carried out in a 250 mL shake flask with baffles. 1% of the primary seed culture was transferred to 50 mL of shake-flask fermentation medium containing the corresponding resistance (Cm, Amp, and Kan), and cultured at 37°C until the OD600 reached approximately 0.6–0.9. IPTG at a final concentration of 0.1 mM and 20% (v / v) of the extractant n-dodecane were added, and the mixture was cultured in a shaker at 30°C. Samples were taken at 24 h and 48 h, and the OD600 was measured using a spectrophotometer. The bisabolene yield was determined by gas chromatography, with three replicates.

[0044] Control group: Single colonies obtained from Comparative Example 1 were selected and bisabolene was synthesized using the shake-flask fermentation method described above. Samples were taken at 24 h and 48 h, and the OD600 was measured by spectrophotometer. The bisabolene yield was determined by gas chromatography. Three replicates were set up.

[0045] As attached Figure 3 As shown, after 48 h of shake-flask fermentation, the bisabolene yields in strains F0, F1, F2, and F3 were 1.02 g / L, 1.29 g / L, 1.69 g / L, and 2.38 g / L, respectively.

[0046] 3. Taking fed-batch fermentation as an example, describe the application of the genetically engineered strain F3 constructed in Example 1 in the synthesis of bisabolene: Primary seed culture: Pick a single colony of the genetically engineered bacteria F3 constructed in Example 1 and put it into 5 mL of LB medium containing the corresponding resistance (Cm / Amp / Kan). Incubate at 37°C in a shaker for 8–12 h to obtain the primary seed culture.

[0047] Secondary seed culture: Transfer 2 mL of primary seed culture to 200 mL of secondary seed culture medium containing the corresponding resistance (Cm / Amp / Kan) and incubate overnight at 37°C in a shaker.

[0048] Antibiotic stock solution, trace element solution, glucose stock solution, and 200 mL of secondary seed culture were added to a 5 L fermenter (BIOTECH-5JG-7000A) containing 2 L of fermentation medium. The aeration rate was set to 3 vvm, the temperature to 37℃, the pH to 6.5, and the fermentation speed was correlated with dissolved oxygen. After the initial sugar was depleted, feeding (70% glucose) was initiated, and the temperature was gradually lowered to 30℃. Once the temperature stabilized, IPTG (final concentration 0.1 mM) and 400 mL of n-dodecane were added. The fermentation speed was gradually reduced to 400 r / min, and fermentation was continued for 120 h before discharge. The pH was adjusted using 50% ammonia during the growth process. Residual sugar, OD600, and bisabolene yield were measured intermittently. (See attached image) Figure 4 As shown, the bisabolene yield reached 29.49 g / L 120 h after induction, exceeding the highest reported yield of 15.5 g / L for microbial synthesis in the current literature.

[0049] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the concept of the present invention should be covered within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An α-bisabolene synthase mutant, characterized in that, The mutant is based on SEQ ID NO.2, with the 806th position H mutated to L.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector containing the gene of claim 2.

4. A recombinant microbial cell containing the gene of claim 2.

5. The use of the mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant microorganism of claim 4 in the preparation of α-bisabolene.

6. A genetically engineered bacterium that produces high levels of α-bisabolene, characterized in that, Using Escherichia coli as the starting strain, the following genes were overexpressed: acetyl-CoA acyltransferase / HMG-CoA reductase mvaE gene, HMG-CoA synthase mvaS gene, mevalonate kinase ERG12 gene, mevalonate-5-phosphate kinase ERG8 gene, mevalonate-5-bisphosphate decarboxylase ERG19 gene, isopentenyl diphosphate isomerase IDI gene, farnesyl diphosphate synthase ispA gene, and the genes described in claim 2.

7. A genetically engineered bacterium that produces high levels of α-bisabolene, characterized in that, Using *Escherichia coli* as the starting strain, the following genes were overexpressed: acetyl-CoA acyltransferase / HMG-CoA reductase mvaE gene, HMG-CoA synthase mvaS gene, mevalonate kinase ERG12 gene, mevalonate-5-phosphate kinase ERG8 gene, mevalonate-5-bisphosphate decarboxylase ERG19 gene, isopentenyl diphosphate isomerase IDI gene, farnesyl diphosphate synthase ispA gene, the genes described in claim 2, and the vgb gene derived from *Vitreoscilla*; the nucleotide sequences of the vgb gene derived from *Vitreoscilla* are shown in SEQ ID NO.

5.

8. A genetically engineered bacterium that produces high levels of α-bisabolene, characterized in that, Using *Escherichia coli* as the starting strain, the following genes were overexpressed: acetyl-CoA acyltransferase / HMG-CoA reductase mvaE gene, HMG-CoA synthase mvaS gene, mevalonate kinase ERG12 gene, mevalonate-5-phosphate kinase ERG8 gene, mevalonate-5-bisphosphate decarboxylase ERG19 gene, isopentenyl diphosphate isomerase IDI gene, farnesyl diphosphate synthase ispA gene, the genes described in claim 2, dCas9 gene, and vgb gene derived from *Vitreoscilla*, while the adenylate cyclase gene cyaA was knocked down; the nucleotide sequence of the adenylate cyclase gene cyaA derived from *E. coli* is shown in SEQ ID NO.

6.

9. The use of the genetically engineered bacteria according to any one of claims 6-8 in the preparation of α-bisabolene.

10. A method for preparing α-bisabolene, characterized in that, The genetically engineered bacteria described in any one of claims 6-8 are added to a fermenter for fermentation.

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