A genetically engineered bacterium for producing amber soft sweet essence in high yield, a construction method and application thereof

Abstract

This invention discloses a genetically engineered bacterium producing high-yield ambergris sweet flavoring, its construction method, and its applications, relating to the field of genetic engineering technology. This engineered strain was prepared by expressing a tobacco-derived diterpenoid synthase, TPS103, in *Escherichia coli*. The amino acid sequence of tobacco diterpenoid synthase TPS103 is shown in SEQ ID NO.1, and the gene sequence is shown in SEQ ID NO.2. The diterpenoid synthase TPS103 was recombined into an *E. coli* expression vector, and then transformed into an engineered bacterium containing both the MVA pathway and the GGPP high-energy synthesis pathway, resulting in a genetically engineered bacterium producing fir alcohol. GC-MS analysis demonstrated that the fir alcohol yield produced by this genetically engineered bacterium was 353 mg / L, solving the problems of insufficient key enzyme activity and low yield in existing fir alcohol synthesizing engineered bacteria.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and relates to the genetic engineering of a high-yield ambergris sweet flavoring, its construction method and application. Background Technology

[0002] Abienol, a natural diterpenoid alcohol, is mainly found in plants such as balsam fir (Abies balsamea) and tobacco. It is an important raw material in the fragrance industry, especially in the field of sustainable alternatives to ambergris, where it has key application value. In tobacco, abienol is one of the core aroma components secreted by leaf glandular trichomes. Taking the flue-cured tobacco variety Da Bai Jin 599 as an example, its surface secretions are rich in abienol, exhibiting a unique aroma characteristic similar to aromatic tobacco, with a significantly superior aroma quality and quantity compared to ordinary flue-cured tobacco. During tobacco processing, curing, and combustion, abienol undergoes oxidative degradation (conversion rate exceeding 90%) to generate C-16 compounds such as ambergris ether and ambergris lactone, releasing a strong ambergris aroma, which is the core substance that gives cigarettes their "ambergris-like sweet aroma." Adding abienol to cigarette products can effectively neutralize the irritating off-flavors produced during combustion, soften the smoke and highlight the natural aroma of tobacco, enhance the sweetness and smoothness, improve the mellowness and reduce the spiciness, and significantly improve smoking comfort.

[0003] Currently, the production of taurine heavily relies on plant extraction, but this technology faces multiple bottlenecks: plants contain low levels of taurine and are accompanied by numerous functionalized derivatives, leading to significant technical difficulties, high production costs, and low efficiency in large-scale extraction and purification. Furthermore, solvent use can easily cause environmental pollution. Therefore, developing alternative production methods has become an urgent need for the industry. Compared with traditional methods, microbial synthesis demonstrates significant potential due to its advantages such as low cost, high yield, simple process, ease of scalability, and lack of seasonal or climatic limitations. *Escherichia coli*, as a model microorganism, is widely used as a biosynthetic host due to its short growth cycle, clear genetic background, and ease of molecular manipulation. However, existing engineered taurine-synthesizing bacteria are limited by factors such as insufficient activity of key enzymes, resulting in yields that have not yet met industrial production requirements and have not been practically applied.

[0004] It is noteworthy that tobacco possesses unique advantages in its ability to synthesize pine resin, but the discovery, identification, and application of key enzymes involved in pine resin synthesis within its genome are still in their early stages. Screening highly active synthetic enzymes from tobacco and optimizing the metabolic flux of engineered strains has become a core technical challenge in overcoming existing yield bottlenecks and achieving efficient pine resin biosynthesis. This invention aims to construct high-yield engineered strains by mining pine resin synthesis-related enzyme genes from tobacco and combining them with metabolic engineering optimization, providing an innovative solution for the green industrial production and application of pine resin. Summary of the Invention

[0005] This invention provides a genetically engineered bacterium that produces high levels of ambergris sweet flavor, along with its construction method and application, offering an innovative solution for the green industrial production and application of tamarind.

[0006] A genetically engineered bacterium for high-yield ambergris sweet flavoring, wherein the genetically engineered bacterium is an *E. coli*-based strain expressing the tobacco diterpene synthase TPS103 with the amino acid sequence shown in SEQ ID NO.2, and the encoding gene of the tobacco diterpene synthase TPS103. TPS103 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] Preferably, the genetically engineered bacteria also express the following gene: acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene. mvaE, 3-Hydroxy-3-methylglutaryl-CoA synthase gene mvaS, Farnesyl pyrophosphate synthase gene Erg20 Geraniol geraniol pyrophosphate synthase gene CrtE, fir alcohol synthase gene abs, Mevalonate kinase gene Erg12 Mevalonate-5-phosphate kinase gene Erg8 Mevalonate-5-bisphosphate decarboxylase gene Erg19 and isopentenyl pyrophosphate isomerase gene IDI1 .

[0008] Tobacco diterpene synthase encoding gene TPS103 Application in engineered bacteria for the preparation of taurine.

[0009] The application of the above-mentioned genetically engineered bacteria in the preparation of azadirachtin.

[0010] Preferably, the method steps are as follows: S1: Gene Cloning: Cloning the Tobacco Diterpenoid Synthesizer Gene TPS103 ; S2: Construction of recombinant plasmid I: The acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene was constructed. mvaE 3-Hydroxy-3-methylglutaryl-CoA synthase gene mvaS The plasmid was cloned into plasmid pACYCDuet-1 to obtain recombinant plasmid I; S2: Construction of recombinant plasmid II: The farnesyl pyrophosphate synthase gene was incorporated into the plasmid. Erg20 Geraniol geraniol pyrophosphate synthase gene CrtE Diterpene synthase gene TPS103 and fir alcohol synthase gene abs The plasmid was cloned into plasmid pCDFDuet-1 to obtain recombinant plasmid II; S3: Construction of Recombinant Plasmid III: Mevalonate kinase geneErg12 Mevalonate-5-phosphate kinase gene Erg8 Mevalonate-5-bisphosphate decarboxylase gene Erg19 and isopentenyl pyrophosphate isomerase gene IDI1 The plasmid was cloned into plasmid pTrcHis2B to obtain recombinant plasmid III; S4: Recombinant bacterial transformation: Recombinant plasmid I, recombinant plasmid II and recombinant plasmid III were co-transformed into competent cells of Escherichia coli BL21(DE3) to obtain engineered bacteria for the preparation of tamarind.

[0011] Preferably, the tobacco-derived diterpene synthase gene TPS103 The steps are as follows: RNA is extracted from tobacco leaf tissue, and reverse transcriptase is used to reverse transcribe the RNA into cDNA. The cDNA is then used as a template for PCR amplification of the target gene. The sequences of the PCR amplification primers are as follows: The forward primer is: 5'-ATGAACACCAGCAACTGGCTGAAAC-3'; The reverse primer is: 5'-TTACACCTGCTGAAACAGCACTTTG-3'.

[0012] A method for producing high yields of tamarind, wherein the method involves producing tamarind using the aforementioned genetically engineered bacteria.

[0013] Preferably, the method involves activating the above-mentioned genetically engineered strain to OD200. 600 Once the culture medium reaches 1.0, the activated strain is inoculated into the fermentation medium at a volume ratio of 1:100, and cultured until the OD reaches 1.0. 600 Reach 0.6, then add IPTG for culture.

[0014] Preferably, the fermentation medium is formulated as follows: yeast powder 5 g / L, glucose 10 g / L, K2HPO4·3H2O 9.8 g / L, citric acid monohydrate 2.1 g / L, ferric ammonium citrate 0.3 g / L; MgSO4·7H2O 0.49 g / L, pH 7.0; and trace elements, kanamycin sulfate 50 μg / mL, chloramphenicol 34 μg / mL and ampicillin 50 μg / mL are added.

[0015] The application of the above-mentioned genetically engineered bacteria in improving the aroma quality of cigarette products.

[0016] Beneficial effects The beneficial effects of this invention are as follows: This invention provides a genetically engineered bacterium that produces high-yield ambergris-sweet flavoring, its construction method, and its applications. Utilizing the tobacco-derived diterpenoid synthase TPS103, a complete firol biosynthetic pathway was constructed within *Escherichia coli*, achieving efficient biosynthesis of firol from glucose. The flavoring prepared using this engineered strain can be used to flavor and enhance cigarette products, enriching tobacco aroma and improving the characteristic sweet and sweet flavor of ambergris and the sensory quality of cigarettes. Attached Figure Description

[0017] Figure 1 For the target gene TPS103 PCR electrophoresis image.

[0018] Figure 2 This is the GC chromatogram for the standard abamectin.

[0019] Figure 3 This is a GC diagram of the fir alcohol synthesized by engineered bacteria. Detailed Implementation

[0020] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, which can be obtained commercially by those skilled in the art.

[0022] Example 1: Cloning of the tobacco diterpenoid synthase gene TPS103 .

[0023] RNA was extracted from tobacco leaf tissue and reverse transcribed into cDNA using reverse transcriptase. The cDNA obtained from the reverse transcription reaction was used as a template. The forward primer was 5'-ATGAACACCAGCAACTGGCTGAAAC-3', and the reverse primer was 5'-TTACACCTGCTGAAACAGCACTTTG-3'. PCR amplification was performed using TakaRa PrimeSTAR Max DNA polymerase, and the cDNA was cloned. TPS103Gene. The PCR reaction system was as follows: 1 μL of cDNA from common tobacco leaves, 1 μL each of forward and reverse primers, 25 μL of PrimeSTAR Max Premix (2×), and 22 μL of H2O. The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 60 s, 33 cycles; extension at 72℃ for 5 min. The PCR products were detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 As shown, Figure 1 M stands for DNA marker DL2000, and the target gene is... TPS103 The fragment size is 2316 bp, which is in line with expectations.

[0024] The target gene fragment was recovered using an agarose gel electrophoresis recovery kit. TPS103 The target fragment was cloned using TA cloning and ligated into the pEASY®-Blunt cloning vector. This vector was then transformed into *E. coli* DH5α clone strains and plated on LB agar plates containing ampicillin (Amp, 100 mg / L) and incubated in the dark at 37°C for 12 hours. Positive clones were screened by colony PCR using the positive clone identification method in the pEASY®-Blunt cloning vector kit. After expanding positive single-clone colonies, plasmids were extracted and sequenced. Sequencing analysis confirmed the cloning of the tobacco diterpenoid synthase gene. TPS103 The nucleotide sequence of the enzyme is shown in SEQ ID NO.1, with a length of 2316 bp. The encoded protein is named diterpene synthase TPS103, consisting of 772 amino acids, the specific amino acid sequence of which is shown in SEQ ID NO.2. The gene for diterpene synthase is thus obtained. TPS103 The recombinant plasmid inserted into the pEASY®-Blunt cloning vector was named pEASY-TPS103.

[0025] SEQ ID NO.1: SEQ ID NO.2: * Example 2: Constructing a fir alcohol-synthesizing engineered bacterium using the tobacco diterpene synthase TPS103 encoding gene.

[0026] (1) Construction of recombinant plasmid I: The acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene ( mvaE (GenBank No. AAG02439.1), 3-hydroxy-3-methylglutaryl-CoA synthase gene ( mvaS(GenBank No. AAG02438.1) was cloned into plasmid pACYCDuet-1 to obtain recombinant plasmid I.

[0027] (2) Construction of recombinant plasmid II: The farnesyl pyrophosphate synthase gene ( Erg20 (GenBank No. NM_001181600.1), Geraniol Geraniol Pyrophosphate Synthase Gene ( CrtE (GenBank No. M87280.1), diterpene synthase gene TPS103 , fir alcohol synthase gene ( abs (GenBank No. HE588140.1) was cloned into plasmid pCDFDuet-1 to obtain recombinant plasmid II.

[0028] (3) Construction of recombinant plasmid III: The mevalonate kinase gene ( Erg12 (GenBank No. NM_001182715.1), mevalonate-5-phosphate kinase gene ( Erg8 (GenBank NO. NM_001182727.1), mevalonate-5-bisphosphate decarboxylase gene ( Erg19 (GenBank NO. X97557.1), isopentenyl pyrophosphate isomerase gene ( IDI1 (GenBank NO. NM_001183931.1) was cloned into plasmid pTrcHis2B to obtain recombinant plasmid III.

[0029] (4) Transformation of recombinant bacteria: Recombinant plasmid I, recombinant plasmid II and recombinant plasmid III were co-transformed into Escherichia coli BL21(DE3) competent cells to obtain engineered bacteria for the synthesis of taurine.

[0030] The specific method is as follows: (1) Construction of recombinant plasmid I.

[0031] Gene mvaE With genes mvaSThe genes were obtained through chemical synthesis by Shanghai Jierui Biotechnology Co., Ltd., and then constructed into the pGH vector to obtain pGH-mvaE and pGH-mvaS plasmids. Using pGH-mvaE as a template, primers mvaE-F (5'-AACTTTAATAAGGAGATATAATGGAGAAAACAGTAGTTATTATTGATGC-3') and mvaE-R (5'-GAGCTCGAATTCGGATCCTTATTGTTTTCTTAAATCATTTAAAATAGCC-3') were used to detect the acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene. mvaE PCR amplification of the 3-hydroxy-3-methylglutaryl-CoA synthase gene was performed using pGH-mvaS as a template with primers mvaS-F (5'-TCCGAATTCGAGCTCAGGAGGTAAAAAAACATGACAATTGGGATTGATAAAATTAGTTTTTTTGTG-3') and mvaS-R (5'-TACGATTACTTTCTGTTCGATTAGTTTCGATAAGAGCGAACGG-3'). mvaS PCR amplification was performed on plasmid pACYCDuet-1 using primers pACY-F (5'-TCGAACAGAAAGTAATCGTATTGTACA-3') and pACY-R (5'-TATATCTCCTTATTAAAGTTAAACAAAATTATTTC-3'). The target DNA fragments were recovered using a gel extraction kit (Omega). The obtained gene fragments were seamlessly ligated using the homologous recombination seamless cloning kit from Beijing TransGen Biotech Co., Ltd., according to... mvaE arrive mvaS The expression sequence was determined, and the two genes were recombined into the multiple cloning restriction site of the pACYCDuet-1 expression vector. The ligation product was then transformed. E. coli DH5α was then plated onto LB solid plates containing 34 mg / L chloramphenicol (Cm). After incubation at 37°C inverted in the dark for 12 h, single-clone plasmids were extracted and sent to a sequencing company for unidirectional sequencing using T7-ter primers: TGCTAGTTATTGCTCAGCGG. The recombinant plasmid I was obtained after confirming that the target gene had been recombined into the pACYCDuet-1 vector.

[0032] (2) Construction of recombinant plasmid II.

[0033] Using the Saccharomyces cerevisiae genome as a template, primers Erg20-F (5'-AACTTTAATAAGGAGATATAATGGCTTCAGAAAAAGAAATTAGGA-3') and Erg20-R (5'-TCCTCCTTAAGCTATTTGCTTCTCTTGTAAACTTTGTTCA-3') were used to detect the farnesyl pyrophosphate synthase gene. Erg20 PCR amplification of the geranylide geranylide pyrophosphate synthase gene was performed using the pGH-CrtE gene synthesized chemically by Suzhou Genewise Biotechnology Co., Ltd. as a template. Primers CrtE-F (5'-AGCAAATAGCTTAAGGAGGAAAAAAACATGGTGAGTGGCAGTAAAGCG-3') and CrtE-R (5'-TCCTCCTTAAGTCAGGCGATTTTCATGACCG-3') were used. CrtE PCR amplification was performed using plasmid pEASY-TPS103 as a template, with primers TPS103-F (5'-ATCGCCTGACTTAAGGAGGAAAAAAAAATGAACACCAGCAACTGGC-3') and TPS103-R (5'-ACCTCCTAAGTTACACCTGCTGAAACAGCACTTT-3') to amplify the diterpenoid synthase gene. TPS103 PCR amplification of the fir alcohol synthase gene was performed using the pGH-abs gene synthesized chemically by Suzhou Genewise Biotechnology Co., Ltd. as a template. Primers abs-F (5'-GCAGGTGTAACTTAGGAGGTAAAAAAAAATGTGCAGCCACAGTACTGCTTC-3') and abs-R (5'-TACGATTACTTTCTGTTCGATTATGGGGAATATTGATTGAGTGG-3') were used. abs PCR amplification was performed using primers pCDF-F (5'-TCGAACAGAAAGTAATCGTATTGTACA-3') and pCDF-R (5'-TATATCTCCTTATTAAAGTTAAACAAAATTATTTC-3') with plasmid pCDFDuet-1 as a template. The target DNA fragments were recovered using a gel extraction kit (Omega). The obtained gene fragments were then seamlessly ligated using the homologous recombination seamless cloning kit from Beijing TransGen Biotech Co., Ltd., according to... Erg20 , CrtE , TPS103 , absThe expression sequence was determined, and the above genes were recombined into the multiple cloning restriction sites of the pCDFDuet-1 expression vector. The ligation product was then transformed. E. coli DH5α was then plated onto LB solid plates containing 50 mg / L streptomycin (Str). After incubation at 37°C inverted in the dark for 12 h, a single-clone plasmid was extracted and sent to a sequencing company for unidirectional sequencing using the T7-ter primers: TGCTAGTTATTGCTCAGCGG. The recombinant plasmid II was obtained by confirming that the target gene was recombined into the pCDFDuet-1 vector.

[0034] (3) Construction of recombinant plasmid III: Genes Erg12, Erg8, Erg19 and IDI1 The gene was obtained by chemical synthesis from Shenzhen BGI Genomics Co., Ltd., and then constructed into the pGH vector to obtain pGH-Erg12, pGH-Erg8, pGH-Erg19, and pGH-IDI1 plasmids. Using pGH-Erg12 as a template, primers Erg12-F and Erg12-R were used to detect the mevalonate kinase gene. Erg12 PCR amplification to obtain the gene Erg12 A fragment; The sequence of primer Erg12-F is as follows: 5'-TAAATAAGGAGGAATAAACCATGTCATTACCGTTCTTAACTTCTGC-3'; The sequence of primer Erg12-R: 5'-AAGGGTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTGTTTC-3'.

[0035] Using pGH-Erg8 as a template, primers Erg8-F and Erg8-R were used to detect the mevalonate-5-phosphate kinase gene. Erg8 PCR amplification to obtain the gene Erg8 A fragment; The sequence of primer Erg8-F is as follows: 5'-TGCGATAGGCCTGCACCCTTAAGGAGGAAAAAAACATGTCAGAGTTGAGAGCCTTCAGTG-3'; The sequence of primer Erg8-R is: 5'-GGGCGAATTCTGCATGCAGCTACCTTAAGTTATTTATCAAGATAAGTTTCCGGATCT-3'.

[0036] Using pGH-Erg19 as a template, primers Erg19-F and Erg19-R were used to detect the mevalonate-5-bisphosphate decarboxylase gene. Erg19 PCR amplification to obtain the gene Erg19 A fragment; The sequence of primer Erg19-F is as follows: 5'-CATGCAGAATTCGCCCTTAAGGAGGAAAAAAAAATGACCGTTTACACAGCATCCG-3' The primer Erg19-R sequence is as follows: 5'-AAGGGCGATGCAGCGAATTGATCTTATTCCTTTGGTAGACCAGTCTTTG-3'.

[0037] Using pGH-IDI1 as a template, primers IDI1-F and IDI1-R were used to detect the isopentenyl pyrophosphate isomerase gene. IDI1 PCR amplification to obtain the gene IDI1 A fragment; The sequence of primer IDI1-F is as follows: 5'-CAATTCGCTGCATCGCCCTTAGGAGGTAAAAAAAAATGACTGCCGACAACAATAGTATGC-3'; The sequence of primer IDI1-R is as follows: 5'-CCATATGGTACCAGCTGCAGTTATAGCATTCTATGAATTTGCCTGTC-3'.

[0038] PCR amplification of plasmid pTrcHis2B was performed using primers pTrc-F (5'-CTGCAGCTGGTACCATATGGG-3') and pTrc-R (5'-GGTTTATTCCTCCTTATTTAATCGATAC-3').

[0039] The target DNA fragments were recovered using a gel extraction kit (purchased from Omega). The obtained gene fragments were then seamlessly ligated using a homologous recombination seamless cloning kit from Beijing TransGen Biotech Co., Ltd., according to... Erg12 , Erg8 , Erg19 and IDI1 The expression sequence was determined, and the above genes were recombined into the multiple cloning restriction site of the pTrcHis2B expression vector. The ligation product was then transformed. E. coliDH5α was then plated onto LB solid plates containing 100 mg / L ampicillin (Amp), and incubated in the dark at 37°C for 12 h. The single-clone plasmid was then extracted and sent to a sequencing company for unidirectional sequencing using the mycHis reverse: GATTTTAATCTGTATCAGG primers. The recombinant plasmid III was obtained by confirming that the target gene was recombined into the pTrcHis2B vector.

[0040] (4) Transformation of recombinant bacteria: Recombinant plasmid I, recombinant plasmid II and recombinant plasmid III were co-transformed into Escherichia coli BL(DE3) competent cells, plated on LB solid plates containing 50 μg / mL Str, 34 μg / mL Cm and 100 μg / mL Amp, and incubated in the dark at 37℃ for 12 h to obtain the engineered strain for the synthesis of taurine.

[0041] Example 3: Fermentation production using engineered strains.

[0042] (1) Fermentation production of pine bark alcohol.

[0043] Pick a single colony obtained in Example 2 and add it to 10 mL of LB liquid medium (containing 50 μg / mL Str, 34 μg / mL Cm, and 100 μg / mL Amp), and incubate at 37 °C in a shaker to activate to OD. 600 The seed culture was obtained at a concentration of 1.0. The seed culture was then amplified. The amplified culture was inoculated into 100 mL of fermentation medium (pH 7.0, yeast extract 5 g / L, glucose 10 g / L, K₂HPO₄·3H₂O 9.8 g / L, citric acid monohydrate 2.1 g / L, ferric ammonium citrate 0.3 g / L; MgSO₄·7H₂O 0.49 g / L) at a volume ratio of 1:100, and 100 μL of trace element stock solution ((NH₄)₆Mo₇O₇) was added. 24 The following concentrations were added: ·4H₂O 3.7 g / L, ZnSO₄·7H₂O 2.9 g / L, H₃BO₄ 24.7 g / L, CuSO₄·5H₂O 2.5 g / L, MnCl₂·4H₂O 15.8 g / L), kanamycin sulfate 50 μg / mL, chloramphenicol 34 μg / mL, and ampicillin 50 μg / mL. The mixture was incubated at 37 ℃ until OD₂O₃ reached its maximum concentration. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.5 mM. The mixture was then cultured at 30 °C for another 48 h. The yield of the fermentation broth was then measured.

[0044] (2) Detection of products: Take 0.5 mL of fermentation broth, add an equal volume of ethyl acetate, mix on a vortex mixer for 2 min, centrifuge at 13000 g for 20 min, take the upper organic phase for filtration, and place it in a liquid chromatography vial for analysis. Detection conditions: GC-MS instrument model: Shimadzu TQ8050; ion source: EI; detector: quadrupole. Detection sample: Take the liquid from the gas chromatography vial for analysis. GC instrument model: Shimadzu GC 2010Pro; separation column model: HP-5; injection volume: 1 μL; detector: flame ionization detector (FID); column temperature: 50℃ for 2.5 min, then increase the temperature to 320℃ at a rate of 10 ℃ / min and hold for 5 min. Qualitative and quantitative analysis of the products by GC-MS showed that ( Figure 2 and Figure 3 The yield of the target product, abacitracin, was 353 mg / L.

[0045] Example 4: Addition of the product to cigarettes and sensory evaluation.

[0046] The extract obtained in Example 3 was concentrated 100 times by nitrogen blowing, and the concentrate was prepared into a 1 mL / L spray solution using anhydrous ethanol as a solvent. A sample of Shandong C3F grade standard tobacco was selected, and 1% of the spray solution was evenly sprayed onto the tobacco at a weight ratio of 1:100, serving as the flavoring treatment group. Anhydrous ethanol was evenly sprayed onto the tobacco at the same ratio, serving as the control group. After rolling the tobacco samples from both the flavoring treatment and control groups using empty tobacco pipes, a sensory evaluation panel composed of seven tobacco industry professionals evaluated the samples. The results are shown in Tables 1 and 2. First, the sensory quality of the cigarette samples was qualitatively described, and the results are shown in Table 1. Compared with the control group, the flavoring group cigarettes had a more delicate and rounded smoke, significantly improved smoothness and sweetness, and enhanced aroma, exhibiting a distinct ambergris-like sweet aroma. Further quantitative evaluation results showed that compared with the control group cigarettes, the flavoring group cigarettes had significant improvements in aroma, smoke, and taste.

[0047] Table 1. Analysis of sample absorption results.

[0048]

[0049] Table 2. Flavoring Effects and Sensory Quality Evaluation of Cigarette Flavoring Raw Materials

[0050] The above description is merely an embodiment of the present invention. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium that produces high levels of ambergris-sweet flavoring, characterized in that, The genetically engineered bacterium is an *E. coli*-based strain expressing the tobacco diterpene synthase TPS103, whose amino acid sequence is shown in SEQ ID NO.

2. The encoding gene for the tobacco diterpene synthase TPS103 is... TPS103 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The genetically engineered bacterium according to claim 1, characterized in that, The genetically engineered bacteria also express the following gene: acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene. mvaE, 3-Hydroxy-3-methylglutaryl-CoA synthase gene mvaS, Farnesyl pyrophosphate synthase gene Erg20 Geraniol geraniol pyrophosphate synthase gene CrtE, fir alcohol synthase gene abs, Mevalonate kinase gene Erg12 Mevalonate-5-phosphate kinase gene Erg8 Mevalonate-5-bisphosphate decarboxylase gene Erg19 and isopentenyl pyrophosphate isomerase gene IDI1 .

3. Gene encoding tobacco diterpene synthase TPS103 Application in engineered bacteria for the preparation of taurine.

4. The use of the genetically engineered bacteria of claim 1 in the preparation of taurine.

5. The method for preparing the genetically engineered bacteria according to claim 1, characterized in that, The method steps are as follows: S1: Gene Cloning: Cloning the Tobacco Diterpenoid Synthesizer Gene TPS103 ; S2: Construction of recombinant plasmid I: The acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene was constructed. mvaE 3-Hydroxy-3-methylglutaryl-CoA synthase gene mvaS The plasmid was cloned into plasmid pACYCDuet-1 to obtain recombinant plasmid I; S2: Construction of recombinant plasmid II: The farnesyl pyrophosphate synthase gene was incorporated into the plasmid. Erg20 Geraniol geraniol pyrophosphate synthase gene CrtE Diterpene synthase gene TPS103 and fir alcohol synthase gene abs The plasmid was cloned into plasmid pCDFDuet-1 to obtain recombinant plasmid II; S3: Construction of Recombinant Plasmid III: Mevalonate kinase gene Erg12 Mevalonate-5-phosphate kinase gene Erg8 Mevalonate-5-bisphosphate decarboxylase gene Erg19 and isopentenyl pyrophosphate isomerase gene IDI1 The plasmid was cloned into plasmid pTrcHis2B to obtain recombinant plasmid III; S4: Recombinant bacterial transformation: Recombinant plasmid I, recombinant plasmid II and recombinant plasmid III were co-transformed into competent cells of Escherichia coli BL21(DE3) to obtain engineered bacteria for the preparation of tamarind.

6. The preparation method according to claim 5, characterized in that, The tobacco-derived diterpene synthase gene TPS103 The steps are as follows: RNA is extracted from tobacco leaf tissue, and reverse transcriptase is used to reverse transcribe the RNA into cDNA. The cDNA is then used as a template for PCR amplification of the target gene. The sequences of the PCR amplification primers are as follows: The forward primer is: 5'-ATGAACACCAGCAACTGGCTGAAAC-3'; The reverse primer is: 5'-TTACACCTGCTGAAACAGCACTTTG-3'.

7. A method for high-yield fir alcohol, characterized in that, The method described herein is to produce afleurone using the genetically engineered bacteria described in claim 1.

8. The method according to claim 7, characterized in that, The method involves activating the genetically engineered strain described in claim 1 to OD200. 600 Once the culture medium reaches 1.0, the activated strain is inoculated into the fermentation medium at a volume ratio of 1:100, and cultured until the OD reaches 1.

0. 600 Reach 0.6, then add IPTG for culture.

9. The method according to claim 8, characterized in that, The fermentation medium was formulated as follows: yeast extract 5 g / L, glucose 10 g / L, K2HPO4·3H2O 9.8 g / L, citric acid monohydrate 2.1 g / L, ferric ammonium citrate 0.3 g / L, MgSO4·7H2O 0.49 g / L, pH 7.0; and trace elements, kanamycin sulfate 50 μg / mL, chloramphenicol 34 μg / mL, and ampicillin 50 μg / mL were added.

10. The application of the genetically engineered bacteria of claim 1 in improving the aroma quality of cigarette products.

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