Patchouli alcohol synthase mutant and method for preparing patchouli alcohol by fermentation with recombinant Escherichia coli

Abstract

The present invention discloses a patchouli alcohol synthase mutant and a method for fermenting patchouli alcohol using recombinant Escherichia coli, belonging to the field of bioengineering. The present invention modifies patchouli alcohol synthase and constructs corresponding recombinant Escherichia coli to obtain a recombinant bacterium capable of efficiently fermenting and producing patchouli alcohol, and a fermentation method thereof. The recombinant strain can ferment and produce high-value-added patchouli alcohol using inexpensive glucose as a substrate. After 96 hours of shake flask fermentation, the patchouli alcohol yield reaches 448.1 mg / L, with a yield of 39.1 mg / g DCW and a volumetric production rate of 112 mg / L / day. In a 5L fermenter, the patchouli alcohol yield can reach 2128 mg / L, with a volumetric production rate of 304 mg / L / day.

Description

Technical Field

[0001] The invention relates to a patchouli alcohol synthase mutant and a method for preparing patchouli alcohol by fermenting recombinant Escherichia coli, and belongs to the field of bioengineering. Background Art

[0002] Patchouli alcohol is a naturally occurring tricyclic sesquiterpene compound that is widely used in the field of daily chemical products. It can be used in the production of perfumes and essential oils, and also has certain potential value in food, medicine, etc.

[0003] The synthesis of patchouli alcohol by microbial fermentation has the advantages of low cost, environmental protection and economic benefits. Currently, Escherichia coli or Saccharomyces cerevisiae are mainly used as host bacteria for fermentation synthesis of patchouli alcohol (Zhou et al. Enhancement of Patchoulol Production in Escherichia coli via Multiple Engineering Strategies. J Agric Food Chem. 2021; 69(27): 7572-7580. Liu et al. High-Level Production of Sesquiterpene Patchoulol in Saccharomyces cerevisiae. ACS Synth Biol. 2021; 10(1): 158-172.), but its yield is still low, and the large-scale fermentation production level of patchouli alcohol has not yet been achieved.

[0004] Patchouliol synthase is a key enzyme that catalyzes the synthesis of patchouliol. Wild-type patchouliol synthase variants from different Pogostemon cablin plants exhibit varying catalytic activities. Previous research has shown that the patchouliol synthase with GenBank accession number KF983531.1 (Frister et al. Characterization of A Recombinant Patchoulol Synthase Variant for Biocatalytic Production of Terpenes. Appl Biochem Biotechnol. 2015; 176:2185-2201.) exhibits significantly higher catalytic activity than other variants. Therefore, molecular engineering of this patchouliol synthase and the construction of recombinant Escherichia coli capable of fermenting and synthesizing patchouliol will help further increase patchouliol yields and promote its industrial production. Summary of the Invention

[0005] In response to the current problems of low patchouli alcohol synthase activity and low patchouli alcohol fermentation production yield, the present invention molecularly modifies patchouli alcohol synthase, screens mutants that efficiently synthesize patchouli alcohol, constructs patchouli alcohol-synthesizing recombinant Escherichia coli, and improves the fermentation process, in order to further increase the yield of patchouli alcohol, reduce costs, and promote large-scale fermentation production of patchouli alcohol.

[0006] The present invention provides a patchouli alcohol synthase mutant. The patchouli alcohol synthase with GenBank No. KF983531.1 is used as a starting sequence, and at least one amino acid at positions 21, 83, 108, 380, 390, 462, 481, 504, 513, 520 and 533 is mutated.

[0007] In one embodiment, the mutant is a mutation of histidine at position 21 to alanine.

[0008] In one embodiment, the mutant is a mutation of valine at position 83 to glutamic acid.

[0009] In one embodiment, the mutant is a mutation of histidine at position 108 to tyrosine.

[0010] In one embodiment, the mutant is a mutation of methionine at position 380 to phenylalanine.

[0011] In one embodiment, the mutant is a mutation of histidine at position 390 to alanine or arginine.

[0012] In one embodiment, the mutant is a mutation of histidine at position 462 to alanine.

[0013] In one embodiment, the mutant is a mutation of valine at position 481 to alanine.

[0014] In one embodiment, the mutant is a mutation of valine at position 504 to threonine.

[0015] In one embodiment, the mutant is a mutation of leucine at position 513 to arginine.

[0016] In one embodiment, the mutant is a mutation of Threonine at position 520 to Valine.

[0017] In one embodiment, the mutant is a mutation of histidine at position 533 to alanine, serine, arginine, glycine or cysteine.

[0018] In one embodiment, the mutant is a mutation of histidine at position 390 to alanine, and a mutation of valine at position 481 to alanine.

[0019] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, and a mutation of histidine at position 108 to tyrosine.

[0020] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, a mutation of histidine at position 108 to tyrosine, and a mutation of valine at position 504 to threonine.

[0021] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, a mutation of histidine at position 108 to tyrosine, a mutation of valine at position 504 to threonine, and a mutation of valine at position 83 to glutamate.

[0022] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, a mutation of histidine at position 108 to tyrosine, a mutation of valine at position 504 to threonine, a mutation of valine at position 83 to glutamic acid, and a mutation of histidine at position 390 to alanine.

[0023] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, a mutation of histidine at position 108 to tyrosine, a mutation of valine at position 504 to threonine, a mutation of valine at position 83 to glutamic acid, and a mutation of histidine at position 390 to tyrosine.

[0024] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, a mutation of histidine at position 108 to tyrosine, a mutation of valine at position 504 to threonine, and a mutation of histidine at position 390 to alanine.

[0025] In one embodiment, the mutant is a mutation of valine at position 481 to alanine, a mutation of histidine at position 108 to tyrosine, a mutation of valine at position 504 to threonine, and a mutation of histidine at position 390 to tyrosine.

[0026] The present invention also provides a gene encoding the mutant.

[0027] The invention also provides a plasmid carrying the gene.

[0028] In one embodiment, the plasmid includes but is not limited to pET series plasmids.

[0029] The present invention also provides a recombinant microbial cell expressing the mutant.

[0030] In one embodiment, the recombinant microbial cell is recombinant Escherichia coli.

[0031] In one embodiment, the recombinant Escherichia coli uses pET28a as a vector to express the mutant and contains the plasmid pBbA5c-MevT(CO)-MBIS(CO,ispA); the Addgene number of the plasmid is 35151.

[0032] The present invention also provides application of the recombinant Escherichia coli in fermentation production of patchouli.

[0033] In one embodiment, the application is to culture the recombinant E. coli in M9-3 medium containing glucose to an OD of 600 When the value reaches 2-2.5, IPTG is added for induction, and glucose, dodecane and CaCO3 are added, and culture is carried out at 15-20℃ for at least 96h.

[0034] In one embodiment, glucose and magnesium sulfate are also fed during the fermentation process.

[0035] The present invention provides use of the patchouli alcohol synthase mutant and / or the recombinant Escherichia coli in preparing a product containing patchouli alcohol.

[0036] Beneficial Effects: The present invention modifies patchouli alcohol synthase and constructs a recombinant Escherichia coli strain with improved patchouli alcohol production efficiency. This recombinant strain is capable of fermenting patchouli alcohol using inexpensive glucose as a substrate to produce the higher-value-added patchouli alcohol. Under shake flask fermentation conditions, after 96 hours of fermentation, the patchouli alcohol yield can reach 448 mg / L, with a yield of 39.1 mg / g cell dry weight and a volumetric production rate of 112 mg / L / day. After 168 hours of fermentation in a 5L fermentor, the patchouli alcohol yield can reach 2128 mg / L, with a volumetric production rate of 304 mg / L / day. This result is currently the highest reported yield of patchouli alcohol synthesis by Escherichia coli using glucose as the sole carbon source. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The diagram shows the results of the PTS3 amino acid sequence alignment; (A): a simulation diagram of patchouli alcohol synthase and substrate docking; (B): an amino acid alignment of multiple enzymes with high homology to PTS3.

[0038] Figure 2 The effect of a single point mutation in patchouli alcohol synthase on the fermentative synthesis of patchouli alcohol; (A): The effect of a single point mutation near the substrate binding site of patchouli alcohol synthase on the fermentative synthesis of patchouli alcohol; (B): The effect of a single point mutation at a non-conserved amino acid site in patchouli alcohol synthase on the fermentative synthesis of patchouli alcohol.

[0039] Figure 3To investigate the effect of combined mutations of patchouli alcohol synthase on the fermentative synthesis of patchouli alcohol.

[0040] Figure 4 SDS-PAGE images of wild-type patchouli alcohol synthase PTS3 and optimal mutant PTS3mut4 expressed in Escherichia coli; M: Protein Molecular Weight Marker; P: cell lysis precipitate; S: cell lysis supernatant.

[0041] Figure 5 This is a comparison chart of by-product analysis in the fermentation products of wild-type patchouliol synthase.

[0042] Figure 6 These are the results of patchouli alcohol fermentation in a 5L fermenter using strain PTS3mut4. DETAILED DESCRIPTION

[0043] (1) Culture medium

[0044] LB liquid culture medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L.

[0045] M9-3 culture medium: Na2HPO4 6.0 g / L, KH2PO4 3.0 g / L, NaCl 0.3 g / L, NH4Cl 1.0 g / L, (NH4)2SO4 5 g / L, glucose 5 g / L, MgSO4 2.0 mM, trace element solution 0.1% (v / v).

[0046] Fermentation medium in the fermenter: glucose 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 4.2 g / L, K2HPO4 11.24 g / L, citric acid 1.7 g / L, MgSO4 0.5 g / L, trace element solution 0.1% (v / v).

[0047] Trace element solution: MnSO4·4H2O 0.5g / L, FeSO4·7H2O 10.0g / L, CaCl2 2.0g / L, (NH4)Mo7O 24 0.1g / L, CuSO4·5H2O 3.0g / L, Na2B4O7·10H2O 0.23g / L, ZnSO4·7H2O 5.25g / L, prepared with 0.1mol / L HCl.

[0048] Corresponding antibiotics were added to the culture medium as needed. The amounts of antibiotics added were: kanamycin at a final concentration of 50 μg / mL, and chloramphenicol at a final concentration of 34 μg / mL.

[0049] (2) Method for inducing expression of patchouli alcohol synthase

[0050] -80℃ glycerol-preserved bacteria were streaked onto plates containing kanamycin and cultured in a 37℃ incubator overnight. A single colony from the plate was inoculated into 5mL of LB liquid medium containing kanamycin and cultured in a shaker at 37℃ and 200r / min for 8h. 2% (v / v) inoculum was transferred to 50mL of LB medium containing kanamycin and shaken at 37℃ and 200r / min for 2-2.5h. When the bacterial concentration reached an absorbance of 0.6-0.8 at 600nm, the inducer IPTG was added to the shake flask to a final concentration of 0.2mmol / L and shaken at 20℃ and 200r / min for 24h.

[0051] (3) Patchouli alcohol fermentation method

[0052] (1) Pre-culture of strains

[0053] The recombinant strain was streaked onto LB plates and cultured at 37°C for 24 h. A single colony from the plate was inoculated into 50 mL of LB liquid medium and cultured at 37°C and 200 rpm for 10 h.

[0054] (2) Shake flask fermentation

[0055] Inoculate 2 mL of bacterial solution into 50 mL of M9-3 medium containing 5 g / L glucose and culture in a shaking incubator at 37°C and 200 rpm. 600 When the value reaches 2-2.5, add IPTG inducer to a final concentration of 0.5 mmol / L. Simultaneously, add 3 mL of 500 g / L glucose, 10 mL of dodecane, and 2 g of CaCO₃. Place the shake flask in a shaker at 20°C and 200 rpm for 96 hours. During this time, add glucose as needed to provide the carbon source required for bacterial growth and fermentation.

[0056] (4) Extraction method of patchouli alcohol

[0057] Patchouli alcohol fermentation sample processing method: centrifuge to collect the dodecane phase of the upper fermentation liquid, moderately dilute with ethyl acetate, add 0.1g anhydrous sodium sulfate to absorb residual water, and filter with a 0.22μm microporous membrane before GC / MS detection.

[0058] (5) Determination method of patchouli alcohol

[0059] The content of patchouli alcohol in the samples was detected by GC / MS gas chromatography-mass spectrometry.

[0060] Chromatographic separation conditions: using a TR-5MS gas chromatography column, the initial column temperature was 50°C, which was kept constant for 1 min; the temperature was increased to 200°C at a rate of 10°C / min; the temperature was then increased to 280°C at a rate of 20°C / min, and the temperature was kept constant for 3 min.

[0061] Mass spectrometry conditions included scanning ions within the m / z range of 35 to 300, an inlet temperature of 280°C, a He flow rate of 1.2 mL / min, an ion source temperature of 280°C, electron ionization mode (60 eV), split injection mode with a split ratio of 4.2, and an injection volume of 1 μL. Quantitative analysis was performed using selected reaction monitoring. The quantitative parent ion had an m / z of 138.2, and the product ions had m / z of 110.1, 95.1, and 123.1, respectively. Collision energies were 8, 14, and 10, respectively.

[0062] Patchouli alcohol standard samples with concentrations of 1, 2, 5, and 10 mg / L were prepared and detected using GC / MS gas chromatography-mass spectrometry, and a standard curve was drawn based on the peak area. 2 =0.9997, indicating that the linear relationship is good within the standard sample concentration range.

[0063] Example 1: Molecular modification of patchouli alcohol synthase

[0064] 1) Construction of mutant enzyme recombinant plasmid

[0065] The molecular modeling of PTS3 was performed using the SWISS-MODEL online server (https: / / swissmodel.expasy.org / ) and the crystal structure of sesquiterpene synthase from Artemisia annua as a template (PDB: 4FJQ.1). Figure 1 A). The H21, H533, and H390 positions near the active center were mutated to representative amino acids such as alanine, arginine, serine, glycine, and cysteine, respectively. Recombinant plasmids harboring the H21A, H390A, H390R, H533A, H533S, H533R, H533G, and H533C mutants were constructed by whole-plasmid PCR using the pET28a-PTS3 plasmid (the PTS3 gene shown in SEQ ID NO. 1 was synthesized and cloned into the BamHI and EcoRI sites of pET28a). The primer sequences are shown in Table 1. DNA sequencing confirmed the successful construction of the recombinant plasmids.

[0066] Using BLAST software, we compared the 10 other protein sequences with the highest similarity to the PTS3 amino acid sequence (homology ranged from 63.36% to 50.19%). We selected sites that were highly conserved in other enzymes but mutated to other amino acids in PTS3 and mutated these sites to conserved sequences in other enzymes to construct mutants: V83E, H108Y, M380F, V481A, V504T, L513R, T520V (e.g. Figure 1 B).

[0067] Table 1 Primers for molecular modification of patchouli alcohol synthase

[0068]

[0069]

[0070] 2) Effect of single-point mutations in patchouli alcohol synthase on the fermentative synthesis of patchouli alcohol

[0071] The patchouli alcohol synthase mutant plasmid constructed in step 1) and the plasmid pBbA5c-MevT(CO)-MBIS(CO,ispA) containing the FPP synthesis metabolic pathway were sequentially transformed into E. coli BL21(DE3) strains to obtain a series of recombinant strains capable of synthesizing patchouli alcohol. Shake flask fermentation was carried out with glucose as the sole carbon source. The bacteria were activated in LB medium to obtain seed liquid, and 2 mL of seed liquid was inoculated into 50 mL of M9-3 medium containing 5 g / L glucose, and cultured in a shaking incubator at 37°C and 200 r / min. The bacterial OD 600 When the value reaches 2-2.5, IPTG inducer is added to a final concentration of 0.5 mmol / L. Simultaneously, 3 mL of 500 g / L glucose, 10 mL of dodecane, and 2 g of CaCO3 are added. The shake flask is placed in a shaker at 20°C and 200 rpm for induction culture for 96 hours. During this period, 500 g / L of glucose is added to maintain the residual sugar concentration in the fermentation system above 10 g / L.

[0072] The results are as follows Figure 2 As shown in Figure 3, single-point mutants H390A, H108Y, H390Y, and V481A effectively increased patchouli alcohol production, increasing patchouli alcohol production by 89.1%, 63.5%, 34.6%, and 78.6%, respectively, relative to the original PTS3 fermentation. This indicates that both mutation site selection strategies used in this study yielded positive mutants with significantly increased yields.

[0073] 3) Multi-point combined mutation of patchouli alcohol synthase

[0074] According to the method of step 1), a combination mutant is further constructed based on the single point mutant, and fermentation verification is performed according to the method of step 2).

[0075] Patchouli alcohol fermentation synthesis level of recombinant strains containing multiple mutations Figure 3 As shown, the patchouliol fermentation yield of the combined mutants was further enhanced. Specifically, the four-point combination mutant H108Y / V481A / V504T / H390A (designated PTS3mut4) increased patchouliol production to 448.1 mg / L, 2.02 times that of the original PTS3. This is also the highest yield reported in shake flask fermentation to date.

[0076] The SDS-PAGE detection results of wild-type patchouli alcohol synthase PTS3 and quadruple mutant PTS3mut4 expressed in E. coli BL21 (DE3) are shown in Figure 2. Figure 4 This indicates that the expression level of the PTS3mut4 mutant in the strain is comparable to that of the original PTS3 enzyme. Therefore, the enhanced catalytic activity of the PTS3mut4 mutant is the main reason for the significant increase in patchouli alcohol fermentation yield.

[0077] In addition to synthesizing patchouliol, patchouliol synthase can also catalyze the formation of more than 20 byproducts from FPP substrates. GC-MS was further used to detect the synthesis levels of the main byproducts in the fermentation products of the PTS3mut4 mutant and the original PTS3. Figure 5 As shown, the product spectrum of PTS3mut4 is similar to that of PTS3, indicating that the amino acid sequence mutation does not significantly change its product spectrum.

[0078] Example 2: Patchouli alcohol fermentation tank fermentation

[0079] Patchouli alcohol was further fermented and synthesized using the PTS3mut4 strain in a 5-L fermentor to test the fermentation effect under controllable scale-up conditions.

[0080] The cells were pre-cultured in LB medium in a shake flask as in Example 1. LB seed solution was transferred to 50 mL of M9-3 medium at a 2% (v / v) inoculation rate and cultured on a shaker at 37°C and 200 rpm for 10 h. 100 mL of M9-3 seed culture solution was inoculated into a 5 L fermentor containing 2 L of medium, and the aeration rate was controlled at 3 L / min and the stirring speed was 200-1000 rpm to control the dissolved oxygen concentration to be greater than 30%. Ammonia water was added to control the pH value of the fermentation liquid to 7. The cell growth phase was carried out at 37°C. After a sudden increase in the dissolved oxygen concentration, feed solution (500 g / L glucose and 5 g / L magnesium sulfate) was added exponentially to meet the exponential growth process of the bacteria. When the cell dry weight reached approximately 15.3 g / L, the patchouli alcohol synthesis phase was initiated, and the culture temperature was set at 20°C. IPTG was added to a final concentration of 0.2 mM, and IPTG was continuously added at a rate of 0.06 mM / h over the next 10 h. A total of 400 mL of dodecane extractant was added at a flow rate of 10 mL / h (for a total of 40 h). Feed solution (500 g / L glucose and 5 g / L magnesium sulfate) was fed, with the glucose feed rate as follows: Figure 5 As shown, the fermentation was completed at 168 h.

[0081] like Figure 6 As shown in the results, the patchouli alcohol yield in the fermentation broth reached 2128 mg / L, and the volumetric production rate was 304 mg / L / d, which were 4.75 times and 2.71 times that of the shake flask experiment, respectively. This result is also the highest reported level of patchouli alcohol fermentation synthesis.

[0082] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A patchoulol synthase mutant, characterized in that: Based on the amino acid sequence shown in GenBank: KF983531.1, any of the following mutations (a) to (d) were performed: (a) Mutation of histidine at position 108 to tyrosine; (b) mutating histidine at position 390 to alanine; (c) mutating histidine at position 390 to tyrosine; (d) The valine at position 481 was mutated to alanine.

2. The mutant according to claim 1, characterized in that Based on the amino acid sequence shown in GenBank: KF983531.1, it has any one of the following mutations (a) to (h): (a) Mutating histidine at position 390 to alanine and valine at position 481 to alanine; (b) mutating valine at position 481 to alanine and histidine at position 108 to tyrosine; (c) mutating valine at position 481 to alanine, histidine at position 108 to tyrosine, and valine at position 504 to threonine; (d) mutating valine at position 481 to alanine, histidine at position 108 to tyrosine, valine at position 504 to threonine, and valine at position 83 to glutamic acid; (e) mutating valine at position 481 to alanine, mutating histidine at position 108 to tyrosine, mutating valine at position 504 to threonine, mutating valine at position 83 to glutamic acid, and mutating histidine at position 390 to alanine; (f) mutating valine at position 481 to alanine, histidine at position 108 to tyrosine, valine at position 504 to threonine, valine at position 83 to glutamic acid, and histidine at position 390 to tyrosine; (g) mutating valine at position 481 to alanine, mutating histidine at position 108 to tyrosine, mutating valine at position 504 to threonine, and mutating histidine at position 390 to alanine; (h) The valine at position 481 was mutated to alanine, the histidine at position 108 was mutated to tyrosine, the valine at position 504 was mutated to threonine, and the histidine at position 390 was mutated to tyrosine.

3. A gene encoding the mutant according to claim 1 or 2.

4. A plasmid carrying the gene according to claim 3.

5. A recombinant microbial cell expressing the mutant according to claim 1 or 2.

6. A recombinant Escherichia coli, characterized in that The mutant according to claim 1 or 2 is expressed using pET28a as a vector and contains the plasmid pBbA5c-MevT(CO)-MBIS(CO, ispA); the Addgene number of the plasmid is 35151.

7. A method for producing patchouli by fermentation, characterized in that: The recombinant Escherichia coli according to claim 6 was cultured in M9-3 medium containing glucose until OD 600 When the value reached 2-2.5, IPTG was added for induction, and glucose, dodecane and CaCO3 were added, and the culture was carried out at 15-20 °C for at least 96 h.

8. The method according to claim 7, characterized in that Glucose and magnesium sulfate were also fed during the fermentation process.

9. Use of the patchouli alcohol synthase mutant according to claim 1 or 2 and / or the recombinant Escherichia coli according to claim 6 in the preparation of a product containing patchouli alcohol.

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