CN115927279A - Patchouli alcohol synthetase mutant and method for preparing patchouli alcohol by recombinant escherichia coli fermentation - Google Patents

Abstract

The invention discloses a patchouli alcohol synthase mutant and a method for preparing patchouli alcohol by fermenting recombinant escherichia coli, belonging to the field of bioengineering. The invention modifies patchouli alcohol synthetase and constructs corresponding recombinant escherichia coli to obtain recombinant bacteria capable of efficiently fermenting and producing patchouli alcohol and a fermentation method thereof. The recombinant strain can use cheap glucose as a substrate to ferment and generate patchouli alcohol with high added value, the yield of the patchouli alcohol reaches 448.1mg/L after the patchouli alcohol is fermented for 96h in a shake flask, the yield is 39.1mg/g DCW, and the volume production intensity is 112mg/L/d. In a 5L fermentation tank, the yield of patchouli alcohol can reach 2128mg/L, and the volume production intensity is 304mg/L/d.

Description

Patchouli alcohol synthetase mutant and method for preparing patchouli alcohol by recombinant escherichia coli fermentation

Technical Field

The invention relates to a patchouli alcohol synthetase mutant and a method for preparing patchouli alcohol by recombinant escherichia coli fermentation, and belongs to the field of bioengineering.

Background

Patchouli alcohol is a natural tricyclic sesquiterpene compound, is widely applied in the field of daily chemicals, can be used for preparing perfume and essential oil, and has certain potential value in the aspects of food, medicines and the like.

The patchouli alcohol is synthesized by microbial fermentation, and has the advantages of low cost, environmental protection, economic benefit creation and the like. At present, escherichia coli or Saccharomyces cerevisiae is mainly used as a host bacterium for fermentation synthesis of patchouli alcohol (Zhou et al. Enhancement of Patchoulol Production in Escherichia coli via Multiple Engineering strains.J. agricultural 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 the yield is still low, and the large-scale fermentation Production Level of patchouli alcohol is not achieved.

Patchouli alcohol synthase is a key enzyme for catalyzing and synthesizing patchouli alcohol. Wild-type variants of patchouli alcohol synthase from different patchouli (Pogostemon cablin) plants have different catalytic activities. The results of the prior studies showed that the patchouli alcohol Synthase with GenBank accession KF983531.1 (Frister et al, characterisation of ARecombiant Patchoulol Synthase Variant for Biocatalytic Production of Terpines. Appl Biochem Biotechnol.2015; 176. Therefore, the molecular modification of the patchouli alcohol synthase and the construction of recombinant escherichia coli for fermenting and synthesizing the patchouli alcohol contribute to further improving the yield of the patchouli alcohol and promoting the industrial production of the patchouli alcohol.

Disclosure of Invention

Aiming at the problems of low activity of the existing patchouli alcohol synthetase, low patchouli alcohol fermentation production yield and the like, the invention carries out molecular modification on the patchouli alcohol synthetase, screens a mutant for efficiently synthesizing the patchouli alcohol, constructs patchouli alcohol synthesis recombinant escherichia coli, and improves the fermentation process so as to further improve the patchouli alcohol yield, reduce the cost and promote the large-scale fermentation production of the patchouli alcohol.

The invention provides a patchouli alcohol synthetase mutant, which takes patchouli alcohol synthetase with the GenBank number of KF983531.1 as a starting sequence and mutates at least one amino acid in 21 st, 83 nd, 108 th, 380 th, 390 th, 462 th, 481 th, 504 th, 513 th, 520 th and 533 th positions.

In one embodiment, the mutant is a mutation of histidine 21 to alanine.

In one embodiment, the mutant has a mutation of valine at position 83 to glutamic acid.

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

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

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

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

In one embodiment, the mutant has a mutation from valine at position 481 to alanine.

In one embodiment, the mutant has a mutation from valine to threonine at position 504.

In one embodiment, the mutant has a mutation of leucine 513 to arginine.

In one embodiment, the mutant has a mutation of threonine 520 to valine.

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

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.

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.

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, and a mutation of valine at position 504 to threonine.

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

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

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

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

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

The invention also provides a gene encoding the mutant.

The invention also provides a plasmid carrying the gene.

In one embodiment, the plasmid includes, but is not limited to, a pET series plasmid.

The invention also provides recombinant microbial cells expressing the mutants.

In one embodiment, the recombinant microbial cell is a recombinant E.coli.

In one embodiment, the recombinant E.coli expresses the mutant with pET28a as a vector and contains plasmid pBbA5c-MevT (CO) -MBIS (CO, ispA); the plasmid was adddge numbered 35151.

The invention also provides application of the recombinant escherichia coli in fermentation production of pogostemon cablin.

In one embodiment, the use is to culture the recombinant E.coli to OD in M9-3 medium containing glucose ₆₀₀ When the value reaches 2-2.5, adding IPTG for induction, and adding glucose, dodecane and CaCO ₃ Culturing at 15-20 deg.c for at least 96 hr.

In one embodiment, glucose and magnesium sulfate are also fed during the fermentation.

The invention provides application of the patchouli alcohol synthetase mutant and/or the recombinant escherichia coli in preparation of patchouli alcohol-containing products.

Has the beneficial effects that: according to the invention, the patchouli alcohol synthetase is modified, and the recombinant escherichia coli strain with improved patchouli alcohol production efficiency is constructed, so that the recombinant strain can be fermented to generate patchouli alcohol with higher added value by taking low-cost glucose as a substrate, the patchouli alcohol is fermented for 96 hours under the condition of shake flask fermentation, the yield of the patchouli alcohol can reach 448mg/L, the yield reaches 39.1mg/g of cell dry weight, and the volume production intensity reaches 112mg/L/d. Fermenting in a 5L fermentation tank for 168h, the patchouli alcohol yield can reach 2128mg/L, and the volume production intensity can reach 304mg/L/d. The result is the highest report that the escherichia coli takes glucose as a sole carbon source to synthesize patchouli alcohol by fermentation at present.

Drawings

FIG. 1 is a diagram showing the alignment results of PTS3 amino acid sequences; (A): patchouli alcohol synthetase and substrate docking simulation diagram; (B): aligning a plurality of enzyme amino acids with high homology with PTS 3.

FIG. 2 shows the effect of single point mutation of patchouli alcohol synthase on patchouli alcohol fermentation synthesis; (A): the single point mutation near the substrate binding site of patchouli alcohol synthase has the function of patchouli alcohol fermentation synthesis; (B): the patchouli alcohol synthetase does not have the function of single point mutation of conservative amino acid sites on the fermentation synthesis of patchouli alcohol.

FIG. 3 shows the effect of patchouli alcohol synthase combination mutation on patchouli alcohol fermentation synthesis.

FIG. 4 is SDS-PAGE pattern of wild type patchouli alcohol synthase PTS3 and optimal mutant PTS3mut4 expressed in E.coli; m: protein Molecular Weight Marker; p: precipitating cell disruption solution; s: cell disruption supernatant.

FIG. 5 is a graph comparing analysis of byproducts in fermentation products of wild-type patchouli alcohol synthase.

FIG. 6 shows the fermentation results of patchouli alcohol in a strain PTS3mut 4L fermentor.

Detailed Description

(I) culture Medium

LB liquid medium: 10g/L of Tryptone (Tryptone), 5g/L of Yeast powder (Yeast extract) and 10g/L of sodium chloride (NaCl).

M9-3 Medium: na (Na) ₂ HPO ₄ 6.0g/L，KH ₂ PO ₄ 3.0g/L，NaCl 0.3g/L，NH ₄ Cl 1.0g/L, (NH 4) 2SO45g/L, glucose 5g/L, mgSO ₄ 2.0mM and 0.1% (v/v) of trace element liquid.

Fermentation medium of fermentation tank: glucose 10g/L, (NH) ₄ ) ₂ SO ₄ 2g/L，KH ₂ PO ₄ 4.2g/L，K ₂ HPO ₄ 11.24g/L, citric acid 1.7g/L, mgSO ₄ 0.5g/L, and 0.1% (v/v) of trace element liquid.

Trace element liquid: mnSO ₄ ·4H ₂ O 0.5g/L，FeSO ₄ ·7H ₂ O 10.0g/L，CaCl ₂ 2.0g/L，(NH ₄ )Mo ₇ O ₂₄ 0.1g/L，CuSO ₄ ·5H ₂ O 3.0g/L，Na ₂ B ₄ O ₇ ·10H ₂ O 0.23g/L，ZnSO ₄ ·7H ₂ O5.25 g/L, prepared with 0.1mol/L HCl.

Corresponding antibiotics are added into the culture medium according to the needs, and the addition amount of the antibiotics is as follows: kanamycin to a final concentration of 50. Mu.g/mL, chloramphenicol to a final concentration of 34. Mu.g/mL.

Method for inducing expression of (di) patchouli alcohol synthetase

The glycerol stocks at-80 ℃ were streaked on plates containing kanamycin and incubated overnight in an incubator at 37 ℃. The plate single colony is inoculated in 5mL LB liquid culture medium containing kanamycin and placed in a shaking table at 37 ℃ and 200r/min for shake culture for 8h. The strain is inoculated into 50mL LB culture medium containing kanamycin with the inoculation amount of 2% (v/v), and the culture medium is placed on a shaker at the temperature of 37 ℃ and 200r/min and shaken for 2 to 2.5 hours. When the light absorption value of the thallus concentration reaching 600nm is 0.6-0.8, adding an inducer IPTG into the shake flask until the final concentration is 0.2mmol/L, and placing the shake flask in a shaking table at 20 ℃ and 200r/min to shake for 24h.

(III) patchouli alcohol fermentation method

(1) Pre-culture of the Strain

The recombinant strain is streaked on an LB plate culture medium and cultured for 24h at 37 ℃. The plate single colony is inoculated in 50mL LB liquid medium and cultured for 10h at 37 ℃ and 200 r/min.

(2) Shake flask fermentation culture

To 50mL of M9-3 containing 5g/L glucose2mL of bacterial liquid is inoculated in the medium, and shaking culture is carried out on a shaking table at 37 ℃ and 200 r/min. Bacterial body OD ₆₀₀ When the value reaches 2-2.5, IPTG inducer is added to the final concentration of 0.5mmol/L, 3mL of 500g/L glucose is added, 10mL of dodecane and 2g of CaCO are added ₃ The shake flask is placed at 20 ℃ and shaken and induced to culture for 96h at 200 r/min. During this period, glucose is added as necessary to supply a carbon source required for growth and fermentation of the cells.

Method for extracting patchouli alcohol

The method for treating the patchouli alcohol fermentation sample comprises the following steps: the dodecane phase in the upper layer of the fermentation broth was collected by centrifugation, diluted moderately with ethyl acetate, and 0.1g of anhydrous sodium sulfate was added to adsorb residual water, and after filtration through a 0.22 μm microporous membrane, GC/MS detection was carried out.

Method for measuring patchouli alcohol

And detecting the content of the patchouli alcohol in the sample by using a GC/MS gas chromatography-mass spectrometer.

Chromatographic separation conditions: using TR-5MS gas chromatographic column, keeping the initial column temperature at 50 deg.C for 1min; heating to 200 ℃ at a heating rate of 10 ℃/min; then the temperature is raised to 280 ℃ at the temperature rise speed of 20 ℃/min, and the constant temperature is kept for 3min.

Mass spectrum conditions: selecting ions with the scanning m/z of 35-300, the temperature of a sample inlet of 280 ℃, the He flow rate of 1.2mL/min, the temperature of an ion source of 280 ℃, the ionization mode of electron ionization (60 EV), selecting a split-flow sample injection mode, the split-flow ratio of 4.2 and the sample injection amount of 1 muL, and carrying out quantitative analysis by selective reaction monitoring. The parent ion m/z was quantified at 138.2, the daughter ion m/z was 110.1, 95.1, and 123.1, respectively, and the collision energy was 8, 14, and 10, respectively.

Preparing patchouli alcohol standard samples with the concentrations of 1, 2, 5 and 10mg/L, detecting by using a GC/MS gas chromatography-mass spectrometer, and drawing a standard curve according to peak areas. R ² =0.9997, indicating a good linear relationship over the range of standard concentrations.

Example 1: molecular modification of patchouli alcohol synthase

1) Construction of mutant enzyme recombinant plasmid

PTS3 was molecularly modeled using SWISS-MODEL on-line server (https:// swisssmall. Expasy. Org /) using the crystal structure of the Artemisia annua-derived sesquiterpene synthase as template (PDB: 4FJQ. 1) (see FIG. 1A). The H21, H533, and H390 sites near the active center are mutated to representative amino acids such as alanine, arginine, serine, glycine, and cysteine, respectively. Recombinant plasmids having H21A, H390R, H533A, H533S, H533R, H533G, and H533C mutants were constructed by whole-plasmid PCR using pET28a-PTS3 plasmid (PTS 3 gene shown in SEQ ID NO.1 was synthesized and cloned at BamHI and EcoRI sites of pET28 a) as a template, and the primer sequences are shown in Table 1. DNA sequencing verification shows that the recombinant plasmid is successfully constructed.

By BLAST software, another 10 protein sequences with the highest similarity to PTS3 amino acid sequence were aligned (homology between 63.36% and 50.19%). And (3) selecting a site which is highly conserved on other enzymes in the homologous alignment result but is mutated into other types of amino acids on PTS3, mutating the site on PTS3 into a conserved sequence of other enzymes, and constructing a mutant: V83E, H108Y, M380F, V481A, V504T, L513R, T520V (as in FIG. 1B).

TABLE 1 patchouli alcohol synthase molecular modification primers

2) Action of single-point mutation of patchouli alcohol synthase on synthesis of patchouli alcohol by fermentation

Sequentially transforming the patchouli alcohol synthase mutant-containing plasmid constructed in the step 1) and the FPP anabolism pathway-containing plasmid pBbA5c-MevT (CO) -MBIS (CO, ispA) into the E.coli BL21 (DE 3) strain to obtain a series of recombinant strains with the capacity of synthesizing patchouli alcohol. And (3) taking glucose as a unique carbon source to carry out shake flask fermentation. The thalli is activated in LB culture medium to obtain seed liquid, 2mL of seed liquid is inoculated into 50mL of M9-3 culture medium containing 5g/L glucose, and shaking culture is carried out on a shaking table at 37 ℃ and 200 r/min. Bacterial body OD ₆₀₀ When the value reaches 2-2.5, IPTG inducer is added to the final concentration of 0.5mmol/L, at the same time, 3mL of 500g/L glucose is added, 10mL of dodecane and 2g of CaCO are added ₃ The shake flask is placed at 20 ℃ and shaken and induced to culture for 96h at 200 r/min. During this time, glucose was added at a concentration of 500g/L to maintain the residual sugar concentration in the fermentation system above 10g/L.

The results are shown in FIG. 2. H390A, H108Y, H390Y and V481A, the single-point mutant can effectively improve the synthesis level of patchouli alcohol, and the synthesis level is respectively improved by 89.1%, 63.5%, 34.6% and 78.6% compared with the original PTS3 patchouli alcohol fermentation synthesis level. It can be seen that both of the two mutation site selection strategies used in this study yielded positive mutants with significantly improved yields.

3) Patchouli alcohol synthase multipoint combined mutation

And (3) further constructing a combined mutant on the basis of the single-point mutant according to the method of the step 1), and performing fermentation verification according to the method of the step 2).

The fermentation synthesis level of the recombinant strain patchouli alcohol containing multiple mutations is shown in figure 3. Therefore, the patchouli alcohol fermentation yield of the combined mutant is further improved, wherein the four-point combined mutant H108Y/V481A/V504T/H390A (named PTS3mut 4) improves the patchouli alcohol yield to 448.1mg/L which is 2.02 times of the original PTS 3. This is also the highest shake flask fermentation yield reported to date.

The results of SDS-PAGE detection of expression of wild-type patchouli alcohol synthase PTS3 and the four-mutant PTS3mut4 in e.coli BL21 (DE 3) are shown in fig. 4. The expression level of the PTS3mut4 mutant in the strain is shown to be similar to that of the PTS3 original enzyme. Therefore, the improvement of the catalytic activity of the PTS3mut4 mutant is the main reason for the remarkable improvement of the fermentation yield of the patchouli alcohol.

In addition to the synthesis of patchouli alcohol, patchouli alcohol synthase can catalyze the formation of over 20 by-products from the FPP substrate. The levels of major by-product synthesis in the PTS3mut4 mutant and the original PTS3 fermentation product were further examined by GC-MS. As shown in figure 5, the PTS3mut4 product profile is similar to PTS3, indicating that amino acid sequence mutations do not significantly alter its product profile.

Example 2: patchouli alcohol fermentation tank fermentation

The patchouli alcohol fermentation synthesis was further performed with PTS3mut4 strain in a 5L fermentor to test the fermentation effect under controlled scale-up conditions.

The cells were pre-cultured in LB medium by shaking in the same manner as in example 1. The LB seed solution was inoculated into 50mL of M9-3 medium at an inoculum size of 2% (v/v), and shake-cultured at 37 ℃ for 10 hours on a shaker at 200 r/min. Inoculating 100mL of M9-3 seed culture solution into a 5L fermentation tank containing 2L of culture medium, controlling the ventilation amount to be 3L/min and the stirring speed to be 200-1000r/min so as to control the dissolved oxygen concentration to be more than 30%; adding ammonia water to control the pH value of the fermentation liquor to be 7; the cell growth stage is carried out at 37 ℃, and after the dissolved oxygen concentration is suddenly increased, a supplement liquid (500 g/L glucose and 5g/L magnesium sulfate) is added in an exponential flow manner to meet the thallus exponential growth process. When the dry cell weight reached about 15.3g/L, the patchouli alcohol synthesis phase was started, with the culture temperature set at 20 ℃; adding IPTG to a final concentration of 0.2mM and continuously supplementing IPTG for the next 10h at a rate of 0.06 mM/h; a total of 400mL of dodecane extractant was added at a flow rate of 10mL/h (40 h total flow); make-up (500 g/L glucose and 5g/L magnesium sulfate) was fed in, wherein the feed rate of glucose was as shown in FIG. 5, and the fermentation was terminated at 168 h.

As shown in FIG. 6, the yield of patchouli alcohol in the fermentation broth reaches 2128mg/L, and the volume production intensity is 304mg/L/d, which is 4.75 times and 2.71 times of the shake flask experiment respectively. This result is also the highest level of patchouli alcohol fermentative synthesis reported to date.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A patchouli alcohol synthase mutant is characterized in that at least one amino acid of 21 st, 83 nd, 108 th, 380 th, 390 th, 462 th, 481 th, 504 th, 513 th, 520 th and 533 th amino acids is mutated on the basis of a starting sequence; the starting sequence has GenBank: KF 983531.1.

2. The mutant according to claim 1, wherein the mutant is characterized in that the mutation is carried out by changing the histidine at position 21 into alanine, or the valine at position 83 into glutamic acid, or the histidine at position 108 into tyrosine, or the methionine at position 380 into phenylalanine, or the methionine at position 390 into alanine or arginine, or the histidine at position 462 into alanine, or the valine at position 481 into alanine, or the valine at position 504 into threonine, or the leucine at position 513 into arginine, or the threonine at position 520 into valine, or the histidine at position 533 into alanine, serine, arginine, glycine or cysteine.

3. The mutant according to claim 1, wherein the mutant is obtained by mutating histidine 390 to alanine and valine 481 to alanine; or

Mutation of valine at position 481 to alanine and mutation of histidine at position 108 to tyrosine; or

Mutating valine at position 481 into alanine, mutating histidine at position 108 into tyrosine, and mutating valine at position 504 into threonine; or

Mutating the 481-th valine to alanine, the 108-th histidine to tyrosine, the 504-th valine to threonine, and the 83-th valine to glutamic acid; or

Mutating the 481 th valine to alanine, mutating the 108 th histidine to tyrosine, mutating the 504 th valine to threonine, mutating the 83 th valine to glutamic acid, and mutating the 390 th histidine to alanine; or

Mutating the 481 th valine to alanine, the 108 th histidine to tyrosine, the 504 th valine to threonine, the 83 th valine to glutamic acid, and the 390 th histidine to tyrosine; or

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; or

Valine at position 481 is mutated to alanine, histidine at position 108 is mutated to tyrosine, valine at position 504 is mutated to threonine, and histidine at position 390 is mutated to tyrosine.

4. A gene encoding the mutant according to any one of claims 1 to 3.

5. A plasmid carrying the gene of claim 4.

6. A recombinant microbial cell expressing a mutant according to any one of claims 1 to 3.

7. A recombinant Escherichia coli, characterized in that pET28a is used as a vector to express the mutant of any one of claims 1 to 3, and the mutant contains plasmid pBbA5c-MevT (CO) -MBIS (CO, ispA); the plasmid was adddge numbered 35151.

8. A method for producing Pogostemon cablin by fermentation, comprising culturing the recombinant Escherichia coli of claim 7 in M9-3 medium containing glucose to OD ₆₀₀ When the value reaches 2-2.5, adding IPTG for induction, and adding glucose, dodecane and CaCO ₃ Culturing at 15-20 deg.c for at least 96 hr.

9. The method of claim 8, wherein glucose and magnesium sulfate are also fed during fermentation.

10. Use of the patchouli alcohol synthase mutants according to any one of claims 1 to 3 and/or the recombinant escherichia coli according to claim 7 for the preparation of patchouli alcohol-containing products.

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