Mutated enzyme of flavin containing monooxygenase with increased indigo production and recombinant microorganism producing the same

Abstract

The present invention relates to a mutated protein of FMO derived from Celeribacter sp. and a gene encoding the same, a vector comprising the gene, a recombinant cell transformed by the vector, a composition for producing indigo comprising them, and a method for increasing indigo production in the recombinant cell using the transformed recombinant cell.

Claims

1. A mutated enzyme having flavin containing monooxygenase activity (FMO), comprising the amino acid sequence of SEQ ID NO: 1.

2. A nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2.

4. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3.

5. A recombinant vector comprising a nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1.

6. A recombinant microorganism which comprises a nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1.

7. The recombinant microorganism of claim 6, wherein the recombinant microorganism is E. coli or yeast.

8. The recombinant microorganism of claim 6, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

9. A method for production of indigo, comprising a step of culturing a recombinant microorganism which comprises a nucleic acid molecule encoding the mutated enzyme having flavin containing monooxygenase activity (FMO) according to claim 1 and produces indigo in the presence of indole or tryptophan.

10. The method for production of indigo of claim 9, wherein the concentration of tryptophan is 0.1 to 0.4% (w/v).

11. The method for production of indigo of claim 9, wherein the recombinant microorganism is cultured at the temperature range of 20 to 40 C.

12. The method for production of indigo of claim 9, wherein the recombinant microorganism is cultured for 20 to 60 hours.

13. The method for production of indigo of claim 9, wherein the culturing is carried out in a medium containing 0.5 to 1.5% (w/v) of NaCl and 0.2 to 0.8% (w/v) of yeast extract.

14. The method for production of indigo of claim 9, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

15. The method for production of indigo of claim 9, wherein the step of culturing a recombinant microorganism is performed under the condition in which the air supply is 0.3 to 1.5 vvm and the stirring speed is 300 to 600 rpm.

16. The method for production of indigo of claim 15, wherein the air supply and the stirring speed are increased with the culture time.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a diagram of the method of production of the recombinant vector into which the mutated fmo gene having a point mutation with T424A is inserted.

(2) FIG. 2 is one example of the mutated enzyme according to the present invention and shows that the threonine (T) at position 424 in the amino acid sequence of the wild-type enzyme is substituted with alanine (A).

(3) FIG. 3 is a graph showing the indigo production as the result of culturing each strain at 30 t for 24 hours. FMO*4 and FMO*12 strains had 57% and 40% higher productivity, respectively, compared to FMOori strain.

(4) FIG. 4 is a graph showing the indigo production when each strain was cultured at 25 C., 30 C. and 37 C., respectively. FMO*4 strain produced 475.5 mg/L indigo at 30 C., and FMO*12 strain produced 329.9 mg/L indigo at 37 C.

(5) FIG. 5 is a simple mimetic diagram of the process of producing indigo from tryptophan.

(6) FIG. 6 is a graph showing the indigo productivity when tryptophan and indole were supplied to FMO*12 strain as a substrate, respectively. FMO*12 strain could use indole as a substrate, but the productivity was significantly lower than tryptophan.

(7) FIG. 7 is a graph showing the indigo productivity when FMO*12 strain was cultured in 5 L Jar. At 24 hours at 37 C., 633.1 mg/L of indigo was produced, and at 29 hours at 30 C., 543.7 mg/L of indigo was produced.

(8) FIG. 8 is the result of comparing indigo productivities by culturing each strain in a 50 L fermenter. FMO*4 strain produced 741 mg/L indigo at 48 hours and FMO*12 strain produced 934 mg/L indigo at 24 hours.

(9) FIG. 9 is a drawing of confirming the indigo productivity in the strains into which an empty vector and the recombinant vector of flavin containing monooxygenase gene derived from Celeribacter sp. were transformed, respectively.

(10) FIG. 10 is a drawing of confirming the degree of indigo productivity depending on the temperature conditions in the strain into which the flavin containing monooxygenase gene derived from Celeribacter sp. was transformed.

(11) FIG. 11 is a graph showing the indigo produced at 24 and 48 hours by mass-culturing (% L) the strain (FMOori) into which fmo (flavin containing monooxygenase) gene derived from Celeribacter sp. was transformed.

(12) FIG. 12 is a graph of the result of qualifying the indigo production by mass-culturing FMO*4 strain in a 50 L fermenter by the method of Example 6.

MODE FOR INVENTION

(13) Hereinafter, the present invention will be described in more detail by the following reference example and examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Reference Example 1. Wild-Type Strain Having Wild-Type Enzyme

(14) Celeribacter sp. TSPH2 is an isolated bacterium which can decompose phenanthrene that is one of poly aromatic hydrocarbons in mudflat in Taean, and it received the accession number KCCM 11874P by depositing to Korean Culture Center of Microorganisms on Jul. 26, 2016.

(15) After culturing using a baffled flask for smooth aeration supply under the condition of 25 C. and 180 rmp in NY (1% (w/v) NaCl, 0.5% (w/v) Yeast extract) medium in which 2.5 mM concentration of indole was added, a part (0.51 ml) of the culture solution was collected and centrifuged, and then the supernatant was removed and an analysis sample in which DMSO indigo was completely dissolved was obtained. As the result of qualifying the indigo production by measuring the OD620 nm value using an ultraviolet ray/visible light spectroscope and obtaining a standard concentration graph with synthesized indigo dissolved in DMSO, the indigo production of 13 mg/L was confirmed (Korean Patent Publication No. 10-2018-0014637).

Example 1. Production of Recombinant Strain Having Wild-Type Enzyme (FMOori)

(16) To express the flavin containing monooxygenase (fmo) gene derived from Celeribacter sp. strain in E. coli by cloning, the experiment as follows was carried out.

(17) In order to clone the flavin containing monooxygenase (fmo) gene derived from Celeribacter sp. strain, the genome DNA of Celeribacter sp. TSPH2 strain was extracted using the known phenol/chloroform method, and PCR was performed by using the extracted genome DNA as a template and using the primer and Pfu polymerase (Bioneer) disclosed in the following Table 1. The PCR condition was denaturation 95 C. for 5 min/denaturation 95 C. for 20 seconds, annealing 55 for 30 seconds, elongation 72 C. for 1 minute 30 seconds (30 cycle repeats)/elongation 72 C. for 5 minutes.

(18) TABLE-US-00001 TABLE1 SEQ ID Name Sequence NO Cfmo-F 5 atgcaagcttaaca 6 (HindIII) cacgctcaaccaac3 Cfmo-R 5 atgcctgcagggac 7 (PstI) gcgaagatcggtta3

(19) For each of the amplified fmo gene derived from Celeribacter sp. strain and pBluescript II KS(+) (Agilent Technologies), HindIII enzyme was reacted at 37 C. for 23 hours, and PstI enzyme was reacted at 37 C. for 23 hours. After the enzyme reaction, it was mixed so that the molar ratio of vector and insert was 1:3, followed by ligation using T4 ligase (Promega), and it was transformed with E. coli DH5 and 40 ul of X-gal (20 mg/ml) was spread in an LB (Luria-Bertani) solid medium comprising 100 ug/ml Ampicillin, and then it was cultured overnight, thereby selecting only the white colony among blue or white colonies.

(20) In order to confirm that the transformed strain selected in Example 1-1 produced indigo, by culturing the colony of the transformed strain selected in Example 1-1 and the colony of the vector-only transformed strain as a substrate in NY medium (0.5% Yeast extract, 1% NaCl) in which 0.2% (w/v) tryptophan was added, it was confirmed that only in the culture solution of flavin containing monooxygenase gene recombinant strain derived from Celeribacter sp., blue materials were mass-produced. The experimental result was shown in FIG. 9.

Example 2. Production of Recombinant Strain Having Mutated FMO Enzyme

(21) The method of production of a recombinant vector comprising the FMO mutated enzyme gene is as shown in FIG. 1. The used strain was E. coli DH5a, and pBluescript II KS(+) (3.0 kb) vector was used.

(22) PCR (TAKARA, ExTaq) for the insert of 1410 bp comprising the fmo gene derived from Celeribacter sp. TSPH2 obtained in Reference example 1 (1344 bp, SEQ ID NO: 5) was conducted by using Taq polymerase. In the PCR amplification method, the same primer pair as Example 1 was used and the PCR condition was also same as Example 1, but the elongation time was 45 seconds. Then, it was subcloned into pGEMTeasy (Promega) vector. The insert was cut by using HindIII and PstI restriction enzymes and pBluescript II KS(+) was cut by using the same restriction enzymes to connect them. By transforming with E. coli DH5a, the recombinant vector and recombinant stain comprising FMO mutation (FMO*4, FMO*12) were produced.

(23) As the result of confirming the insert sequence to Genotech, Inc. to confirm the accurate sequence of the clone of which insert size was confirmed, FMO*4 and FMO*12 in which adenine (a) at the 1270th position in fmo gene of 1344 bp of SEQ ID NO: 5 that was the sequence of the gene of the wild-type enzyme was substituted with guanine (g) were confirmed. Through this, as can be seen in FIG. 2, the 434th threonine of the FMO enzyme was replaced with alanine (T424A). In addition, in case of FMO*12, after the stop codon, 34 bp that was a portion of T-vector was inserted. It is presumed that this plays a role of a terminator in expression of fmo, and it is presumed that the mutated FMO enzyme is stably expressed at the temperature of 37 C. to enable indigo production.

(24) FMOori recombinant strain: E. coli DH5a/pBluescript II KS(+)::fmo from Celeribacter sp. TSPH2 (recombinant E. coli strain expressing the wild-type enzyme according to Example 1)

(25) FMO*4 recombinant strain: E. coli DH5a/pBluescript II KS(+)::fmo*(T424A) Celeribacter sp. TSPH2 (recombinant E. coli strain expressing the mutated enzyme according to Example 2)

(26) FMO*12 recombinant strain: E. coli DH5a/pBluescript II KS(+)::fmo*(T424A) Celeribacter sp. TSPH2 and a portion of T-vector at the 3 end, 34 bp additional insertion (recombinant E. coli strain expressing the mutated enzyme according to Example 2)

Example 3: Indigo Production of Recombinant Strain

(27) 3-1: Indigo Qualification Method

(28) The strain culture solution 0.3 to 1 mL was collected and centrifuged at 13,000 rpm for 5 minutes to obtain blue precipitates, and it was washed by aliquoting sterilized triple distilled water 1 mL, and then it was centrifuged at 13,000 rpm for 5 minutes again. After dissolving blue precipitates in dimethyl sulfoxide (DMSO), insoluble microbial cells-indigo lumps were dissolved by ultrasound wave irradiation. Then, to remove the microbial cells, it was centrifuged at 13,000 rpm at 20 C. for 10 minutes, and then for the supernatant in which only indigo was dissolved purely, the 620 nm value was measured by using a ultraviolet ray/visible light spectrophotometer (V630-Bio UV-Vis Spectrophotometer, JASCO). If necessary, the sample was diluted with DMSO and the 620 nm value was measured, and then the dilution factor was multiplied. Indigo was qualified by using a synthetic indigo dissolved in DMSO (sigma-aldrich, 229296, 95%) as a standard concentration graph.

(29) 3-2: Flask Culture of Recombinant Strain

(30) As a seed culture, FMOori strain obtained in Example 1 and FMO*4 and FMO*12 strains of Example 2 were cultured at 37 C. or 30 C. overnight (O/N) by adding 100 u/ml Ampicillin into LB (BD) medium of 10 ml in a 50 ml conical tube and then inoculating a single colony.

(31) As a main culture, 30 ml of medium was aliquoted in a 125 ml Erlenmeyer flask, and 0.3 ml of 1% of the obtained culture solution was inoculated and cultured at 25 C., 30 C. and 37 C., respectively. It was cultured by using a baffled flask for 24 hours for smooth aeration supply under the condition of 30 C. and 180 rpm in NY medium in which tryptophan was added. The medium composition for the main culture comprised yeast extract (ACCUMEDIA) 0.5% (w/v), NaCl (Samchun Chemicals) 1% (w/v) and tryptophan (Daejung Chemicals) 0.2% (w/v).

(32) The indigo production qualified in the culture of the three strains was shown in FIG. 3. FIG. 3 is a graph showing the indigo production obtained as the result of culturing each strain at 30 C. for 24 hours.

(33) As shown in FIG. 3, as the result of measuring the indigo production of the recombinant strains, FMOori strain of Example 1 was 303.1 (mg/L), and FMO*4 recombinant strain of Example 3 was 475.5 mg/L, and FMO*12 recombinant strain of Example 4 was 425.7 mg/L. In other words, when the indigo production (mg/L) of FMOori strain producing the wild-type enzyme was set at 100%, FMO*4 and FMO*12 strains producing the mutated enzyme showed the indigo productivity of 157% and 140%, respectively.

(34) 3-3: 5 L Fermenter Culture

(35) FMOori strain obtained in Example 1 and FMO*12 strain of Example 2 were seed cultured with the same medium used in Example 3-2.

(36) The medium was added to 5 L JAR (KoBioTech) so that the volume of the main culture became 3 L and the seed culture solution of 30 ml (1%) was inoculated, and it was cultured under the condition of pH 7, 0.8 vvm aeration amount and 500 rpm, and indigo was qualified.

(37) It was confirmed that 207.2 mg/L of indigo was produced when culturing FMOori recombinant strain for 24 hours, and it was confirmed that 369.3 mg/L of indigo was produced when culturing for 48 hours (FIG. 11).

(38) FMO*12 strain, as shown in FIG. 7, produced 633.1 mg/L of indigo at 37 C. at 24 hours and 543.7 mg/L at 30 C. at 29 hours, as the result of comparing the productivities after culturing for 24 hours and 29 hours at 30 C. and 37 C. in the 5 L Jar (FIG. 7).

(39) 3-4: 50 L Fermenter Culture

(40) The productivity was confirmed by culturing FMO*4 strain and FMO*12 strain of Example 2 by using a 50 L fermenter at 30 C. and 37 C., respectively.

(41) Specifically, the O/N primary seed culture was performed by adding 100 ug/ml of Ampicillin to an LB medium by the substantially same method as Example 3-2, and the O/N secondary seed culture was conducted by adding 100 ug/ml of Ampicillin to the LB medium o 300 ml in a 1 L flask. As a main culture, to a 50 L fermenter, 30 L medium (1% (w/v) NaCl, 0.5% (w/v) Yeast extract, 0.2% (w/v) Tryptophan) was prepared and 300 ml of the secondary seed culture solution was inoculated, and it was cultured under the condition of Air 1 vvm, 500 rpm and pH7 for 48 hours.

(42) As shown in FIG. 8, it was confirmed that as the result of culturing FMO*4 strain and FMO*12 strain at 30 C. and 37 C., respectively, FMO*4 strain produced 741 mg/L indigo at 30 C. at 48 hours, and FMO*12 strain produced 934 mg/L at 37 C. at 24 hours. These are 2 times and 2.5 times increased values, respectively, compared to FMOori strain of Example 1 which produced 370 mg/L indigo at 30 C. at 48 hours (FIG. 8).

Example 4: Indigo Production with Culture Temperature

(43) FMOori strain obtained in Example 1 and FMO*4 and FMO*12 strains of Example 2 were cultured by using a baffled flask for 24 hours for smooth aeration supply at 180 rpm under the condition of 25 C., 30 C. and 37 C. temperatures in NY medium in which 0.2% (w/v) concentration of tryptophan was added. The indigo production of the culture was qualified by the same method as Example 3-1, and thereby the indigo production qualified in the cultures of the three strains was shown in FIG. 4 and FIG. 10.

(44) FIG. 4 is a graph showing the indigo production when each strain of three kinds was cultured at 25 C., 30 C. and 37 C., respectively, and FIG. 10 is a graph showing the indigo productivity according to the temperature condition of FMOori. FMO*4 strain produced 475.5 mg/L of indigo at 30 C., and FMO*12 produced 329.9 mg/L of indigo at 37 C. In FMOori strain, the indigo productivity at 25 C. and 37 C. except for the temperature of 30 C. was very poor (FIG. 10).

(45) As the result of confirmation in FMO*4 and FMO*12 strains expressing the FMO mutated enzyme, FMOori strain hardly produced indigo at 25 C., but FMO*4 strain and FMO*12 strain produced 83.9 mg/L and 47.5 mg/L, respectively. In addition, in case of 37 C., the level of 329.9 mg/L was shown in FMO*12 strain, and this is an about 5.5 times increased value, compared to 51.0 mg/L of the indigo production of FMOori strain (FIG. 4, FIG. 10).

Example 5. Indigo Production for Indole Substrate

(46) Tryptophan was used as a substrate for indigo production. Tryptophan is decomposed into indole by tryptophanase, and indole is oxidized to indoxyl by FMO, and then indigo is produced by a natural oxidation reaction (FIG. 5). The indigo productivity of each strain when adding indole reacting with FMO directly as a substrate was confirmed.

(47) FMO*4 and FMO*12 strains obtained in Example 2 were cultured by using a baffled flask for smooth aeration supply under the condition of 30 C. and 37 C. and 180 rpm in NY medium in which 2 mM concentration of indole was added. The medium composition for the main culture was yeast extract (ACCUMEDIA) 0.5% (w/v) and NaCl (Samchun Chemicals) 1% (w/v), and Indole (Sigma) 2 mM was also used as a substrate to replace tryptophan.

(48) As the result of culturing FMO*12 strain of Example 2 under the condition of 30 C. and 37 C. after adding 2 mM indole, indole could be used as a substrate for indigo production, but the indigo productivity was significantly low compared to tryptophan (FIG. 6, Table 2).

(49) TABLE-US-00002 TABLE 2 Indigo Indigo Culture Culture production (mg/L) production (mg/L) temperature time with Tryptophan with Indole ( C.) (hr) substrate substrate 30 24 365.9 144.8 30 48 485.1 151.3 37 24 429.5 131.8 37 48 442.9 143.3

Example 6. Indigo Mass-Production Using Recombinant Strain

(50) The indigo productivity was confirmed by performing mass-culture for indigo production by optimizing the culture condition in 50 L JAR, using FMO*4 strain of Example 2 (DH5a/pBluescript::fmo4 recombinant strain). In Table 3, the culture conditions from the seed culture to the main culture were described.

(51) Specifically, the primary seed culture was performed by inoculating a single colony of FMO*4 strain in a 50 mL tube in which 10 ml of LB medium comprising Ampicillin 100 ug/mL was filled, and culturing under the condition of 30 C. and 180 rpm for about 15 hours overnight. Then, the secondary seed culture was performed under the same temperature and rpm conditions as the primary seed culture for 8 hours by putting 350 mL of LB medium in which Ampicillin 100 ug/mL was added into a IL flask, and inoculating 3.5 mL of the primary seed culture, to use for inoculation of the main culture (Table 3 below).

(52) The main culture was performed by putting a 35 L medium to a 50 L fermenter, and the medium was composed of 1% (w/v) NaCl (Samchun), 0.2% (w/v) tryptophan (CJ) and 0.5% (w/v) yeast extract (Neogen). In the medium, 350 mL of the secondary seed culture was inoculated and the cultured for 40 hours, and it was cultured under varying conditions of air supply and stirring speed (rpm). Specifically, as the air supply and stirring speed with time, under the condition of filtered air 20 L/min (0.57 vvm) and 350 rpm until 16 hours after the start of the culture, the condition of filtered air 30 L/min (0.86 vvm) and 450 rpm over 16 hours below 24 hours, and the condition of filtered air 35 L/min (1 vvm) and 500 rpm in 24 hours after the culture, the fermenter culture was performed. The culture temperature was maintained as 30 C., and the pH was maintained at pH 7 by adding IM phosphoric acid.

(53) TABLE-US-00003 TABLE 3 Classification Condition Specific description Primary seed Medium LB medium + Amp100 ug/ml culture Culture 30 C., 180 rpm condition Inoculation 10 ml/50 ml tube: single colony inoculation Culture Overnight culture (O/N, 15 hr) time Secondary seed Medium LB medium + Amp100 ug/ml culture Culture 30 C., 180 rpm condition Inoculation 350 ml/1 L flask: primary seed culture 3.5 ml inoculation Culture 8.5 hr time Main culture Medium 1% NaCl (350 g), 0.2% Tryptophan (70 g), 0.5% Yeast extract (175 g) Culture 30 C., pH7/Air (L/min) 20.fwdarw.30 (16 hr).fwdarw. condition 35 (24 hr) RPM 350.fwdarw.450 (16 hr).fwdarw. 500 (24 hr) Inoculation 35 L/50 L Fermenter: Secondary seed culture 350 ml (1%) inoculation Culture 40 hr time

(54) After 12 hours from the culture, sampling was performed for about 10 ml into a 15 ml falcon tube once every 4 hours, and the indigo sampled in the culture was qualified by the same method as the indigo qualification method of Example 3-1, and the result was shown in the following Table 4.

(55) In the following Table 4 and FIG. 12, the result of qualifying indigo according to the indigo qualification method from the sampled indigo was shown.

(56) TABLE-US-00004 TABLE 4 Culture time (hr) 16 20 28 40 OD620 1.576 0.425 0.928 1.277 Diluted indigo concentration 18.13 4.90 10.68 14.69 (ug/ml) Indigo concentration in 18.13 48.97 106.78 146.90 culture solution (ug/ml) Sampling volume (ml) 1.00 3.00 3.00 3.00 Final indigo concentration 60.4 489.7 1067.8 1469.0 (mg/L)

(57) As shown in the Table 4 and FIG. 12, as the result of mass-culturing FMO*4 strain of Example 2 by varying the air supply and stirring speed (rpm) conditions, the indigo productivity was increased by 1,469 mg/L at 40 hours, and thus about 4 times increased indigo productivity was shown in a shorter time, compared to 370 mg/L of the indigo productivity of FMOori strain of Example 1 when cultured at 30 C. for 48 hours. Thus, it can be seen that the indigo productivity of FMO*4 mutated enzyme protein was significantly increased, compared to the wild-type enzyme protein.

(58) In addition, in Example 3-4 in which the air supply and stirring speed were maintained constant, when culturing FMO*4 strain under the condition of Air 1 vvm and 500 rpm at 30 C. for 48 hours, the indigo productivity of 741 mg/L was shown, but in case of culturing for 40 hours of Example 6 in which the air supply and stirring speed were gradually increased, the indigo productivity was increased by 1469 mg/L, and therefore the indigo productivity was increased about 1.9 times, compared to the case where the stirring speed and air supply were constant. Thus, when culturing the recombinant strain, a higher indigo yield can be obtained by increasing the air supply and stirring speed gradually. The mechanism of this result has not been clear yet, but it is predicted that it is because the stirring speed and air supply are gradually increased to prevent reduction of indigo produced already.