CN110643525B

CN110643525B - Clostridium butyricum strain for producing 1, 3-propylene glycol and screening method and application thereof

Info

Publication number: CN110643525B
Application number: CN201910688714.9A
Authority: CN
Inventors: 齐向辉; 杨苗苗; 员君华; 张国艳; 袁娇; 张宇飞; 王洋; 郭齐
Original assignee: Jiangsu University
Current assignee: Zhenjiang Baitai Biotechnology Co.,Ltd.
Priority date: 2019-07-29
Filing date: 2019-07-29
Publication date: 2022-07-22
Anticipated expiration: 2039-07-29
Also published as: CN110643525A

Abstract

The invention relates to the technical field of bioengineering, in particular to a clostridium butyricum strain for producing 1, 3-propylene glycol and a screening method and application thereof. The strain has the preservation number in China Center for Type Culture Collection (CCTCC) as follows: no. M2019459, designated Clostridium butyricum AYM 014. The clostridium butyricum strain is obtained by performing mutagenesis treatment on a physicochemical compound mutagenesis mutant strain C.butyricum QXYZ514 serving as an original strain. The breeding method of the strain comprises the following steps: preparing bacterial suspension and carrying out plasma mutagenesis; directional screening of the mutagenized strains; and (4) identifying the yield of the mutant strain. The clostridium butyricum strain bred and obtained by the invention has higher tolerance to 1, 3-propanediol, and the yield of the obtained 1, 3-propanediol reaches 40.8 g/L, which is about 42.4 percent higher than that of the original strain. The invention has the advantages of simple mutagenesis equipment, simple operation and low operation cost, provides a high-efficiency strain for producing 1, 3-propylene glycol by a biological method, and has wide industrial application prospect.

Description

Clostridium butyricum strain for producing 1, 3-propylene glycol and screening method and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a clostridium butyricum strain for producing 1, 3-propylene glycol and a screening method and application thereof.

Background

1, 3-propanediol (1, 3-propentadiol, 1, 3-PD) is a colorless viscous organic compound having the structural formula CH₂OH-CH₂-CH₂OH is a nonflammable and low-toxicity liquid. The 1,3-PD has unique bifunctional groups, can participate in various polycondensation reactions to synthesize polyesters such as polytrimethylene terephthalate (PTT) and the like, and has wide application; can be used for producing related compounds in food industry and pharmaceutical industry, and can be used as cosmetic additive, and emollient and humectant. Therefore, the development of an economical and efficient production mode of 1,3-PD is of great significance to various industrial developments. At present, the conventional methods for synthesizing 1,3-PD are by hydration of acrolein and hydroformylation of ethylene oxide, and these chemical synthesis methods have disadvantages of high cost and environmental unfriendliness, both from an economic and ecological point of view. The enzymatic conversion of glycerol to 1,3-PD developed by DuPont opens a new era of 1,3-PD synthesis, and strains capable of naturally producing 1,3-PD include Klebsiella pneumoniae, Clostridium butyricum, Lactobacillus brevis, Citrobacter freundii and the like, but the fermentation process using the natural strains has certain defects, so that the production of 1,3-PD by a microbial fermentation method cannot meet the requirements of industrial production.

Bioconversion with glycerol as substrate is the only way for 1,3-PD production, and the main factors restricting this fermentation method are: low tolerance of the producing strain to substrate glycerol, accumulation of toxic by-products in the metabolic pathway and partial strain towards coenzyme B during transformation₁₂Of the cell surface. Among them, the low tolerance of the cells themselves to glycerol substrates directly restricts the increase in the yield of 1,3-PD, and therefore, optimization is requiredThe fermentation conditions of natural strains or the method for developing novel strains for fermentation are used for modifying natural production bacteria so as to improve the yield of 1,3-PD produced by a biological method.

Disclosure of Invention

In view of the above, the present invention aims to provide a clostridium butyricum having high tolerance to substrate glycerol during the production of 1, 3-propanediol without adding coenzyme B, and a screening method and applications thereof₁₂The yield of 1, 3-propanediol can be improved.

In order to realize the aim of the invention, the technical scheme adopted by the invention is as follows:

the invention provides clostridium butyricum which is preserved in China center for type culture collection with the preservation number as follows: CCTCC number M2019459, classified and named as Clostridium butyricum AYM014, and the preservation unit address is as follows: wuhan university 299 in the Wuchang area of Wuhan city, Hubei province, Wuhan university Collection center; the preservation date is as follows: 6 and 17 months in 2019.

The strain Clostridium butyricum AYM014 has the morphological characteristics of milk white bacterial colony which is irregular round and large, and is arranged regularly in microscopic form and in an expanded spindle shape.

The strain is obtained by taking Clostridium butyricum QXYZ514 as an original strain and screening after mutagenesis treatment.

The invention also provides a screening method of the clostridium butyricum with the preservation number of CCTCC number M2019459, which comprises the following steps:

(1) activating Clostridium butyricum QXYZ514 serving as a starting strain, diluting the activated bacterium liquid to a CFU of 106, putting the CFU into an ARTP mutagen for mutagenesis, coating the bacterium liquid after mutagenesis on a glycerol RCM agar plate culture medium containing a certain concentration for screening culture, repeatedly carrying out mutagenesis and screening culture under the same mutagenesis condition, and gradually increasing the glycerol concentration in the culture medium during screening culture to obtain a primary screening mutant strain;

(2) respectively carrying out basic culture on single colonies of the primarily screened mutant strains obtained in the step (1), then inoculating the single colonies on a 96-well plate for rescreening, and rescreening mutant strains of the first five biomass strains by taking biomass as an index;

(3) and (3) respectively inoculating the mutant strains obtained in the step (2) into a glycerol RCM liquid culture medium with the concentration of 60 mM for culture to obtain fermentation seed liquid, inoculating the fermentation seed liquid into the fermentation culture medium for anaerobic culture to collect fermentation liquid, and detecting the content of 1, 3-propanediol in the fermentation liquid, so as to screen out the mutant strains with the highest 1, 3-propanediol yield.

Preferably, the activation in the step (1) is to streak the starting strain in an RCM agar plate culture medium, put the streaked starting strain into an anaerobic jar for anaerobic culture, select a single colony, inoculate the streaked starting strain into an RCM liquid culture medium which is boiled and cooled to 40-55 ℃, cover paraffin when the streaked starting strain is hot, and culture the streaked starting strain for a period of time to obtain activated bacterial liquid.

Preferably, the screening culture condition in the step (1) is anaerobic culture at 37 ℃ for 12 h.

Preferably, the mutagenesis condition in the step (1) is a treatment distance of 2 mm, the radio frequency power input is 100-120W, the helium gas flow rate is 10L/min, and the treatment time is 180-200 s.

Preferably, the concentration of the glycerol in the step (1) is 100-120 g/L.

Preferably, the fermentation medium in step (3) comprises (per liter): 100-120 g/L of glycerol, 1-2 g/L of yeast powder and K₂HPO₄×3H₂O 1g，KH₂PO₄ 0.5 g，(NH₄)₂SO₄ 2 g，MgSO₄×7H₂O 0.2 g，CaCl₂×2H₂0.02 g of O, 1.0 mL of trace element solution and 1.0 mL of iron solution.

Wherein the microelement solution comprises the following components (contained in each liter): ZnCl₂ 70 mg，MnCl₂×4H₂O 100mg，H₃BO₃ 60 mg，CoCl₂×6H₂O 200 mg，CuCl₂×2H₂O 20 mg，NiCl₂×6H₂O 25 mg，Na₂MoO₄×2H₂O35 mg, 37% HCl (v/v) 0.9 mL; the iron solution composition (per liter): FeSO₄×7H₂O 5 g，37%HCl 4 mL。

Preferably, the inoculation amount in the step (3) is 8%, and the anaerobic culture is anaerobic culture at 37 ℃ for 20-30 h.

The Clostridium butyricum mutant strain Clostridium butyricum mutant mAYM014 takes glycerol as a substrate, and the yield of 1,3-PD reaches 41.3 g/L.

The invention also provides application of the clostridium butyricum with the preservation number of CCTCC number M2019459 in producing 1, 3-propylene glycol.

In addition, the invention also provides a production method of 1, 3-propylene glycol, which comprises the steps of inoculating clostridium butyricum with the preservation number of CCTCC number M2019459 into an RCM liquid culture medium containing glycerol with the final concentration of 60 mM for culture to obtain a fermentation seed solution, and then transferring the fermentation seed solution into the fermentation culture medium for fermentation.

Description of biological preservation

The biological material Clostridium butyricum QXYZ514 is preserved in China center for type culture Collection with the preservation number: CCTCC number M2019460, the address of the preservation unit is: wuhan university 299 in the Wuchang area of Wuhan city, Hubei province, Wuhan university Collection center; the preservation date is as follows: 6 and 17 months in 2019.

The biological material Clostridium butyricum AYM014 is preserved in the China center for type culture Collection, and the preservation numbers are as follows: CCTCC number M2019459, the address of the preservation unit is: wuhan university 299 in the Wuchang area of Wuhan city, Hubei province, Wuhan university Collection center; the preservation date is as follows: 6 and 17 months in 2019.

Compared with the prior art, the invention has the following beneficial effects:

the Clostridium butyricum with the preservation number of CCTCC number M2019459 uses Clostridium butyricum QXYZ514 as an initial strain, improves the tolerance to glycerol through ARTP repeated mutagenesis, obtains the 1, 3-propanediol highest-yield strain Clostridium butyricum AYM014 through batch fermentation after rescreening, and the yield of 1,3-PD produced by the Clostridium butyricum AYM014 reaches 41.3 g/L and is obviously improved compared with the yield of the initial strain 1,3-PD, and coenzyme B does not need to be added in the conversion process₁₂Greatly reduces the production cost, has theory and practice on the modification of natural production strains of the 1,3-PD and the biological industrial production of the 1,3-PDPractice the meaning of the reference. Compared with the traditional mutagenesis method, the ARTP mutagenesis method adopted by the invention can effectively cause various denaturation of DNA, has high mutation ratio, can stably transmit the mutation gene to filial generation, and has the advantages of low running cost, convenient operation, no generation of toxic and harmful substances and the like.

Drawings

FIG. 1 is a graph showing the lethality at various ARTP mutagenesis times of the starting strain Clostridium butyricum QXYZ514 in example 5.

Detailed Description

The clostridium butyricum has higher tolerance to substrate glycerol in the process of producing 1, 3-propylene glycol without adding coenzyme B₁₂The yield of 1, 3-propanediol can be improved. Those skilled in the art can modify the process parameters appropriately in view of the disclosure herein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate variations and combinations of the methods and applications described herein may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention is further illustrated by the following examples:

example 1: the invention relates to a primary screen of Clostridium butyricum AYM014

Activating Clostridium butyricum QXYZ514, wherein the specific steps of activating are as follows: the strain of Clostridium butyricum QXYZ514 stored at-80 ℃ is streaked in an RCM agar plate culture medium, the RCM agar plate is placed in a 2.5L anaerobic tank for anaerobic culture, then a single colony is selected and inoculated into an RCM liquid culture medium which is boiled and cooled to 40-55 ℃, paraffin is covered on the RCM liquid culture medium when the RCM agar plate is hot, the inoculation amount is 2%, and the culture condition is that the RCM agar plate is cultured for 10-14 hours at 37-40 ℃ to obtain activated bacterial liquid. The components of the RCM agar plate culture medium are as follows: 3 g/L of yeast extract, 10 g/L of beef extract, 10 g/L of tryptone, 5 g/L of glucose, 1 g/L of soluble starch, 5 g/L of NaCl, 3 g/L of sodium acetate trihydrate, 0.15 g/L of cysteine hydrochloride and 15 g/L of agar. The RCM liquid medium was composed in the same manner as the above-mentioned RCM agar plate medium except that no agar was added.

Diluting the activated bacterial liquid to CFU of about 10⁶Then the solution is dripped on a stainless steel slide prepared by an ARTP mutagen, and the slide is put into a normal pressure room temperature plasma mutagen (ARTP-IIS, Qingtian wood biotechnology limited without tin source), and the set parameters are as follows: the treatment distance is 2 mm, the radio frequency power input is 100-120W, the helium gas flow rate is 10L/min, the treatment time is 180-200 s, plasma irradiation mutagenesis treatment is carried out, and the bacterium liquid after mutagenesis is repeatedly eluted and resuspended in an RCM liquid culture medium; coating 100 mu L of the mutagenized bacterial liquid on an RCM agar plate culture medium containing glycerol for screening culture, wherein the initial glycerol concentration is 100 g/L, performing anaerobic culture at 37 ℃ for 12h, and collecting thalli; performing secondary mutagenesis of ARTP by the same method as the above steps except that the concentration of glycerol in RCM agar plate culture medium is 110 g/L during screening culture of the mutagenized bacteria liquid; the third mutagenesis of ARTP was carried out in the same manner as in the above-mentioned step except that the glycerol concentration in the RCM agar plate medium was 110 g/L in the screening culture of the mutagenized bacterial liquid.

After three times of repeated mutagenesis, 29 primarily screened mutant strains are obtained and named as AYM 001-029, and morphological characteristics of colonies are recorded; respectively selecting single colonies of the primary screening mutant strains, performing basal culture in an RCM liquid culture medium to obtain liquid cultures, inoculating the liquid cultures into a 96-well plate added with the RCM liquid culture medium, and performing secondary screening, wherein the inoculation amount is 2% of the volume of the culture medium; because the biomass of the strain has positive correlation with the yield of the 1,3-PD, the biomass is taken as an index, and the mutant strain with the biomass in the top five in the same culture time is screened for carrying out the batch fermentation for subsequently producing the 1, 3-propanediol. Optical Density (OD) of biomass at wavelength of 600 nm₆₀₀) The factor of 0.34 multiplied is converted into Dry Cell Weight (CDW), and the first five mutant strains are AYM003, AYM007, AYM013, AYM014 and AYM022 respectively. The CDW values of the first five mutant strains are shown in table 1 below, and the relative biomass was calculated based on AYM014 strain, which had the highest biomass.

TABLE 1 Biomass of five mutant strains

Mutant strains	CDW	Relative biomass
			AYM003	2.62±0.21	94.2%
AYM007	2.70±0.16	97.1%
			AYM013	2.55±0.09	91.7%
AYM014	2.78±0.22	100.0%
			AYM022	2.62±0.11	94.2%

Example 2: 1,3-PD fermentation of mutant strains

The five mutant strains obtained in example 1 were inoculated into RCM broth containing glycerol at a final concentration of 60 mM and cultured at 37 ℃ for 24 hours to obtain fermentation seed solutions, and glycerol was added to induce the expression of enzymes involved in glycerol metabolism. Then toRespectively inoculating 8% of the inoculum size into a fermentation medium for batch fermentation, wherein the fermentation place is a 500 mL triangular flask, and the fermentation volume is 400 mL; and (3) carrying out anaerobic culture for 20-30 h at 37 ℃ under the fermentation condition, and collecting fermentation liquor. The components in the fermentation medium (per liter contained): 100-120 g of glycerol, 1-2 g of yeast powder and K₂HPO₄×3H₂O 1 g，KH₂PO₄ 0.5 g，(NH₄)₂SO₄ 2 g，MgSO₄×7H₂O 0.2 g，CaCl₂×2H₂0.02 g of O, 1.0 mL of trace element solution and 1.0 mL of iron solution.

Wherein, the trace element solution comprises the following components (per liter): ZnCl₂ 70 mg，MnCl₂×4H₂O 100mg，H₃BO₃60 mg，CoCl₂×6H₂O 200 mg，CuCl₂×2H₂O 20 mg，NiCl₂×6H₂O 25 mg，Na₂MoO₄×2H₂O35 mg, 37% HCl (v/v) 0.9 mL; iron solution composition (per liter): FeSO₄×7H₂O 5 g，37%HCl 4 mL。

And (3) determining the yield of the 1, 3-propanediol in the fermentation liquor by adopting a high performance liquid chromatography method, and screening out the clostridium butyricum mutant strain with the highest 1,3-PD yield. The final yields of 1,3-PD of the five mutants are shown in Table 2, and relative yields were calculated based on AYM014 strain with the highest 1,3-PD yield.

TABLE 2 Final yields of 1,3-PD from five mutants

Mutant strain	Yield (g/L) of 1,3-PD	Relative yield
			AYM003	40.2±0.99	97.3%
AYM007	40.2±1.07	97.3%
			AYM013	38.7±0.57	93.7%
AYM014	41.3±0.84	100.0%
			AYM022	39.5±1.21	95.6%

As shown in Table 2, the optimal mutant strain Clostridium butyricum AYM014 showed that the final yield of 1,3-PD after 24 hours of fermentation reached 41.3 g/L. Culturing spore of mutant strain Clostridium butyricum AYM014 on RCM liquid medium at constant temperature of 37 deg.C for 1 d to obtain clear milky colony, which is irregular round and large, and has regular microsomal arrangement and enlarged spindle shape; and continuously culturing for 1 d to obtain spores, ovate and secondary growth. The mutant strain Clostridium butyricum AYM014 has the advantages of high growth speed, high tolerance to glycerol substrate, good genetic stability, and high yield of 1,3-PD obtained by using glycerol as the sole substrate. The optimal mutant strain Clostridium butyricum AYM014 is preserved in China center for type culture Collection in 2019, 6 and 17 months, with the preservation number of CCTCC number M2019459, and the preservation unit address is Wuhan university No. 299 in Wuchang district, Wuhan university, Hubei province.

Example 3: 1,3-PD fermentation of starting strains

The starting strain of the invention is fermented by 1,3-PD according to the same production method as that of the embodiment 2, the fermentation environment and the inoculation amount are the same, the final yield of the 1,3-PD in the fermentation liquor is measured to be 28.7 g/L by adopting high performance liquid chromatography, and the yield of the 1,3-PD of the mutant strain Clostridium butyricum AYM014 is improved by 43.9 percent compared with the yield of the starting strain Clostridium butyricum QXYZ 514.

Example 4: glycerol tolerance assay of the starting Strain

Clostridium butyricum QXYZ514 was activated by the same procedure as described in example 1. The activated colonies were placed in RCM liquid media containing different glycerol concentrations (60, 70, 80, 90, 100, 110, 120 g/L), anaerobic incubation was performed at 37 ℃ for 24 h with the RCM liquid media without glycerol as a control, absorbance of each group of cultures at 600 nm was measured, and the tolerance of Clostridium butyricum QXYZ514 to glycerol was 80 g/L, and the survival rate of bacteria was 85-90% at this concentration.

Example 5: ARTP mutagenesis timing of starting strains

Clostridium butyricum QXYZ514 was activated by the same procedure as described in example 1. Culturing the activated bacterial colony to an exponential phase, diluting the bacterial liquid to 106 CFU, dropwise adding the bacterial liquid to a stainless steel slide prepared by a mutagen, and then putting the slide into an ARTP mutagen, wherein the set parameters are as follows: the treatment distance is 2 mm, the radio frequency power input is 100W, the helium gas flow rate is 10L/min, the treatment time is respectively set to be 0, 60, 90, 120, 150, 180, 210 and 240 s, the lethality rate at different time is calculated, and a lethality curve is manufactured; FIG. 1 is a graph showing the lethality of the starting strain Clostridium butyricum QXYZ514 ARTP at various times during mutagenesis; as shown in FIG. 1, the lethality of the strain increased with the increasing time of ARTP mutagenesis, and increased exponentially as the time increased from 120 s to 160 s, and reached about 90% as it continued to 180 s, while the lethality was found to be 100% after treating the strain for 240 s. Generally, it is considered that the treatment conditions are appropriate when the lethality reaches about 90%, and the ARTP mutagenesis time is selected to be 180 to 200 s, and the optimum ARTP mutagenesis time is determined to be 180 s.

The embodiments of the invention are not limited to the applications set forth in the description and the embodiments, but may be modified in practice by those skilled in the art. Therefore, the invention is not to be limited to the specific details and embodiments shown herein without departing from the general concept as defined by the appended claims and their equivalents.

Claims

1. The strain of Clostridium butyricum has a preservation number of CCTCC number M2019459 in China center for type culture collection, and is classified and named Clostridium butyricum AYM 014.

2. Use of clostridium butyricum according to claim 1 for the production of 1, 3-propanediol.

3. A production method of 1, 3-propanediol is characterized in that clostridium butyricum with the preservation number of CCTCC number M2019459 is inoculated into an RCM liquid culture medium containing 60 mM of glycerol to be cultured to obtain a fermentation seed solution, the fermentation seed solution is inoculated into the fermentation culture medium to be subjected to anaerobic culture, and the 1, 3-propanediol is collected.