CN117925747A

CN117925747A - Application of Serratia marcescens in co-production of prodigiosin and diltiazem intermediate

Info

Publication number: CN117925747A
Application number: CN202311835867.4A
Authority: CN
Inventors: 吴凌天; 杨铠忆; 周子奕; 崔巍; 葛楠; 陈�峰; 徐渊美; 胡西琪
Original assignee: Changshu Institute of Technology
Current assignee: Changshu Institute of Technology
Priority date: 2023-12-28
Filing date: 2023-12-28
Publication date: 2024-04-26

Abstract

The invention discloses an application of Serratia marcescens in co-production of prodigiosin and diltiazem intermediates, and belongs to the technical field of bioengineering. According to the invention, molasses is used as a carbon source, tryptone is used as a nitrogen source, prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM are fermented and synthesized, the yield of prodigiosin obtained by fermentation is up to 27.6g/L, and the yield of diltiazem intermediate (2R, 3S) - (-) -MPGM is 17.3g/L. The operation method disclosed by the invention is simple, has a short production period, has a great industrial popularization and application prospect, and simultaneously provides a novel process for biosynthesis of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM.

Description

Application of Serratia marcescens in co-production of prodigiosin and diltiazem intermediate

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to application of Serratia marcescens in co-production of prodigiosin and diltiazem intermediates.

Background

Prodigiosin (Prodigiosins, PG) is a natural red pigment produced by microorganisms and is a secondary metabolite. There is a great deal of attention for their anti-inflammatory, antibacterial, antioxidant, various biological activities, antitumor effects, etc. The main production method of the prodigiosin is a microbial fermentation method at present, and the prodigiosin yield is improved by optimizing the components of fermentation liquor, the culture conditions, the culture environment and the culture time because the prodigiosin yield is closely related to the composition of a culture medium and the culture conditions.

Diltiazem is a cardiovascular and cerebrovascular medicine commonly used in clinic, has good efficacy and wide clinical applicability, and has considerable market prospect at home and abroad. Diltiazem is generally synthesized by chemical resolution, asymmetric synthesis and biological resolution. However, the asymmetric synthesis reaction conditions are harsh, the resolution reagent is expensive, the cost is high, and the requirements of industrial production are not met, so that the diltiazem is synthesized by a resolution method which is easy to operate and environment-friendly. The method is suitable for large-scale industrial production, high in efficiency, low in toxicity, low in cost and simple in operation, and general attention in the pharmaceutical field is paid. And the lipase produced by Serratia marcescens can be used for resolution of trans-4-p-methoxyphenyl glycidyl acid methyl ester [ (+ -) -MPGM ] on the synthetic route to obtain p-methoxyphenyl glycidyl acid methyl ester [ (2R, 3S) - (-) -MPGM ], which is a key intermediate for synthesizing diltiazem. The (+/-) -MPGM is directly split by using Serratia marcescens fermentation broth, and high-yield prodigiosin and diltiazem key intermediate (2R, 3S) - (-) -MPGM can be obtained, so that the production period is shortened, and the production cost is reduced.

Through searching, no related report of the coproduction of the prodigiosin and the diltiazem intermediate by Serratia marcescens is currently found.

Disclosure of Invention

The invention aims to: the invention aims to solve the problem of providing a method for co-producing prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM by utilizing serratia marcescens, preparing prodigiosin by utilizing serratia marcescens fermentation, and simultaneously resolving trans-4-p-methoxyphenyl glycidic acid methyl ester [ (+ -) -MPGM ] by utilizing lipase of lipase in serratia marcescens fermentation liquid to obtain p-methoxyphenyl glycidic acid methyl ester [ (2R, 3S) - (-) -MPGM ], thereby improving production efficiency and reducing production cost.

The technical scheme is as follows: in order to solve the problems, the invention adopts the following technical scheme:

Serratia marcescens is applied to coproduction of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM.

The Serratia marcescens is named as Serratia marcescens LT-7 (SERRATIA MARCESCENS LT-7) which is preserved in China center for type culture Collection (CCTCC for short), and the preservation address is: eight ways in the mountain area of the city of Wuhan in Hubei province, wuhan university mountain country collection of typical cultures, post code: 430072, the preservation number is CCTCC NO: M20231646, and the preservation date is 2023, 9 and 7. The strain was used as a production strain in the following.

The specific application method comprises inoculating Serratia marcescens LT-7 in a solid culture medium, transferring to a seed culture medium, inoculating to a fermentation culture medium, performing aerobic culture, and adding (+ -) -MPGM and toluene into the fermentation broth to obtain prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM.

The Serratia marcescens LT-7 and the application of the Serratia marcescens LT-7 in preparing prodigiosin and (2R, 3S) - (-) -MPGM sequentially comprise the following steps:

1. Preparing a culture medium:

(1) Slant culture medium components: 10g/L of tryptone, 10g/L of NaCl, 5g/L of yeast powder, 20g/L of agar and the balance of water, wherein the pH value is 6.0-7.0, preferably 6.8;

(2) Solid seed medium: 10g/L of tryptone, 10g/L of NaCl, 5g/L of yeast powder, 20g/L of agar, pH value of 6.8, the balance of water, and pH value of 6.0-7.0, preferably 6.8;

(3) Liquid seed medium: 15g/L of tryptone, 10g/L of maltose, pH value of 6.8 and the balance of water, wherein the pH value is 6.4-8.0, preferably 6.8;

(4) Fermentation medium: 5-15 g/L of carbon source, 10-20 g/L of nitrogen source, 0.005-1 g/L of metal salt, 6.4-8.0 of pH value, 2-5 g/L of proline and water as solvent.

Wherein the carbon source is any one or the combination of more than one of sugarcane juice, molasses, sucrose, mannose and glycerol; molasses is preferred, and the addition amount of the molasses is preferably 10g/L;

The nitrogen source is any one or a combination of a plurality of tryptone, soybean peptone, peanut cake powder and soybean cake powder, preferably tryptone, and the addition amount of the tryptone is preferably 15g/L;

The metal salt is magnesium sulfate, and most preferably 0.8g/L of magnesium sulfate;

The most preferred fermentation medium is as follows: 10g/L molasses, 15g/L tryptone, 0.8g/L magnesium sulfate, 25g/L tween-80 and 3g/L proline, and the solvent is water.

2. And (3) strain selection:

the deposited strain Serratia marcescens LT-7 was selected.

3. Activating strains:

Inoculating Serratia marcescens LT-7 strain into slant culture medium, standing at 20-30 deg.c for 18-34 hr, and then picking single colony into common solid culture medium for three-area line and standing at 20-30 deg.c for 18-34 hr to obtain activated strain.

4. Seed liquid preparation:

And (3) inoculating the activated strain obtained in the step (3) into a shaking bottle of the seed liquid under the aseptic condition, and culturing the seed liquid in a shaking table with the temperature of 30 ℃ and the rotating speed of 200rpm for 12 hours in a dark place to obtain the seed liquid.

5. Shake flask fermentation culture:

Inoculating the seed solution obtained in the step 4 into a fermentation culture shake flask with an inoculum size of 3% under aseptic condition, and culturing in dark for 36h at a temperature of 32deg.C and a rotation speed of 250 rpm. Fermentation is stopped when the concentration of prodigiosin and (2R, 3S) - (-) -MPGM in the fermentation broth is substantially no longer rising.

6. Fermentation and culture in a fermentation tank:

Inoculating the seed liquid obtained in the step 4 into a 50L fermentation tank with an inoculum size of 3% under aseptic condition, wherein the total liquid loading amount of the fermentation liquid is 35L, the fermentation temperature is 32 ℃, the stirring speed is 250rpm, the pH is 6.8, the ventilation amount is 0.9-1.1 VVM, and the fermentation time is 46h; fermentation is stopped when the concentration of prodigiosin and (2R, 3S) - (-) -MPGM in the fermentation broth is substantially no longer rising.

7. Preparation of (2R, 3S) - (-) -MPGM:

When fermentation is carried out for 38 hours, 5-20 g/L (+ -) -MPGM and 0.5-1 times volume of toluene of the fermentation broth are added into the culture broth, and the culture is stopped after continuous culture for 2-8 hours at the temperature of 32 ℃ and the rpm of 250 on a shaking table.

8. Extraction of prodigiosin and (2R, 3S) - (-) -MPGM:

(1) Preparing a crude product: centrifuging Serratia marcescens LT-7 fermentation liquor, extracting thallus with 2 times methanol until thallus becomes white, mixing with supernatant, concentrating 3-5 times, adding equal volume of ethyl acetate for overnight extraction, filtering organic phase, and rotary evaporating to concentrate into extract-like coarse body.

(2) Extraction of prodigiosin and (2R, 3S) - (-) -MPGM: the organic phase of (1) was washed with 5% NaHSO ₃ and then with 5% NaHCO ₃, and dried over magnesium sulfate to give (2R, 3S) - (-) -MPGM. 200-300 mesh silica gel is used as a stationary phase, and petroleum ether: ethyl acetate=7: 3 as mobile phase, dissolving the residual organic phase with methanol, loading, purifying the prodigiosin by adopting a silica gel column chromatography method, collecting the component with the corresponding Rf value, rotationally evaporating, concentrating, and freeze-drying for 24 hours to obtain the prodigiosin.

9. Prodigiosin content determination:

The prodigiosin standard with a constant dry weight was precisely weighed and prepared as standard solutions of 0.05, 0.10, 0.15, 0.20, 0.25 and 0.30mg/mL, and absorbance was measured at 535 nm. And drawing a standard curve by taking absorbance as an ordinate and concentration as an abscissa to obtain a regression equation, and calculating the content of the prodigiosin according to the standard curve as shown in the figure.

10. Identification of prodigiosin

And (3) identifying Serratia marcescens LT-7 fermentation products by adopting a high performance liquid chromatograph, an infrared spectrometer and a nuclear magnetic resonance spectrometer.

10Mg of prodigiosin purified product is dissolved in 100ml of methanol, and the pH is adjusted to 3, so that prodigiosin methanol solution is obtained, and the liquid chromatography result is shown in figure 1.

And (3) identifying an infrared spectrum of the purified product of the serratia marcescens LT-7 fermented prodigiosin by using an infrared spectrometer, grinding and tabletting 0.2mg of a sample and a small amount of KBr to prepare the sample, and measuring the infrared spectrum of the sample by using the infrared spectrometer, wherein the scanning range is 4000-400 cm ^-1 (figure 2). NMR ¹ H and NMR ¹³ C were performed on Serratia marcescens LT-7 fermentation purified product using CD ₃ Cl as solvent. The results are shown in fig. 3 and 4.

The beneficial effects are that:

According to the invention, the serratia marcescens S.marcescens LT-7 capable of co-producing prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM is obtained through first screening, the highest concentration of prodigiosin in a fermentation medium is 27.6g/L, the highest concentration of diltiazem intermediate (2R, 3S) - (-) -MPGM is 17.3g/L, the highest conversion rate can reach 86.5%, the production period is greatly shortened, the process is simple, and the cost is reduced, so that the serratia marcescens S.marcescens has important social and economic significance for producing prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM.

Drawings

FIG. 1 is a phylogenetic tree of Serratia marcescens LT-7.

FIG. 2 is a high performance liquid chromatogram of Serratia marcescens LT-7 prodigiosin.

FIG. 3 is a Fourier infrared spectrum of Serratia marcescens LT-7 prodigiosin.

FIG. 4 is a NMR ¹ H analysis of Serratia marcescens LT-7 prodigiosin.

FIG. 5 is a NMR ¹³ C analysis chart of Serratia marcescens LT-7 prodigiosin.

FIG. 6 is an effect of carbon source species on co-production of prodigiosin and diltiazem intermediates.

FIG. 7 is an effect of carbon source concentration on co-production of prodigiosin and diltiazem intermediates.

FIG. 8 is an effect of nitrogen source species on co-production of prodigiosin and diltiazem intermediates.

FIG. 9 is an effect of nitrogen source concentration on co-production of prodigiosin and diltiazem intermediates.

Figure 10 is an effect of metal salt species and their concentration on co-production of prodigiosin and diltiazem intermediates.

FIG. 11 is the effect of Tween-80 concentration on co-production of prodigiosin and diltiazem intermediates.

FIG. 12 is an effect of temperature on co-production of prodigiosin and diltiazem intermediates.

FIG. 13 is an effect of pH on co-production of prodigiosin and diltiazem intermediates.

Figure 14 is the effect of inoculum size on co-production of prodigiosin and diltiazem intermediate.

FIG. 15 is an effect of an intermediate preparation process on co-production of prodigiosin and diltiazem intermediates.

The intermediate preparation conditions of figure 16 are the effect on co-production of prodigiosin and diltiazem intermediates.

FIG. 17 is a plot of the progress of 50L fermentor fed-batch co-production of prodigiosin and diltiazem intermediates.

FIG. 18 is a graph of the progress of fed-batch co-production of prodigiosin and diltiazem intermediates in a 1t fermenter.

The specific embodiment is as follows:

The invention may be better understood from the following examples, which are set forth to illustrate, but are not to be construed to limit the invention to the details of the claims, as will be apparent to those skilled in the art.

Example 1: isolation and screening of Serratia marcescens LT-7

The composition of the medium used in this example is as follows:

LB solid medium: 10g/L of peptone, 10g/L of sodium chloride, 5g/L of yeast powder, 20g/L of agar and the balance of water, and sterilizing for 20min at 121 ℃.

The specific operation procedure of this example is as follows:

The procedure for screening pigment-producing bacteria was as follows: microbial pigment-producing bacteria were selected from 60 parts of soil from various places throughout the country, 1g each was added to a triangular flask containing sterile enrichment medium, and the mixture was subjected to enrichment culture at 30℃on a shaker at 200rpm for 48 hours. 1mL of the culture solution is transferred to the same liquid enrichment culture medium for culturing for 48 hours under the same conditions. Under the aseptic condition, diluting the culture solution to 10 ^-6 and 10 ^-7, respectively taking 0.2mL of the culture solution to be coated in LB solid culture medium, culturing for 48 hours at 30 ℃, screening positive strains according to the surface color of bacterial colonies, separating and purifying, inoculating the positive strains into a fermentation culture medium, culturing for 48 hours at 30 ℃ and 200rpm, and measuring the yield of pigment of the primary screened strain to obtain the strain with the highest yield. In subsequent experiments with various physiological and biochemical assays, it was found that a certain amount of lipase was contained therein. The lipase can split (+ -) -MPGM to obtain the diltiazem key intermediate (2R, 3S) - (-) -MPGM.

Example 2: identification of Serratia marcescens LT-7.

Extracting genome DNA of Serratia marcens LT-7 by using a bacterial genome DNA extraction kit, amplifying a 16S rDNA sequence by using an upstream primer 27F and a downstream primer 1492R PCR, carrying out gel recovery and purification on the PCR amplified product, and sending the gel recovery and purification product to Suzhou gold and other intelligent biotechnology Co. The length of the nucleotide sequence of the 16S rDNA gene of the strain is 1471bp, and the gene sequence is shown as SEQ ID No.1: as shown. The sequences were compared for homology from the Gene Bank database using the BLAST program, and a 16SrDNA full sequence-based phylogenetic tree was constructed (FIG. 1). The results show that: the strain has 100% homology with Serratia marcescens JW-CZ 2. According to the morphological observation of the strain and the analysis result of physiological and biochemical experiments, serratia marcescens, specifically Serratia marcescens LT-7, is adopted.

Example 3: identification of the fermentation product prodigiosin of Serratia marcescens LT-7

Seed liquid culture mediums of Serratia marcescens LT-7 are respectively inoculated into the same fermentation culture mediums (the composition of the fermentation culture mediums is as follows, molasses is 10g/L, tryptone is 15g/L, magnesium sulfate is 0.8g/L, tween-80 is 25g/L and proline is 3 g/L) according to the inoculation amount of 3.5 percent, the pH value is adjusted to 6.8, the culture is carried out at 32 ℃ under the shaking condition of 250rpm, the liquid content of the fermentation culture mediums is 50mL/250mL conical flask, and the fermentation culture is stopped after 38 hours.

Centrifuging the LT-7 pigment fermentation liquor at 9000rpm, adding 1-2 times of methanol into the sediment for leaching, concentrating the supernatant into 1/5 of the original volume by rotary evaporation at 55-65 ℃, adding 3-5 times of methanol, standing overnight in a refrigerator at 4 ℃, combining with the settled methanol solution, centrifuging, filtering, adding 1-2 times of water for extraction, and concentrating the organic phase to obtain a prodigiosin crude product; the crude product is dissolved in 2 times of methanol for standby. The prodigiosin is separated and purified by adopting a silica gel chromatographic column, and petroleum ether is adopted as a mobile phase: acetone (7:3), silica gel with 200-300 meshes, and concentrating the purified solution by rotary evaporation at 50 ℃ and freeze-drying to obtain prodigiosin powder.

10Mg of prodigiosin purified product was dissolved in 10mL of acidic methanol to obtain prodigiosin methanol solution, and the liquid chromatography result thereof was shown in FIG. 2.

And (3) identifying an infrared spectrum of the purified product of the serratia marcescens LT-7 fermented prodigiosin by using an infrared spectrometer, grinding and tabletting 0.2mg of a sample and a small amount of KBr, and measuring the infrared spectrum of the sample by using the infrared spectrometer, wherein the scanning range is 4000-400 cm ^-1 (figure 3). The purified product of Serratia marcescens LT-7 fermentation was subjected to (NMR) ¹ H and (NMR) ¹³ H detection using CD ₃ Cl as a solvent. The results are shown in fig. 4 and 5.

Example 4: optimization of the carbon source types of the combined production of prodigiosin and diltiazem intermediates by Serratia marcescens.

This example illustrates the effect of different carbon source species on the yield of prodigiosin and diltiazem intermediates prepared by fermentation of the strain.

Seed liquid culture medium is respectively inoculated into fermentation culture medium containing 15g/L (total sugar concentration in fermentation liquid) of Sugarcane Juice (SJ), molasses (CM), sucrose (Suc), mannose (Mal) and glycerin (Gly) in an inoculum size of 3% (v/v) (other components of the fermentation culture medium are as follows: tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 25g/L and proline 3 g/L), initial pH value is 7.0, shaking culture is carried out at 28 ℃ and 250rpm, liquid loading amount of the fermentation culture medium is 50mL/250mL conical flask, diltiazem intermediate (2R, 3S) - (-) -MPGM is prepared after fermentation culture is carried out for 38 hours, and 15g/L of (+ -) -MPGM and 25mL toluene are respectively added to carry out continuous reaction on a shaking table for 2 hours. And (3) taking fermentation solutions of different carbon sources for content measurement, and calculating the yield of the prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The highest yield was found when the carbon source was chosen as molasses. Prodigiosin yields were about 7.1g/L, production rates as high as 0.19g/L/h, and diltiazem intermediates yields were about 5.42g/L (FIG. 6).

Example 5: and (3) optimizing the carbon source concentration of the combined prodigiosin and diltiazem intermediate of Serratia marcescens.

This example illustrates the effect of different carbon source concentrations on the yields of prodigiosin and diltiazem intermediates prepared by fermentation of the strains, seed solution medium was inoculated in an inoculum size of 3% (v/v) into fermentation medium containing 0g/L, 5g/L, 10g/L, 15g/L, 20g/L molasses (other components of the fermentation medium are as follows: tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 25g/L, proline 3 g/L), initial pH value was 7.0, shaking culture was carried out at 28℃at 250rpm, the medium volume was 50mL/250mL Erlenmeyer flask, diltiazem intermediate (2R, 3S) - (-) -MPGM) was prepared after fermentation culture for 38 hours, 15g/L (. + -.) -MPGM and 25mL toluene were added respectively, and the reaction was continued on a shaker for 2 hours. Measuring the content of fermentation liquid with different carbon sources, and calculating the yield of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The highest yield was found when the carbon source concentration was selected to be 10 g/L. Prodigiosin yields were about 8.1g/L, production rates as high as 0.21g/L/h, and diltiazem intermediates yields were about 7.77g/L (FIG. 7).

Example 6: optimization of the nitrogen source species of Serratia marcescens co-produced prodigiosin and diltiazem intermediates.

This example illustrates the effect of different nitrogen sources on the yields of prodigiosin and diltiazem intermediates prepared by fermentation of the strains, seed solution medium was inoculated in an inoculum size of 3% (v/v) into a fermentation medium containing 10g/L tryptone (A), soybean peptone (B), peanut cake (C) and soybean cake (D) (other components of the fermentation medium are as follows: molasses 10g/L, magnesium sulfate 0.8g/L, tween-80 25g/L, proline 3 g/L), initial pH was 7.0, shaking culture was carried out at 28℃and 250rpm, the medium volume was a 50mL/250mL Erlenmeyer flask, and after fermentation culture for 38 hours, diltiazem intermediate (2R, 3S) - (-) -MPGM was prepared by adding 15g/L of (+ -) -MPGM and 25mL toluene, respectively, and continuing the reaction on a shaker for 2 hours. And (3) taking fermentation solutions of different nitrogen sources for content measurement, and calculating the yield of the prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The highest yield was found when the nitrogen source was selected as tryptone. Prodigiosin yields were about 12.6g/L, production rates as high as 0.33g/L/h, and diltiazem intermediates yields were about 7.92g/L (FIG. 8).

Example 7: and (3) optimizing nitrogen source concentration of the combined prodigiosin and diltiazem intermediate of Serratia marcescens.

This example illustrates the effect of different nitrogen source concentrations on the yields of prodigiosin and diltiazem intermediates prepared by fermentation of the strain, seed solution medium was inoculated in an inoculum size of 3% (v/v) into a fermentation medium containing 5g/L, 10g/L, 15g/L and 20g/L tryptone (other components of the fermentation medium are as follows: molasses 10g/L, magnesium sulfate 0.8g/L, tween-80 25g/L and proline 3 g/L), initial pH was 7.0, shaking culture was carried out at 28℃at 250rpm, the liquid loading of the fermentation medium was a 50mL/250mL Erlenmeyer flask, diltiazem intermediate (2R, 3S) - (-) -MPGM was prepared after fermentation culture for 38 hours, and 15g/L of (+ -) -MPGM and 25mL toluene were added, respectively, and the reaction was continued on a shaker for 2 hours. Measuring the content of fermentation liquid with different nitrogen sources, and calculating the yield of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The highest yield was found when the nitrogen source concentration was selected to be 15 g/L. Prodigiosin yields were about 13.3g/L, production rates as high as 0.35g/L/h, and diltiazem intermediates yields were about 8.4g/L (FIG. 9).

Example 8: and (3) optimizing the concentration of metal salts required by the Serratia marcescens for co-producing prodigiosin and diltiazem intermediate.

This example illustrates the effect of different metal salts and their concentrations on the yield of prodigiosin and diltiazem intermediates prepared by fermentation of strains, seed culture broth was inoculated in an inoculum size of 3% (v/v) into a medium containing the following magnesium sulfate concentrations (other components of the medium are as follows: 10g/L molasses, 15g/L tryptone, 25g/L tween-80, 3g/L proline) respectively, as follows: magnesium sulfate: 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2g/L and control, the optimal concentration of magnesium sulfate is 0.8g/L; for the fermentation culture medium of the experimental group, 3 groups of parallel controls are arranged, the initial pH value is 7.0, the culture medium is subjected to shaking culture at 28 ℃ and 250rpm, the liquid loading amount of the culture medium is a 50mL/250mL conical flask, 15g/L (+ -) -MPGM and 25mL toluene are respectively added into the fermentation culture medium for fermentation culture for 38 hours, the reaction is continued for 2 hours on a shaking table, the fermentation liquid under each metal salt concentration is taken for content measurement, and the yield of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM) is calculated. When the metal salt was selected to have a magnesium sulfate concentration of 0.8g/L, the prodigiosin yield was about 14.7g/L, the production rate was as high as 0.39g/L/h, and the diltiazem intermediate yield was about 7.8g/L (FIG. 10).

Example 9: and (3) optimizing the concentration of the combined prodigiosin and diltiazem intermediate Tween-80 by Serratia marcescens.

This example illustrates the effect of different concentrations of Tween-80 on the production of prodigiosin and diltiazem intermediate by fermentation of the strain, seed solution medium was inoculated in an inoculum size of 3% (v/v) into 15g/L, 20g/L, 25g/L, 30g/L, 35g/L Tween-80 fermentation medium (other ingredients of the fermentation medium were as follows: molasses 10g/L, tryptone 15g/L, magnesium sulfate 0.8g/L, proline 3 g/L), initial pH was 7.0, shaking culture was carried out at 28℃at 250rpm, the medium volume was a 50mL/250mL Erlenmeyer flask, and diltiazem intermediate (2R, 3S) - (-) -MPGM) was prepared after 38h fermentation culture, and reaction was continued on a shaker with 15g/L (+ -) -MPGM and 25mL toluene, respectively, for 2h. The fermentation broths of Tween-80 with different concentrations are used for content measurement, and the yields of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM are calculated. The highest yield was found when Tween-80 was selected at a concentration of 25 g/L. Prodigiosin yields were about 16.9g/L, production rates as high as 0.44g/L/h, and diltiazem intermediates yields were about 8.3g/L (FIG. 11).

Example 10: optimization of temperature of Serratia marcescens co-produced prodigiosin and diltiazem intermediate.

This example illustrates the effect of culturing at different temperatures on the yields of prodigiosin and diltiazem intermediates prepared by fermentation of the strain, seed liquid medium was inoculated in an inoculum size of 3% (v/v) into the same fermentation medium (composition of fermentation medium is as follows: molasses 10g/L, tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 25g/L, proline 3 g/L), initial pH was 7.0, shaking culture was carried out at 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃,250rpm, the volume of the fermentation medium was 50mL/250mL Erlenmeyer flask, diltiazem intermediate (2R, 3S) - (-) -MPGM was prepared after fermentation culture for 38 hours, and 15g/L of (+ -) -MPGM and 25mL toluene were added, respectively, and the reaction was continued on a shaker for 2 hours. The fermentation broths cultured at different temperatures are taken for content measurement, and the yields of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM are calculated. The highest yield was found at a selection temperature of 32 ℃. Prodigiosin yields were about 18.2g/L, production rates as high as 0.48g/L/h, and diltiazem intermediates yields were about 9.6g/L (FIG. 12).

Example 11: and (3) optimizing the pH value of the combined prodigiosin and diltiazem intermediate of Serratia marcescens.

This example illustrates the effect of culturing in different pH values on the yields of prodigiosin and diltiazem intermediates prepared by fermentation of the strain, seed liquid medium was inoculated in the same fermentation medium in an inoculum size of 3% (v/v) respectively (composition of fermentation medium is as follows: molasses 10g/L, tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 g/L, proline 3 g/L), pH values were adjusted to 6.4, 6.8, 7.2, 7.6, 8.0 at 32 ℃, shaking culture was carried out at 250rpm, the liquid loading amount of fermentation medium was 50mL/250mL Erzem flask, diltiazem intermediate (2R, 3S) - (-) -MPGM was prepared after fermentation culture for 38 hours, and 15g/L of (+ -) -MPGM and 25mL toluene were added respectively, and the reaction was continued on a shaking table for 2h. Measuring the content of fermentation liquids with different pH values, and calculating the yield of the prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The highest yield was found when the pH was chosen at 6.8. Prodigiosin yields were about 19.4g/L, production rates as high as 0.51g/L/h, and diltiazem intermediates yields were about 10.5g/L (FIG. 13).

Example 12: optimization of the inoculum size of the combined production of prodigiosin and diltiazem intermediate by Serratia marcescens.

This example illustrates the effect of different inoculum size on the yield of prodigiosin and diltiazem intermediates prepared by fermentation of the strain, seed solution medium was inoculated in the same fermentation medium in an inoculum size of 2%, 2.5%, 3%, 3.5%, 4% (v/v) (composition of fermentation medium is as follows: molasses 10g/L, tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 g/L, proline 3 g/L), pH was adjusted to 6.8 at 32 ℃, shaking culture was carried out at 250rpm, fermentation medium was filled in a 50mL/250mL Erlenmeyer flask, diltiazem intermediate (2R, 3S) - (-) -MPGM was prepared after 38h fermentation culture, and reaction was continued on shaking table-type MPGM and 25mL toluene for 2h, respectively. The different precursor fermentation broths are taken for content measurement, and the yields of prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM are calculated. The highest yield was found when the inoculum size was 3.5%. Prodigiosin yields were about 21.5g/L, production rates as high as 0.57g/L/h, and diltiazem intermediates yields were about 10.1g/L (FIG. 14).

Example 13: the preparation method of the diltiazem intermediate produced by Serratia marcescens fermentation is optimized.

This example illustrates the effect of substrate concentration addition on conversion of a fermentatively produced diltiazem intermediate. Inoculating the seed solution culture medium into the same fermentation culture medium (the composition of the fermentation culture medium is as follows: molasses 10g/L, tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 25g/L and proline 3 g/L) respectively, adjusting the pH value to 6.8, carrying out shaking culture at 32 ℃ and 250rpm, wherein the liquid loading amount of the fermentation culture medium is 50mL/250mL conical flask, preparing diltiazem intermediate (2R, 3S) - (-) -MPGM after 38h of fermentation culture, and respectively adding 0, 5, 10, 15, 20, 25 and 30g/L (+ -) -MPGM and 25mL toluene to continuously react on a shaking table for 2h. And (3) taking different fermentation broths for content measurement, and calculating the yield of the prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The highest yield was found at a (+ -) -MPGM concentration of 20 g/L. Prodigiosin yield was about 23.1g/L, production rates were as high as 0.61g/L/h, diltiazem intermediate yield was about 14.9g/L, and conversion was 73.0% (FIG. 15).

Example 14: the preparation condition of the diltiazem intermediate produced by Serratia marcescens fermentation is optimized.

This example illustrates the effect of a toluene-water system on the conversion of a fermentatively produced diltiazem intermediate. Inoculating the seed solution culture medium into the same fermentation culture medium (the composition of the fermentation culture medium is as follows: molasses 10g/L, tryptone 15g/L, magnesium sulfate 0.8g/L, tween-80 25g/L and proline 3 g/L) respectively, adjusting the pH value to 6.8, carrying out shaking culture at 32 ℃ and 250rpm, wherein the liquid loading amount of the fermentation culture medium is 50mL/250mL conical flask, preparing diltiazem intermediate (2R, 3S) - (-) -MPGM after 38h fermentation culture, and respectively adding 20g/L of (+ -) -MPGM and 10, 15, 20, 25 and 30mL of toluene to continuously react on a shaking table for 2h. And (3) taking different fermentation broths for content measurement, and calculating the yield of the prodigiosin and diltiazem intermediate (2R, 3S) - (-) -MPGM. The yield was found to be highest when 25mL toluene was added. Prodigiosin yield was about 23.7g/L, production rates were as high as 0.62g/L/h, diltiazem intermediate yield was about 16.3g/L, and conversion was 81.5% (FIG. 16).

Example 15: the 50L fermentor is fed in batches to co-produce prodigiosin and diltiazem intermediates.

Inoculating Serratia marcescens LT-7 strain into a solid culture medium, standing at 28 ℃ for 24 hours, picking single bacterial colony again for full-drawing on the solid culture medium, standing at 28 ℃ for 24 hours, taking the full-drawing strain, hanging 1 ring in a shake flask containing a seed liquid culture medium under aseptic condition, and culturing for 24 hours on a shake flask with the rotating speed of 200rpm and the temperature of 30 ℃ to obtain fermentation seed liquid; inoculating the seed solution into a fermentation medium with an inoculum size of 5%, and regulating the pH value to 6.8 by using dilute hydrochloric acid; the total liquid loading amount of the fermentation tank is 35L, the fermentation temperature is 32 ℃, the stirring speed is 250rpm, and the ventilation amount is 1.0VVM for fermentation; the fermentation pH value is constant at 6.8, an automatic pH control device is started in the fermentation process, and the pH is controlled by dilute hydrochloric acid; 10g/L of molasses is supplemented when fermentation is carried out for 30 hours, and the total fermentation time is 38 hours. After completion of fermentation, (2R, 3S) - (-) -MPGM was prepared by sampling and measuring the yield of prodigiosin in the fermentation broth every 2 hours. Assay analysis: taking the fermentation liquor, centrifuging at 10000rpm for 3min, taking supernatant to dilute 500 times, and detecting that the prodigiosin yield in the fermentation liquor after one feeding is about 25.4g/L, the production rate is as high as 0.67g/L/h, the yield of diltiazem intermediate is about 16.9g/L, and the conversion rate is 84.5% (shown in figure 17).

Example 16: batch feeding of 1t tank to co-produce prodigiosin and diltiazem intermediate

Inoculating Serratia marcescens LT-7 strain into a solid culture medium, standing at 28 ℃ for 24 hours, picking single bacterial colony again for full-drawing on the solid culture medium, standing at 28 ℃ for 24 hours, taking the full-drawing strain, hanging 1 ring in a shake flask containing a seed liquid culture medium under aseptic condition, and culturing for 24 hours on a shake flask with the rotating speed of 200rpm and the temperature of 30 ℃ to obtain fermentation seed liquid; inoculating the seed solution into a fermentation medium with an inoculum size of 5%, and regulating the pH value to 6.8 by using dilute hydrochloric acid; the total liquid loading amount of the fermentation tank is 700L, the fermentation temperature is 32 ℃, the stirring rotation speed is 250rpm, and the ventilation amount is 1.0VVM for fermentation; the fermentation pH value is constant at 6.8, an automatic pH control device is started in the fermentation process, and the pH is controlled by dilute hydrochloric acid; 10g/L of molasses is supplemented when fermentation is carried out for 30 hours, and the total fermentation time is 38 hours. The yield of prodigiosin in the fermentation broth was determined by sampling every 2h and (2R, 3S) - (-) -MPGM was prepared after the fermentation was completed. Assay analysis: taking the fermentation liquor, centrifuging at 10000rpm for 3min, taking supernatant to dilute 500 times, and detecting that the prodigiosin yield in the fermentation liquor after one feeding is about 27.6g/L, the production rate is as high as 0.73g/L/h, the yield of diltiazem intermediate is about 17.3g/L, and the conversion rate is 86.5% (shown in figure 18).

Finally, it should also be noted that the above list is only a specific embodiment of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications which can be directly derived or suggested by the person skilled in the art from the present disclosure should be considered as the protection scope of the present invention.

Claims

1. The application of Serratia marcescens in the co-production of prodigiosin and diltiazem intermediate is characterized in that the Serratia marcescens is classified as Serratia marcescens SERRATIA MARCESCENS LT-7, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20231646 and the preservation date of 2023, 9 and 7 days.

2. The use according to claim 1, wherein serratia marcescens LT-7 is inoculated into a fermentation medium for aerobic fermentation, and (±) -MPGM and toluene are added into the fermentation broth to prepare prodigiosin and diltiazem intermediate (2 r,3 s) - (-) -MPGM.

3. The use according to claim 2, wherein the fermentation medium comprises the following components:

1-20 g/L of molasses, 5-20 g/L of tryptone, 0.2-1.2 g/L of magnesium sulfate, 15-35 g/L of tween-80 and 2-5 g/L of proline, and adjusting the initial pH value of fermentation liquor to 6.0-7.0 by using water as a solvent.

4. Use according to claim 3, characterized in that the initial pH of the fermentation broth is adjusted to 6.0-7.0 with sodium bicarbonate.

5. The use according to claim 2, wherein the aerobic fermentation is carried out under the following conditions: the temperature is 26-34 ℃, the speed is 220-250 rpm, and the culture time is 24-38 h.

6. The use according to claim 5, wherein when the aerobic fermentation is shake flask culture, the seed liquid is inoculated in an amount of 2 to 4% by volume of the fermentation liquid, and the culture time is 24 to 38 hours.

7. The use according to claim 5, wherein when the aerobic culture is a fermenter culture, the seed liquid is inoculated in an amount of 3 to 6% by volume of the fermentation liquid, and the aeration rate is 1.0 to 1.4VVM.

8. The use according to claim 2, wherein the aerobic fermentation is fed-batch fermentation.

9. The use according to claim 8, wherein the fed-batch fermentation is a fermentation to 30 hours with the addition of molasses at 5-10 g/L.

10. The use according to claim 2, characterized in that (±) -MPGM in an amount of 5-20 g/L and toluene in an amount of 0.5-1 times the total volume of the fermentation broth are added to the fermentation broth when the yield of prodigiosin in the fermentation broth is no longer increased, and then the culture is continued at 32 ℃ for 2-3 hours under 250rpm to prepare diltiazem intermediate (2 r,3 s) - (-) -MPGM.