Gene acid reductase and application thereof in synthesis of indole-3-propionic acid

Abstract

The invention belongs to the technical field of biological enzymes, and particularly relates to olefine acid reductase and application thereof in synthesis of indole-3-propionic acid. The invention relates to a clostridium sporogenes source cinnamic acid reductase, which is characterized in that the amino acid sequence of the clostridium sporogenes source cinnamic acid reductase is as shown in SEQ ID NO: 1. The clostridium sporogenes source cinnamate reductase is obtained by screening different enzyme sources, and compared with the previously reported acrylate reductase, the clostridium sporogenes source cinnamate reductase does not need the assistance of electron transfer protein, can reduce the energy load of an engineering bacterium host and promote the generation efficiency of a target product, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioenzyme technology, and in particular relates to an enoic acid reductase and its application in the synthesis of dole-3-propionic acid. Background Technology

[0002] Indole-3-propionic acid (IPA) is a naturally occurring indole compound produced from tryptophan metabolism. It is primarily produced by intestinal bacteria in humans and animals through the metabolism of tryptophan. Studies have shown that, in addition to improving animal gut health, enhancing growth performance and feed conversion efficiency, IPA also possesses numerous physiological benefits, including antioxidant, anticancer, lipid-lowering, neuroprotective, metabolic-regulating, and anti-aging effects, making it a promising candidate for applications in pharmaceuticals, food, and animal health.

[0003] Currently, IPA is mainly produced through chemical synthesis, which has drawbacks such as demanding reaction conditions, severe environmental pollution, and difficulty in obtaining some key reagents. Fermentation of substrates using engineered bacteria such as *E. coli* can efficiently synthesize the target product. Currently, only a few studies have reported on the whole-cell catalytic synthesis of IPA using engineered bacteria. Research on the direct fermentation of IPA using engineered bacteria remains scarce.

[0004] Oleic acid reductase, a key enzyme catalyzing the direct conversion of precursors to IPA, is currently reported to require the assistance of two electron transfer proteins to reduce indole-3-acrylic acid to IPA. Patent document CN202311235220.8 discloses a biosynthetic pathway for IPA including enoic acid reductase and two electron transfer proteins. However, this whole-cell catalytic synthesis only achieved a yield of 2.62% (based on a 2 g / L tryptophan substrate), indicating an extremely low yield of the target product. Furthermore, the expression of these electron transfer proteins increases the energy load of the engineered bacterial host, thus affecting the formation of the target product.

[0005] There are no reports on the direct catalysis of the reduction of indole-3-acrylic acid to IPA by enoic acid reductases that do not require the assistance of electron transfer proteins. Summary of the Invention

[0006] This invention provides an engineered strain containing cinnamic acid reductase derived from Clostridium sporogenes, which can directly catalyze the reduction of indole-3-acrylic acid to IPA without the assistance of electron transfer proteins.

[0007] This invention provides a Clostridium sporogenes-derived cinnamic acid reductase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] This invention provides a gene encoding cinnamic acid reductase derived from Clostridium sporogenes;

[0009] The nucleotide sequence of the gene for cinnamic acid reductase from Clostridium sporogenes is shown in SEQ ID NO:2.

[0010] This invention provides a recombinant expression vector containing the gene for cinnamic acid reductase derived from Clostridium sporogenes.

[0011] This invention provides an engineered bacterial strain containing a recombinant expression vector.

[0012] This invention provides the application of an engineered strain in the preparation of indole-3-propionic acid.

[0013] Furthermore, the engineered strain was inoculated into a culture medium containing ampicillin and indole-3-acrylic acid for fermentation to obtain indole-3-propionic acid.

[0014] This invention provides a method for preparing indole-3-propionic acid using engineered strains, comprising the following steps:

[0015] The engineered strain was inoculated into a culture medium containing ampicillin and indole-3-acrylic acid and fermented to obtain indole-3-propionic acid;

[0016] Furthermore, the culture medium also includes 0.5–20 mM ascorbic acid, 0.5–20 mM sodium thiosulfate, or 1.2 mL / L mercaptoacetic acid.

[0017] This invention provides a method for preparing indole-3-propionic acid using engineered strains, characterized by comprising the following steps:

[0018] The engineered strain was inoculated into a culture medium containing ampicillin and indole-3-acrylic acid for fermentation. IPTG was added to a final concentration of 0.2 mM and thioglycolate was added to the fluid fermentation culture at a volume ratio of 50% to obtain indole-3-propionic acid.

[0019] Furthermore, the thioglycolate fluid accounts for 50% of the culture medium volume.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention uses an engineered strain of Escherichia coli BL21(DE3) for direct fermentation to prepare IPA. Gene manipulation becomes relatively reliable, culture conditions are simple, and the large-scale extraction, separation, and purification processes required in industrial production are avoided. It also eliminates the need for high-temperature, high-pressure synthesis conditions, making it environmentally friendly and easy to control production costs. Only a simple culture medium is needed as a raw material, making it suitable for industrial production.

[0022] 2. This invention obtains cinnamic acid reductase from Clostridium sporogenes through screening of different enzyme sources. Compared with previously reported acrylate reductase, it does not require the assistance of electron transfer proteins, which can reduce the energy load of the engineered bacterial host and promote the production efficiency of the target product, showing good prospects for industrial application.

[0023] 3. The reducing reaction system composed of thioglycolate fluid culture medium used in this invention significantly improves the catalytic activity of cinnamic acid reductase, improves the yield of IPA, and is conducive to large-scale production of IPA, thus promoting its production and preparation. Attached Figure Description

[0024] Figure 1 This is a phylogenetic tree of enoic acid reductases from different sources in Example 1.

[0025] Figure 2 This is a graph showing the yield analysis of IPA produced by indole-3-acrylic acid catalyzed by enoic acid reductases from different sources in Example 1.

[0026] Figure 3 The diagram shows the reaction process of indole-3-acrylic acid reduced to IPA by acrylate reductase from different sources in Example 1; Figures A and B are catalytic reaction process diagrams of acrylate reductase and cinnamic acid reductase, respectively.

[0027] Figure 4 The figure shows the effect of different reducing reaction systems on the yield of indole-3-acrylic acid to IPA catalyzed by cinnamic acid reductase in Example 2; Figures A and B show the effect of different types of reducing agents and different concentrations of ascorbic acid on the yield of IPA produced by the enzyme.

[0028] Figure 5 The effect of the reducing reaction system consisting of 50% by volume thioglycolate fluid medium as in Example 3 on the yield of cinnamic acid reductase-catalyzed reduction of indole-3-acrylic acid to IPA. Detailed Implementation

[0029] Example 1

[0030] Performance analysis of enoic acid reductases from different sources catalyzing the formation of IPA from indole-3-acrylic acid.

[0031] 1. Twenty-five enoic acid reductases were selected from the uniprot database. Based on their nucleotide sequences, the codons of the *E. coli* expression system were optimized by a biotechnology company, and then the NdeI and XhoI restriction enzyme sites of the pETDuet-1 vector were synthesized and constructed. The synthesized recombinant plasmid was transformed into host cells *E. coli* BL21(DE3) (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0032] 2. After the transformed *E. coli* BL21(DE3) from step 1 has grown into clearly visible colonies on LB solid medium, positive transformants of single colonies of *E. coli* (including engineered strains containing cinnamic acid reductase from *Clostridium sporogenes*) are directly selected and inoculated into LB medium containing 100 μg / mL ampicillin. The culture is then incubated overnight at 37 °C with shaking to obtain the seed culture. The seed culture is then inoculated at a volume ratio of 1:100 into LB medium containing 100 μg / mL ampicillin and 100 mg / L indole-3-acrylic acid, and cultured at 37 °C and 220 r / min until the bacterial OD reaches the target value. 600 Once the concentration reached 0.6, IPTG was added to each bacterial culture to a final concentration of 0.2 mM. The culture was fermented in a shaker at 30 °C and 220 r / min for 48 h. The E. coli bacterial culture sample was taken, centrifuged at 8000 r / min for 5 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane and then transferred to a liquid chromatography bottle. The yield of IPA was then detected by HPLC.

[0033] The yield of IPA was calculated based on the standard curve. All fermentation experiments were repeated three times, and the results are expressed as the mean ± standard deviation of the three experiments.

[0034] The HPLC detection conditions for IPA were as follows: Alphasil ES-C18 column (5 μm, 100 Å), detection wavelength 280 nm, gradient elution, mobile phase B changing from 10% to 20% linearly over 5 min, from 20% to 40% linearly over 5 to 10 min, from 40% to 90% linearly over 10 to 25 min, from 90% to 80% linearly over 25 to 30 min, from 80% to 10% linearly over 30 to 31 min, and remaining constant at 10% linearly over 31 to 35 min. Phase A was 0.2% formic acid in water, phase B was acetonitrile, column temperature was 40 ℃, flow rate was 1.0 mL / min, and injection volume was 10 μL.

[0035] IPA standard curve: IPA standard solutions of 78.125, 39.063, 19.53, 9.76, 4.88 and 2.44 mg / L were prepared, and the peak areas were measured by HPLC analysis to plot the standard curve.

[0036] The results are as follows Figure 1 and Figure 2 As shown, by screening 25 enoic acid reductases, only the enoic acid reductase from *Clostridium sporogenes* showed a significant ability to convert 100 mg / L indole-3-acrylic acid to IPA, with a yield of 10.48 mg / L. +The concentration of 2.34 mg / L indicates that Clostridium sporogenes-derived enoic acid reductase has a higher potential for IPA synthesis through fermentation.

[0037] Further identification of the enzyme types of Clostridium sporogenes-derived enoic acid reductase, such as... Figure 3 As shown, it was found to be a cinnamate reductase that does not require the assistance of electron transfer proteins, which is significantly different from the previously reported acrylate reductases that require the assistance of electron transfer proteins.

[0038] The amino acid sequence of cinnamic acid reductase derived from Clostridium sporogenes is shown in SEQ ID NO:1:

[0039] ,

[0040] The nucleotide sequence of cinnamic acid reductase derived from Clostridium sporogenes is shown in SEQ ID NO:2:

[0041]

[0042] Example 2

[0043] Investigating the effect of different reducing reaction systems on the yield of indole-3-acrylic acid to IPA catalyzed by Clostridium sporogenes cinnamic acid reductase.

[0044] The engineered strain containing cinnamic acid reductase from *Clostridium sporogenes* prepared in Example 1 was inoculated at a bacterial culture ratio of 0.5–3% into LB broth containing 100 μg / mL ampicillin. The culture was incubated overnight at 37 °C with shaking to obtain a seed culture. This seed culture was then inoculated at a volume ratio of 1:100 into LB broth containing 100 μg / mL ampicillin, 100 mg / L indole-3-acrylic acid, and either 2 mM ascorbic acid, 2 mM sodium thiosulfate, or 1.2 mL / L mercaptoacetic acid. The culture was incubated at 37 °C and 220 r / min until the bacterial culture reached its OD value. 600 To reach a concentration of 0.6, IPTG was added to each vial of bacterial culture to a final concentration of 0.2 mM. Fermentation was carried out at 30 °C and 220 r / min on a shaker for 48 h. E. coli bacterial culture samples were collected, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane. After filtration, the supernatant was transferred to a liquid chromatography vial, and the IPA yield was determined by HPLC under the detection conditions described in Example 1. The IPA yield was calculated based on the standard curve. All fermentation experiments were repeated three times, and the results are expressed as the mean ± standard deviation of the three experiments.

[0045] like Figure 4 As shown in Figure A, ascorbic acid was found to promote the yield of indole-3-acrylic acid to IPA catalyzed by cinnamic acid reductase to a certain extent, with the yield reaching 25.90%. + The yield of IPA was 1.96%, significantly higher than the control and other experimental groups, while sodium thiosulfate and mercaptoacetic acid did not significantly promote the yield of IPA. Further optimization of the ascorbic acid concentration is needed, such as... Figure 4 As shown in Figure B, the yield of IPA reached its highest level of 39.70% when the concentration of ascorbic acid added was 1 mM. + 2.91%.

[0046] Example 3

[0047] The effect of a reducing reaction system consisting of a 50% (v / v) thioglycolate fluid medium on the yield of cinnamic acid reductase-catalyzed reduction of indole-3-acrylic acid to IPA was investigated.

[0048] The engineered strain containing cinnamic acid reductase from *Clostridium sporogenes* prepared in Example 1 was inoculated at a bacterial culture ratio of 0.5–3% into LB broth containing 100 μg / mL ampicillin. The culture was incubated overnight at 37 °C with shaking to obtain a seed culture. This seed culture was then inoculated at a volume ratio of 1:100 into LB broth containing 100 μg / mL ampicillin and 100 mg / L indole-3-acrylic acid. The culture was incubated at 37 °C and 220 r / min until the bacterial culture reached its OD value. 600 To achieve a concentration of 0.6, IPTG was added to each vial of bacterial culture to a final concentration of 0.2 mM and 50% (v / v) thioglycolate. The culture was then incubated at 30 °C and 220 r / min in a shaker for 48 h. E. coli bacterial culture samples were collected, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane. The filtered product was then transferred to a liquid chromatography vial, and the IPA yield was determined by HPLC under the conditions described in Example 1. The IPA yield was calculated based on the standard curve. All fermentation experiments were repeated three times, and the results are expressed as the mean ± standard deviation of the three experiments.

[0049] like Figure 5 As shown, thioglycolate fluid medium significantly promoted the yield of indole-3-acrylic acid to IPA catalyzed by cinnamic acid reductase, achieving a yield of 92.45%. + 1.79%.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A cinnamic acid reductase derived from Clostridium sporogenes, characterized in that, The amino acid sequence of the Clostridium sporogenes-derived cinnamic acid reductase is shown in SEQ ID NO:

1.

2. A gene encoding cinnamic acid reductase from Clostridium sporogenes as described in claim 1, wherein the nucleotide sequence of the gene for cinnamic acid reductase from Clostridium sporogenes is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, It contains the gene as described in claim 2.

4. An engineered bacterial strain, characterized in that, It includes the recombinant expression vector as described in claim 3.

5. The use of the engineered strain as described in claim 4 in the preparation of indole-3-propionic acid.

6. The application as described in claim 5, characterized in that, The engineered strain was inoculated into a culture medium containing ampicillin and indole-3-acrylic acid and fermented to obtain indole-3-propionic acid.

7. A method for preparing indole-3-propionic acid using the engineered strain as described in claim 4, characterized in that, Includes the following steps: The engineered strain was inoculated into a culture medium containing ampicillin and indole-3-acrylic acid and fermented to obtain indole-3-propionic acid; The culture medium also includes 0.5–20 mM ascorbic acid, 0.5–20 mM sodium thiosulfate, or 1.2 mL / L mercaptoacetic acid.

8. A method for preparing indole-3-propionic acid using the engineered strain as described in claim 4, characterized in that, Includes the following steps: The engineered strain was inoculated into a culture medium containing ampicillin and indole-3-acrylic acid for fermentation. IPTG was added to a final concentration of 0.2 mM and thioglycolate was added to the fluid fermentation culture at a volume ratio of 50% to obtain indole-3-propionic acid. The thioglycolate fluid accounts for 50% of the culture medium volume.

Images