A method for increasing Bacillomycin D fermentation yield

Abstract

The present invention provides a method for increasing the fermentation yield of Bacillomycin D, using Bacillus subtilis fmbJ as the starting strain and adding the growth factor VC to increase the yield of Bacillomycin D. In the method of the present invention, adding VC to the fermentation medium can increase the relative expression of the Bacillomycin D synthase gene, promote the transcription of key enzyme genes in the amino acid precursor synthesis pathway, and promote degQ 、 comA 、 comP 、 comQ 、 sigM and spo0A The expression of positive regulatory signal factors involved in the synthesis of secondary metabolites was upregulated, while some negative regulatory factors such as rapC and April , which increased the yield of Bacillomycin D to 1.44 times that of the control group; at the same time, the method of the present invention has low cost, simple and convenient operation, can effectively reduce the production cost of lipopeptides, improve production efficiency, and is expected to be used for large-scale production applications.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation and relates to a method for improving the fermentation yield of Bacillomycin D. Background Art

[0002] Bacillomycin D belongs to the iturin family and is a type of lipopeptide synthesized by Bacillus subtilis, Bacillus amyloliquefaciens and other Bacillus microorganisms through non-ribosomal pathways. It consists of two parts: lipid and peptide. The lipid part is a β-amino fatty acid chain (C 11 -C 17 The peptide's amino acid sequence is L-Asn, D-Tyr, D-Asn, L-Pro, L-Glu, D-Ser, and L-Thr, connected by a threonyl-β-amino bond. Bacillomycin D has a strong inhibitory effect against pathogenic fungi such as Aspergillus flavus, Aspergillus ochraceus, and Rhizoctonia suspensa. It has the potential to be a highly effective and safe food preservative for food and grain preservation.

[0003] However, fermentation of the wild fungus Bacillus subtilis using a basic fermentation medium resulted in low Bacillomycin D production. Researchers optimized the basic medium, using inulin as a carbon source, to significantly increase Bacillomycin D production, with the yield increasing by 3.12-fold after fed-batch fermentation. However, the high cost of the optimized medium hindered the widespread use of Bacillomycin D in preventing and controlling fungal contamination of grains. Additionally, as reported in Chinese patent applications CN109022474A and CN109136250A, overexpressing signaling factors involved in Bacillomycin D synthesis also significantly increased production, but this approach involved complex genetic manipulation.

[0004] The search has not yet found any reports on increasing Bacillomycin D production by adding growth factor VC. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides a method for increasing the fermentation yield of Bacillomycin D. The present invention can increase Bacillomycin D yield by at least 27%, and with the optimal addition of VC, yield can be increased by 44%. Furthermore, VC is inexpensive and readily available, making it ideal for large-scale production applications.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for increasing the fermentation yield of Bacillomycin D comprises adding a growth factor VC to a fermentation medium while transferring a Bacillomycin D-producing Bacillus seed solution to the fermentation medium at the initial stage of fermentation.

[0008] In this method, the addition of the growth factor VC of the present invention promotes the upregulation of the Bacillomycin D synthase expression gene, and also promotes the upregulation of the expression of some signal factors involved in the synthesis of secondary metabolites, thereby increasing the yield of Bacillomycin D.

[0009] Preferably, the amount of VC added is 0 g / L-0.75 g / L, and more preferably the amount of VC added is 0.6 g / L.

[0010] When the VC addition amount was 0.6 g / L, the production of Bacillomycin D could be increased to 1.44 times that of the control group.

[0011] Preferably, the Bacillus is Bacillus subtilis or Bacillus amyloliquefaciens.

[0012] More preferably, the Bacillus is Bacillus subtilis fmbJ, which was independently screened by the Enzyme Engineering Laboratory of Nanjing Agricultural University and has a strain accession number of CGMCC No. 0943.

[0013] Among them, due to the advancement of identification technology, Bacillus subtilis fmbJ was later renamed Bacillus amyloliquefaciens fmbJ. Therefore, in this application, both Bacillus subtilis fmbJ and Bacillus amyloliquefaciens fmbJ refer to the strain with the deposit number CGMCC No. 0943.

[0014] Specifically, the method includes the following specific steps:

[0015] (1) Take Bacillus subtilis fmbJ and culture it on a plate;

[0016] (2) transferring the Bacillus subtilis fmbJ in step (1) into a seed culture medium for seed liquid culture;

[0017] (3) The seed solution in step (2) is transferred to a fermentation medium, and growth factor VC is added to the fermentation medium to perform fermentation culture to prepare the antimicrobial lipopeptide Bacillomycin D.

[0018] Preferably, the seed culture medium is: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, and distilled water to 1000 mL, pH 7.0-7.2.

[0019] Preferably, the seed solution culture conditions are 37° C., 180 rpm, and culture until OD600 reaches 0.8-1.0.

[0020] Preferably, the culturing time in step (2) is about 3 hours.

[0021] Preferably, the fermentation medium comprises: 20.0 g glucose, 5.0 g L-glutamic acid, 1.0 g yeast extract, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.15 mg CuSO4·5H2O, 1.2 mg FeSO4·7H2O, 5 mg MnSO4, and distilled water to 1000 mL, with a pH of 7.0. The fermentation conditions are: 33°C, 180 rpm, and a temperature of 72-150 h, preferably 72-120 h, and more preferably 120 h.

[0022] The present invention also protects the application of VC in increasing the yield of antibacterial lipopeptide Bacillomycin D.

[0023] Among them, the added concentration of VC is 0g / L-0.75g / L.

[0024] Beneficial effects

[0025] The present invention enhances Bacillomycin D production by adding the growth factor VC to the basal fermentation medium. VC promotes lipopeptide synthesis by upregulating the expression of the Bacillomycin D synthase gene. Furthermore, VC upregulates the expression of several signaling proteins closely associated with Bacillomycin D growth and metabolism, such as the signaling protein degQ that regulates iturin A synthesis, spoθA that regulates spore synthesis and antimicrobial secretion, and the two-component regulatory system comA-comP. It also downregulates signaling factors that are detrimental to lipopeptide synthesis, such as rapC and abrB. Experimental verification has shown that Bacillomycin D yield can be increased by at least 27%, and at the optimal VC addition level, yield can be increased by 44%. This method is simple to manufacture, low-cost, and easy to operate, effectively reducing lipopeptide production costs and improving production efficiency, making it promising for large-scale production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of VC on the production of Bacillomycin D;

[0027] Figure 2The effect of exogenous VC on the relative expression of Bacillomycin D synthase gene;

[0028] Figure 3 The effect of exogenous VC on the relative expression of key enzymes in amino acid precursor synthesis;

[0029] Figure 4 The effect of exogenous VC on the relative expression of Bacillomycin D synthesis-related signaling factors;

[0030] Figure 5 The effects of exogenous addition of growth factors VB1, VB12, lipoic acid, VK1, VB2, VH, VB6, and VPP on Bacillomycin D production. DETAILED DESCRIPTION

[0031] The present invention is further described below with reference to examples. Experimental methods in the following examples where specific conditions are not specified are generally performed in accordance with known methods in the art or the conditions recommended by the manufacturer.

[0032] Example 1

[0033] (1) Plate activation: Prepare LB solid medium (10.0 g peptone, 10.0 g NaCl, 5.0 g yeast extract, 20.0 g agar, dilute to 1000 mL, sterilize at 121°C for 20 min), take Bacillus subtilis fmbJ glycerol culture, streak it on the LB solid plate, and incubate it upside down in a 37°C constant temperature incubator for 16 h.

[0034] (2) Seed Culture: A single colony was picked from a Bacillus subtilis fmbJ plate and inoculated into a 500 mL Erlenmeyer flask containing 150 mL of seed culture medium. The culture was incubated at 37°C and 180 rpm for approximately 3 h, until the OD600 reached 0.8-1.0. The seed culture medium consisted of: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, and distilled water to 1000 mL, pH 7.0-7.2.

[0035] (3) Preparation of VC mother solution: Weigh 3 g of vitamin C powder (analytical grade) and dissolve it in 10 ml of sterile water. Filter through a 0.22 μm filter membrane to sterilize the solution and use it immediately.

[0036] (4) Fermentation flask culture: The Bacillus subtilis fmbJ seed solution obtained in step (2) was inoculated at a volume ratio of 5% into a 1000 ml Erlenmeyer flask containing 200 ml of fermentation medium. VC was added at a concentration gradient of 0 g / L, 0.15 g / L, 0.30 g / L, 0.45 g / L, 0.60 g / L, and 0.75 g / L. The culture was incubated at 33°C, 180 rpm for 120 h. The fermentation medium consisted of 20.0 g of glucose, 5.0 g of L-glutamic acid, 1.0 g of yeast extract, 0.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 0.15 mg of CuSO4·5H2O, 1.2 mg of FeSO4·7H2O, and 5 mg of MnSO4. The volume was adjusted to 1000 ml with distilled water, pH 7.0.

[0037] (5) Sample RNA Extraction: 1 mL of the fermentation broth from step (4) (with VC added at 0 g / L and 0.60 g / L) after 36 h of fermentation was taken and total RNA was extracted using Trizol reagent. The specific steps were as described in the instructions. RNA concentration was determined using a NanoDrop 2000, and RNA quality was assessed on a 2% agarose gel.

[0038] (6) Preparation of a crude methanol extract containing Bacillomycin D: The fermentation broth in step (4) was centrifuged at 8000 rpm for 20 min to remove the bacterial cells, and the supernatant was collected. The pH of the supernatant was adjusted to 2.0 with 6 M HCl. After standing at 4° C. overnight, the supernatant was centrifuged at 9000 rpm for 20 min, and the supernatant was discarded to obtain a precipitate. Chromatographic grade methanol was added for dissolution, and the pH was then adjusted to 7.0 with NaOH. The supernatant was centrifuged at 9500 rpm for 20 min, and the supernatant was collected to obtain a methanol solution containing a crude Bacillomycin D extract.

[0039] (7) HPLC detection of methanol solution containing crude Bacillomycin D extract: The crude Bacillomycin D extract was filtered through a 0.22 μm filter membrane and then subjected to HPLC detection. Column: Shim-pack GWS C18 (5 μm × 4.6 mm × 250 mm); mobile phase A: pure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient elution conditions: 0-15 min, A 70%-55%, B 30%-45%, 15-40 min, A 55%-45%, B 45%-55%; detection wavelength: 207 nm; flow rate: 0.6 mL / min. The final yield of Bacillomycin D was calculated according to the following standard curve: y = 7.6396x - 2.3576, R 2= 0.9999, x, Bacillomycin D concentration, mg / L; y, peak area, mAU·h.

[0040] Results see Figure 1 As the amount of VC added increased, the production of Bacillomycin D continued to increase. When the addition amount was 0.6 g / L, the production of Bacillomycin D reached a peak of 320.39 mg / L, which was 1.44 times that of the control group.

[0041] (8) qPCR experiment: reverse transcribe the RNA in step (5) into cDNA. The specific operation method is shown in IIIRT SuperMix for qPCR (+gDNA wiper) kit instructions. The fluorescence quantitative PCR system is as follows: 10μL SYBR Premix Ex TaqII, 1μL cDNA, 1μL forward primer (10μM) and 1μL reverse primer (10μM), ddH2O is added to 20μL, and gently mixed. The two-step amplification program is as follows: 95℃, 30s; 40 cycles: 95℃, 5s; 60℃, 30s. 16S rRNA was used as the internal reference gene, and the expression of the target gene was analyzed by the 2-ΔΔCt method. The primer design is shown in Table 3-1. The experimental results are shown in Figure 2 、 Figure 3 and Figure 4 shown. Figure 2 The effect of exogenous VC addition on Bacillomycin D synthase gene was shown. Compared with the control group, the expression levels of bam A, bamB and TE were significantly increased, and the expression levels of bam C and bam D were also increased, but not significantly. Figure 3 The effect of exogenous VC addition on key genes in amino acid metabolism showed that the relative expression levels of asparagine synthetase asnB involved in Asn synthesis, glutamate dehydrogenase GDH involved in glu synthesis, glutamine synthetase glnA and phosphoserine phosphorylase rsbX involved in ser synthesis were significantly upregulated, while threonine synthetase thrC involved in the final step of Thr synthesis and another phosphoserine phosphorylase rsbU also involved in ser synthesis also increased, but not significantly. Figure 4 The effect of exogenous VC addition on signal factors involved in the secondary metabolic pathway of Bacillus was shown. The relative expression levels of pleiotropic regulatory protein DegQ, two-component regulatory system ComP / ComA, SigA / SigM encoding RNA polymerase σ factor, spo0A involved in spore formation and regulatory protein ComQ were significantly upregulated, while the relative expression levels of RapC, RapI, RapF and transition state regulatory protein AbrB in the aspartate phosphatase protein family were significantly downregulated.

[0042] Table 1 Primers used in RT-qPCR

[0043] Table1 Oligonucleotide primers for RT-qPCR

[0044]

[0045]

[0046] Comparative Example 1

[0047] (1) Plate activation: Prepare LB solid medium (10.0 g peptone, 10.0 g NaCl, 5.0 g yeast extract, 20.0 g agar, dilute to 1000 mL, sterilize at 121°C for 20 min), take Bacillus subtilis fmbJ glycerol culture, streak it on the LB solid plate, and incubate it upside down in a 37°C constant temperature incubator for 16 h.

[0048] (2) Seed Culture: A single colony was picked from a Bacillus subtilis fmbJ plate and inoculated into a 500 mL Erlenmeyer flask containing 150 mL of seed culture medium. The culture was incubated at 37°C and 180 rpm for approximately 3 h. The seed culture medium consisted of 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, and distilled water to 1000 mL, pH 7.0-7.2.

[0049] (3) Fermentation flask culture: The Bacillus subtilis fmbJ seed solution obtained in step (2) was inoculated into a 1000 mL triangular flask containing 200 mL fermentation medium at a volume ratio of 5%, and growth factors VB1, VB12, lipoic acid, VK1, VB2, VH, VB6 and VPP were added at the same time, wherein the addition gradient of VB1 was 0.0 mg / L, 1.0 mg / L, 2.0 mg / L, 3.0 mg / L, 4.0 mg / L, and 5.0 mg / L; the addition gradient of VB12, lipoic acid and VK1 was 0.0 mg / L, 2.0 mg / L, 4.0 mg / L, and 5.0 mg / L. The addition rates of VB2 and VH were 0.0 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, and 1.0 mg / L, respectively; the addition rate of VB6 was 0.0 mg / L, 0.67 mg / L, 1.33 mg / L, 2.00 mg / L, 1.67 mg / L, and 3.33 mg / L, respectively; the addition rate of hemin and VPP was 0.0 mg / L, 3.0 mg / L, 6.0 mg / L, 9.0 mg / L, 12.0 mg / L, and 15.0 mg / L. The culture was incubated at 33°C and 180 rpm for 120 h. The fermentation medium comprises: 20.0 g of glucose, 5.0 g of L-glutamic acid, 1.0 g of yeast extract, 0.5 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 0.15 mg of CuSO4·5H2O, 1.2 mg of FeSO4·7H2O, 5 mg of MnSO4, and distilled water to 1000 mL, with a pH of 7.0.

[0050] (4) Preparation of a crude methanol extract containing Bacillomycin D: The fermentation broth in step (3) was centrifuged at 8000 rpm for 20 min to remove the bacterial cells, and the supernatant was collected. The pH of the supernatant was adjusted to 2.0 with 6 M HCl. After standing at 4° C. overnight, the supernatant was centrifuged at 9000 rpm for 20 min, and the supernatant was discarded to obtain a precipitate. Chromatographic grade methanol was added for dissolution, and the pH was then adjusted to 7.0 with NaOH. The supernatant was centrifuged at 9500 rpm for 20 min, and the supernatant was collected to obtain a methanol solution containing a crude Bacillomycin D extract.

[0051] (5) HPLC detection of methanol solution containing crude Bacillomycin D extract: The crude Bacillomycin D extract was filtered through a 0.22 μm filter membrane and then subjected to HPLC detection. Column: Shim-pack GWS C18 (5 μm × 4.6 mm × 250 mm); mobile phase A: pure water + 0.1% trifluoroacetic acid, mobile phase B: acetonitrile + 0.1% trifluoroacetic acid; gradient elution conditions: 0-15 min, A 70%-55%, B 30%-45%, 15-40 min, A 55%-45%, B 45%-55%; detection wavelength: 207 nm; flow rate: 0.6 mL / min. The final yield of Bacillomycin D was calculated according to the following standard curve: y = 7.6396x - 2.3576, R 2 = 0.9999, x, Bacillomycin D concentration, mg / L; y, peak area, mAU·h.

[0052] The results are as follows Figure 5 As shown, the addition of growth factors VH and VB12 also had a significant improvement effect, but the maximum production of BacillomycinD was only 1.14 times that of the control; the addition of other growth factors VPP, VK1, VB6, VB1, VB2 and lipoic acid had no significant improvement effect, and the addition of hemin directly inhibited the growth of strain fmbJ.

[0053] In summary, the effects of nine growth factors on Bacillomycin D production compared in Example 1 and Comparative Example 1 demonstrate that specific concentrations of VC can significantly increase Bacillomycin D production. Targeting VC, which exhibits the highest yield, we investigated its effects on the Bacillomycin D synthase gene, key genes for amino acid precursor synthesis, and related signaling factors in the metabolic pathway to explore the mechanism by which VC regulates Bacillomycin D synthesis. The results showed that exogenous VC could, on the one hand, promote the synthesis of amino acid precursors by up-regulating key enzyme genes involved in the biosynthesis pathway of Bacillomycin D amino acid precursors, thereby promoting the synthesis of amino acid precursors and thus promoting the synthesis of Bacillomycin D; on the other hand, it could promote the transcription of pleiotropic regulatory protein DegQ, two-component regulatory system ComP / ComA, SigA encoding RNA polymerase σ factor RpoD, SigM encoding RNA polymerase σ factor rpoE, spo0A involved in spore formation and regulatory protein ComQ, thereby inhibiting the transcription of RapC, RapI, RapF and transition state regulatory protein AbrB in the aspartate phosphatase protein family, thereby promoting the up-regulation of Bacillomycin D synthesis gene expression and increasing Bacillomycin D production.

[0054] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for increasing the fermentation yield of Bacillomycin D, characterized in that: At the beginning of fermentation, the Bacillus D-producing Bacillus seed solution is transferred to the fermentation medium, and the growth factor VC is added, and fermentation culture is then carried out; Wherein, the VC addition amount is 0.15 g / L - 0.75 g / L; The Bacillus is Bacillus subtilis fmbJ, which was independently screened by the Enzyme Engineering Laboratory of Nanjing Agricultural University and has a strain collection number of CGMCC No. 0943.

2. The method for increasing the fermentation yield of Bacillomycin D according to claim 1, wherein: The amount of VC added is 0.6 g / L.

3. The method for increasing the fermentation yield of Bacillomycin D according to claim 1, wherein: The method comprises the following specific steps: (1) Take Bacillus subtilis fmbJ and culture it on a plate; (2) Transfer the Bacillus subtilis fmbJ from step (1) to the seed culture medium and culture the seed liquid at 37°C and 180 rpm until the OD600 reaches 0.8-1.0; (3) The seed solution in step (2) was transferred to the fermentation medium, and the growth factor VC was added to the fermentation medium for fermentation to prepare the antimicrobial lipopeptide Bacillomycin D. The fermentation conditions were: 33°C, 180 rpm, 72-150 h.

4. The method for increasing the fermentation yield of Bacillomycin D according to claim 3, wherein: The seed culture medium comprises: 3.0 g beef extract, 10.0 g peptone, 5.0 g NaCl, and distilled water to a volume of 1000 mL, with a pH of 7.0-7.

2.

5. The method for increasing the fermentation yield of Bacillomycin D according to claim 3, wherein: The fermentation medium is composed of 20.0 g glucose, 5.0 g L-glutamic acid, 1.0 g yeast extract, 0.5 g KH2PO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.15 mg CuSO4·5H2O, 1.2 mg FeSO4·7H2O, and 5 mg MnSO4, and the volume is adjusted to 1000 mL with distilled water, and the pH value is 7.

0.

6. Application of VC to increase the yield of the antimicrobial lipopeptide Bacillomycin D. The VC was added at a concentration of 0.15 g / L to 0.75 g / L. The strain used to prepare the antimicrobial lipopeptide Bacillomycin D was Bacillus subtilis fmbJ, which was independently screened by the Enzyme Engineering Laboratory of Nanjing Agricultural University and has a strain accession number of CGMCC No. 0943.

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