Escherichia coli chassis bacterium, recombinant Escherichia coli and application of recombinant Escherichia coli in synthesis of 2, 5-furandicarboxylic acid

Abstract

The invention relates to the technical field of microorganisms, in particular to an Escherichia coli chassis bacterium, recombinant Escherichia coli and application of the Escherichia coli chassis bacterium in synthesis of 2, 5-furandicarboxylic acid. The invention provides an Escherichia coli chassis bacterium which has an optimized curli system and can efficiently display target protein on the surface of Escherichia coli. Based on the chassis bacterium, the invention provides recombinant escherichia coli which has remarkably improved target protein surface display efficiency and can be used as a biocatalyst for chemical synthesis. The invention also provides a 2, 5-furandicarboxylic acid synthesis method using the recombinant Escherichia coli as a biocatalyst, and the method has the advantages of simplicity, high efficiency, low cost and the like, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a *Escherichia coli* basal strain, recombinant *Escherichia coli*, and their application in the synthesis of 2,5-furandicarboxylic acid. Background Technology

[0002] Bacterial surface display technology is a technique that utilizes bacterial surface proteins to display exogenous proteins or peptides, and it is widely used in fields such as target protein expression, screening, and biocatalysis. With in-depth research on the *E. coli* curli pili system, researchers have used this system to directly display target proteins onto *E. coli* biofilms. The synthetic pathway of *E. coli* curli pili mainly includes CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG (…). Figure 1 The curli pili system is a key component of the *E. coli* biofilm. CsgA is the main component; CsgB acts as an anchor point on the biofilm, accumulating CsgA protein; CsgG is a channel protein; CsgE and CsgF are accessory proteins; CsgC is responsible for removing misfolded proteins from the membrane; and CsgD regulates the expression of CsgA and CsgB. Although there are reports on expressing target proteins on the surface of *E. coli* using the curli pili system, the display levels achievable using this system are currently low, limiting its application in fields such as biocatalysis. Therefore, it is necessary to improve the protein display levels achieved by the curli pili system.

[0003] Lignocellulose is widely distributed and is a representative renewable biomass resource. Currently, numerous studies have explored various methods to convert lignocellulose into higher value-added bio-based platform compounds. One such method involves using 5-hydroxymethylfurfural (HMF), a sugar derivative obtained from the degradation of lignocellulose, as a substrate to produce 2,5-furandicarboxylic acid (FDCA). FDCA is an important bio-based C6 platform compound that can replace terephthalic acid (TPA) in the production of polyethylene terephthalate (PET), used to produce a biodegradable and higher-performance bio-based material—polyethylene furan-dicarboxylate (PEF). In the preparation of bio-based platform compounds, biocatalysts offer advantages over traditional catalysts, including fewer byproducts, lower emissions, and milder reaction conditions, thus demonstrating broader application prospects.

[0004] In the biocatalytic production of FDCA, 5-hydroxymethylfurfural oxidase (HMFO) can achieve a three-step oxidation from HMF to FDCA using a single enzyme, showing great promise for application. However, current research on the production of HMFO catalysts is limited. Immobilizing HMFO on the cell surface using bacterial surface display technology can avoid problems such as enzyme purification and substrate transport. Cells can be collected and repeatedly catalyzed through simple operations such as centrifugation, which can significantly reduce costs in industrial applications. Summary of the Invention

[0005] This invention provides a *Escherichia coli* basal strain, recombinant *Escherichia coli*, and their application in the synthesis of 2,5-furandicarboxylic acid.

[0006] To address the issue of low protein content in the E. coli curli system, this invention utilizes gene editing technology to regulate the E. coli curli system, thereby increasing the content of CsgA protein on its surface and thus improving surface display efficiency.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a *Escherichia coli* spore strain in which the CsgA gene is inactivated and the expression of the CsgB, CsgG, CsgE and CsgF genes is enhanced.

[0009] This invention has found that the expression levels of CsgB, CsgG, CsgE, and CsgF genes affect the content of CsgA protein on the surface of Escherichia coli, thereby affecting the surface display efficiency of the target protein. Enhancing the expression of CsgB, CsgG, CsgE, and CsgF genes in Escherichia coli can significantly increase the surface display amount of the target protein.

[0010] In this invention, enhancing the expression of CsgB, CsgG, CsgE, and CsgF genes can be achieved through the following methods (1) and / or (2): (1) Increase the copy number of the gene; (2) Enhance the activity of transcriptional and / or translational regulatory elements of the gene.

[0011] In (1) above, increasing the copy number of the gene can be achieved by inserting the gene into the genome of Escherichia coli, or by introducing an expression plasmid carrying the gene.

[0012] In (2) above, the transcriptional regulatory elements include promoters, enhancers, etc. The translational regulatory elements include ribosome binding sites, etc. Enhancing the activity of the transcriptional and / or translational regulatory elements of the gene includes replacing the promoter of the gene with a more active promoter.

[0013] Preferably, the expression of the CsgB, CsgG, CsgE, and CsgF genes is enhanced by introducing an overexpression vector containing the CsgB, CsgG, CsgE, and CsgF genes.

[0014] The present invention also found that, compared with expressing CsgB, CsgG, CsgE and CsgF genes using a polycistronic structure, controlling the expression intensity of CsgB, CsgG, CsgE and CsgF genes at specific levels, i.e. using promoters with specific different activities to control the overexpression of the above genes, can further increase the content of CsgA protein on the surface of Escherichia coli, thereby increasing the surface display of the target protein fused with it.

[0015] Preferably, the overexpression vector comprises a first promoter and CsgB connected downstream thereto, a second promoter and CsgG connected downstream thereto, and a third promoter and CsgE and CsgF connected downstream thereto; wherein the first promoter has stronger activity than the second promoter, and the second promoter has stronger activity than the third promoter. Controlling the overexpression of CsgB, CsgG, CsgE, and CsgF according to the above promoter activity intensity order is beneficial to increasing the content of CsgA protein on the surface of *E. coli*, thereby increasing the surface display amount of the target protein fused with it.

[0016] Preferably, the first promoter, the second promoter, and the third promoter are all compositional promoters.

[0017] In some embodiments of the present invention, the first promoter is a trc promoter, the second promoter is an spc promoter, and the third promoter is a pBAD promoter.

[0018] Preferably, the nucleotide sequence of the trc promoter is shown in SEQ ID NO.1, the nucleotide sequence of the spc promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the pBAD promoter is shown in SEQ ID NO.3.

[0019] Preferably, the overexpression vector is constructed using pCDFDuet-1 plasmid as the starting plasmid.

[0020] The overexpression vector described above is obtained by connecting a first promoter and CsgB connected downstream thereto, a second promoter and CsgG connected downstream thereto, and a third promoter and CsgE and CsgF connected downstream thereto to the vector backbone.

[0021] In this invention, the inactivation of the CsgA gene can be achieved by inserting, deleting, or replacing one or more bases in the CsgA gene.

[0022] In some embodiments of the present invention, the CsgA gene is inactivated by knocking out the CsgA gene.

[0023] In some embodiments of the present invention, the CsgA gene is knocked out in the Escherichia coli basalis strain and the above-mentioned overexpression vector is present.

[0024] In some embodiments of the present invention, the starting strain of the *Escherichia coli* basal plate is *Escherichia coli* K12 or a derivative thereof. Preferably, it is *Escherichia coli* BL21 or a derivative thereof. More preferably, it is *Escherichia coli* BL21(DE3).

[0025] Secondly, the present invention provides a method for constructing the above-described Escherichia coli chassis bacteria, the method comprising: knocking out the CsgA gene and introducing it into the above-described overexpression vector.

[0026] Thirdly, the present invention provides the application of the above-described Escherichia coli basal bacteria in the Escherichia coli surface display technology; the application includes: displaying target proteins on the surface of the Escherichia coli basal bacteria.

[0027] Preferably, the application includes: introducing the gene encoding the fusion protein into the Escherichia coli basalis bacteria; wherein the fusion protein comprises CsgA protein and the target protein.

[0028] In some embodiments of the present invention, the fusion protein is obtained by fusing CsgA protein and target protein.

[0029] Preferably, the introduction of the coding gene for the fusion protein is achieved by introducing an expression vector containing the coding gene for the fusion protein.

[0030] Optional expression vectors include expression vectors suitable for Escherichia coli, including but not limited to pET series expression vectors, such as: pET22 series (pET-22b, pET-22a), pET28 series (pET-28a), pET32 series (pET-32b, pET-32a), etc.

[0031] In some embodiments of the present invention, the expression vector is pET-22b(+).

[0032] Fourthly, the present invention provides a recombinant Escherichia coli, which is obtained by introducing a gene encoding a fusion protein into the aforementioned Escherichia coli basal strain; the fusion protein comprises CsgA protein and a target protein.

[0033] The Escherichia coli substrate strain provided by this invention is a curli-enhanced Escherichia coli, which can achieve a higher CsgA protein content on its surface, thereby significantly improving the surface display efficiency of the target protein.

[0034] Preferably, the introduction of the coding gene for the fusion protein is achieved by introducing an expression vector containing the coding gene for the fusion protein.

[0035] In some embodiments of the present invention, the fusion protein is obtained by fusing CsgA protein and target protein.

[0036] In some embodiments of the present invention, the CsgA protein is fused to the N-terminus of the target protein, and the CsgA protein and the target protein are linked by a GS-rich short peptide linker. A preferred linker is GGGGS (SEQ ID NO. 10).

[0037] Preferably, the introduction of the coding gene for the fusion protein is achieved by introducing an expression vector containing the coding gene for the fusion protein.

[0038] Preferably, in the expression vector, the promoter for initiating the expression of the gene encoding the fusion protein is the T7 promoter.

[0039] Optional expression vectors include expression vectors suitable for Escherichia coli, including but not limited to pET series expression vectors, such as: pET22 series (pET-22b(+), pET-22a(+)), pET28 series (pET-28a(+)), pET32 series (pET-32b(+), pET-32a(+)), etc.

[0040] In some embodiments of the present invention, the expression vector is pET-22b(+).

[0041] The advantage of the *E. coli* basal plate strain of this invention lies in its ability to more efficiently display target proteins on the surface, a function achieved by regulating the *E. coli* curli system. Therefore, this invention does not limit the types of target proteins in principle.

[0042] In some embodiments of the present invention, the target protein is 5-hydroxymethylfurfural oxidase.

[0043] In this invention, the 5-hydroxymethylfurfural oxidase can be any species-derived 5-hydroxymethylfurfural oxidase. Preferably, it is derived from *Methylovorus sp.* MP688. Preferably, the amino acid sequence of the 5-hydroxymethylfurfural oxidase is shown in SEQ ID NO.4.

[0044] This invention utilizes the E. coli surface curli system to directly display 5-hydroxymethylfurfural oxidase onto the E. coli biofilm, constructing a biocatalyst that can be used to catalyze the production of FDCA.

[0045] In some embodiments of the present invention, the recombinant Escherichia coli is obtained by introducing an expression vector containing a fusion protein encoding gene into the Escherichia coli basal strain; the fusion protein is a fusion protein of CsgA protein and 5-hydroxymethylfurfural oxidase.

[0046] In this invention, the amino acid sequences and their encoding gene sequences of the CsgA, CsgB, CsgG, CsgE, and CsgF proteins of *Escherichia coli* can be obtained from publicly available databases. Specifically, the accession number for the CsgA gene is NC_415560.1; the accession number for the CsgB gene is NC_415559.1; the accession number for the CsgG gene is NC_415555.1; the accession number for the CsgE gene is NC_415557.1; and the accession number for the CsgF gene is NC_415556.1.

[0047] In some embodiments of the present invention, the amino acid sequences of CsgA, CsgB, CsgG, CsgE, and CsgF proteins are shown in SEQ ID NO.5-9, respectively.

[0048] Fifthly, the present invention provides the application of the above-described recombinant Escherichia coli on the bacterial surface display of the target protein or as a biocatalyst.

[0049] Sixthly, the present invention provides the application of the above-described recombinant Escherichia coli in the preparation of 2,5-furandicarboxylic acid.

[0050] In a seventh aspect, the present invention provides a method for preparing 2,5-furandicarboxylic acid, the method comprising: using the recombinant Escherichia coli as a biocatalyst to catalyze the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural.

[0051] To address the problems of easy enzyme inactivation and low catalytic efficiency in the production of 2,5-furandicarboxylic acid, as well as the complex metabolic pathways and numerous byproducts of most whole-cell catalysts, this invention provides a method for preparing 2,5-furandicarboxylic acid based on a surface-display strain of *E. coli* containing 5-hydroxymethylfurfural oxidase. The method uses a surface-display system to express 5-hydroxymethylfurfural oxidase on the surface of *E. coli* during fermentation, and uses the bacterial cells as a biocatalyst to catalyze the production of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural.

[0052] Preferably, the method includes: mixing the biocatalyst with 5-hydroxymethylfurfural and carrying out catalysis at 28-32°C under aerobic conditions.

[0053] Preferably, the above preparation method includes the preparation of a biocatalyst and the use of the biocatalyst to catalyze the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural.

[0054] The preparation of the biocatalyst includes: inoculating the recombinant Escherichia coli into a seed culture medium for cultivation to obtain a seed solution; and inoculating the seed solution into a fermentation medium and culturing it to OD0.05. 600 =0.6-0.8, add IPTG, and induce culture at 16-18℃. After induction culture, collect the bacterial cells and wash to obtain the biocatalyst. The washing solution can be a buffer such as PBS.

[0055] In some embodiments of the present invention, the seed culture medium is LB liquid medium. The fermentation medium is TB liquid medium. The seed culture time is 10-14 hours, and the induction culture time is 20-30 hours.

[0056] The above-mentioned method for synthesizing 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural using the biocatalyst includes: adding the biocatalyst to a 5-hydroxymethylfurfural solution and carrying out catalysis at 29-31°C under aerobic conditions.

[0057] The beneficial effects of this invention include at least the following: This invention provides a *E. coli* basal plate bacterium with an optimized curli system, capable of efficiently displaying target proteins on the surface of *E. coli*, significantly improving the surface display efficiency of the target protein, and suitable as a basal plate bacterium for *E. coli* surface display technology. Based on this basal plate bacterium, this invention provides recombinant *E. coli*, which has a significantly improved surface display amount of target proteins and can be used as a biocatalyst for chemical synthesis. This invention also provides a method for synthesizing 2,5-furandicarboxylic acid using the above-mentioned recombinant *E. coli* as a biocatalyst. This method solves the problems of complex enzyme purification processes and high costs in enzyme-catalyzed production of 2,5-furandicarboxylic acid, and has advantages such as short catalytic time and high efficiency compared to other biocatalysts, showing good application prospects. Attached Figure Description

[0058] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0059] Figure 1 This is the gene composition of the Escherichia coli curli fimbriae system in the background technology of this invention.

[0060] Figure 2 This is a schematic diagram of the pCDFDuet-curli(+) expression vector in Example 1 of the present invention.

[0061] Figure 3 This is a schematic diagram of the CsgA-HMFO fusion expression vector in Embodiment 2 of the present invention. Detailed Implementation

[0062] In a specific embodiment of the present invention, a method for constructing a *E. coli* basal strain is provided, comprising: knocking out the CsgA gene in the genome of *E. coli* K-12 using CRISPR-Cas9 gene editing technology to obtain *E. coli* BL21(DE3)-CsgA(-); and overexpressing CsgB, CsgG, CsgE, and CsgF in *E. coli* BL21(DE3)-CsgA(-) to obtain a curli-enhanced *E. coli*.

[0063] In the aforementioned expression vectors for CsgB, CsgG, CsgE, and CsgF, constitutive promoters of varying strengths are used to overexpress the CsgB, CsgG, CsgE, and CsgF genes. Preferably, in these expression vectors, the promoter activity of CsgB is stronger than that of CsgG, the promoter activity of CsgG is stronger than that of CsgE, and CsgE and CsgF share the same promoter. The gene expression levels of the expression vectors are CsgB > CsgG > CsgE, with CsgE and CsgF expression levels being roughly equivalent.

[0064] The CsgB, CsgG, CsgE, and CsgF genes contained in the expression vector are endogenous CsgB, CsgG, CsgE, and CsgF genes of the starting strain.

[0065] In some embodiments of the present invention, the starting strain of the chassis bacteria is Escherichia coli BL21(DE3).

[0066] In a specific embodiment of the present invention, a recombinant *Escherichia coli* is provided, which is obtained by introducing a CsgA-HMFO fusion protein expression vector into the *Escherichia coli* basal strain. The CsgA-HMFO fusion protein expression vector is constructed by inserting the CsgA gene upstream of the HMFO gene, with the CsgA gene and the HMFO gene linked by a GS-rich short peptide linker sequence.

[0067] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0068] In the following embodiments, the formula for calculating FDCA yield is as follows:

[0069] Example 1: Construction of Escherichia coli chassis bacteria

[0070] This embodiment provides a method for constructing a chassis strain of *Escherichia coli* as follows: S1: The CsgA gene in the genome of Escherichia coli BL21(DE3) was knocked out using CRISPR-Cas9 gene editing technology to obtain E.coliBL21(DE3)-CsgA(-).

[0071] S2: Using pCDFDuet-1 plasmid as the starting plasmid, the expression vector pCDFDuet-curli(+) was constructed, and its structural diagram is shown below. Figure 2 As shown, CsgB was expressed using the trc promoter (SEQ ID NO.1), CsgG was expressed using the spc promoter (SEQ ID NO.2), and CsgE and CsgF were expressed using the pBAD promoter (SEQ ID NO.3). pCDFDuet-curli(+) was transferred into E.coliBL21(DE3)-CsgA(-) to obtain E.coliBL21(DE3)-curli(+), which is E. coli basal bacteria.

[0072] Example 2 Construction of recombinant Escherichia coli

[0073] This embodiment provides a recombinant Escherichia coli strain capable of displaying 5-hydroxymethylfurfural oxidase (HMFO) on its surface. The construction method is as follows: The CsgA-HMFO fusion expression vector was constructed using the pET-22b(+) plasmid and transformed into E. coliBL21(DE3)-curli(+) to obtain recombinant Escherichia coli.

[0074] Specifically, the structural diagram of the CsgA-HMFO fusion expression vector is shown below. Figure 3 As shown, the construction method is as follows: the CsgA gene is inserted upstream of the HMFO gene, and a GS-rich short peptide linker (GGGGS) coding sequence is introduced between the CsgA gene and the HMFO gene to obtain the fusion protein gene. The fusion protein gene is then linked into the pET-22b(+) plasmid to obtain the CsgA-HMFO fusion expression vector.

[0075] The CsgA-HMFO fusion expression vector was transformed into the E. coli BL21(DE3)-curli(+) constructed in Example 1 to obtain recombinant E. coli.

[0076] Example 3 Preparation of biocatalyst

[0077] The biocatalyst was prepared using the recombinant Escherichia coli constructed in Example 2, as follows: S1: Select single colonies of recombinant Escherichia coli and incubate them in 4 mL of LB medium containing 100 μg / mL ampicillin sodium and 50 μg / mL kanamycin sulfate at 37°C and 220 rpm for 12-16 h to obtain seed culture.

[0078] S2: The seed culture obtained in S1 was transferred to fermentation medium (TB medium) containing 100 μg / mL ampicillin sodium and 50 μg / mL kanamycin sulfate and cultured at 37°C until OD. 600 When the concentration is 0.6-0.8, add the inducing agent and induce culture at 17℃ for 24 hours.

[0079] S3: Centrifuge to collect bacterial cells, wash with PBS buffer to obtain the biocatalyst.

[0080] Example 4: Synthesis of FDCA from HMF using a biocatalyst

[0081] This embodiment provides a method for synthesizing FDCA using a biocatalyst catalyzing HMF. The steps are as follows: 200 g / L (wet weight of bacterial cells) of biocatalyst (prepared by the method in Example 3) and 0.5% (v / v) catalase are added to 2 mM HMF solution, and oxygen is introduced for catalysis at 30°C for 24 h.

[0082] Calculations show that the FDCA yield of the method in this embodiment is 85%.

[0083] Example 5

[0084] This embodiment provides a method for synthesizing FDCA from HMF using a biocatalyst. The only difference between this method and the method in Example 4 is that, in the recombinant Escherichia coli used as the biocatalyst, the expression vector pCDFDuet-curli(+) utilizes a polycistronic structure and the spc promoter to directly express the CsgB, CsgG, CsgE, and CsgF genes.

[0085] Calculations show that the FDCA yield of the method in this embodiment is 70%.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Escherichia coli spore, characterized in that, The CsgA gene was inactivated in the Escherichia coli basal strain, and the expression of the CsgB, CsgG, CsgE and CsgF genes was enhanced.

2. The *Escherichia coli* basal plate bacteria according to claim 1, characterized in that, The expression of the CsgB, CsgG, CsgE, and CsgF genes was enhanced by introducing an overexpression vector containing the CsgB, CsgG, CsgE, and CsgF genes.

3. The *Escherichia coli* basal plate bacteria according to claim 1 or 2, characterized in that, The overexpression vector includes a first promoter and CsgB connected downstream thereto, a second promoter and CsgG connected downstream thereto, and a third promoter and CsgE and CsgF connected downstream thereto. Among them, the first promoter is more active than the second promoter, and the second promoter is more active than the third promoter; Preferably, the first promoter is a TRC promoter, the second promoter is an SPC promoter, and the third promoter is a pBAD promoter; More preferably, the nucleotide sequence of the trc promoter is shown in SEQ ID NO.1, the nucleotide sequence of the spc promoter is shown in SEQ ID NO.2, and the nucleotide sequence of the pBAD promoter is shown in SEQ ID NO.

3.

4. The *Escherichia coli* spp. according to any one of claims 1 to 3, characterized in that, The overexpression vector was constructed using pCDFDuet-1 plasmid as the starting plasmid.

5. The application of the Escherichia coli basal plate bacteria as described in any one of claims 1 to 4 in the Escherichia coli surface display technology; The application includes: displaying the target protein on the surface of the Escherichia coli basal plate; Preferably, the application includes: introducing the gene encoding the fusion protein into the Escherichia coli basalis bacteria; The fusion protein comprises CsgA protein and the target protein.

6. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli was obtained by introducing the gene encoding the fusion protein into the Escherichia coli spp. as described in any one of claims 1 to 4. The fusion protein comprises CsgA protein and the target protein; Preferably, the introduction of the coding gene for the fusion protein is achieved by introducing an expression vector containing the coding gene for the fusion protein.

7. The recombinant Escherichia coli according to claim 6, characterized in that, The target protein is 5-hydroxymethylfurfural oxidase.

8. The use of the recombinant Escherichia coli according to claim 6 or 7 in the bacterial surface display of the target protein or as a biocatalyst.

9. The use of the recombinant Escherichia coli according to claim 7 in the preparation of 2,5-furandicarboxylic acid.

10. A method for preparing 2,5-furandicarboxylic acid, characterized in that, The method includes: using the recombinant Escherichia coli of claim 7 as a biocatalyst to catalyze the synthesis of 2,5-furandicarboxylic acid from 5-hydroxymethylfurfural; Preferably, the method includes: mixing the biocatalyst with 5-hydroxymethylfurfural and catalyzing it under aerobic conditions at 28-32°C.

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