Recombinant bacteria with high yield of pyrroloquinoline quinone and construction method and application thereof

Abstract

This invention discloses a recombinant bacterium producing high levels of pyrroloquinoline quinone, its construction method, and its applications. The invention involves introducing the pqq gene cluster or the pqq gene itself into denitrifying mycelial microbes via a shuttle expression vector to construct a recombinant bacterium producing high levels of pyrroloquinoline quinone. The pqq gene cluster is either the pqqABCDE gene cluster or the pqqADE gene cluster, and the pqq gene is any one of pqqA, pqqB, pqqC, pqqD, and pqqE. The recombinant bacterium constructed by this invention can increase the PQQ anabolic flux without the addition of an inducer, effectively increasing the yield of pyrroloquinoline quinone and the methanol conversion rate. Experiments show that after fermentation in a 5L fermenter for 134 h, the recombinant bacterium achieved a PQQ yield of 1809±14 mg / L, an increase of 49.51% compared to the starting strain. The recombinant bacterium of this invention has the advantages of simple process, high production efficiency, and low production cost, and has good prospects for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to recombinant bacteria that produce high levels of pyrroloquinoline quinone, their construction methods, and applications. Background Technology

[0002] Pyrroloquinoline quinone (PQQ) is a compound containing an ortho-quinone structure and is the third class of redox enzyme cofactors discovered after NAD / NADP and FMN / FAD. It is widely found in animal and plant tissues. PQQ exhibits significant application potential in multiple dimensions: at the animal physiological level, PQQ not only participates in the formation and functional regulation of mitochondria but also plays a crucial role in maintaining cellular redox homeostasis; in agroecology, PQQ can promote crop growth and improve agricultural productivity; in the field of bioanalysis, PQQ, with its excellent electron transport capabilities, has become an important component in constructing efficient electrochemical biosensors, widely used for the precise detection of insulin, enzyme activity, and glucose. Clinical evidence further indicates that long-term intake of PQQ helps improve cognitive performance and overall health. Regulatory agencies such as the US FDA, Japan's MHLW, the EU EFSA, and my country's National Health Commission have successively approved PQQ as a dietary supplement, a new food ingredient, and a new feed additive.

[0003] Methyltrophic bacteria are Gram-negative bacteria that produce the most PQQ. Among them, *Denitrifying mycelium* is a typical methyltrophic bacterium in nature, capable of using C1 compounds such as methanol as its sole carbon and energy source. Its natural metabolic pathway produces and secretes PQQ, making it a major strain for PQQ production via fermentation. Unlike de novo synthetic cofactors such as NAD and FMN, PQQ is a novel ribosomally synthesized and post-translational modified peptide (RiPP). The PQQ biosynthesis pathway in methyltrophic bacteria involves 5-9 genes from 1-2 gene clusters. pqqABCDE Five genes are essential for its biosynthesis. However, the regulatory mechanism of PQQ remains unclear, and gene editing systems for methyltrophic bacteria are immature. There are few reports of significantly increasing PQQ production in methyltrophic bacteria through rational design. Traditional breeding programs remain the main means of obtaining high-yielding strains, but the increase in PQQ production is limited.

[0004] Chinese invention patent CN105624084 A discloses a method for the targeted domestication and selection of methyltrophic bacteria that produce high levels of pyrroloquinoline quinone. This method involves domesticating denitrifying filamentous microbes for eight rounds (more than two months), ultimately resulting in a mutant strain with a maximum yield of 63.3 mg / L. An earlier Chinese invention patent by the inventor, CN106086052A, discloses a bacterium that produces pyrroloquinoline quinone and its applications. This patent clones the pqq gene from denitrifying filamentous microbes into the plasmid pBAD / hisB, introduces it into the host bacterium *E. coli* K12ΔiscR, and constructs a recombinant *E. coli* strain heterologously expressing the PQQ synthesis gene cluster. However, this recombinant bacterium uses *E. coli* as its host, and *E. coli* itself lacks the ability to metabolize methanol, making it impossible to utilize methanol, a cheap carbon source, for PQQ production. Furthermore, this system requires the addition of L-arabinose as an inducer, increasing production costs and process complexity. Chinese invention patent CN121320216 A uses denitrifying mycelial bacteria as a host and overexpresses the *Acetobacter* pqqABCDE gene cluster using the T7 and lac promoters, achieving a PQQ yield of 2.6 g / L. However, the host needs to be modified to express T7 RNA polymerase, and IPTG must be added as an inducer during the culture process.

[0005] In summary, the existing strategies for constructing high-yield recombinant PQQ strains mainly have the following technical bottlenecks: (1) Heterologous expression systems do not have methanol metabolism capabilities and cannot utilize inexpensive carbon sources; (2) Whether it is heterologous expression or homologous expression systems, most require the addition of inducers such as IPTG, which increases costs and may affect strain growth; (3) Existing schemes all adopt the strategy of overexpressing the complete pqqABCDE gene cluster, which involves a large number of genes, complex plasmid construction, and failure to analyze which genes are rate-limiting steps.

[0006] Therefore, developing a strategy for constructing a high-yield recombinant PQQ strain based on the PQQ gene derived from denitrifying filamentous microbes, utilizing a methanol-inducible promoter, requiring no exogenous inducers, and identifying key rate-limiting genes, is of great significance for reducing PQQ production costs and meeting the growing market demand. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a recombinant strain for high-yield pyrroloquinoline quinone and its construction method, and applies it to the production of pyrroloquinoline quinone, which can achieve high yield of pyrroloquinoline quinone and high methanol conversion rate, laying the foundation for the green and efficient production of pyrroloquinoline quinone.

[0008] To achieve the above objectives, the present invention provides the following technical solution: One of the objectives of this invention is to provide a method for constructing a recombinant bacterium that produces high levels of pyrroloquinoline quinone, characterized in that the recombinant bacterium is obtained by introducing the pqq gene cluster or the pqq gene into denitrifying mycelial microbes using a shuttle expression vector.

[0009] Furthermore, the nucleotide sequence of the pqqABCDE gene cluster is shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No. 4.

[0010] Furthermore, the nucleotide sequence of the pqqADE gene cluster is shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No. 8.

[0011] Furthermore, the pqq gene is at least one of the pqqA gene, pqqB gene, pqqC gene, pqqD gene, and pqqE gene; The nucleotide sequence of the pqqA gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42; The nucleotide sequence of the pqqB gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14; The nucleotide sequence of the pqqC gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18; The nucleotide sequence of the pqqD gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, and SEQ ID No. 34; The nucleotide sequence of the pqqE gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, and SEQ ID No. 38.

[0012] As a preferred embodiment, the pqq gene is a combination of the pqqA gene and the pqqE gene, and the nucleotide sequence is SEQ ID No. 9 and SEQ ID No. 23 or SEQ ID No. 9 and SEQ ID No. 24 or SEQ ID No. 9 and SEQ ID No. 25 or SEQ ID No. 9 and SEQ ID No. 26 or SEQ ID No. 9 and SEQ ID No. 5 or SEQ ID No. 9 and SEQ ID No. 36 or SEQ ID No. 9 and SEQ ID No. 37 or SEQ ID No. 9 and SEQ ID No. 38 or SEQ ID No. 10 and SEQ ID No. 23 or SEQ ID No. 10 and SEQ ID No. 24 or SEQ ID No. 10 and SEQ ID No. 25 or SEQ ID No. 10 and SEQ ID No. 26 or SEQ ID No. 10 and SEQ ID No. 35 or SEQ ID No. 10 and SEQ ID No. 36 or SEQ ID No. 10 and SEQ ID No. 37 or SEQ ID No. 10 and SEQ ID No. 38 or SEQ ID No. 10 and SEQ ID No. 38. SEQ ID No. 27 and SEQ ID No. 23 or SEQ ID No. 27 and SEQ ID No. 24 or SEQ ID No. 27 and SEQ ID No. 25 or SEQ ID No. 27 and SEQ ID No. 26 or SEQ ID No. 27 and SEQ ID No. 35 or SEQ ID No. 27 and SEQ ID No. 36 or SEQ ID No. 27 and SEQ ID No. 37 or SEQ ID No. 27 and SEQ ID No. 38 or SEQ ID No. 28 and SEQ ID No. 24 or SEQ ID No. 28 and SEQ ID No. 25 or SEQ ID No. 28 and SEQ ID No. 26 or SEQ ID No. 28 and SEQ ID No. 35 or SEQ ID No. 28 and SEQ ID No. 36 or SEQ ID No. 28 and SEQ ID No. 37 or SEQ ID No. 28 and SEQ ID No. 38 or SEQ ID No. 29 and SEQ ID No. 24 or SEQ ID No. 29 and SEQ ID No. 25 or SEQ ID No. 29 and SEQ ID No. 28. No. 26 or SEQ ID No. 29 and SEQ ID No. 35 or SEQ ID No. 29 and SEQ ID No. 36 or SEQ ID No. 29 and SEQ ID No. 37 or SEQ ID No.29 and SEQ ID No. 38 or SEQ ID No. 30 and SEQ ID No. 24 or SEQ ID No. 30 and SEQ ID No. 25 or SEQ ID No. 30 and SEQ ID No. 26 or SEQ ID No. 30 and SEQ ID No. 35 or SEQ ID No. 30 and SEQ ID No. 36 or SEQ ID No. 30 and SEQ ID No. 37 or SEQ ID No. 30 and SEQ ID No. 38 or SEQ ID No. 39 and SEQ ID No. 24 or SEQ ID No. 39 and SEQ ID No. 25 or SEQ ID No. 39 and SEQ ID No. 26 or SEQ ID No. 39 and SEQ ID No. 35 or SEQ ID No. 39 and SEQ ID No. 36 or SEQ ID No. 39 and SEQ ID No. 37 or SEQ ID No. 39 and SEQ ID No. 38 or SEQ ID No. 40 and SEQ ID No. 24 or SEQ ID No. 40 and SEQ ID No. 25 or SEQ ID No. 40 and SEQ ID No. 38 No. 26 or SEQ ID No. 40 and SEQ ID No. 35 or SEQ ID No. 40 and SEQ ID No. 36 or SEQ ID No. 40 and SEQ ID No. 37 or SEQ ID No. 40 and SEQ ID No. 38 or SEQ ID No. 41 and SEQ ID No. 24 or SEQ ID No. 41 and SEQ ID No. 25 or SEQ ID No. 41 and SEQ ID No. 26 or SEQ ID No. 41 and SEQ ID No. 35 or SEQ ID No. 41 and SEQ ID No. 36 or SEQ ID No. 41 and SEQ ID No. 37 or SEQ ID No. 41 and SEQ ID No. 38 or SEQ ID No. 42 and SEQ ID No. 24 or SEQ ID No. 42 and SEQ ID No. 25 or SEQ ID No. 42 and SEQ ID No. 26 or SEQ ID No. 42 and SEQ ID No. 35 or SEQ ID No. 42 and SEQ ID No. 36 or SEQ ID No. 42 and SEQ ID No. 37 or SEQ ID The cDNA molecules or genomic DNA shown in SEQ ID No. 42 and SEQ ID No. 38.

[0013] As a preferred embodiment, the pqq gene is a combination of the pqqA gene, the pqqD gene, and the pqqE gene, and its nucleotide sequence is any three of the gene combinations shown in the above-mentioned pqqA gene nucleotide sequence, pqqD gene nucleotide sequence, and pqqE gene nucleotide sequence, which are cDNA molecules or genomic DNA.

[0014] Furthermore, the promoter that initiates the transcription of the pqq gene cluster or the pqq gene in the shuttle expression vector is the mxaF promoter.

[0015] The second objective of this invention is to provide a recombinant strain that produces high levels of pyrroloquinoline quinone according to the above-described construction method.

[0016] Furthermore, the recombinant bacteria can overexpress one or more of the following: precursor pqqA, hydroxylase pqqB, oxidase pqqC, chaperone protein pqqD, and SAM radical enzyme pqqE.

[0017] Furthermore, the precursor pqqA is derived from denitrifying mycelial microbes and encodes a DNA molecule from a11 to a13: a11, whose nucleotide sequence is the cDNA molecule or genomic DNA shown in SEQ ID No. 9 or SEQ ID No. 10 or SEQ ID No. 27 or SEQ ID No. 28 or SEQ ID No. 29 or SEQ ID No. 30 or SEQ ID No. 39 or SEQ ID No. 40 or SEQ ID No. 41 or SEQ ID No. 42; a12. A cDNA molecule or genomic DNA that hybridizes under stringent conditions with the DNA molecule defined in a11 and encodes the precursor pqqA; a13. A cDNA molecule or genomic DNA that has 90% or more identity with the DNA molecule defined by a11 or a12 and encodes the precursor pqqA; Furthermore, the hydroxylase pqqB is derived from denitrifying mycelial microbes, and its encoding gene is any one of the DNA molecules b11-b13: b11, whose nucleotide sequence is the cDNA molecule or genomic DNA shown in SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, or SEQ ID No. 14; b12. A cDNA molecule or genomic DNA that hybridizes under stringent conditions with the DNA molecule defined in b11 and encodes the hydroxylase pqqB. b13, a cDNA molecule or genomic DNA that has 90% or more identity with the DNA molecule defined by b11 or b12 and encodes the hydroxylase pqqB.

[0018] Furthermore, the oxidase pqqC is derived from denitrifying mycelial microbes, and its encoding gene is any one of the DNA molecules c11-c13: c11, whose nucleotide sequence is the cDNA molecule or genomic DNA shown in SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, or SEQ ID No. 14; c12, a cDNA molecule or genomic DNA that hybridizes under stringent conditions with the DNA molecule defined by c11 and encodes the oxidase pqqC; c13, a cDNA molecule or genomic DNA that has 90% or more identity with the DNA molecule defined by c11 or c12 and encodes the oxidase pqqC; Furthermore, the chaperone protein pqqD is derived from denitrifying filamentous microbes, and its encoding gene is any one of the DNA molecules d11-d13: d11, whose nucleotide sequence is the cDNA molecule or genomic DNA shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, or SEQ ID No. 34; d12. A cDNA molecule or genomic DNA that hybridizes under stringent conditions to the DNA molecule defined by d11 and encodes the chaperone protein pqqD; d13, a cDNA molecule or genomic DNA that has 90% or more identity with the DNA molecule defined by d11 or d12 and encodes the said chaperone protein pqqD.

[0019] Furthermore, the SAM free radical enzyme pqqE is derived from denitrifying filamentous microbes, and its encoding gene is any one of the DNA molecules e11-e13: e11, whose nucleotide sequence is the cDNA molecule or genomic DNA shown in SEQ ID No. 23 or SEQ ID No. 24 or SEQ ID No. 25 or SEQ ID No. 26 or SEQ ID No. 35 or SEQ ID No. 36 or SEQ ID No. 37 or SEQ ID No. 38; e12, a cDNA molecule or genomic DNA that hybridizes under stringent conditions with the DNA molecule defined in e11 and encodes the SAM radical enzyme pqqE; e13, a cDNA molecule or genomic DNA that has 90% or more identity with the DNA molecule defined by e11 or e12 and encodes the SAM radical enzyme pqqE.

[0020] The 90% or higher identity can be at least 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity; the term "identity" as used refers to the percentage of identical or identical sequences when compared and aligned using nucleotide or amino acid residue sequence comparison algorithms or by visual inspection to achieve the highest possible correspondence. In other words, the identity of nucleotide or amino acid sequences can be defined using a ratio that represents the proportion of the number of identical nucleotides or amino acids in the total number of nucleotides or amino acids in the aligned portion, assuming the maximum number of identical nucleotides or amino acids and omitting gaps as needed.

[0021] The third objective of this invention is to provide the application of the above-mentioned recombinant bacteria in the fermentation of high-yield pyrroloquinoline quinone.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention constructs a novel high-yield recombinant bacterium for pyrroloquinoline quinone. The pqq gene cluster or the pqq gene itself is introduced into denitrifying mycelial microbes via a shuttle vector to form the recombinant bacteria. Using the pqq gene derived from the denitrifying mycelial microbes themselves and the methanol-inducible mxaF promoter, the recombinant bacteria can increase the PQQ anabolic flux without the addition of additional inducers, effectively improving the yield of pyrroloquinoline quinone and the methanol conversion rate. Experimental results show that the recombinant bacteria overexpressing the pqq gene, after fermentation in a 5L fermenter for 134 h, achieved a PQQ yield of 1809±14 mg / L, which is 49.51% higher than the starting strain. The process for producing pyrroloquinoline quinone using the recombinant bacteria provided by this invention is simple, has high synthesis efficiency, and low production cost, showing good prospects for industrial application. Attached Figure Description

[0023] Figure 1 This is a fluorescence micrograph of fluorescent protein expressed in denitrifying mycelial microbe FJNU-6 using a shuttle expression vector in Example 1 of the present invention; Figure 2 The fermentation culture curve of the recombinant bacteria overexpressing the pqq gene cluster in Example 11 of this invention; Figure 3 This is the fermentation culture curve of the pqq gene overexpressing recombinant bacteria in Example 12 of the present invention. Detailed Implementation The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges. These numerical ranges should be considered as specifically disclosed herein.

[0024] Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0025] The molecular biology experiments not specifically described in the following examples include vector construction, enzyme digestion, ligation, preparation of competent cells, transformation, and culture medium preparation, which are mainly performed with reference to "Molecular Cloning: A Laboratory Manual" (3rd edition); PCR amplification experiments are performed according to the reaction conditions or kit instructions provided by the vector or DNA template supplier.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] In the quantitative experiments in the following examples, three replicate experiments were set up, and the results were taken as the arithmetic mean.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] The denitrifying microbe (Hyphomicrobium denitrificans) FJNU-6 used in the following examples was deposited on April 17, 2014, at the China General Microbiological Culture Collection Center (CGMCC), with the registration number CGMCC No. 1.12893.

[0030] Hyphomicrobium denitrificans FJNU-R8 was deposited on March 13, 2015, at the China General Microbiological Culture Collection Center (CGMCC), with the registration number CGMCC No. 10620.

[0031] Hyphomicrobium denitrificans DSM 1869 was purchased from the German Center for Microbiology and Cell Culture Collection on December 16, 2016, order number: A1613843-1.

[0032] In the examples below, the liquid culture medium consists of 20 g / L methanol, 3 g / L ammonium sulfate, 2 g / L potassium dihydrogen phosphate, 4 g / L disodium hydrogen phosphate, 1 g / L magnesium sulfate, 2 mL / L trace element solution, and water, with an initial pH of 6.8-7.0. The trace element solution consists of 80 g / L ferrous sulfate, 22.5 g / L zinc sulfate, 40 g / L manganese sulfate, 5 g / L copper sulfate, 15 g / L sodium chloride, 0.3 g / L sodium molybdate, 0.3 g / L potassium chloride, 0.03 g / L cobalt chloride, 3 g / L boric acid, 300 g / L calcium chloride, and water.

[0033] The seed culture medium consisted of 5 g / L methanol, 2 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 4 g / L disodium hydrogen phosphate, 1 g / L magnesium sulfate, and water.

[0034] Example 1 This embodiment provides a recombinant bacterium that produces high levels of pyrroloquinoline quinone. The specific steps of its construction method are as follows: The constitutive derepressed lac promoter of the bacterial broad-host vector pBBRMCS-2 was replaced with the methanol-inducible mxaF promoter of the universal methyltrophic bacteria vector pCM110. The three fluorescent protein encoding genes were cloned into the multiple cloning sites of vectors pBBRMCS-2, pCM110, and the methyltrophic bacteria-specific expression vector pTE102, respectively, using a seamless cloning ligation kit, resulting in plasmids pBBRMCS-2-RFP, pCM110-GFP, and pTE102-CFP. These three plasmids were then chemically transformed into *E. coli* S17-1 λpir competent cells to obtain three donor bacteria: pBBRMCS-2-RFP / S17-1 λpir, pCM110-GFP / S17-1 λpir, and pTE102-CFP / S17-1 λpir. The donor bacteria were then mixed with the recipient bacteria. H. denitrificans Conjugation transfer experiments were performed on FJNU-6, and conjugates were obtained through double-antibody plate selection. Plasmids were extracted from the obtained conjugates and re-transformed into *E. coli* T1. A second plasmid extraction was performed, and PCR was conducted using universal primers with the second extracted plasmid as a template. Nucleic acid electrophoresis verified the target size, and gene sequencing analysis confirmed that the constructed plasmids could stably exist in FJNU-6, thus successfully constructing the recombinant bacteria pBBRMCS-2-RFP / FJNU-6, pCM110-GFP / FJNU-6, and pTE102-CFP / FJNU-6. Subsequently, methanol culture broths of the three recombinant bacteria were processed and slides were prepared for observation under a fluorescence microscope; the results are as follows. Figure 1 As shown, this indicates that all three fluorescent proteins can indeed be expressed in FJNU-6.

[0035] Recombinant bacteria pBBRMCS-2-RFP / FJNU-R8, pCM110-GFP / FJNU-R8, pTE102-CFP / FJNU-R8, pBBRMCS-2-RFP / DSM 1869, pCM110-GFP / DSM 1869, and pTE102-CFP / DSM1869 were constructed using the methods described above. The results were consistent with... Figure 1 The consistency indicates that both FJNU-R8 and DSM 1869 can express the fluorescent protein genes of the three recombinant plasmids.

[0036] Following the method described above, the fluorescent protein encoding gene was replaced with the nucleotide sequence shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or SEQ ID No. 4 of the pqqABCDE gene cluster to construct pqq gene cluster overexpression plasmids pBBRMCS-2-pqqABCDE, pCM110-pqqABCDE, and pTE102-pqqABCDE, which were then introduced into the pqq gene cluster. H. denitrificans Three strains of pqqABCDE overexpression recombinant bacteria were obtained from FJNU-6, FJNU-R8, and DSM 1869.

[0037] Example 2 This embodiment provides the application of recombinant bacteria in the fermentation production of pyrroloquinoline quinone, and the specific steps are as follows: The pqqABCDE series overexpressing recombinant bacteria of the three strains obtained in Example 1 were subjected to shake-flask batch fermentation culture, and the PQQ content in the fermentation broth was determined by HPLC.

[0038] The batch fermentation process is as follows: The recombinant bacteria are streaked onto solid fermentation plates and incubated at 30°C for 7-10 days. Then, single clones are picked and transferred to Erlenmeyer flasks containing seed culture medium and incubated at 220 rpm and 30°C for 3-4 days. When the OD... 650When the inoculum reached approximately 1.5%, it was transferred at a 5% (v / v) inoculation rate to a 250 mL Erlenmeyer flask containing 50 mL of liquid culture medium and cultured at 220 rpm and 30℃ for 96 h. The PQQ yield of the recombinant bacteria is shown in Table 2. Overexpression of the pqqABCDE gene cluster from four sources using three shuttle vectors in recombinant strain FJNU-6 resulted in an average increase of 28.21% in PQQ yield; overexpression of the pqqABCDE gene cluster from four sources using three shuttle vectors in FJNU-R8 resulted in an average increase of 22.29% in PQQ yield; and overexpression of the pqqABCDE gene cluster from four sources using three shuttle vectors in DSM 1869 resulted in an average increase of 34.05% in PQQ yield. The recombinant strain carrying the pCM110 plasmid overexpression plasmid showed the greatest increase in PQQ yield. Using pCM110 to express the pqqABCDE gene cluster from four sources, the average yield of PQQ increased by 30.89%; using pTE102 and pBBRMCS-2 to express the pqqABCDE gene cluster from four sources, the average yield of PQQ increased by 27.32% and 25.20%, respectively.

[0039] Table 1. PQQ yield of recombinant bacteria constructed with different pqq gene cluster sequences.

[0040] Example 3 Following the construction method described in Example 1, the pqqABCDE gene cluster was replaced with the nucleotide sequence shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, or SEQ ID No. 8 to construct overexpression plasmids pBBRMCS-2-pqqADE, pCM110-pqqADE, and pTE102-pqqADE expressing the pqq gene cluster. H. denitrificansThe recombinant strains FJNU-6, FJNU-R8, and DSM 1869 were cultured in shake flasks for 96 h. The PQQ yields of each strain are shown in Table 2. FJNU-6, using three shuttle vectors to overexpress the pqqADE gene clusters from four sources, showed an average increase of 20.45% in PQQ yield; FJNU-R8, using three shuttle vectors to overexpress the pqqADE gene clusters from four sources, showed an average increase of 11.23% in PQQ yield; and DSM1869, using three shuttle vectors to overexpress the pqqADE gene clusters from four sources, showed an average increase of 19.95% in PQQ yield. The recombinant strain carrying the pCM110 plasmid overexpression plasmid showed the greatest increase in PQQ yield. Using pCM110 to express the pqqADE gene cluster from four sources, the average PQQ yield was increased by 20.36%; while using pTE102 and pBBRMCS-2 to express the pqqADE gene cluster from four sources, the average PQQ yield was only increased by 17.43% and 13.83%, respectively.

[0041] Table 2. PQQ yield of recombinant bacteria constructed from different pqq gene cluster sequences

[0042] Example 4 The difference between this embodiment and Embodiment 3 is that the nucleotide sequence shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, or SEQ ID No. 42 is used to replace the pqqADE gene cluster, and the pqqA gene overexpression plasmid pCM110-pqqA is constructed. H. denitrificans The recombinant strain FJNU-R8, after 96 hours of culture, produced PQQ yields ranging from 75.42 to 85.49 mg / L, which was an average increase of 27.02% compared to the original strain FJNU-R8.

[0043] Example 5 The difference between this embodiment and Embodiment 4 is that the nucleotide sequence of the pqqB gene, i.e., the sequence shown in SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, or SEQ ID No. 14, is used to replace the pqqA gene, and the pqqB gene overexpression plasmid pCM110-pqqB is constructed. H. denitrificans The recombinant strain FJNU-R8, after 96 h of culture, produced PQQ yields ranging from 56.73 to 68.53 mg / L.

[0044] Example 6 The difference between this embodiment and Embodiment 4 is that the nucleotide sequence of the pqqC gene (i.e., the sequence shown in SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, or SEQ ID No. 18) is used to replace the pqqA gene, and the pqqC gene overexpression plasmid pCM110-pqqC is constructed. H. denitrificans The recombinant strain FJNU-R8, after 96 h of culture, produced PQQ yields ranging from 57.12 to 69.04 mg / L.

[0045] Example 7 The difference between this embodiment and Example 4 is that the nucleotide sequence of the pqqD gene (i.e., the sequence shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, or SEQ ID No. 34) is used to replace the pqqA gene, and the pCM110-pqqD overexpression plasmid is constructed. H. denitrificans The recombinant strain FJNU-R8, after 96 h of culture, produced PQQ yields ranging from 64.83 to 72.98 mg / L.

[0046] Example 8 The difference between this embodiment and Example 4 is that the pqqA gene is replaced with the nucleotide sequence shown in SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, or SEQ ID No. 38 to construct the pqqE gene overexpression plasmid pCM110-pqqE. H. denitrificans The recombinant strain FJNU-R8, after culturing for 96 h, produced PQQ yields ranging from 99.58 to 115.73 mg / L.

[0047] Example 9 The difference between this embodiment and Embodiment 4 is that the pqqAE gene is used, i.e., SEQ ID No. 9 and No. 23 or SEQ ID No. 9 and No. 24 or SEQ ID No. 9 and No. 25 or SEQ ID No. 9 and No. 26 or SEQ ID No. 9 and No. 35 or SEQ ID No. 9 and No. 36 or SEQ ID No. 9 and No. 37 or SEQ ID No. 9 and No. 38 or SEQ ID No. 10 and No. 23 or SEQ ID No. 10 and No. 24 or SEQ ID No. 10 and No. 25 or SEQ ID No. 10 and No. 26 or SEQ ID No. 10 and No. 35 or SEQ ID No. 10 and No. 36 or SEQ ID No. 10 and No. 37 or SEQ ID No. 10 and No. 38 or SEQ ID No. 27 and No. 23 or SEQ ID No. 27 and No. 24 or SEQ ID No. 27 and No. 25 or SEQ ID No. 27 and No. 26 or SEQ ID No. 27 and No. 35 or SEQ ID No. 27 and No. 36 or SEQ ID No. 27 and No. 37 or SEQ ID No. 27 and No. 38 or SEQ ID No. 28 and No. 24 or SEQ ID No. 28 and No. 25 or SEQ ID No. 28 and No. 26 or SEQ ID No. 28 and No. 35 or SEQ ID No. 28 and No. 36 or SEQ ID No. 28 and No. 37 or SEQ ID No. 28 and No. 38 or SEQ ID No. 29 and No. 24 or SEQ ID No. 29 and No. 25 or SEQ ID No. 29 and No. 26 or SEQ ID No. 29 and No. 35 or SEQ ID No. 29 and No. 36 or SEQ ID No. 29 and No. 37 or SEQ ID No. 29 and No. 38 or SEQ ID No. 30 and No. 24 or SEQ ID No. 30 and No. 25 or SEQ ID No. 30 and No. 26 or SEQ ID No. 30 and No. 35 or SEQ ID No. 30 and No. 36 or SEQ ID No. 30 and No. 37 or SEQ ID No. 30 and No. 38 or SEQ ID No. 39 and No. 24 or SEQ ID No. 39 and No. 25 or SEQ ID No. 39 and No. 26 or SEQ ID No. 39 and No. 35 or SEQ ID No. 39 and No. 36 or SEQ ID No. 39 and No. 37 or SEQ ID No. 39 and No. 24.38 or SEQ ID No. 40 and No. 24 or SEQ ID No. 40 and No. 25 or SEQ ID No. 40 and No. 26 or SEQ ID No. 40 and No. 35 or SEQ ID No. 40 and No. 36 or SEQ ID No. 40 and No. 37 or SEQ ID No. 40 and No. 38 or SEQ ID No. 41 and No. 24 or SEQ ID No. 41 and No. 25 or SEQ ID No. 41 and No. 26 or SEQ ID No. 41 and No. 35 or SEQ ID No. 41 and No. 36 or SEQ ID No. 41 and No. 37 or SEQ ID No. 41 and No. 38 or SEQ ID No. 42 and No. 24 or SEQ ID No. 42 and No. 25 or SEQ ID No. 42 and No. 26 or SEQ ID No. 42 and No. 35 or SEQ ID No. 42 and No. 36 or SEQ ID No. 42 and No. 37 or SEQ ID The nucleotide sequences shown in No. 42 and No. 38 were used to replace the pqqA gene to construct the pCM110-pqqAE overexpression plasmid. H. denitrificans The recombinant strain FJNU-R8, after 96 h of culture, produced PQQ yields ranging from 87.44 to 103.95 mg / L.

[0048] Example 10 The difference between this embodiment and Embodiment 4 is that it uses the pqqADE gene, i.e., any nucleotide sequence of the pqqA gene shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, or SEQ ID No. 42, and the pqqD gene shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, or SEQ ID No. 34, and the pqqE gene shown in SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, or SEQ ID No. 42. Combine any of the nucleotide sequences shown in No. 38, and replace the pqqA gene with the combined nucleotide sequences to construct the pqqADE gene overexpression plasmid pCM110-pqqADE2. H. denitrificans The recombinant strain FJNU-R8, after culturing for 96 h, produced PQQ yields ranging from 81.96 to 98.27 mg / L.

[0049] Example 11 The recombinant strains pCM110-pqqABCDE / FJNU-R8 and pCM110-pqqABCDE / DSM 1869 expressing the SEQ ID No. 1 gene cluster constructed in Example 2, and the recombinant strain pCM110-pqqADE / FJNU-R8 expressing the SEQ ID No. 5 gene cluster constructed in Example 3, with the recombinant strain pCM110-GFP / FJNU-R8 constructed in Example 1 as the control strain, were subjected to fed-batch fermentation in a 1L quadruple fermenter to verify the PQQ synthesis performance of the recombinant strains overexpressing the pqq gene cluster. The PQQ content in the fermentation broth was determined by HPLC, and the biomass of the fermentation broth was determined by spectrophotometry. The verification experiment was repeated three times. The fed-batch fermentation process was as follows: the recombinant bacteria were streaked on solid plates of fermentation medium and cultured at 30°C for 7-10 days. Then, single clones were picked and placed into seed culture medium test tubes, and cultured at 220 rpm and 30℃ for 3-4 days. When the OD650 reached approximately 1.2-1.4, these were used as primary seed culture. A 1% (v / v) inoculum was then transferred to a 500 mL Erlenmeyer flask containing 100 mL of liquid culture medium and cultured at 220 rpm and 30℃ for 24-30 h. When the OD650 reached 1.8, a 10% (v / v) inoculum was added to a 1 L fermenter with an initial liquid culture medium volume of 700 mL. The initial fermentation speed was 300 rpm, and the initial aeration rate was 1 L / min. During fermentation, pH was automatically regulated using 6.25% (v / v) ammonia: pH 6.5 during cell growth and stable at 7.0 during PQQ synthesis. Methanol control during fermentation involved real-time manual adjustment to keep the remaining methanol level extremely low. After the initial methanol was consumed, a 25% (g / g) methanol aqueous solution was used for replenishment. Dissolved oxygen is controlled in two stages: in the early stage, the dissolved oxygen level is controlled at 60%, and in the later stage, the dissolved oxygen level is controlled at 40%.

[0050] PQQ production and biomass of recombinant bacteria, such as Figure 2 As shown, overexpression of the pqq gene cluster did indeed increase PQQ yield. After 134 h of fermentation, FJNU-R8 overexpressing the pqqABCDE and pqqADE gene clusters resulted in PQQ yields of 1524±15 mg / L and 1360±26 mg / L, respectively, representing increases of 31.38% and 17.28%. DSM 1869 overexpressing the pqqABCDE gene cluster resulted in a PQQ yield of 1215±21 mg / L, an increase of 42.11%.

[0051] Example 12 The pCM110-pqqA / FJNU-R8 strain expressing the SEQ ID No. 9 gene (constructed in Example 4), the pCM110-pqqE / FJNU-R8 strain expressing the SEQ ID No. 23 gene (constructed in Example 8), and the pCM110-pqqAE / FJNU-R8 strain expressing both SEQ ID No. 9 and SEQ ID No. 23 genes (constructed in Example 9) were used as control strains. The PQQ synthesis performance of the pqq gene-overexpressing recombinant strains was verified using the fed-batch fermentation method described in Example 11. The PQQ yield and biomass of different recombinant strains are shown below. Figure 3 As shown. After fermentation for 134 h, FJNU-R8 overexpression of pqqA, pqqE and pqqAE genes resulted in PQQ yields of 1464±8 mg / L, 1809±14 mg / L and 1660±18 mg / L, respectively, representing increases of 20.99%, 49.51% and 37.19%.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for constructing a recombinant bacterium that produces high levels of pyrroloquinoline quinone, characterized in that, The recombinant bacteria were obtained by introducing the pqq gene cluster or the pqq gene into denitrifying mycelial microbes using a shuttle expression vector.

2. The method for constructing a recombinant bacterium producing high levels of pyrroloquinoline quinone according to claim 1, characterized in that, The pqq gene cluster is the pqqABCDE gene cluster, and the nucleotide sequence of the pqqABCDE gene cluster is shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3 or SEQ ID No.

4.

3. The method for constructing a recombinant bacterium producing high levels of pyrroloquinoline quinone according to claim 1, characterized in that, The pqq gene cluster is the pqqADE gene cluster, and the nucleotide sequence of the pqqADE gene cluster is shown in SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 or SEQ ID No.

8.

4. The method for constructing a recombinant bacterium producing high levels of pyrroloquinoline quinone according to claim 1, characterized in that, The pqq gene is at least one of the pqqA gene, pqqB gene, pqqC gene, pqqD gene, and pqqE gene; The nucleotide sequence of the pqqA gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, and SEQ ID No. 42; The nucleotide sequence of the pqqB gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14; The nucleotide sequence of the pqqC gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 18; The nucleotide sequence of the pqqD gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, and SEQ ID No. 34; The nucleotide sequence of the pqqE gene is any one of the cDNA molecules or genomic DNA shown in SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 37, and SEQ ID No.

38.

5. The method for constructing a recombinant bacterium producing high levels of pyrroloquinoline quinone according to claim 1, characterized in that: The promoter that initiates the transcription of the pqq gene cluster or the pqq gene in the shuttle expression vector is the mxaF promoter.

6. A recombinant bacterium that produces high levels of pyrroloquinoline quinone, constructed according to any one of the construction methods described in claims 1 to 5.

7. The recombinant strain producing high levels of pyrroloquinoline quinone according to claim 6, characterized in that, The recombinant bacteria can overexpress one or more of the following: precursor pqqA, hydroxylase pqqB, oxidase pqqC, chaperone protein pqqD, and SAM free radical enzyme pqqE.

8. The use of the recombinant strain as described in claim 6 in the fermentation production of pyrroloquinoline quinone.

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