New method for increasing yield of erythromycin

Abstract

The invention discloses a method for increasing the yield of strain erythromycin. The method comprises the following step: over-expressing ATP synthetase beta subunit by using erythromycin producing strain. The saccharopolyspora erythraea overexpression ATP synthetase beta subunit AtpD can significantly improve the erythromycin fermentation level, and has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and relates to a new method for improving erythromycin yield, and specifically to a method for improving erythromycin fermentation level by causing a strain to overexpress ATP synthase beta subunit. Background Art

[0002] Erythromycin is a macrolide antibiotic, known for its broad-spectrum antibacterial effect, and is mainly produced by fermentation of Saccharopolyspora erythraea. Erythromycin is widely used in many fields such as medicine, agriculture and animal husbandry. The main components of erythromycin include erythromycin A, B, C and D, among which erythromycin A has the best antibacterial effect. Various erythromycin derivatives such as clarithromycin, azithromycin and roxithromycin have been widely used in clinical practice, with annual sales of billions of dollars. Therefore, improving the fermentation yield of erythromycin is of great significance both at the social and economic levels.

[0003] Traditionally, the method of improving erythromycin production by optimizing fermentation conditions is not only time-consuming but also costly. In contrast, using genetic engineering to knock out or enhance the expression of key genes in Saccharopolyspora erythromycin can effectively cultivate high-yield strains. This method has the advantages of simple operation, short time, low cost and easy control, showing broad application prospects. Summary of the invention

[0004] In the study of obtaining erythromycin high-yield strains by genetic engineering directed gene change, we found that the steps involved in the biosynthesis of erythromycin, such as acyl transfer, hydroxylation and glycosylation, all require ATP participation to maintain the normal progress of the synthesis reaction. On the other hand, ATP also plays an important role in the activation and transportation of precursor substances, such as activating precursor glycosyl compounds into nucleotide sugar derivatives, thereby becoming part of the erythromycin molecule. In addition, ATP levels also indirectly affect the metabolic balance in cells, optimizing the synthesis efficiency of erythromycin by regulating metabolic pathways and energy distribution. Therefore, sufficient ATP supply is the basis for ensuring the efficient biosynthesis of erythromycin, so improving the synthesis of ATP may increase the yield of erythromycin. We conducted a single factor investigation on proteins and genes related to ATP generation, and unexpectedly found that the ATP synthase β subunit AtpD (or atpD) (GenBank: WP_211898549.1) from Saccharopolyspora erythraea can effectively promote the biosynthesis of erythromycin, and the strain overexpressing the gene atpD can increase the erythromycin fermentation level by more than 50%. Specifically, the present invention includes the following technical solutions.

[0005] The first aspect of the present invention provides a method for increasing the erythromycin production of a strain, comprising the following steps: causing the erythromycin-producing bacteria to overexpress an ATP synthase β subunit, or a conservative variant polypeptide having more than 95% homology, preferably more than 96% homology, preferably more than 97% homology, preferably more than 98% homology, more preferably more than 99% homology, and having ATP synthase function.

[0006] Preferably, the ATP synthase β subunit is the ATP synthase β subunit AtpD from Saccharopolyspora erythraea (GenBank: WP_211898549.1), whose amino acid sequence is shown in SEQ ID NO: 1, and may also be written as atpD.

[0007] In one embodiment, the erythromycin producing bacterium is Saccharopolyspora erythraea. More preferably, the erythromycin producing bacterium is Saccharopolyspora erythraea ATCC 11635.

[0008] Preferably, the nucleotide sequence of the gene atpD encoding the ATP synthase β subunit is SEQ ID NO: 2, or a nucleotide sequence having more than 95% homology, preferably more than 96% homology, preferably more than 97% homology, preferably more than 98% homology, and more preferably more than 99% homology with SEQ ID NO: 2.

[0009] Optionally, the overexpression of the ATP synthase β subunit is implemented by:

[0010] A. inserting a strong promoter upstream of the ATP synthase β subunit in the genome of the erythromycin producing bacteria; and / or

[0011] B. integrating one or more copies, preferably two or more, three or more, four or more, six or more, or eight or more copies of the gene encoding the β subunit of the ATP synthase into the genome of the erythromycin-producing bacteria; and / or

[0012] C. Express the gene encoding the β-subunit of ATP synthase in the genome of the erythromycin producing host bacteria in the form of a plasmid.

[0013] The above steps A or B can be achieved by transforming the recombinant plasmid containing the ATP synthase β subunit encoding gene into the host bacteria. Preferably, the plasmid vector is a plasmid vector suitable for expression in Saccharopolyspora erythrae, such as the integrative vector pIB139 with the strong promoter ermE*P.

[0014] The method of inserting the strong promoter in the above step A and the method of integrating the ATP synthase β subunit encoding gene in the genome in step B can be selected from plasmid transformation and gene editing technology.

[0015] The above-mentioned gene editing technology can be selected from the following groups: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT (multiplex genome editing by natural transformation), etc.

[0016] The second aspect of the present invention provides a recombinant plasmid, which is a plasmid suitable for expressing foreign genes in Saccharopolyspora erythraea and contains the ATP synthase β subunit encoding gene atpD.

[0017] Preferably, the plasmid vector of the above-mentioned recombinant plasmid is an integrative vector pIB139 with a strong promoter ermE*P (see Xinjuan Ning, Xinran Wang, Yuanting Wu, Qianjin Kang*and Linquan Bai*: Identification and Engineering of Post-PKS Modification Bottlenecks for Ansamitocin P-3Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280. Biotechnology Journal 2017, 12, 1700484.), etc., but is not limited thereto.

[0018] Optionally, the nucleotide sequence of the above recombinant plasmid is shown in SEQ ID NO:3.

[0019] The third aspect of the present invention provides an engineered bacterium of Saccharopolyspora erythrae that overexpresses the ATP synthase β subunit as described above, wherein the engineered bacterium of Saccharopolyspora erythrae has the ATP synthase β subunit encoding gene as described above integrated into the genome of Saccharopolyspora erythrae; or the engineered bacterium of Saccharopolyspora erythrae is a transformant in which the recombinant plasmid as described above is transformed in a host bacterium.

[0020] The fourth aspect of the present invention provides the use of the above-mentioned Saccharopolyspora erythraea engineered bacteria in the production of erythromycin by fermentation.

[0021] The present invention firstly discovered that ATP synthase β subunit AtpD (GenBank: WP_211898549.1) has a new function of enhancing the erythromycin metabolic pathway. By overexpressing the ATP synthase β subunit gene atpD in Saccharopolyspora erythraea, the erythromycin yield can be increased by 55.6%, providing technical support for increasing the erythromycin fermentation yield in industrial production, and having good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a structural map of the recombinant plasmid pB023-1 used to express ATP synthase β subunit AtpD (GenBank: WP_211898549.1).

[0023] Figure 2 It is a comparison chart of erythromycin fermentation yield of wild-type Saccharopolyspora erythraea, empty plasmid expression strain and atpD overexpression engineered strain. DETAILED DESCRIPTION

[0024] Based on the study of genes positively correlated with ATP biosynthesis, we screened out the ATP synthase β subunit AtpD (GenBank: WP_211898549.1) that can promote erythromycin biosynthesis. We overexpressed the atpD gene in Saccharopolyspora erythraea ATCC11635 through genetic engineering and obtained a genetically engineered strain with high erythromycin production.

[0025] In this article, for the sake of simplicity, a certain protein, such as ATP synthase β subunit AtpD (GenBank: WP_211898549.1), and its encoding gene (DNA) name are sometimes used interchangeably. Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for AtpD (atpD), when used to describe the function or category of ATP synthase β subunit protein, it refers to the protein; when described as a gene, it refers to the gene encoding the protein.

[0026] The terms "Saccharopolyspora erythrae (genetic) engineered bacteria", "engineered bacteria (strain)" and "recombinant bacteria (strain)" herein have the same meaning and can be used interchangeably. Similarly, the terms "recombinant vector" and "recombinant plasmid" have the same meaning and can be used interchangeably.

[0027] Correspondingly, for the convenience of description, the starting strain that has not overexpressed atpD, such as Saccharopolyspora erythraea ATCC 11635, can be referred to as the "host strain" or "wild type (strain)".

[0028] Through genetic engineering modification, such as integrating a single copy or multiple copies of the atpD gene into the genome of the erythromycin-producing bacteria and / or placing it under the control of a strong promoter to promote the overexpression of the ATP synthase β subunit, the erythromycin production of the strain can be effectively increased.

[0029] The terms "(erythromycin production) increase", "enhance" or "enhance" may mean an increase of at least 10% compared to a reference level (such as a starting strain / host bacterium), for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10% and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0030] In the description of the technical solutions of the present invention, the terms used, such as "A and / or B", "A and / or B", "and / or", "and / or", are intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to cover each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0031] Based on the above-mentioned special function of ATP synthase β subunit AtpD, those skilled in the art can expect that it is reasonable that conservative variant polypeptides with high homology (identity) to the amino acid sequence SEQ ID NO:1, for example, more than 90%, have the same or similar erythromycin biosynthesis promoting function.

[0032] As used herein, the term "conservative variant polypeptide" refers to a polypeptide that substantially maintains the same biological function or activity of the ATP synthase β subunit AtpD. The variation refers in particular to a small amount of amino acid mutations, and the "mutation" includes but is not limited to replacement, deletion, insertion, and chemical modification of amino acid residues, preferably a forward mutation, i.e., a mutation that improves the function of GluA1-L1. The substitution may be a non-conservative substitution, a conservative substitution, or a combination of a non-conservative substitution and a conservative substitution. "Conservative" amino acid substitutions or mutations refer to the interchangeability of residues with similar side chains, and therefore generally include substitutions of amino acids in the same or similar amino acid definition categories in a polypeptide. However, as used herein, if a conservative mutation can be replaced by aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or a restricted residue to a restricted residue, then the conservative mutation does not include a substitution of hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or a small residue to a small residue. It is well known in the art that common cases of conservative substitutions include: mutual substitutions between aromatic amino acids F, W, and Y; mutual substitutions between hydrophobic amino acids L, I, and V; mutual substitutions between polar amino acids Q and N; mutual substitutions between basic amino acids K, R, and H; mutual substitutions between acidic amino acids D and E; and mutual substitutions between hydroxyl amino acids S and T. In addition, A, V, L, or I can be conservatively mutated to another aliphatic residue or another non-polar residue. Exemplary conservative substitutions can be performed, for example, according to the following table.

[0033] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0034] The construction of genetically engineered bacteria overexpressing atpD can be achieved by cloning the AtpD encoding gene (atpD), such as SEQ ID NO: 2, into the host bacterial genome using conventional recombinant plasmid transformation methods or gene editing techniques.

[0035] When using above-mentioned coding gene atpD to build recombinant plasmid, can add any enhanced promoter or constitutive promoter before its transcription initiation nucleotide.In addition, when using this coding gene atpD to build recombinant plasmid, can also use enhancer, comprise translation enhancer or transcription enhancer, these enhancer regions can be ATG start codon or adjacent region start codon etc., but must be identical with the reading frame of coding sequence, to guarantee the correct translation of whole sequence.The source of described enhancer is extensive, can be natural, also can be synthetic.The translation initiation region can be from transcription initiation region or structural gene.

[0036] By using the integrative vector pIB139 with a strong promoter ermE*P to insert more than one copy of the gene atpD on the chromosome, the erythromycin yield of the control strain (blank vector integrated strain) was increased by 55.6% at the laboratory shake flask level, indicating that the technical solution of the present invention can significantly increase the yield of erythromycin and provide a new transformation strategy for constructing industrial strains.

[0037] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0038] Example

[0039] The examples herein involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0040] In the examples herein, if no specific description is given for the reaction temperature or the operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0041] For experimental methods without specific conditions specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0042] Materials and methods

[0043] The plasmid pIB139 involved in the embodiment has been recorded in the SCI database document "Xinjuan Ning, Xinran Wang, Yuanting Wu, Qianjin Kang* and Linquan Bai*: Identification and Engineering of Post-PKS Modification Bottlenecks for Ansamitocin P-3 Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280. Biotechnology Journal 2017, 12, 1700484".

[0044] All primer synthesis and sequencing involved were completed by Shanghai Qingke Biotechnology Co., Ltd.

[0045] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0046] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0047] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium plus 20 g / L agar powder).

[0048] 2× spore pre-germination medium: yeast extract 10 g / L, casamino acid 10 g / L, calcium chloride 0.01 M, sterilized at 121°C for 20 min, used for spore germination culture in conjugation transfer experiments.

[0049] TES (0.05M, pH 8.0) solution: 1.15 g N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid was diluted to 100 mL pure water and the pH value was adjusted to 8.0. Sterilized at 121°C for 20 min and used for conjugation transfer of Saccharopolyspora erythrae spore germination.

[0050] Tryptone Soy Broth (TSB) culture: 3% TSB, used for the culture of Saccharopolyspora erythraea.

[0051] MS medium: 20g / L soybean cake powder infusion, 20g / L mannitol, 20g / L agar powder, used for conjugation and transfer of red Saccharopolyspora culture.

[0052] Slant culture medium: corn slurry 13 g / L, starch 18 g / L, NaCl 3 g / L, (NH4)2SO4 3 g / L, CaCO3 5 g / L, agar powder 20 g / L.

[0053] Seed culture medium: corn slurry 13g / L, starch 35g / L, soybean cake powder 15g / L, NaCl 3g / L, (NH4)2SO4 2g / L, CaCO3 6g / L, soybean oil 5mL.

[0054] Fermentation medium: starch 35 g / L, dextrin 40 g / L, soybean cake powder 35 g / L, (NH4)2SO4 2 g / L, CaCO3 6 g / L, soybean oil 10 mL.

[0055] Example 1: Construction of engineered bacteria overexpressing atpD gene

[0056] This example is a specific process for preparing an engineered strain B023-01 that overexpresses the atpD gene encoding the β subunit of ATP synthase. Specifically, the following steps are included:

[0057] Step 1: Construction of plasmid pB023-01: Using the genomic DNA of Saccharopolyspora erythrae ATCC 11635 as a template, primers atpD-F / R with NdeI / EcoRI restriction sites at both ends were used to amplify the atpD gene fragment by PCR. The amplified fragment (i.e., atpD gene fragment) after restriction digestion was inserted into the NdeI / EcoRI site downstream of the artificial strong promoter ermEp* in plasmid pIB139 to obtain plasmid pB023-01. The plasmid map is shown in the figure. Figure 1 The nucleotide sequence is shown in SEQ ID NO:3.

[0058] The endonuclease recognition sites (enzyme cutting sites) involved in the first step are as follows:

[0059] NdeI recognition site: EcoRI recognition site:

[0060] 5'...CA^TATG...3' 5'...G^AATTC...3'

[0061] 3'...GTAT^AC...5' 3'...CTTAA^G...5'

[0062] The primer sequences used in the first step are:

[0063]

[0064] PCR system and conditions used for gene fragment preparation in the first step:

[0065] PCR reaction system: DNA template 30ng, primer 20pmol, 50% DMSO 4μL, PrimerSTAR max, add pure water to make up to 50μL;

[0066] PCR conditions: 95°C for 5 min; 95°C for 15 s, 60°C for 15 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0067] Step 2: The overexpression plasmid vector pB023-01 constructed in the first step is introduced into the recipient bacterium Saccharopolyspora erythraea ATCC 11635 through conjugation transfer for site-specific recombination, and the correct conjugants are screened through resistance and PCR verification to obtain a mutant strain with overexpression of the atpD gene. Specifically, the following steps are included:

[0068] The gene overexpression plasmid pB023-01 was transferred into ET12567 (pUZ8002) to obtain the donor strain. The inoculated donor strain was cultured in LB medium containing apramycin, kanamycin and chloramphenicol at final concentrations of 50 μg / mL, 50 μg / mL and 25 μg / mL, respectively, and cultured at 37°C with shaking for 20 hours. Subsequently, the bacteria were rinsed with fresh LB solution to remove residual antibiotics. Fresh spores of Saccharopolyspora erythrae on the plate were collected, centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded and the spores were resuspended with 5 mL of TES solution (0.05 M, pH 8.0). The centrifuge tube was placed in a 50°C water bath for heat shock for 10 minutes, and then cooled to room temperature with tap water. 5 mL of 2× spore pre-germination medium was added and incubated at 37°C and 200 rpm for 3 hours. After incubation, the cells were centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the spores were rinsed with fresh LB solution.

[0069] The treated spores were mixed with the donor strain at a ratio of about 1:10, evenly spread on MS solid medium containing 10mM MgCl2, and cultured at 37°C for 20 hours. Afterwards, apramycin (final concentration 50μg / mL) and nalidixic acid (final concentration 40μg / mL) were mixed in 1mL sterile water, covered on the surface of MS solid medium, and transferred to a 30°C incubator for inverted culture after the medium dried. Usually after 3 to 5 days, conjugants will appear on the plate, which will be transferred to MS solid medium containing the same concentration of apramycin and nalidixic acid for further culture. Through mycelium PCR verification screening, a mutant strain with doubled atpD gene was obtained, named B023-01. The same method was used to transform the empty plasmid pIB139 red sugar polyspora ATCC 11635 bacteria, and the obtained strain was named B023-00 as a control strain.

[0070] In the second step, the PCR system and conditions used to screen mutants by PCR verification are as follows:

[0071] PCR system: DNA template 10-100 ng, primer 10 pmol, 50% DMSO 2 μL, 2×Mix buffer 10 μL, add pure water to make up to 20 μL;

[0072] PCR conditions: 95°C for 10 min; 9°C for 15 s, 60°C for 15 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0073] Example 2: Strain fermentation

[0074] This example is a process of producing erythromycin by fermentation using the engineered strain B023-01 in which the atpD gene is overexpressed.

[0075] The specific steps are as follows: the engineered bacteria B023-01 with overexpression of atpD gene, its control strain B023-00 and the starting strain ATCC 11635 are respectively applied on a solid slant culture medium for activation, cultured at 30°C for 7 days, scraped half of the spores and inoculated into a seed culture medium, cultured at 34°C and 200-220rpm for 48h, transferred to a fermentation culture medium at an inoculum rate of 10% v / v, fermented at 30°C and 200-220rpm, added 1% n-propanol after 24h, and collected the fermentation liquid for erythromycin detection after 120h.

[0076] Example 3: Fermentation product detection

[0077] This embodiment is a method for detecting erythromycin using liquid phase. Specifically, 0.5 mL of fermentation liquid was taken, and the mixture was incubated at 12000 rpm for 10 min. 100 μL of supernatant was taken and appropriately diluted with methanol. The mixture was then filtered through a membrane and then subjected to liquid phase detection.

[0078] Erythromycin detection conditions: Agilent 1260 liquid chromatograph, chromatographic column C18 (4.6×250 mm, 5 μm), mobile phase: dipotassium hydrogen phosphate aqueous solution pH 7-8, 45% acetonitrile, detection wavelength: 215 nm, column temperature: 33°C, flow rate: 1 mL / min, injection volume: 20 μL.

[0079] Figure 2 The diagram is a comparative diagram of erythromycin fermentation yields of the atpD overexpressing engineered bacteria, the starting strain and the control strain. The results show that at the laboratory shake flask level, the yield of the engineered strain B023-01 of the present invention is increased by 60.68% and 55.6% compared with the starting strain ATCC 11635 and the control strain B023-00, respectively.

[0080] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments and features in the embodiments herein can be combined with each other at will.

Claims

1. A method for increasing the erythromycin production of a strain, comprising the following steps: causing the erythromycin producing strain to overexpress an ATP synthase β subunit, or a conservative variant polypeptide having more than 95% homology thereto and having ATP synthase function.

2. The method according to claim 1, characterized in that The ATP synthase β subunit is the ATP synthase β subunit AtpD (GenBank: WP_211898549.1) derived from Saccharopolyspora erythraea, and its amino acid sequence is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that The erythromycin producing bacterium is Saccharopolyspora erythraea.

4. The method according to claim 2, characterized in that The nucleotide sequence of the gene atpD encoding the ATP synthase β subunit is SEQ ID NO: 2 or a nucleotide sequence having more than 95% homology with SEQ ID NO:

2.

5. The method according to claim 1, characterized in that Overexpression of the ATP synthase β subunit is carried out in the following manner: A. inserting a strong promoter upstream of the ATP synthase β subunit in the genome of the erythromycin producing bacteria; and / or B. integrating more than one copy of the gene encoding the β subunit of the ATP synthase into the genome of the erythromycin-producing bacteria; and / or C. Express the gene encoding the β-subunit of ATP synthase in the genome of the erythromycin producing host bacteria in the form of a plasmid.

6. The method according to claim 5, characterized in that Step A or B is achieved by transforming the recombinant plasmid containing the ATP synthase β subunit encoding gene into the host bacteria. Preferably, the plasmid vector is a plasmid vector suitable for expression in Saccharopolyspora erythrae, such as the integrative vector pIB139 with the strong promoter ermE*P.

7. A recombinant plasmid as claimed in claim 5 or 6, characterized in that: The plasmid vector is a plasmid suitable for expressing foreign genes in Saccharopolyspora erythraea, and contains the ATP synthase beta subunit encoding gene atpD.

8. The recombinant plasmid according to claim 7, characterized in that Its nucleotide sequence is shown in SEQ ID NO:

3.

9. An engineered bacterium of Saccharopolyspora erythraea that overexpresses the β subunit of the ATP synthase as claimed in claim 1, characterized in that: The engineered Saccharopolyspora erythrae has the ATP synthase β subunit encoding gene as described above integrated into the genome of the Saccharopolyspora erythrae; or the engineered Saccharopolyspora erythrae is a transformant in which the recombinant plasmid as described in claim 7 or 8 is transformed in a host bacterium.

10. Use of the engineered Saccharopolyspora erythraea as claimed in claim 9 in the production of erythromycin by fermentation.

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