CN116515879B - Induction expression system, strain and application for improving erythromycin yield by controlling intracellular NADH (NADH) level of rhodosporidium saccharopolyspora - Google Patents

Abstract

The invention relates to the technical field of synthetic biology, and provides an induction expression system for improving erythromycin yield by controlling intracellular NADH (NADH) level of rhodosporidium saccharopolyspora, which comprises a plasmid pSETcd-1905-cmt-sgRNA constructed by a SACE_1905 gene; wherein the nucleotide sequence of the SACE_1905 gene is shown as SEQ ID NO. 1. The invention also provides an erythromycin high-yield engineering strain and application thereof, wherein the strain comprises the induction expression system. The invention designs and constructs an induction system for dynamically controlling intracellular reducing force by means of CRISPRi technology and an induction system based on SACE_1905 and the correlation of primary growth and secondary metabolism of strains and by means of a synthetic biology method, thereby providing technical support for improving erythromycin yield in industrial production.

Description

Induction expression system, strain and application for improving erythromycin yield by controlling intracellular NADH (NADH) level of rhodosporidium saccharopolyspora

Technical Field

The invention relates to the technical field of synthetic biology, in particular to an induction expression system, a strain and application for improving erythromycin yield by controlling intracellular NADH (NADH) level of rhodosporidium saccharopolyspora.

Background

Rhodotorula saccharopolyspora is a main producing strain of a broad-spectrum macrolide antibiotic-Erythromycin (Erythrocycline), and plays an important role in industrial production of Erythromycin. Since most biosynthetic gene clusters in actinomycetes are silent under routine experimental conditions, antibiotic production is not very ideal. At present, the strategy of optimizing the secondary metabolic process by carrying out genetic engineering on rhodosporidium to improve the fermentation yield of erythromycin is widely applied. However, such static regulation results in a lack or redundancy of certain substances within the cell, and thus, the yield is limited. In order to improve such limitation, in recent years, a "dynamic regulation strategy" has been proposed which allows recombinant cells to dynamically sense and process specific signals for adaptive regulation by designing and constructing gene pathway units.

Research shows that in the streptomyces avermitilis, NADH can weaken the inhibition of the negative regulatory factor Rex synthesized by the avermectin to the target gene, and promote the biosynthesis of the avermectin; in rhodosporidium saccharopolyspora, overexpression of NADH oxidase or overexpression of ATPase genes can lead to intracellular NADH/NAD of the strain ⁺ The ratio decreases and the yield of erythromycin increases. The biosynthesis of erythromycin involves a series of redox reactions in which the participation of NADH or NADPH dependent ketoreductase, enoyl reductase, aldehyde ketoreductase and the like is required. It can be seen that NADH is an important cofactor for erythromycin biosynthesis enzymes whose intracellular levels are closely related to erythromycin synthesis, and therefore, control of intracellular reducing power levels of the production strain may also be an effective strategy for increasing erythromycin yield.

The growth curve of actinomycetes is mainly divided into three stages: transient primary metabolic phases, transition phases and lengthy secondary metabolic phases. However, since actinomycetes often require a complicated stage of the conversion process from primary metabolism to secondary metabolism, this seriously affects the biosynthesis yield of secondary metabolites. Thus, starting from the primary and secondary metabolism switching, controlling the switching phase between the two is a key point for optimizing the production of secondary metabolites. The prior-stage discovery of the laboratory is that,SACE_1905gene encoding alcohol dehydrogenase, increaseThe gene copy number can improve the primary growth of the strain and the erythromycin yield to a certain extent, suggesting thatSACE_1905Plays an important role in the primary metabolism and the secondary metabolism of rhodosporidium saccharopolyspora.

Accordingly, the present invention attempts to build bySACE_1905The expression system is induced, and the intracellular NADH level of the strain is dynamically controlled to balance the primary growth and secondary metabolism of the strain, so that the erythromycin yield is further improved.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for improving the yield of erythromycin by controlling the intracellular NADH level of rhodosporidium saccharopolysporaSACE_1905Inducible expression systems, strains and uses.

The invention adopts the following technical scheme to solve the technical problems:

an inducible expression system for increasing erythromycin production by controlling intracellular NADH levels of rhodosporidium saccharopolyspora comprisingSACE_1905Plasmid pSETcd for gene construction1905-cmt-sgRNA; wherein the saidSACE_1905The nucleotide sequence of the gene is shown as SEQ ID NO. 1.

As one of the preferable modes of the invention, theSACE_1905The amino acid sequence of the gene code is shown as SEQ ID NO. 2.

As one of the preferable embodiments of the present invention, the plasmid pSETcd-1905-cmtThe construction method of the sgRNA is as follows:

(1) will bedCas9Fragments, synthetic DNA fragmentscmtInserting the recombinant plasmid pSETcd into an enzyme cutting site of the plasmid pSET152 to obtain a recombinant plasmid pSETcd;

(2) will P _ermE* Fragments(s),SACE_1905The gene is inserted into the enzyme cutting site of the recombinant plasmid pSETcd to obtain the recombinant plasmid pSETcd-1905；

(3) Amplification targetingSACE_1905Obtaining a sgRNA-containing fragment; inserting the sgRNA-containing fragment into a recombinant plasmid pSETcd-1905Restriction enzyme sites to obtain recombinant plasmid pSETcd1905-sgRNA；

(4) Amplification ofcmtElement and apply the samecmtInsertion of the fragment into the recombinant plasmid pSETcd-1905The cleavage site of sgRNA to obtain the desired engineering plasmid pSETcd1905-cmt-sgRNA。

As one of the preferable embodiments of the present invention, the plasmid pSETcd-1905-cmt-sgrnas for induction of expressionSACE_1905The gene and the erythrocin yield are improved by dynamically controlling the intracellular reducing power level of the rhodosporidium saccharopolyspora.

An erythromycin high-yield engineering strain, comprising the induction expression system.

As a preferred embodiment of the present invention, the plasmid pSETcd of the inducible expression system is prepared1905Introducing cmt-sgRNA into erythromycin industrial strain.

The application of the erythromycin high-yield engineering strain is as follows: fermenting and producing erythromycin by using the erythromycin high-yield engineering strain, and dynamically controlling genes in a mode of adding an inducer and optimizing induction conditionsSACE_1905The expression of the strain balances the primary metabolism growth and the secondary metabolism of the strain, and the erythromycin yield is improved.

As one of the preferable modes of the present invention, the inducer is cumate (4-isopropylbenzoic acid ); the induction conditions are as follows: at the time of 96h fermentation, a final concentration of 60. Mu. Mol/L of cumate solution was added.

The design idea is as follows:

the invention adopts the method of adding in the erythromycin high-yield engineering strain in the early stage of the laboratorySACE_1905The copy number method improves erythromycin yield and shows a certain effect. However, this approach ignores the balance between primary and secondary metabolism of actinomycetes, making yield improvement practically limited. The invention further discovers that the polysaccharide red fungus is over expressed on the basisSACE_1905Will result in intracellular NADH/NAD ⁺ The ratio decreases, suggesting thatSACE_1905It is possible to influence secondary metabolic production by perturbing intracellular reducing power levels. Thus, attempts can be made to construct the erythromycin industrial strain by constructing itSACE_1905And the induction expression system balances the primary metabolism growth and the secondary metabolism of the strain by optimizing the induction condition, thereby breaking the limit and further improving the erythromycin yield.

Compared with the prior art, the invention has the advantages that:

(1) The invention is based onSACE_1905And the correlation of the primary growth and secondary metabolism of the strain, and the induction system for dynamically controlling the intracellular reducing force is designed and constructed by means of the CRISPRi technology and the inducible system through a synthetic biological method, so that technical support is provided for improving the erythromycin yield in industrial production;

(2) The invention applies the induction system in the erythromycin industrial strain, constructs the engineering strain, and improves the erythromycin yield by 78.2 percent compared with WB by optimizing the addition time point and the addition concentration of the inducer in the fermentation process, and the highest yield reaches 1497.5 mg/mL;

(3) Compared with single over-expression in rhodosporidium saccharopolysporaSACE_1905The method for improving the yield of the erythromycin can further improve the yield of the erythromycin by 15.4 percent; this suggests dynamic control of rhodosporidium saccharolyticumSACE_1905The expression level of (2) can further improve the fermentation yield of erythromycin.

Drawings

FIG. 1 is a schematic diagram of a conventional gas turbineSACE_1905A map of the location information of genes and surrounding adjacent genes on the chromosome;

FIG. 2 is intracellular NADH/NAD of the relevant strain ⁺ Ratio determination results (strains A226, A226/pIB139 and A226/p)1905Intracellular NADH/NAD fermenting 48 h ⁺ Content detection results; in the figure, "×":P< 0.001; "ns": no significant difference);

FIG. 3 shows the results of SACE_1905 protease activity (SACE_1905 protein catalyzes a substrate reaction, and the detection of OD by a microplate reader) ₃₄₀ A change in value);

FIG. 4 is a schematic diagram of the construction of an inducible expression system plasmid;

FIG. 5 shows the results of erythromycin production assay of the strain after addition of the inducer cumate (in the figure, A is the strain WB/. DELTA.1906-pSETcd-1905-cmt-the sgrnas were added with final concentration of 20 μmol/L inducer cumate at fermentation time 0h, 48 h and 96h, respectively, and fermentation continued until the erythromycin yield detection result at day seven; "ns": no significant difference; "***”：P< 0.001; panel B shows strain WB/delta1906-pSETcd-1905-cmt-the erythromycin yield test results after further fermentation of sgrnas to seventh day after fermentation 96h with addition of cuamete solutions at final concentrations of 20, 40 and 60 μmol/L, respectively; "***":P＜0.001）；

FIG. 6 is a strain after addition of the inducer cumateSACE_1905Transcript level and intracellular NADH/NAD ⁺ Ratio analysis (in the figure, A is strain WB/delta)1906-pSETcd-1905-cmtAfter fermentation 96h of the sgRNA with addition of the cuamet solution,SACE_1905qRT-PCR detection results of the transcription level; "**":P＜0.01；“***”：P＜0.001；“****”：P< 0.0001; panel B shows strain WB/delta1906-pSETcd-1905-cmtAfter fermentation of 96h with addition of the cuame solution, intracellular NADH/NAD ⁺ Ratio detection results; "*":P＜0.05；“**”：P＜0.01）。

Detailed Description

The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.

The strains and plasmids used in the examples below are shown in Table 1, and the primer sequences synthesized are shown in Table 2. Wherein, the original strain, namely rhodosporidium saccharatum A226 (CGMCC 8279), and the erythromycin industrial high-yield strain WB (CGMCC 8280) are all strains which can be directly purchased.

Meanwhile, E.coli used in the following examples was cultured on a liquid LB medium at 37℃or on a solid LB plate with 1.25% agar added. Erythromyces polyspora erythraea was cultured on 30 ℃ Tryptone Soy Broth (TSB) medium or on R3M plates containing 2.2% agar.

Among the materials used in the examples below, PEG3350, lysozyme, TES, casamino acid, apramycin were purchased from Sigma. TSB, yeast extract, peptone were purchased from Oxoid corporation. Glycine, agar powder, sodium chloride and other biological agents are all purchased from reagent companies. General procedures for E.coli and Rhodotorula saccharopolyspora are standard procedures. Primer synthesis and DNA sequencing were accomplished by general biosciences, inc.

TABLE 1 bacterial species, plasmids and their main properties according to the invention

TABLE 2 primers according to the invention

Example 1

SACE_1905Gene-related information:

gene annotation information based on the KEGG database (https:// www.kegg.jp /) in the rhodosporidium saccharopolyspora genomeSACE_1905The positional information of the peripheral genes and the potential gene functions are shown in FIG. 1.SACE_1905The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

Example 2

A226/p1905Intracellular NADH/NAD of the relevant strain ⁺ And (3) ratio detection:

a226, A226/pIB139 and A226/p1905The strain is inoculated to a TSB culture medium, cultured at 30 ℃ and 220rpm for 48 h, then inoculated to an R5 fermentation culture medium, and a fermentation 48 h sample is taken. Centrifuging the fermentation liquor, discarding supernatant, sequentially adding-80 ℃ precooled methanol, -20 ℃ precooled chloroform and 4 ℃ precooled water, vibrating 20 s, standing at-20 ℃ for 1 h, centrifuging, and taking the uppermost organic phase to obtain intracellular NADH and NAD ⁺ . Then, using Biyundian NAD ⁺ NADH detection kit (WST-8 method) "detection A226, A226/pIB139 and A226/p1905NADH/NAD in ⁺ Ratio of the two.

The results are shown in FIG. 2, which demonstratesSACE_1905Overexpression will result in intracellular NADH/NAD ⁺ The ratio decreases.

Example 3

Determination of SACE_1905 enzyme activity:

(1) Amplification Using 1905-28a-P1 and 1905-28a-P2 as primers and A226 genome as templateSACE_ 1905And (3) a gene. UsingNdeI andHindIII endonuclease pairsSACE_1905Double enzyme digestion is carried out on the gene fragment and the pET28a plasmid, and then the enzyme-digested gene fragment and the plasmid are connected by using T4 ligase to obtain pET28a-SACE_1905A plasmid.

(2) Using CaCl ₂ pET28 a-mediated transformation methodSACE_1905The plasmid was introduced into BL21 (DE 3) competence and plated on LB plates containing kanamycin resistance, and the positive monoclonal obtained was designated BL21 (DE 3)SACE_ 1905。

(3) BL21 (DE 3) is addedSACE_1905Culturing in LB liquid medium containing kanamycin resistance, and adding IPTG to induce protein expression. The cells were collected by centrifugation, sonicated, and purified using a nickel ion column. Finally, SACE_1905 protein was eluted using 500mM imidazole.

(4) The enzyme activity reaction system is as follows: 1 mL of 0.1-mol/L Tris-HCl (pH 7.6), 5. Mu.L of 5-mu.L DTT, 42.85. Mu.L of 5 mg/mL NADH and 20. Mu.L of 40% aqueous acetaldehyde.

After mixing well, 100. Mu.L of the mixed reaction system was added to each well of the 96-well plate, and 10. Mu.L of SACE_1905 protein was added to each well before detection to initiate the reaction, and the OD value was measured at 340 nm wavelength every 20 s. As shown in FIG. 3, OD after addition of SACE_1905 protein ₃₄₀ The value drops rapidly, indicating that NADH is required for the SACE_1905 protein to catalyze the reaction.

Example 4

SACE_1905Induction system (pSETcd)1905-cmt-sgRNA) construction:

(1) The transformation was based on plasmid pSET 152. First, the primer pair dCAS9-P1/P2 was used to amplify the nucleic acid sequences containingNdeI andEcoRV restriction enzyme sitedCas9Fragments; and then is usedXbaI andEcoRV restriction endonuclease double-enzyme digestion of plasmid pSET152 is usedXbaI andNdei restriction endonuclease pair comprisingXbaI andNdei synthetic DNA fragment of cleavage sitecmtDouble enzyme cutting is carried out byNdeI andEcoRV restriction enzyme pairdCas9Performing double enzyme digestion on the fragments; subsequently, the digested gene fragment and plasmid were ligated with T4 ligase to give a recombinant plasmid designated pSETcd.

(2) Amplification Using A226 genome as template and primer pair 1905-P1/P2SACE_1905And (3) a gene. Meanwhile, using pSETcd as a template and using a primer pair ermE P-P1/P2 to amplify P _ermE* Fragments. UsingXbaI andKpni double enzyme digestionSACE_ 1905，EcoRV andXbai double enzyme cleavage P _ermE* ，EcoRV andKpni double restriction plasmid pSETcd. Then the plasmid after double enzyme digestion is connected with two fragments by T4 ligase to obtain recombinant plasmid which is named pSETcd-1905。

(3) Design of targeting using sgRNA design software sgRNA cas9 (v 3.0)SACE_1905The primers sgRNA-P1 and sgRNA-P2 were used to amplify the sgRNA-containing fragments. UsingSpeI andEcoRI endonucleases double-enzyme digestion of sgRNA fragment and plasmid pSETcd-1905Then the fragment and plasmid which are cut by the T4 ligase are used for connecting enzyme to obtain plasmid pSETcd-1905-sgRNA。

(4) With plasmid pSET-cmtdCAS9 as template and primers cmt-P1 and cmt-P2 as primers for amplificationcmtA component. UsingKpnI andSpei endonuclease cleavagecmtFragment and plasmid pSETcd-1905-sgRNA, linked by T4 ligase, dynamic controlSACE_1905Engineering plasmid pSETcd for gene expression1905-cmtSgrnas, as shown in figure 4.

Example 5

WB/Δ1906-pSETcd-1905-cmtConstruction of the sgRNA engineering strain:

pSETcd constructed in example 4 was transformed using PEG-mediated protoplast transformation1905-cmtIntroduction of the sgRNA plasmid into WB/deltaSACE_1906Protoplasts. Screening by using apramycin resistance, and carrying out PCR identification on sgRNA-P1/P2 by using a primer to obtain engineering strain WB/delta1906-pSETcd-1905-cmt-sgRNA。

In this embodiment, the host is employedThe erythromycin high-producing strain WB/delta is usedSACE_1906"deletion in WB" for laboratory pre-constructionSACE_1906Strains of genes,SACE_1906the nucleotide sequence of the gene is shown as SEQ ID NO. 3.

Example 6

WB/delta 1906-pSETcd before and after cumate addition1905-cmtErythromycin yield analysis of sgRNA strains:

WB, WB/delta1906-pSETcd-1905-cmtThe sgRNA strain is inoculated into an industrial seed culture medium, and is cultured at 30 ℃ and 220rpm for 48 h. And transferring the seed liquid to an industrial fermentation culture medium, and continuing to culture under the same conditions. Then fermenting 0, 48 and 96h to WB/delta1906-pSETcd-1905-cmtThe sgRNA strains were each added with a final concentration of 20. Mu. Mol/L cumate solution and fermentation continued until day seven. Erythromycin extraction was performed by adding chloroform to each flask of fermentation broth and the yield was checked using HPLC to determine the inducer action time point.

Further, WB/Δ is to be1906-pSETcd-1905-cmtThe sgRNA strain was inoculated into an industrial seed medium, cultured at 30℃and 220rpm for 48 h. And transferring the seed liquid into an industrial fermentation culture medium, and continuing to culture under the same conditions. Then fermenting 96h to WB/delta1906-pSETcd-1905-cmtThe final concentrations of 20, 40 and 60. Mu. Mol/L cumate solution were added to the sgRNA strain, respectively, and fermentation was continued until the seventh day. Erythromycin extraction was performed by adding chloroform to each flask of fermentation broth and the yield was checked using HPLC to determine the optimal concentration of inducer.

As shown in FIG. 5, the addition of cumate at fermentation 96h was effective in increasing erythromycin yield, and the addition of final concentration of 60. Mu. Mol/L at fermentation 96h was most effective.

Is increased only in cell with earlier stage of laboratorySACE_1905Copy number allows the highest yield of erythromycin to reach 1297.9 mg/mL comparison (WB/delta in the erythromycin industry high-yielding strain)SACE_1906Single over-expression based on (a)SACE_1905) Control using the dynamic control system of the present inventionSACE_1905Expression can lead the fermentation yield of erythromycin to reach1497.5 mg/mL further increased the yield of erythromycin by 15.4%, thus it was seen that this approach increased the yield of erythromycin more significantly.

Example 7

WB/delta 1906-pSETcd before and after cumate addition1905-cmt-sgRNA strainsSACE_1905Transcript and intracellular NADH/NAD ⁺ And (3) ratio detection:

strains WB and WB/delta1906-pSETcd-1905-cmtThe sgrnas were inoculated into TSB seed medium, 30 ℃ and 220rpm for 48 h, respectively. And transferring the seed liquid into an R5 fermentation medium, and culturing at 30 ℃ and 220 rpm. Samples of the fermentation broth were taken after fermentation 96h (no cuamete added) and 120 h (after addition of cuamete 24 h), RNA was extracted and analyzed by qRT-PCR before and after addition of cumateSACE_1905Is a transcriptional change of (a). As shown in fig. 6A, after adding cumate 24 h,SACE_1905the transcript level was significantly reduced.

With reference to the fermentation method, 96h (with no addition of cuamete) and 120 h (with addition of cuamete 24 h) are fermented, supernatant is taken after centrifugation, pre-cooled methanol at-80 ℃ and pre-cooled chloroform at-20 ℃ and pre-cooled water at 4 ℃ are sequentially added, shaking is carried out for 20 s, standing is carried out at-20 ℃ for 1 h, and the uppermost organic phase is taken after centrifugation to obtain intracellular NADH and NAD ⁺ . Re-using the Biyundian NAD ⁺ Detection kit of NADH (WST-8 method) "detection of the Induction agent cumate-added Pre-and post-Strain WB/delta1906-pSETcd-1905-cmtNADH/NAD in sgRNA ⁺ Is a ratio of (2). As shown in FIG. 6B, intracellular NADH/NAD after addition of cumate ⁺ The ratio rises, indicatingSACE_ 1905Is inhibited.

It can be seen from the above that:

1. the invention realizes the alignment by means of the CRISPRi technology and the inducible system through a synthetic biological methodSACE_1905Dynamic control of gene expression to obtain erythromycin high-yield engineering strain WB/delta1906-pSETcd-1905-cmt-sgRNA。

2. By intracellular NADH/NAD ⁺ And SACE_1905 enzyme activity detection, foundSACE_1905Overexpression can result in intracellular NADH/NAD ⁺ The ratio is reduced, and SACE_1905 protein catalyzes the reaction of acetaldehyde as a substrateNADH-consuming biochemical reactions implyingSACE_ 1905The biosynthesis of erythromycin is affected by affecting intracellular NADH levels.

3. For the geneSACE_1905By means of CRISPRi, inducible system, etc., and through adding inducer, the expression of the gene is controlled dynamically to constitute engineering plasmid pSETcd1905-cmtsgRNA, which is then introduced into WB/deltaSACE_1906Engineering mutant strain WB/delta is obtained in protoplast1906-pSETcd-1905-cmt-sgRNA。

Further comparing and analyzing engineering strain WB/delta by adding inducers with different final concentrations at each fermentation time point1906-pSETcd-1905-cmtErythromycin yield change of sgRNA, which shows that the engineering strain had the highest erythromycin yield after addition of final concentration of 60. Mu. Mol/L cumate solution at 96h fermentation. Is increased only in cell with earlier stage of laboratorySACE_ 1905Copy number to maximize erythromycin yield to 1297.9 mg/mL comparison, controlled using the dynamic control system of the present inventionSACE_1905The expression can lead the fermentation yield of the erythromycin to reach 1497.5 mg/mL, and further lead the yield of the erythromycin to be increased by 15.4 percent.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims (2)

1. A method for improving erythromycin yield of rhodosporidium, which is characterized by constructing a plasmid pSETcd-1905-cmt-sgRNA comprising SACE_1905 gene, wherein the plasmid pSETcd-1905-cmt-sgRNA is used for inducing and expressing SACE_1905 gene, and improving erythromycin yield by dynamically controlling intracellular reducing power level of rhodosporidium; the nucleotide sequence of the SACE_1905 gene is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2; meanwhile, the construction method of the plasmid pSETcd-1905-cmt-sgRNA is as follows:

(1) inserting dCS 9 fragment and synthetic DNA fragment cmt into corresponding restriction enzyme sites of plasmid pSET152 to obtain recombinant plasmid pSETcd; wherein the cmt module is located between the XbaI and NdeI cleavage sites of plasmid pSET152 and is ligated to the dCAS9 fragment carrying the fd terminator after the NdeI cleavage site;

(2) will P _ermE* Inserting the fragment and SACE_1905 gene into corresponding restriction enzyme sites of the recombinant plasmid pSETcd to obtain the recombinant plasmid pSETcd-1905; wherein P is _ermE* Located between the EcoRV and XbaI cleavage sites of the plasmid pSETcd, and following the XbaI cleavage site, linked to the SACE_1905 fragment carrying the T0 terminator;

(3) amplifying the sgRNA of the targeted SACE_1905 to obtain a fragment containing the sgRNA; inserting the sgRNA-containing fragment between SpeI and EcoRI cleavage sites of the recombinant plasmid pSETcd-1905 to obtain a recombinant plasmid pSETcd-1905-sgRNA;

(4) the cmt element was amplified and the cmt fragment was inserted between KpnI and SpeI cleavage sites of the recombinant plasmid pSETcd-1905-sgRNA to obtain the desired engineering plasmid pSETcd-1905-cmt-sgRNA.

2. Use of the method for improving erythromycin yield of rhodosporidium saccharopolyspora according to claim 1, characterized in that erythromycin is produced by fermenting an erythromycin high-yield engineering strain containing plasmid pSETcd-1905-cmt-sgRNA, and expression of a gene sace_1905 is dynamically controlled in a mode of adding an inducer and optimizing induction conditions, primary metabolic growth and secondary metabolism of the strain are balanced, and erythromycin yield is improved; wherein, the inducer adopts cumate; the induction conditions are as follows: at 96h of fermentation, a final concentration of 60. Mu. Mol/L of cumate solution was added.