CN114317581B - Method for enhancing erythromycin production capacity of saccharopolyspora erythraea and strain obtained by method - Google Patents

Abstract

The present invention relates to Saccharopolyspora erythraeaSACE_3478The application of gene disturbance in promoting erythromycin yield, and is especially method of enhancing erythromycin production capacity of saccharopolyspora erythraea, and the method includes gene engineering means to select original strain saccharopolyspora erythraeaSACE_3478Use of a gene that is knocked out, inactivated, reduced in expression, or reduced or lost in activity of the encoded protein to significantly increase the production of erythromycin.

Description

Method for enhancing erythromycin production capacity of saccharopolyspora erythraea and strain obtained by method

Technical Field

The present invention belongs to the field of microbe and biological technology. More particularly, the invention relates to saccharopolyspora erythraeaSACE_3478Application of gene perturbation to improvement of erythromycin yield, and more particularly relates to application of gene perturbation to encoding of long-chain fatty acid coenzyme A ligase in saccharopolyspora erythraeaSACE_3478The gene is knocked down by using a genetic engineering means, and the application of the gene can obviously improve the yield of the erythromycin.

Background

Erythromycin is prepared from Saccharopolyspora erythraea (A)Saccharopolyspora erythraea) The produced macrolide antibiotics have wide antibacterial spectrum and have strong inhibiting effect on gram-positive bacteria such as staphylococcus and streptococcus pneumoniae, gram-negative bacteria such as haemophilus influenzae and bacillus dysenteriae, and the like, so the macrolide antibiotics have important application value. Erythromycin is one of the antibiotics with the greatest demand in China, but the production capacity is still poor, and the work of increasing the yield of erythromycin is still in progress. At present, work aiming at the improvement of erythromycin titer mainly focuses on random mutagenesis to obtain high-yield mutant strains, the improvement of culture medium components, the optimization of fermentation process and the like, and rational design and gene modification on the genome level are lacked, so that the modification and industrial application of erythromycin production strains are greatly restricted.

Erythromycin is a secondary metabolite of Saccharopolyspora erythraea, a biosynthetic gene cluster thereoferyLack of pathway-specific transcriptional regulators, which makes it impossible to rationally increase erythromycin production by conventional regulatory factor engineering. But in addition to transcriptionIn addition to regulation, factors such as precursor supply, byproduct competition, adaptive expression of biosynthetic genes and the like can also greatly influence the yield of secondary metabolites, so that related genes and pathways can also become targets for rationally modifying strains. Biosynthesis of 1 molecule of erythromycin requires the use of 1 molecule of propionyl-coa and 6 molecules of methylmalonyl-coa small molecule precursors produced by primary metabolic processes, the supply of which directly affects the production of secondary metabolites. The method can be used as an important way for reasonably designing and modifying the strain to improve the yield of the erythromycin by selectively regulating the flow of intracellular metabolism to different ways.

The principle of the antisense RNA inhibition strategy is that a small RNA (sRNA) sequence which is reversely complementary with a target gene is designed and expressed to be combined with mRNA of the target gene, so that the mRNA cannot be combined with ribosome to start a translation process, and the mRNA is induced to be spontaneously degraded, thereby achieving the purpose of knocking down the expression level of the target gene. The method has the advantages that the function of the target gene can be researched without gene knockout, the construction period is shorter, the method is particularly suitable for researching essential genes causing cell death by gene knockout, and the down regulation strength of the genes can be regulated by regulating the copy number of plasmids and the strength of sRNA pre-promoters.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a target modification site for rationally modifying erythromycin producing bacteria to improve the yield.

The invention is realized by the following technical scheme:

step one, finding out a mutation site shared by high-yield strains through fermentation and genome sequence comparison of an original strain for producing erythromycin and a plurality of high-yield mutant strains;

step two, constructing a target gene knockdown strain and a control strain containing empty plasmids by an antisense RNA inhibition strategy;

step three, performing 24-pore plate fermentation on the original strain, the control strain and the knockdown strain;

and step four, measuring the erythromycin titer of each strain by using a chemical color development method, and summarizing the influence of the target genes on the erythromycin biosynthesis.

Preferably, the saccharopolyspora erythraeaSACE_3478In the application of gene knockdown to the improvement of erythromycin yield, in the first step, the second step, the third step and the fourth step, the original strain for erythromycin production and the original strain of each mutant and modified strain are Saccharopolyspora erythraea NRRL23338 or mutant strains thereof.

Preferably, the saccharopolyspora erythraeaSACE_3478In the application of gene knockdown to the improvement of erythromycin yield, in the second step, the method for verifying the function of the target gene and the method for constructing a knockdown strain are antisense RNA inhibition strategies. In addition, however, other useful genetic modifications, such as double crossover, CRISPR-mediated gene inactivation, and the like, can also be used for this purpose.

Preferably, the saccharopolyspora erythraeaSACE_3478In the fourth step of the application of gene knockdown to the improvement of erythromycin yield, the fermentation time is 7 d.

Wherein the fermentation medium for culturing the strain contains starch, dextrin, soybean meal and bean cake meal as carbon-nitrogen source. Preferably, the formulation of the fermentation medium is: 20 g/L of starch, 20 g/L of dextrin, 15 g/L of bean cake powder, 4 g/L of ammonium sulfate, 6 g/L of calcium carbonate and 5 mL/L of soybean oil. The formula of a seed culture medium for culturing the strain is as follows: 10 g/L glucose, 4 g/L peptone, 4 g/L yeast extract, 0.5 g/L magnesium sulfate, 2 g/L potassium dihydrogen phosphate, and 4 g/L dipotassium hydrogen phosphate.

The invention has the advantages and beneficial effects that:

in the present invention, the encoding long-chain fatty acid coenzyme A ligase in erythromycin producing bacteria was identifiedSACE_3478The gene has negative regulation effect on the biosynthesis of erythromycin, so that the competitive relationship between the fatty acid synthesis path and the biosynthesis of erythromycin is shown, the gene and the related path are favorable to be used as modification targets, and the yield of erythromycin is improved by reasonably modifying strains.

Drawings

FIG. 1 shows the titer of erythromycin original strain S0 and 6 high-producing strains 1-A6, 5-A3, 1-A2, B4, B5 and C2 in the first embodiment of the present invention;

FIG. 2 is a schematic diagram illustrating the metabolic flux of acetyl-CoA in a cell according to a first embodiment of the present invention;

FIG. 3 shows a second embodiment of the invention in a target orientationSACE_3478A schematic diagram of construction of antisense RNA suppression plasmid pSET152-hyg-SACE _3478-sRNA of gene;

FIG. 4 is a standard curve graph showing the relationship between erythromycin concentration and absorbance at 483 nm in the fourth embodiment of the present invention;

FIG. 5 shows the titer of 24-well plate fermentations of the original strain S0, the control strain S0/pSET152-hyg and the knockdown strain S0/pSET152-hyg-SACE _3478-sRNA in example four of the present invention.

Detailed Description

In order that those skilled in the art will better understand the technical solutions of the present invention, the technical solutions of the present invention are further described below with reference to specific examples.

EXAMPLE A comparison of Titers and genomic sequence analysis of erythromycin Primary and high Producers

Early in the period from the original strainSaccharopolyspora erythraea S0 (which is obtained by subculture of NRRL 23338) is subjected to random mutagenesis and screening to obtain 6 erythromycin high-producing strains 1-A6, 5-A3, 1-A2, B4, B5 and C2, wherein the erythromycin titer is remarkably improved compared with S0, and the improvement ratio is respectively 26.9% ((the improvement ratio is 26.9%) (the improvement ratio isp=0.016），27.3%（p=0.042），38.4%（p=0.00094），68.2%（p=0.00023），62.4%（p= 0.00024) and 62.6%, (p= 0.00087) (fig. 1) (see operations in examples three and four, below, for specific experimental procedures).

The 6 high producing strains were subjected to genome sequencing with reference to the original strain S0 and analyzed for mutation sites relative to the original strain, and foundSACE_3478Mutations of the gene (NCBI reference genomic sequence: NC-009142.1) were present in 6 high producing mutants at the same time.SACE_3478（fal) The gene has the total length of 564 bp, codes 187 aa long-chain fatty acid coenzyme A ligase, and deletes 4 bp in 21 bp from 119 bp to 139 bp in the first quarter of the gene to cause frame shift mutation, therebyRendering its encoded product non-functional. Known wild-type is found by searching the NCBI genome poolSaccharopolyspora erythraea NRRL23338 and mutants derived therefrom have this gene in their genome. Since this mutation is shared by a plurality of erythromycin high-producing strains, and inactivation thereof results in high-producing erythromycin by the mutant strain, it is presumed that the long-chain fatty acid coenzyme A ligase encoded by this gene has a negative regulatory effect on erythromycin biosynthesis. Acetyl-coa, as a key node of primary and secondary metabolism in cells, flows into different metabolic pathways: it can generate propionyl coenzyme A and methylmalonyl coenzyme A precursor molecules required by erythromycin biosynthesis through a propionic acid metabolic pathway, and can also flow into a fatty acid synthesis pathway to generate long-chain fatty acid CoA and further generate lipid molecules required by cells such as phospholipid and the like. Inactivation of the long-chain fatty acid CoA ligase results in a decrease in metabolic flux in the fatty acid synthesis pathway, resulting in more acetyl-CoA entering the propionate metabolic pathway to generate erythromycin precursors, which in turn increases erythromycin production (fig. 2).

Example twoSACE_3478Construction of antisense suppression strain S0/pSET-hyg-SACE _3478-sRNA

To verifySACE_3478For the effect of erythromycin production, the present invention knockdown the expression of this gene in S0 strain using an antisense RNA inhibition strategy. The gene containing the j23119 promoter (promoter sequence from Liu Y,et al, A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora erythraeaErythromycin Biosynthetic Gene Cluster with Knock-inBidirectional Promoters, ACS Synth. Biol. 2019, 8, 1134−1143）、SACE_3478gene antisense sequences, upstream and downstream hairpin structures, and the sRNA expression module fragment of fd terminator (sRNA cassette). This fragment was ligated to pSET152-hyg backbone by in vitro homologous recombination to give plasmid pSET-hyg-SACE _3478-sRNA (FIG. 3).

Empty plasmids pSET152-hyg andSACE_3478gene antisense suppression plasmid pSET152-hyg-SACE _3478-sRNA plasmid was transformed into ET 12567-In pUZ8002 strain, plasmid was transformed into Saccharopolyspora erythraea S0 by intergeneric conjugative transfer to give a control strain S0/pSET152-hyg with empty plasmid integrated and a control strain S0/pSET152-hyg with empty plasmid integratedSACE_3478Knock-down strain of gene antisense suppression plasmid S0/pSET152-hyg-SACE _ 3478-sRNA. The strains used and constructed in the present invention are shown in Table 1, and the primers used in constructing plasmids are shown in Table 2.

TABLE 1 strains used and constructed in the present invention

Bacterial strains	Feature(s)
		S. erythraea S0	Erythromycin-producing strain, original strain
S0/pSET152-hyg	Control Strain, hygromycin resistance, integrating empty plasmid pSET152-hyg
		S0/pSET152-hyg-SACE_3478-sRNA	Is integrated withSACE_3478Knock-down strain of gene antisense suppression plasmid pSET152-hyg-SACE _3478-sRNA, hygromycin resistance
Escherichia coliET12567/pUZ8002	Combined with transfer helper strain, carrying pUZ8002 plasmid, chloramphenicol, kanamycin resistance

Table 2 primers used in the examples

Example three 24-well plate fermentation of knockdown strains

Spores of the original strain S0, the control strain S0/pSET152-hyg and the knockdown strain S0/pSET152-hyg-SACE _3478-sRNA were respectively scraped and inoculated into 3 mL of seed medium, and cultured at 32 ℃ and 250 rpm for 3 days. Then, 300. mu.L of the seed solution was inoculated into 3 mL of the fermentation medium, and the mixture was further cultured at 32 ℃ and 250 rpm for 7 days to complete the fermentation. For each strain, at least 3 biological replicates were set up to ensure accurate experiments. Wherein, the formula of the seed culture medium is as follows: 10 g/L glucose, 4 g/L peptone, 4 g/L yeast extract, 0.5 g/L magnesium sulfate, 2 g/L potassium dihydrogen phosphate and 4 g/L dipotassium hydrogen phosphate. The formula of the fermentation medium is as follows: 20 g/L of starch, 20 g/L of dextrin, 15 g/L of bean cake powder, 4 g/L of ammonium sulfate, 6 g/L of calcium carbonate and 5 mL/L of soybean oil.

Example four assay of erythromycin Titer by chemical colorimetry

And (4) performing titer determination on the fermentation sample by using a chemical colorimetric method. A standard curve was first plotted for absorbance at 483 nm as a function of erythromycin standard at different concentrations. The specific operation method comprises the following steps: dissolving the precisely weighed erythromycin standard with methanol, diluting with water to different molar concentrations, adding 8M sulfuric acid solution at the same volume, holding in 50 deg.C water bath for 30 min, taking out, and measuring absorbance at 483 nm with enzyme-labeling instrument. As absorbance (A)_483nm) Plotted as ordinate and concentration (mg/L) as abscissa, a standard curve was obtained with the following linear equation: y =0.0012x +0.0656, R²=0.9834 (fig. 4).

After fermentation is finished, treating the samples and measuring the erythromycin titer of each sample, wherein the specific operation method comprises the following steps: and standing the fermentation liquor to settle hyphae, taking 100 mu L of supernatant into a 1.5 mL centrifuge tube, adding 400 mu L of potassium carbonate solution with the mass concentration of 0.35%, uniformly mixing, adding 250 mu L of butyl acetate, performing shaking extraction, centrifuging, taking the upper organic phase, and transferring the upper organic phase into another clean 1.5 mL centrifuge tube. Then, 300. mu.L of 0.1M hydrochloric acid solution was added thereto and extracted by shaking, and after centrifugation, 150. mu.L of the lower aqueous phase was transferred to another clean 1.5 mL centrifuge tube. And finally adding 8M concentrated sulfuric acid with the same volume, uniformly mixing, heating for 30 min under the condition of 50 ℃ water bath, and measuring the absorbance of the mixture at 483 nm. The measured values were fitted to a standard curve and the titer of each sample was calculated.

The measurement results of the three strains show that the original strain S0 titer is 832.7 +/-34.4 mg/L, the control strain S0/pSET152-hyg titer is 871.0 +/-30.3 mg/L, and the three strains have no significant difference compared with the original strain (the titer of the original strain is 832.7 +/-34.4 mg/L and the titer of the control strain S0/pSET152-hyg titer is 871.0 +/-30.3 mg/L), (the three strains have no significant difference: (p= 0.3027), while the knockdown strain S0/pSET152-hyg-SACE _3478-sRNA titer is 1146.5 ± 40.2 mg/L, which is 31.6% higher than the control strain, and has significant difference (S) ((0.3027)p= 0.0015) (fig. 5). This result is illustrative of the fact that,SACE_3478the gene-coded long-chain fatty acid coenzyme A ligase has negative effect on the biosynthesis of the erythromycin, can be used as a key target for rationally modifying an erythromycin producing strain to improve the yield, and can be knocked down in saccharopolyspora erythraea by an antisense RNA inhibition strategySACE_3478The expression of the gene can effectively improve the yield of the erythromycin.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

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Claims (9)

1. A method for enhancing erythromycin production ability of Saccharopolyspora erythraea is characterized in that gene encoding long chain fatty acid coenzyme A ligase in Saccharopolyspora erythraea of original strain is subjected to gene engineeringSACE_3478Knock-out, inactivation, reduced expression of a gene, or reduced or lost activity of an encoded protein; the starting strain saccharopolyspora erythraea is saccharopolyspora erythraea for producing erythromycin.

2. The method of claim 1, wherein the starting strain Saccharopolyspora erythraea NRRL23338 is saccharomyces cerevisiae or a mutant thereof, and said mutant is still capable of producing erythromycin.

3. The method of claim 1, wherein the method comprisesSACE_3478The nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.

4. The method of claim 1, wherein the genetic engineering means is an antisense RNA inhibition strategy, double crossover, CRISPR mediated gene editing, site directed mutagenesis.

5. The method of any one of claims 1 to 4, further comprising the step of further screening for a mutant strain that produces high erythromycin production.

6. A strain of Saccharopolyspora erythraea having enhanced erythromycin production ability obtained by the method according to any one of claims 1 to 5.

7. A method for producing erythromycin using the strain of claim 6, comprising the steps of culturing the strain and collecting erythromycin.

8. The method of claim 7, wherein the fermentation medium for culturing said strain comprises starch, dextrin, soybean meal, soybean cake meal as a carbon-nitrogen source.

9. The method of claim 7, wherein the seed medium formulation for culturing said strain is: 10 g/L glucose, 4 g/L peptone, 4 g/L yeast extract, 0.5 g/L magnesium sulfate, 2 g/L potassium dihydrogen phosphate and 4 g/L dipotassium hydrogen phosphate.

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