CN109504689B - Transporter coding gene involved in regulating and controlling ACT yield in streptomycete - Google Patents
Transporter coding gene involved in regulating and controlling ACT yield in streptomycete Download PDFInfo
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Abstract
The application belongs to the technical field of microbial fermentation engineering, and particularly relates to a gene related to the yield of Actinorhodin (ACT) of a polyketide in streptomyces. The application particularly relates to the application of transporter genes in the aspect of regulating and controlling the yield of the streptomyces ACT, wherein the number of transporter coding genes is 6; the method comprises the following steps:SCO2254、SCO2534、SCO3765、SCO6160、SCO2519、SCO3185(ii) a After the gene mutation, the gene has regulation and control influence on the yield of streptomyces ACT. The inventors have mutagenized Streptomyces coelicolor M145 and screened strains with altered actinorhodin production. And (3) positioning the position of the transposon on the genome of the screened positive clone by enzyme digestion, self-ligation and sequencing, eliminating spontaneous mutant genes by the principle of repeatedly screening and positioning the same gene, and finally determining the gene influencing the yield of the actinorhodin.
Description
Technical Field
The application belongs to the technical field of microbial fermentation engineering, and particularly relates to a gene related to the yield of Actinorhodin (ACT) of a polyketide in streptomyces.
Background
The existing research generally considers that small molecular compounds generated by the secondary metabolism of microorganisms have important significance on human health. Statistics indicate that more than 60% of the current FDA-approved anti-infective and anti-tumor drugs are derived from microbial secondary metabolites. As more and more genome sequencing work of microorganisms is carried out, genome sequence analysis shows that the microorganisms have great potential for generating various secondary metabolites, but partial research shows that many secondary metabolites of the microorganisms cannot be expressed under the laboratory culture condition, and most environmental microorganisms cannot be effectively cultured under the laboratory condition, so that the development of the secondary metabolites of the microorganisms is seriously hindered.
In the existing research, streptomyces is an important antibiotic producing bacterium, and the production of the antibiotic is controlled by environmental signals and self-regulating genes. The streptomyces coelicolor is a model strain for researching the differentiation and secondary metabolism of streptomyces and can produce two colored antibiotics actinorhodin (ACT blue) and undecylprodigiosin (Red Red), and ACT and RED synthetases belong to type I polyketide synthase (I-PKS) and type II polyketide synthase (II-PKS), respectively. The 2 secondary metabolites in the streptomyces coelicolor are used as a model for researching the synthesis and regulation of the secondary metabolism of the streptomyces coelicolor, and aiming at the screening of the 2 secondary metabolite regulation genes, a good foundation can be laid for the genetic modification of antibiotic producing bacteria, the yield improvement of antibiotic industrial strains or the activation and expression of silent gene clusters.
In the prior art, the inventor uses transposition mutagenesis technology to research the functional genome of streptomyces coelicolor A3(2) in early stage and initially constructs a streptomyces coelicolor A (3)2 mutant library (specifically, refer to Xuzhong, research the functional genome of streptomyces coelicolor A3(2) by using transposition mutagenesis, 2011 university of Master thesis in Huazhong agriculture), but because of complexity and uncertainty of related gene regulation functions, further intensive research and discussion are needed for specific genes related to regulation of secondary metabolites.
Disclosure of Invention
The application aims to provide the screened part of genes related to ACT metabolic regulation in microorganisms, thereby laying a foundation for the research of streptomycete secondary metabolites and the development of novel drugs.
The technical solution adopted in the present application is described in detail as follows.
Genes influencing ACT yield in streptomycete, wherein the genes have a regulating and controlling effect in the synthesis of a streptomycete polyketide ACT; examples of the microorganism include Streptomyces coelicolor (S. coelicolor)Streptomyces coelicolor) (ii) a The above-mentionedGenes are classified into four major groups according to their existing gene annotation, specifically:
cells are synthesized in regulatory classes:
a total of 7, including genesSCO1525、SCO2097、SCO2132、SCO3150、SCO4440、SCO4878、SCO5174(ii) a According to the existing gene annotation, in particular:
SCO1525 and SCO2132 are involved in cell wall phosphatidylinositol mannosylation modification;
SCO2097 is involved in cell wall synthesis;
SCO3150 is involved in cell lysis;
SCO4440 is involved in cell membrane phosphatidylinositol signal regulation;
SCO4878 and SCO5174 are involved in cell wall glycosylation modification;
in the present application, after the mutation (gene deletion or inactivation) of the gene, the expression level (yield) of ACT is decreased, in other words, the gene has a positive regulation effect on the expression level (yield) of ACT; that is, after the gene is mutated (gene deletion or inactivation), the expression quantity (yield) of the ACT is reduced, and after the copy number of the gene is increased or the gene is over-expressed, the gene has the potential of regulating the expression quantity (yield) of the ACT;
amino acid metabolism classes:
7 in total; involving association with amino acid metabolismSCO2241、SCO2528、SCO3345、SCO5281、SCO5512、SCO2999、SCO3962(ii) a According to the existing gene annotation, in particular:
SCO2241 participates in the synthesis of glutamic acid;
SCO2528 is involved in leucine synthesis;
SCO3345 is involved in the synthesis of valine and isoleucine;
SCO5281 α -ketoglutarate decarboxylase;
SCO5512 is involved in branched amino acid metabolism;
the SCO2999 is involved in the metabolism of glutamic acid,
SCO3962 is involved in prephenate metabolism;
in the present application, after the mutation of the gene (gene deletion or inactivation), the gene has uncertain regulation and control effects on ACT expression level (yield), and further:
SCO2241、SCO2528、SCO3345、SCO5281andSCO5512after any one of the 5 genes is mutated (gene deletion or inactivation), the expression level (yield) of ACT is reduced, in other words, the 5 genes have positive regulation effect; that is, any one of the 5 genes can regulate and control the reduction of the expression quantity (yield) of the ACT after mutation (gene deletion or inactivation), and has the potential of regulating and controlling the increase of the expression quantity (yield) of the ACT after the copy number of the gene is increased or the gene is over-expressed;
SCO2999andSCO3962after any one of the 2 genes is mutated (gene deletion or inactivation), the expression level (yield) of ACT is increased, in other words, the 2 genes have a negative regulation effect; that is, any of the 2 genes can regulate the increase of the expression level (yield) of the ACT after mutation (gene deletion or inactivation), and the possibility of regulating the decrease of the expression level (yield) of the ACT can be realized after the copy number of the genes is increased or even the genes are over-expressed;
stress, signal and transcriptional regulation classes:
a total of 14, including genes:SCO0871、SCO1663、SCO2832、SCO2987、SCO3571、SCO4204、SCO4215、SCO4358、SCO6994、SCO1728、SCO2686、SCO3008、SCO5220、SCO6636；
according to the existing gene annotation, in particular:
the SCO0871 participates in the regulation of signals,
SCO1663, SCO4204 and SCO2987 participate in environmental stress regulation,
SCO2832, SCO3571, SCO4215, SCO4358, SCO6994, SCO1728, SCO2686, SCO3008, SCO5220 and SCO6636 are transcription regulation proteins;
in the present application, after the mutation of the gene (gene deletion or inactivation), the gene has uncertain regulation and control effects on ACT expression level (yield), and further:
SCO0871、SCO1663、SCO2832、SCO2987、SCO3571、SCO4204、SCO4215、SCO4358andSCO6994after any one of the 9 genes is mutated (gene deletion or inactivation), the expression level (yield) of ACT is reduced, in other words, the 9 genes have positive regulation effect; that is, any one of the 9 genes can regulate and control the reduction of the expression quantity (yield) of the ACT after mutation (gene deletion or inactivation), and has the potential of regulating and controlling the increase of the expression quantity (yield) of the ACT after the copy number of the gene is increased or the gene is over-expressed;
SCO1728、SCO2686、SCO3008、SCO5220andSCO6636after any one of the 5 genes is mutated (gene deletion or inactivation), the expression level (yield) of ACT is increased, in other words, the 5 genes have negative regulation effect; that is, any of the 5 genes can regulate the increase of the expression level (yield) of the ACT after mutation (gene deletion or inactivation), and the regulation of the expression level (yield) of the ACT can be reduced after the genes are complemented back, copy number is increased or overexpressed;
transporters:
6 in total; the method comprises the following steps:SCO2254、SCO2534、SCO3765、SCO6160、SCO2519、SCO3185(ii) a According to the existing gene annotation, in particular:
SCO2254 is a transmembrane channel protein;
SCO2534, SCO3765 and SCO3185 participate in ion transport;
SCO2519 is involved in lipid transport and antibiotic efflux;
SCO6160 is involved in protein transport;
in the present application, after the mutation of the gene (gene deletion or inactivation), the gene has uncertain regulation and control effects on ACT expression level (yield), and further:
SCO2254、SCO2534、SCO3765andSCO6160after mutation (gene deletion or inactivation) of any of these 4 genes, the ACT expression level (yield) is decreased, in other words, these 4 genesThe product has positive regulation and control effect; that is, any one of the 4 genes can regulate and control the reduction of the expression quantity (yield) of the ACT after mutation (gene deletion or inactivation), and has the potential of regulating and controlling the increase of the expression quantity (yield) of the ACT after the copy number of the gene is increased or the gene is over-expressed;
SCO2519andSCO3185after any one of the 2 genes is mutated (gene deletion or inactivation), the expression level (yield) of ACT is increased, in other words, the 2 genes have a negative regulation effect; that is, any of these 2 genes may regulate the increase of the expression level (yield) of ACT after mutation (gene deletion or inactivation), and may regulate the decrease of the expression level (yield) of ACT after complementation, copy number increase, or overexpression of the gene.
In the application, in order to systematically excavate a series of genes influencing and controlling the yield of Actinorhodin (ACT) in an antibiotic-producing strain streptomyces coelicolor, the inventor adopts an in-vivo transposition system based on mini-Tn5 to mutate streptomyces coelicolor M145 and further screens strains with variable Actinorhodin yield. The experimental results show that: the vector (pHL 734) used in the mutagenesis system does not contain a Streptomyces replicon and an integration site, and only one transposition event can occur after the vector is transferred into a Streptomyces cell by conjugation, so that each positive clone genome screened only contains one randomly inserted mini-Tn5 copy. And further positioning the position of the transposon on the genome of the screened positive clone by enzyme digestion, self-ligation and sequencing, and eliminating a spontaneous mutant gene by the principle of repeatedly screening and positioning the same gene, thereby finally determining the gene influencing the yield of the actinorhodin. Further taking the wild type strain as a reference, and finally determining that the related genes influence the yield of the actinorhodin through fermentation culture. Based on the screening results, a foundation can be laid for the regulation of the yield of related metabolites or the synthesis of new secondary metabolites by further using a genetic engineering technical means.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
Biological material:
streptomyces coelicolor M145 (Streptomyces coelicolor M145) (prototrophic wild type strain A3 (2)) as a transposable mutagenesis starting strain;
escherichia coli ET12567/pUZ8002 (Escherichia coliET12567/pUZ 8002) for introducing plasmid pHL734 into streptomyces coelicolor M145;
plasmid pHL734 carries transposable element Mini-Tn5 and transposase, and is used for transposable mutagenesis of Streptomyces coelicolor M145;
experimental reagent:
LB culture medium is used for culturing Escherichia coli at 37 ℃;
YEME culture medium and TSBY culture medium are used for culturing streptomycete liquid,
adding MgSO (MgSO) into SFM medium4(20 mM) for conjugal transfer of Streptomyces;
the YBP culture medium is used for streptomycete solid culture and fermentation, and the culture temperature of the streptomycete is 30 ℃;
apramycin (Apramycin, Apr), resistance geneAcc(3)IVThe use concentration is 50 mug/mL;
trimethoxybenzylaminopyrimidine (TMP) was used at a concentration of 50. mu.g/mL.
Example 1
This example is briefly described below with respect to the mutagenesis procedure of S.coelicolor M145 using the mini-Tn5 transposition system. It should be noted that the detailed process is described in "study of functional genome of Streptomyces coelicolor A3(2) by transposable mutagenesis" (Xuzhong, 2011 Master thesis at Huazhong university of agriculture).
Transposons transposable mutagenesis of Streptomyces coelicolor
Firstly, referring to the prior art, a transposon plasmid pHL734 is introduced into streptomycete by conjugal transfer of an escherichia coli strain ET12567/pUZ8002 and streptomyces coelicolor, and the ratio of recipient bacteria to donor bacteria is 1: 1;
theoretically, after entering the streptomycete, the transposase Tnp (5) starts to express, the mini-Tn5 fragment is cut and randomly inserted into the genome of the streptomycete, and the plasmid disappears after the transposition occurs because the plasmid cannot replicate and integrate sites in the streptomycete;
covering the cells with TMP and Apr (screening mutants with transposition insertion through an apramycin resistance gene inside Mini-Tn 5) about 12-14 hours after conjugation transfer, and culturing for about 4-5 days; the resulting conjugative transfer clones were transferred to YBP medium (each mutant contains a transposition insert) and mutants with varying secondary metabolite production were selected.
(II) transposition insertion mapping of mutant strains
Respectively extracting the total DNA of the mutant strain with the change of the secondary metabolite yield in the step (I), and carrying out enzyme digestion on the total DNA by using ApaI restriction enzyme;
introducing the digested DNA sample into escherichia coli DH5 alpha through self-ligation and chemical transformation;
screening clone containing mini-Tn5 fragment by using apramycin, sequencing to obtain genome sequences at two ends of mini-Tn5 and a transposition insertion site thereof, and positioning the position of the transposition insertion on a chromosome through sequence comparison.
(III) complementation of mutant strains
Selecting 5 genes related to transcription regulationSCO2832、SCO4215、SCO4358、SCO2686AndSCO3008the back-supplementing verification is carried out,
CDS of the genes and upstream 250 bp promoter region sequences thereof are amplified by PCR and connected to a streptomycete integration vector pMT3, after the sequencing is verified to be correct, escherichia coli ET12567/pUZ8002 is transformed and transferred into a streptomycete mutant strain through the joint of the escherichia coli and the streptomycete;
after the genome of the 5-gene mutant strain is respectively introduced with corresponding normal gene copies, the actinorhodin yield is restored to the wild level, which shows that the regulation and control of the actinorhodin yield by the genes repeatedly screened for many times are 100 percent correct.
(IV) determination of ACT production by mutant strains
The transposable mutant and the original strain are respectively cultured in solid YBP for 84 hours at 30 ℃, and 0.5 g of each culture product is used for ACT yield determination; ACT yield determination:
0.5 mL of 1M NaOH was added to 0.5 g of the culture product, disrupted by a homogenizer (5,000 rpm, 15 s; twice), centrifuged (12000 Xg, 5 min) to take the supernatant, and the absorbance of the supernatant was measured by UV ray at 633 nm to obtain the relative yield of ACT of the Mutant strain (Mutant 633/WT 633).
Example 2
This example is briefly described below with respect to the screening results of example 1.
On the basis of the screening method of example 1, 988 strains with changed actinorhodin production were screened from 50000 mutants as a result, and the transposition insertion sites of the strains were located to show that the mutant strains relate to 551 genes; some of the genes having a regulatory effect on the production of actinorhodin are specifically explained below.
Cells are synthesized in regulatory classes:
a total of 7, including genesSCO1525、SCO2097、SCO2132、SCO3150、SCO4440、SCO4878、SCO5174；
Note (the following tabular data are synonymous and will not be described): in the table, relative ACT yields indicate the ratio of ACT yields of mutant to wild-type, less than 1 indicates a reduced ACT yield of the mutant, 0 indicates no ACT production was detected, and values are "spaced" to indicate multiple mutant strains of the same gene, which have different mini-Tn5 insertion sites in the gene.
Note that, in the above table data, the annotation contents are obtained by integrating StrepDB data (http:// StrepDB. streptomyces. org. uk) and literature reports; ACT relative yields were calculated from the mean values for each mutant (data in the tables below are synonymous and are not described).
Analysis of the data in the above table shows that the actinorhodin production is reduced after 7 cells are inactivated by genes related to synthesis, which indicates that the integrity of the cell envelope is necessary for the synthesis of ACT, and the mutation (inactivation) of the genes has a positive correlation with the actinorhodin production.
Amino acid metabolism classes:
7 in total; involving association with amino acid metabolismSCO2241、SCO2528、SCO3345、SCO5281、SCO5512、SCO2999、SCO3962；
Analysis can see that:
SCO2241、SCO2528、SCO3345、SCO5281andSCO5512all mutant strains of the genes had reduced ACT production, indicating that the products encoded by these genes favor ACT synthesis,
whileSCO2999AndSCO3962all mutant strains of the genes showed elevated ACT production, indicating that the encoded products of these genes are detrimental to ACT synthesis.
Stress, signal and transcriptional regulation classes:
a total of 14, including genesSCO0871、SCO1663、SCO2832、SCO2987、SCO3571、SCO4204、 SCO4215、SCO4358、SCO6994、SCO1728、SCO2686、SCO3008、SCO5220、SCO6636；
Analysis can see that:
SCO0871、SCO1663、SCO2832、SCO2987、SCO3571、SCO4204、SCO4215、SCO4358andSCO6994all mutant strains of the genes have reduced ACT yield, which indicates that the coded products of the genes are favorable for ACT synthesis;
SCO1728、SCO2686、SCO3008、SCO5220andSCO6636all mutant strains of the genes showed elevated ACT production, indicating that the encoded products of these genes are detrimental to ACT synthesis.
Transporters:
a total of 6, including genesSCO2254、SCO2534、SCO3765、SCO6160、SCO2519、SCO3185；
Analysis can see that:
SCO2254、SCO2534、SCO3765andSCO6160all mutant strains of the genes have reduced ACT yield, which indicates that the coded products of the genes are favorable for ACT synthesis;
SCO2519andSCO3185all mutant strains of the genes showed elevated ACT production, indicating that the encoded products of these genes are detrimental to ACT synthesis.
In conclusion, based on the effect of the different genes on the yield of ACT, one or more of the genes can be subjected to independent mutation or combined mutation to inactivate the genes, or overexpression is performed by using transgenic engineering, so that the yield of ACT is specifically regulated, and the production performance of antibiotics is further improved.
Claims (1)
1. The application of the transport protein coding gene in the aspect of regulating and controlling the yield of the streptomycete ACT is characterized in that the number of the transport protein coding genes is 5; the method comprises the following steps: SCO2254, SCO2534, SCO3765, SCO6160, SCO 2519;
after the gene mutation, the gene has regulation and control influence on the yield of streptomyces ACT;
the regulation and control influence is specifically as follows:
after any one of the 4 genes SCO2254, SCO2534, SCO3765 and SCO6160 is inactivated and mutated, the ACT yield is reduced;
after the inactivation and mutation of the SCO2519 gene, the yield of ACT is increased;
the streptomyces is streptomyces coelicolor.
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