CN116515882A - Bacillus subtilis double-module transcription optimization system based on T7RNA polymerase - Google Patents

Bacillus subtilis double-module transcription optimization system based on T7RNA polymerase Download PDF

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CN116515882A
CN116515882A CN202310668197.5A CN202310668197A CN116515882A CN 116515882 A CN116515882 A CN 116515882A CN 202310668197 A CN202310668197 A CN 202310668197A CN 116515882 A CN116515882 A CN 116515882A
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刘龙
陈坚
吕雪芹
堵国成
李江华
刘延峰
武耀康
李洋
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Jiangnan University
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Abstract

The invention discloses a bacillus subtilis double-module transcription optimization system based on T7RNA polymerase, and belongs to the technical field of biology. The T7-BOOST system constructed by the invention comprises two parts: (1) T7RNA polymerase driving module: the module obtains a chassis strain for stably inducing and expressing the T7RNAP by integrating the T7RNAP controlled by the inducible promoter and a repressor expression frame corresponding to the inducible promoter on a genome; (2) an expression control module: the module comprises a P gene with a heterozygous T7 promoter T7‑inducer Is specifically recognized by T7RNAP T7 And an operator corresponding to the inducible promoter. The bacillus subtilis double-module transcription optimization system based on the T7RNA polymerase constructed by the invention has the advantages of low leakage, high expression strength, wide dynamic range and effectivenessThe expression protein and the regulation of the synthesis of metabolic products are of great significance to the construction of bacillus subtilis cell factories and the research of synthetic biology.

Description

Bacillus subtilis double-module transcription optimization system based on T7RNA polymerase
Technical Field
The invention relates to a bacillus subtilis double-module transcription optimization system based on T7RNA polymerase, and belongs to the technical field of biology.
Background
Bacillus subtilis (Bacillus subtilis) has been used as a model microorganism for studying gram-positive bacteria with its clear genetic background and mature genetic manipulation techniques. In addition, the strain does not produce harmful products such as endotoxin, and is therefore a recognized food-safe microorganism, which is certified as GRAS (Generally recognized as safe) strain by the U.S. Food and Drug Administration (FDA). And because B.subtilis has the excellent characteristics of fast growth rate, strong protein secretion capability, no obvious codon preference, difficult phage infection and the like, the B.subtilis is widely applied to the construction of cell factories to produce various industrial products, such as recombinant proteins, platform chemicals, biopolymers and the like. The construction of a plant of subilis cells depends on expression systems that efficiently express proteins or regulate key genes in the synthetic pathway. Gene expression systems are generally classified into constitutive expression and inducible expression systems, and the inducible expression system has advantages in that controllable gene expression can be achieved, and expression levels can be regulated as required, as compared with the constitutive expression system, to avoid the burden on host cells caused by the expression of some proteins that have an influence on growth in the early stage of growth.
The T7 expression system consists of T7RNA polymerase (T7 RNAP) derived from T7 phage and its corresponding T7 promoter, and is widely used for the production of recombinant proteins in e.coli. T7RNAP has higher specificity to T7 promoter, does not act on the native promoter of the host, and even on a similar phage T3 promoter; moreover, the extension rate is about 5 times faster than that of E.coli endogenous RNA polymerase. In addition, the T7 promoter can produce very long transcripts, which are advantageous for expressing polycistronic genes, especially gene clusters of some natural products. In addition, an operon (e.g., lacO) may be inserted into the T7 promoter for inducing transcription of the gene of interest. T7 expression system has been used in various hosts and even cell-free systems due to its simple genetic composition, high promoter specificity and good controllability.
There have been many studies in B.subtilis of T7 RNAP-based protein expression and gene regulatory base system construction and use (Castillo-Hair et al, 2019; chen et al, 2010;Conrad et al, 1996; ji et al, 2021; ye et al, 2022). However, they all have problems such as toxicity to cells and some leaky expression of the whole system by using the P43 promoter for constitutive expression of T7RNAP, lack of optimization of the expression of the whole system and limitation of the expression of proteins, and are not used for modification and regulation of metabolic pathways.
Disclosure of Invention
In order to solve the problems, the invention provides a bacillus subtilis double-module transcription optimization system (two-module T7-based optimized output strategy for transcription (T7-BOOST) system) based on T7RNA polymerase. The T7-BOOST system consists of a T7RNA polymerase (T7 RNAP) driving module and an expression control module, can be combined with any one inducible promoter and an operator thereof, and can be used for high-efficiency expression of proteins and regulating and controlling synthetic pathways of metabolites through high-efficiency transcription capacity and specificity identification capacity of the T7RNA polymerase in the T7RNAP driving module. The T7-BOOST system is combined with two most commonly used inducible expression systems (IPTG inducible and xylose inducible expression systems) in the hay, so that the performance of the system is improved, the whole system is expressed in an ultralow leakage way under the condition of no inducer, and the system is expressed in a high strength after the inducer is added.
The first object of the invention is to provide a bacillus subtilis double-module transcription optimization system based on T7RNA polymerase, which comprises a constitutive expression repressor protein expression frame, a T7RNA polymerase expression frame regulated by an inducible promoter and a target gene regulated by a heterozygous T7 promoter; wherein the hybrid T7 promoter comprises a T7 promoter and an operator, and the hybrid T7 promoter is subjected to negative regulation by a repressor protein and positive regulation by a T7RNA polymerase.
Further, the repressor expression cassette, the inducible promoter, and the T7RNA polymerase expression cassette are located on a first vector for integration on the genome; the heterozygous T7 promoter and the target gene are positioned on a second vector and are used for free expression of the target gene.
Further, the inducible promoter is selected from the group consisting of P hy-spank Or P xylA 。
Further, the repressor is selected from the repressor lacI of lactose promoter or the repressor xylR of xylose promoter.
Further, the operon is selected from lactose operon lacO or xylose operon xylO.
Further, an RBS sequence is linked to the second vector.
Further, the inducible promoter P hy-spank The nucleotide sequence of (2) is shown as SEQ ID NO. 7.
Further, the inducible promoter P xylA The nucleotide sequence of (2) is shown as SEQ ID NO. 8.
Further, the Genbank accession number of the T7RNA polymerase is NC_001604.1, and the amino acid sequence of the Genbank accession number is shown as SEQ ID NO. 9.
Further, the amino acid sequence of the repressor protein lacI of the lactose promoter is shown as SEQ ID NO. 10.
Further, the amino acid sequence of the repressor xylR of the xylose promoter is shown as SEQ ID NO. 11.
Further, the nucleotide sequence of the T7 promoter is shown as SEQ ID NO. 12.
Further, the nucleotide sequence of the lactose operon lacO is shown as SEQ ID NO. 13.
Further, the nucleotide sequence of the xylose operon xylO is shown as SEQ ID NO. 14.
Further, the nucleotide sequence of the RBS sequence is shown as SEQ ID NO. 15.
It is a second object of the present invention to provide the use of the above described two-module transcription optimization system in biosynthesis.
Further, the two-module transcription optimization system is used for protein expression of bacillus subtilis.
Further, the genetically engineered bacterium containing the two-module transcription optimization system is cultured under proper conditions, and the culture system comprises an inducer.
Further, the inducer is selected according to the kind of inducible promoter and operator, such as xylose isopropyl-beta-D-thiogalactoside (IPTG) or xylose, etc.
Further, the genetically engineered bacteria are cultured in an LB culture medium under the following culture conditions: culturing at 35-37deg.C and 200-220rpm for 10-15 hr to obtain seed solution; inoculating the seed solution into a TB fermentation medium in an inoculum size of 2-5%, wherein the culture conditions are as follows: culturing at 35-37 deg.C and 200-220rpm for 14-96 hr.
Further, the LB medium is a conventional medium in the art, and each liter of the composition comprises: 5-15g of peptone, 1-10g of yeast powder and 5-15g of NaCl.
Further, the TB medium is a medium conventional in the art comprising, per liter of components: 5-20g of peptone, 20-30g of yeast powder, 5-15g of NaCl, 1-10mL of glycerol and 1-10g of KH 2 PO 4 ，10-20g K 2 HPO 4 。
A third object of the present invention is to provide the use of the above described two-module transcription optimization system in metabolic regulation.
The fourth object of the invention is to provide a bacillus subtilis capable of performing metabolic regulation, wherein the bacillus subtilis contains the two-module transcription optimization system.
The fifth object of the present invention is to provide a method for metabolic synthesis control of bacillus subtilis, which comprises introducing the above-mentioned two-module transcription optimization system. Specifically, according to metabolic synthesis needs, different inducible promoters and operators regulated by the inducible promoters are replaced, so that the expression regulation of genes to be regulated is realized.
The sixth object of the present invention is to provide a method for inducing low leakage and high-intensity expression of a target gene after induction, wherein the following expression system is introduced into bacillus subtilis: the expression system comprises a constitutively expressed repressor expression frame, a T7RNA polymerase expression frame regulated by an inducible promoter and a target gene regulated by a heterozygous T7 promoter; wherein the inducible promoter is selected from the group consisting of P hy-spank Or P xylA The hybrid T7 promoter includes a T7 promoter and an operon that is positively regulated by an inducible promoter and negatively regulated by a repressor protein.
The T7-BOOST system constructed by the invention comprises two parts: (1) a T7RNA polymerase (T7 RNAP) driver module: the module integrates a strict inducible promoter (IPTG inducible promoter P) on the genome by using a CRISPR/Cpf 1-based bacillus subtilis polygene editing system hy-spank And xylose inducible promoter P xylA ) Controlled T7RNAP and the corresponding repressor expression cassette (P penP -lacI and P xylR -xylR) to obtain chassis strains BST7L and BST7R stably inducing expression of T7 RNAP; (2) an expression control module: the module comprises a T7 promoter P with heterozygosity T7-inducer (P T7lac Or P T7xyl ) Is specifically recognized by T7RNAP T7 Operons (lacO and xylO) corresponding to the inducible promoter. T7RNAP and P in the absence of an inducer T7-inducer Are all inhibited by constitutively expressed repressor proteins, and double inhibition reduces leakage expression of the system. After addition of inducer (IPTG or xylose), P T7-inducer The target gene is expressed with high intensity under the drive of the specific T7RNAP, and has a large dynamic range.
Further, the copy number of the target gene is not higher than 50, more preferably not higher than 20.
Further, B.subtilis G600 was used as a starting strain. The starting strain is prepared by inactivating 6 extracellular protease genes of a wild B.subtilis 168 strain on the basis of the wild B.subtilis 168 strain, has high conversion efficiency and can effectively produce protein; this strain is described by Li et al in Agenetic toolkit for efficient production of secretory protein in Bacillus subtilis Bioresource Technology, 2022.
The invention has the beneficial effects that:
the invention constructs a bacillus subtilis double-module transcription optimization system (two-module T7-based optimized output strategy for transcription (T7-BOOST) system) based on T7RNA polymerase. The T7-BOOST system consists of two parts: (1) a T7RNA polymerase (T7 RNAP) driver module: the module integrates a strict T7RNAP controlled by an inducible promoter and a repressor protein expression frame corresponding to the inducible promoter on a B.subtilis G600 genome by utilizing a CRISPR/Cpf 1-based bacillus subtilis polygene editing system to obtain a chassis strain for stably inducing and expressing the T7 RNAP; (2) an expression control module: the module consists of three shuttle plasmids pHT-T7 (low copy), pADK-T7 (medium copy) and pSTOP-T7 (high copy) with different copy numbers, all of which carry the hybrid T7 promoter P T7-inducer . The T7-BOOST system can be combined with any one inducible promoter and any one operator, and the whole system can be expressed in ultralow leakage under the condition of no inducer by the high-efficiency transcription capability of the T7RNA polymerase and the specific recognition capability of the T7 promoter in the T7RNAP driving module, and the inducer is added for high-strength expression. The T7-BOOST system is combined with two most commonly used inducible expression systems (IPTG inducible and xylose inducible expression systems) in the hay, so that the T7 system for the induction expression of the IPTG and the xylose is successfully constructed, the leakage is low, the expression intensity is high, the dynamic range is wide, and the protein can be effectively expressed and the synthesis of metabolic products can be regulated and controlled. Has important significance for construction of bacillus subtilis cell factories and research on synthesis biology.
Drawings
FIG. 1 is a schematic diagram of a two-module transcription optimization system (T7-BOOST) of Bacillus subtilis based on T7RNA polymerase.
FIG. 2 is a schematic construction of CRISPR related plasmids.
FIG. 3 is a characterization of the IPTG-and xylose-induced T7-BOOST systems. (a) Expression time curves of different induction times of the IPTG induced T7-BOOST system on plasmids with different copy numbers; (b) Induction curves of IPTG-induced T7-BOOST systems on plasmids of different copy numbers; (c) Expression time curves of different induction times of the xylose-induced T7-BOOST system on plasmids with different copy numbers; (d) Induction curves of xylose-induced T7-BOOST system on different copy number plasmids.
FIG. 4 shows the application of the T7-BOOST system in whole cell catalysis and protein expression. (a) The T7-BOOST system expresses GADB from escherichia coli and is used for producing gamma aminobutyric acid by whole cell catalysis; (b) T7-BOOST system for expressing human alpha S1 Casein; (c) T7-BOOST system expresses human lactoferrin.
FIG. 5 shows the use of the T7-BOOST system for controlling lycopene production. (a) lycopene synthesis pathway; (b) yield of lycopene expressed using different promoters.
FIG. 6 shows the use of the T7-BOOST system for regulating riboflavin production. (a) a riboflavin synthesis pathway; (b) Replacement of different promoter schematic diagrams by riboflavin-expressing gene clusters; (c) The riboflavin expression gene clusters used the yields of expression from different promoters.
FIG. 7 shows IPTG and xylose inducible promoter P hy-spank And P xylA Is characterized by (3). (a) IPTG inducible promoter P hy-spank Expression time curves for different induction times on different copy number plasmids; (b) IPTG-inducible promoter P hy-spank Induction curves on different copy number plasmids; (c) Xylose inducible promoter P xylA Expression time curves for different induction times on different copy number plasmids; (d) Xylose inducible promoter P xylA Induction curves on different copy number plasmids.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The invention relates to a material and a method as follows:
(one) Strain and vector
Plasmid construction was performed in E.coli DH 5. Alpha. And the plasmid construction was transformed into B.subtilis G600 for integration. The vectors used, pHT-xcR6 and pcrF19NM2, are described by Wu et al in CAMERS-B: CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus sublis.Biotechnology and Bioengineering 2020,117:1817-1825 (available from molecular plasmids sharing platform under accession numbers MC_0068418 and MC_0101256, respectively). The vectors used, pHT-01, pAD123 and pSTOP1622, are commercial plasmids.
(II) Medium
Both the subtitle seeds and E.coli were cultivated using LB medium (10 g tryptone, 5g yeast powder and 10g NaCl per liter). TB Medium (g/L): peptone 12, yeast powder 24, naCl 10,4mL glycerol, 2.31KH 2 PO 4 ，12.54K 2 HPO 4 . Shake flask fermentation medium (g/L): glucose 85, tryptone 15, yeast powder 15, urea 6, glycerol 5 and MgSO 4 ·7H 2 O 3，K 2 HPO 4 ·3H 2 O 12.5，KH 2 PO 4 2.5. Glucose and MgSO 4 ·7H 2 O needs to be sterilized separately from other components and added before inoculation.
(III) fluorometry
The relative fluorescence intensity of sfGFP was measured using a multi-purpose microplate detector Cystation 3 (Botanus instruments Co., ltd.) with reference to the methods of Yang S, liu Q, zhang Y, et al construction and characterization of broad-spectrum promoters for synthetic biology [ J ]. ACS Synthetic Biology,2018,7 (1): 287-291.
(IV) Whole cell catalysis
The GAD fermentation culture was collected and centrifuged (8000 Xg, 20 min). The cells were concentrated and heated at 55℃for 45min. 4mL of 5 Xacetic acid-sodium acetate buffer solution, 10mL of 320g/L L-glutamic acid solution and appropriately concentrated cells were mixed to give 20mL of a reaction system, final OD 600 10. The pH was adjusted to 5.0 with acetate and the volume was adjusted to 20ml with deionized water. The whole cell catalytic reaction is then carried out at 40℃and 150rpmThe reaction was completed in a 250ml Erlenmeyer flask; the pH was adjusted to 5.0 with acetic acid every 8 hours. After completion of the reaction, the supernatant was appropriately diluted by centrifugation at 12000 Xg for 5 minutes, and passed through an aqueous filter having a pore size of 0.22. Mu.m, followed by HPLC-OPAprecollumn derivatization. The conversion of glutamic acid to GABA was calculated using the formula: GABA conversion (%) =the number of moles actually produced by GABA/theoretical number of moles of GABA produced by glutamate conversion x 100.
Method for measuring (penta) lycopene
Sample treatment: taking 600 mu L of bacterial liquid, washing, adding 600 mu L of ethyl acetate into the bacterial liquid, carrying out ultrasonic crushing, centrifuging for 8min at 12000 Xg, sucking the upper ethyl acetate, diluting properly, and then passing through a membrane to enter a liquid phase bottle; directly feeding into a liquid phase bottle after passing through the membrane. Liquid phase detection: CI8 column, sample injection 10 μl, column temperature 40 ℃, mobile phase acetonitrile: methanol: isopropyl alcohol=5:3:2, flow rate 1.0mL/min, detection time 30min, ultraviolet absorption wavelength 474nm.
Method for measuring riboflavin
200. Mu.L of the fermentation culture was added to 800. Mu.L of 0.05M NaOH. After centrifugation at 10,000Xg for 2 minutes, the supernatant was collected and diluted with 0.1M acetic acid-sodium acetate buffer (pH 4.42). Subsequently, the absorption at 444nm was measured. The riboflavin concentration calculation uses a standard equation, y= (OD 444 -0.0203)×DF/0.0163(R 2 ＝0.9997；OD 444 Absorbance at 444 nm; y, riboflavin concentration (mg/L); DF, dilution factor; OD after dilution 444 Controlled to be in the range of 0.1 to 0.8).
(seventh) sequence
Genbank accession number of glutamate decarboxylase (GADB) is CAD6014267.1, a gene obtained directly from E.coli MG1655 by clone PCR;
the Genbank accession number of Human Lactoferrin (HLF) is AAA59511.1, the gene is optimized according to the codon preference of B.subtilis, and the nucleotide sequence and the amino acid sequence are respectively shown as SEQ ID NO.16 and SEQ ID NO. 17;
human alpha S1 Genbank accession number of casein (HA) is P47710, the gene HAs been optimized according to the codon preference of B.subtilis, and the nucleotide sequence and the amino acid sequence are shown as SEQ ID NO.18 and SEQ ID NO.19 respectively；
The regulation and expression of lycopene, an exogenous metabolite, is realized by connecting a low copy expression plasmid pHT-T7 with lycopene expression gene cluster crtEBI. The gene cluster crtbi consists of the synthesis-related pathway genes pyrophosphoric acid synthase gene crtE, phytoene synthase gene crtB and phytoene dehydrogenase gene crtI. This cluster is described by Li et al in Agenetic toolkit for efficient production of secretory protein in Bacillus subtilis Bioresource Technology, 2022.
(eighth) in the examples below, low copy represents copy numbers of 15-20, medium copy represents copy numbers of 40-100, and high copy represents copy numbers greater than 400.
EXAMPLE 1 construction of CRISPR series plasmid in T7-BOOST System
Plasmid pHT-XCR6 (ampicillin resistance in E.coli and chloramphenicol resistance in B.subtilis) is a Cpf1 expression vector, wherein Cpf1 is under the induction control of xylose; in addition, the plasmid also contains NgAgo protein genes, which are used for improving the efficiency of homologous recombination in the process of gene editing. Plasmid pcrF19NM2 (kanamycin resistance in both E.coli and B.subtilis) is a temperature-sensitive plasmid in B.subtilis, cannot replicate when cultured at a temperature above 37 ℃, and is used as a crRNA expression vector for expressing crRNA; and a homologous repair template may be inserted therein, the homologous template insertion region of which contains the mCherry gene for insertion screening.
As shown in FIG. 2, a total of 5 plasmids of the pcrF19NM2 series consisting of a plasmid backbone, upstream homology arms, downstream homology arms, insert (replacement) sequences, were constructed, and plasmid construction methods, primer design, and verification primer design were all described by Wu et al in CAMERS-B: CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus peptides, biotechnology and Bioengineering 2020, 117:1817-1825.
(one) construction of plasmid primers
1)P penP -lacI-P hy-spank T7RNAP expression cassette integration plasmid pcrF19Construction of T7RNAPL1R, insertion sequence consisting of constitutively expressed repressor expression cassette P penP -lacI and T7RNAP expression cassette P hy-spank T7RNAP, the nucleotide sequence of which is shown in SEQ ID NO. 1. The integration site is an S1 site, and the primers used for constructing the plasmid are as follows:
2)P xylR -xylR-P xylA t7RNAP expression cassette integration plasmid pcrF19-T7RNAPL2R, insert sequence consisting of constitutively expressed repressor expression cassette P xylR -xylR and T7RNAP expression cassette P xylA T7RNAP, the nucleotide sequence of which is shown in SEQ ID NO. 2. The integration site is an S1 site, and the primers used for constructing the plasmid are as follows:
3) The promoter of the riboflavin synthesis endogenous gene cluster ribDEAHT is P veg Integration plasmid pcrF19-P veg Rib can use the strong promoter P veg Replacement of endogenous promoters (P) veg Nucleotide sequence SEQ ID NO. 3), the primers used for constructing the plasmid are:
4) The promoter of the riboflavin synthesis endogenous gene cluster ribDEAHT is P T7lac Integration plasmid pcrF19-P T7lac -rib available promoter P T7lac Replacement of endogenous promoters (P) T7lac Nucleotide sequence SEQ ID No. 4), the primers used for constructing the plasmid are:
5) The promoter of the riboflavin synthesis endogenous gene cluster ribDEAHT is P T7xyl Integration plasmid pcrF19-P T7xyl -rib available promoter P T7xyl Replacement of endogenous promoters (P) T7-xylO Nucleotide sequence SEQ ID No. 5), the primers used for constructing the plasmid are:
6) Other validation primers were as follows:
construction method of (II) plasmid
1) CrRNA ligation
Ligation of single crrnas: at this point, crRNA can be directly passed through a pair of primers designed with overlapping regions, and denatured and annealed to form primer dimers with cohesive ends. Using Biyun 5XAnnealing Buffer for DNAOligos, the primer concentration was 10uM, and each of the upstream and downstream primers (Gene name-cr-F/R) was 20uL,Annealing Buffer 10uL in 50uL system. The reaction conditions are as follows: 2min at 98 ℃, and then preserving heat after cooling to 4 ℃ at 0.1 ℃/S. Diluting the dimer by 10 times, and connecting 1uL with the vector pcrF19NM2-Li after the enzyme digestion of Eco 31I.
After crRNA ligation, it was transferred into e.coli DH5 a competence and colony PCR verified using the verification primer mcherry-VR and the gene name-cr-F.
2) Amplification and ligation of homology arms
Using gene name-U1000-F and gene name-U1000-R, and using bacillus subtilis genome as template to respectively amplify upstream homologous arms of different genes; the downstream homology arms of the different genes were amplified separately using the gene name-D1000-F and the gene name-D1000-R, with the Bacillus subtilis genome as a template. Using lacI-F/R, RNAP-F1/R1, xyl R-F/R, RNAP-F2/R2, pveg-F/R, P T7-lacO -F/R、P T7-xylO -F/R amplification of the integrated expression cassette; the PCR product was recovered using a DNA purification kit.
Primers F19-LiF and F19-LiR were used to linearize the 5 plasmids that successfully ligated crRNA. Vector, upstream and downstream homology arms, insert (replacement) gene fragments of 5 plasmids were ligated using Biyundian Seamless Cloning Kit (seamless cloning kit) and transferred into E.coli DH 5. Alpha. Competence. Transformants were verified and sequenced using Pveg-VF and Hominsert-R.
Example 2 construction of T7BOOST System Chassis Strain
Cpf1 protein expression plasmid pHT-XCR6 of the CRISPR/Cpf1 system was transferred into the competence of Bacillus subtilis G600 and plated onto LB plates containing chloramphenicol resistance until single colonies developed. Then the pHT-XCR6 transformed strain G600-XCR6 was made competent, and the pcrF19-T7RNAPL1R, pcrF-T7 RNAPL2R and pcrF19-P constructed in example 1 were transformed veg Rib, plasmid was added to competent culture for two hours and then not directly plated, but after centrifugation (4000 rmp,2 min) the bacterial solution was resuspended in 500. Mu.L LB containing chloramphenicol, kanamycin and 3% xylose overnight for culture, and the next day was concentrated by centrifugation to 150. Mu.L LB plates with chloramphenicol, kanamycin and 3% xylose added. After a single colony is grown, colony PCR can be performed to verify whether gene editing is completed. Single colonies which were confirmed to be successful were inoculated into 2mL of LB containing 0.006% SDS for overnight culture, streaked on LB plates, and spot-plated for pcrF19-T7RNAPL1R, pcrF-T7 RNAPL2R, pcrF-P, respectively veg Whether the rib plasmid and pHT-XCR6 plasmid are deleted. The strains with successful elimination of 2 plasmids were designated BST7L, BST R and G600-P veg -rib; only the strains from which the pcrF19-T7RNAPL1R, pcrF19-T7RNAPL2R plasmid was successfully deleted were designated BST7L-XCR6 and BST7R-XCR6 for the next step. Integration of plasmid pcrF19-P Using promoter T7lac Rib and pcrF19-P T7xyl The rib is transformed into BST7L-XCR6 and BST7R-XCR6, respectively. The above steps (transformation-post culture-plating-colony PCR verification-plasmid elimination) are followed to obtain the riboflavin expression engineering strain BST7L-P T7lac Rib and BST7R-P T7xyl -rib。
Example 3 construction of expression plasmid for T7BOOST System
1) T7BOOST system expression plasmid
2) The plasmids pHT-GP0s, pADK-GP0s and pSTOP-GP0s are prepared by using pHT01, pAD123 and pSTOP1622 as templates and connecting P veg The promoter, riboj ribozyme, RBS, sfGFP expression frame, nucleotide sequence as SEQ ID NO.6, and the plasmid as template constitute other plasmids with the following required primers:
with P T7lac LiF/R and P T7xyl PCR was performed on pHT-GP0s, pADK-GP0s and pSTOP-GP0s, respectively, by LiF/R to obtain all sfGFP expression plasmids; on this basis, fragments were amplified from the synthesized genes using T7 galB-F/R, T7HLF-F/R, T7HA-F/R and T7EBI-F/R by PCR, linearized fragments corresponding to the T7 expression plasmid were obtained by PCR amplification using T7-liF/R, linearized fragments corresponding to the T7 expression plasmid with a 6 XHis tag were obtained by PCR amplification using T7-liF/T7-6His-liR, and the PCR products were recovered with a DNA purification kit and ligated using Biyun Seamless Cloning Kit (seamless cloning kit). Transfer into E.coli DH 5. Alpha. Competence. Transformants were verified and sequenced using the verification primers.
EXAMPLE 4 construction and characterization of sfGFP-inducible expression engineering Strain
The constructed sfGFP expression plasmid was transferred to the corresponding strain, inoculated into a 96-well plate at 37℃in an inoculum size of 1%, and light absorption (OD) at 600nm was measured using a microplate reader with the addition of an inducer (1 mM IPTG and 0.5g/L xylose) at 0,3,6,9h under conditions of 750rpm in a 37℃well plate shaker 600 ) And sfGFP fluorescence values (excitation light 480nm,516nm emission) and normalized relative fluorescence values were calculated using the formula:
the induction concentration curve of the induction expression system is fitted by using Hill equation, and the fitting formula is as follows:
where n is the correlation, K a Is the threshold value, y is the relative expression intensity (y max And y min Maximum and minimum expression intensity, respectively), x is the concentration of IPTG or xylose.
As shown in FIG. 3, the optimal induction time for BST7L-pHT-T7Ls and BST7L-pSTOP-T7Ls was 6h, and BST7L-pADK-T7Ls c The optimal induction time of (2) is 3h; the maximum induction concentration is between 0.4 and 0.6 mM. The optimal induction time for both BST7R-pHT-T7Rs and BST7L-pADK-T7Rs was 6h, and the optimal induction time for BST7R-pSTOP-T7Rs was 0h. In both induction systems, the expression levels of the low-copy and high-copy plasmids are similar and far greater than the expression intensity of the medium-copy plasmid.
Example 5T7-BOOST System for expression of GADB
The strains corresponding to the GAD expression plasmid in example 3 were transferred into BST7L and BST7R, respectively, and the commonly used pP43NMK-GAD transferred into G600 strain was used as a control, and the strain BST7L/pHT-P was fermented by shaking T7lac -GAD,BST7L/pADK-P T7lac -GAD,BST7R/pHT-P T7xyl -GAD,BST7R/pADK-P T7xyl GAD, and G600/pP43NMK-GAD. The fermentation process is as follows: (1) plate culture: inoculating the preserved strain at-80deg.C on an activation plate, culturing at 37deg.C for 12 hr, and passaging once; (2) shake flask seed culture: scraping a ring of inclined seeds by an inoculating loop, inoculating the inclined seeds into a 50mL triangular flask filled with 10mL of seed culture medium, sealing a nine-layer gauze, and culturing at 37 ℃ for 10h at 220 rpm; (3) shake flask fermentation culture: inoculating into a 250mL triangular flask (final volume of 40 mL) filled with TB culture medium according to the inoculum size of 4% of the volume of the seed culture solution, sealing with nine layers of gauze, performing shake culture at 37 ℃ under 200r/min, and respectively adding induction IPTG and xylose for fermentation period of 60h. The expression level of GAD was verified by whole cell catalysis, as shown in FIG. 4, BST7L/pHT-P T7lac -GAD,BST7L/pADK-P T7lac -GAD,BST7R/pHT-P T7xyl -GAD,BST7R/pADK-P T7xyl The yields of gamma-aminobutyric acid for GAD and G600/pP43NMK-GAD were 109.8G/L,101.1G/L,105.0G/L,80.9G/L and 110.2G/L, respectively. The low copy expression series plasmid of T7-BOOST has the same combined expression amount as the most commonly used high copy strong promoter in the hay in the GAD expression.
Example 6T7-BOOST System for expression of HLF and HA
The HLF expression plasmid and the HA expression plasmid constructed in example 3 were transferred into BST7L and BST7R, respectively, and the conventional pP43NMK-HLF and pP43NMK-HA were transferred into G600 strain as a control, and subjected to shake flask fermentation. The fermentation process is as follows: (1) plate culture: inoculating the preserved strain at-80deg.C on an activation plate, culturing at 37deg.C for 12 hr, and passaging once; (2) shake flask seed culture: scraping a ring of inclined seeds by an inoculating loop, inoculating the inclined seeds into a 50mL triangular flask filled with 10mL of seed culture medium, sealing a nine-layer gauze, and culturing at 37 ℃ for 10h at 220 rpm; (3) shake flask fermentation culture: inoculating into a 250mL triangular flask (final volume of 40 mL) filled with TB culture medium according to the inoculum size of 4% of the volume of the seed culture solution, sealing with nine layers of gauze, performing shake culture at 37 ℃ under 200r/min, and respectively adding induction IPTG and xylose for fermentation period of 60h. Collecting cell sediment from the fermentation broth for 24 hours, and crushing the cell sediment by using an ultrasonic crusher to obtain cell crushing liquid for Western blot detection and ELISA detection.
As shown in FIG. 4, the HA-expressing strain BST7L/pHT-P T7lac HA and G600/pP43NMK-HA are banded at 22.7kDa and 45.4 kDa. While HLF expressing strains have BST7R/pHT-P alone T7xyl HLF is banded at 79.0kDa, and the expression level of HLF is detected using ELISA kit, BST7L/pHT-P T7lac -HLF,BST7L/pADK-P T7lac -HLF,BST7R/pHT-P T7xyl -HLF,BST7R/pHT-P T7xyl HLF expression levels of-HLF and G600/pP43NMK-HLF were 12.7,8.3,34.6,10.6,and 7.6. Mu.g/L, respectively. This result demonstrates that the T7-BOOST system is capable of efficiently expressing HLF.
Example 7T7-BOOST System regulates lycopene Synthesis
Lycopene expression plasmid pHT-P constructed in example 3 veg -EBI，pHT-P T7lac -EBI and pHT-P T7xyl EBI, respectively transferring into engineering strain G600,BST7L and BST7R give G600-pHT-P veg -EBI，BST7L-pHT-P T7lac -EBI and BST7R-pHT-P T7xyl -EBI, shake flask fermentation. The fermentation process is as follows: (1) plate culture: inoculating the preserved strain at-80deg.C on an activation plate, culturing at 37deg.C for 12 hr, and passaging once; (2) shake flask seed culture: scraping a ring of inclined seeds by an inoculating loop, inoculating the inclined seeds into a 50mL triangular flask filled with 10mL of seed culture medium, sealing a nine-layer gauze, and culturing at 37 ℃ for 10h at 220 rpm; (3) shake flask fermentation culture: inoculating into a 250mL triangular flask (final volume of 40 mL) filled with a fermentation culture medium according to the inoculum size of 4% of the volume of the seed culture solution, sealing a nine-layer gauze, performing shake culture at 37 ℃ under 200r/min, adding an inducer for 12h for induction, and performing fermentation period for 60h;
as shown in FIG. 5, the strain G600-pHT-P was subjected to 60h shaking flask fermentation veg -EBI，BST7L-pHT-P T7lac -EBI and BST7R-pHT-P T7xyl Lycopene production by EBI was 0,8.53 and 5.06mg/g DCW. The results indicate that relative to promoter P veg The T7-BOOST system can effectively drive the production of lycopene.
Example 8T7-BOOST System regulates Riboflavin Synthesis
The riboflavin-producing strain constructed in example 2 was G600-P veg -rib，BST7L-P T7lac Rib and BST7R-P T7xyl The rib is subjected to shake flask fermentation. The fermentation process is as follows: (1) plate culture: inoculating the preserved strain at-80deg.C on an activation plate, culturing at 37deg.C for 12 hr, and passaging once; (2) shake flask seed culture: scraping a ring of inclined seeds by an inoculating loop, inoculating the inclined seeds into a 50mL triangular flask filled with 10mL of seed culture medium, sealing a nine-layer gauze, and culturing at 37 ℃ for 10h at 220 rpm; (3) shake flask fermentation culture: inoculating into a 250mL triangular flask (final volume of 40 mL) filled with fermentation medium according to the inoculum size of 4% of the volume of the seed culture solution, sealing with nine layers of gauze, performing shake culture at 37 ℃ under 200r/min, and respectively adding induction IPTG and xylose for fermentation period of 60h.
As shown in FIG. 6, the concentration OD of glucose and the concentration OD of cells in shake flask fermentation were respectively 600 And a plot of riboflavin production versus time. Relative to the strong promoter P veg Expression Strain G600-P veg 16.8mg/L of rib, BST7L-P T7lac Rib and BST7R-P T7xyl The final yields of rib were 26.6mg/L and 54.7mg/L, respectively strain G600-P veg 1.58 and 3.26 fold in rib, the results indicate that relative to promoter P veg The T7-BOOST system can effectively regulate and control the production of the riboflavin.
Comparative example 1 expression Properties of common inducible promoters
In order to show the superiority of the induction performance of T7-BOOST, we constructed the common induction type expression vector pHT-P of IPTG and xylose hy-spank -sfGFP、pADK-P hy-spank -sfGFP、pHT-P xylA -sfGFP and pADK-P xylA sfGFP. The induction performance of the transformed strain G600 is characterized, and the induction time and the induction curve are shown in FIG. 7. Compared with a common induction type expression system, the IPTG induced T7-BOOST system has the advantages that the maximum expression intensity and the dynamic range of the low-copy pHT expression system are respectively improved by 4.24 times and 4.26 times under the condition that the leakage expression quantity is not improved; while the maximum expression intensity of the medium copy pADK expression system is reduced, the dynamic range is improved by 2.79 times. Compared with a common induction type expression system, the maximum expression intensity of the xylose induced T7-BOOST system is improved by 20.22 times; whereas the leakage intensity of the medium copy pADK expression system was reduced and the dynamic range was improved by a factor of 1.27.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (17)

1. A bacillus subtilis double-module transcription optimization system based on T7RNA polymerase is characterized in that: comprises a constitutively expressed repressor expression cassette, a T7RNA polymerase expression cassette regulated by an inducible promoter, and a gene of interest regulated by a hybrid T7 promoter; wherein the hybrid T7 promoter comprises a T7 promoter and an operator, and the hybrid T7 promoter is subjected to negative regulation by a repressor protein and positive regulation by a T7RNA polymerase.
2. The bacillus subtilis dual-module transcription optimization system according to claim 1, wherein: the repressor expression cassette, the inducible promoter and the T7RNA polymerase expression cassette are located on a first vector, and the hybrid T7 promoter and the gene of interest are located on a second vector.
3. Use of the bacillus subtilis two-module transcription optimization system according to claim 1 or 2 in biosynthesis.
4. A use according to claim 3, characterized in that: culturing the genetically engineered bacteria containing the bacillus subtilis double-module transcription optimization system.
5. The use according to claim 4, characterized in that: the culture system contains inducer.
6. The use according to claim 4, characterized in that: culturing the genetically engineered bacteria in a seed culture medium to obtain seed liquid, and inoculating the seed liquid into a fermentation culture medium for fermentation production.
7. The use according to claim 6, characterized in that: the seed culture medium is LB culture medium.
8. The use according to claim 6, characterized in that: the fermentation medium is a TB medium.
9. Use of the bacillus subtilis two-module transcription optimization system according to claim 1 or 2 in metabolic regulation.
10. A bacillus subtilis capable of performing metabolic regulation and control is characterized in that: the bacillus subtilis contains the bacillus subtilis dual-module transcription optimization system according to claim 1 or 2.
11. A method for metabolic synthesis regulation of bacillus subtilis is characterized by comprising the following steps: introducing the bacillus subtilis two-module transcription optimization system according to claim 1 or 2.
12. The method according to claim 11, wherein: the control is performed by replacing the inducible promoter and the operon.
13. A method for high-strength expression of a target gene after induction without low leakage, which is characterized in that the following expression system is introduced into bacillus subtilis: the expression system comprises a constitutively expressed repressor expression frame, a T7RNA polymerase expression frame regulated by an inducible promoter and a target gene regulated by a heterozygous T7 promoter; wherein the inducible promoter is selected from the group consisting of P hy-spank Or P xylA The hybrid T7 promoter includes a T7 promoter and an operon that is positively regulated by an inducible promoter and negatively regulated by a repressor protein.
14. The method according to claim 13, wherein: the repressor is selected from the repressor of lactose promoter or the repressor of xylose promoter.
15. The method according to claim 13, wherein: the operon is selected from lactose operon or xylose operon.
16. The method according to claim 13, wherein: b.subtilis G600 was used as starting strain.
17. The method according to claim 13, wherein: the inducible promoter P hy-spank The nucleotide sequence of (2) is shown as SEQ ID NO. 7; the inducible promoter P xylA The nucleotide sequence of (2) is shown as SEQ ID NO. 8.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020118261A2 (en) * 2018-12-06 2020-06-11 William Marsh Rice University Bacillus expression system
US20210163963A1 (en) * 2018-12-17 2021-06-03 Jiangnan University Bacillus Subtilis Efficiently-Induced Expression System Based on Artificial Series Promoter
CN114645062A (en) * 2022-04-18 2022-06-21 江南大学 Dehydrated tetracycline induced escherichia coli-bacillus subtilis universal induced expression system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020118261A2 (en) * 2018-12-06 2020-06-11 William Marsh Rice University Bacillus expression system
US20210163963A1 (en) * 2018-12-17 2021-06-03 Jiangnan University Bacillus Subtilis Efficiently-Induced Expression System Based on Artificial Series Promoter
CN114645062A (en) * 2022-04-18 2022-06-21 江南大学 Dehydrated tetracycline induced escherichia coli-bacillus subtilis universal induced expression system

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