WO2018171488A1 - 一种基于转录组测序筛选的棒杆菌组成型表达载体启动子及其筛选方法和应用 - Google Patents

一种基于转录组测序筛选的棒杆菌组成型表达载体启动子及其筛选方法和应用 Download PDF

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WO2018171488A1
WO2018171488A1 PCT/CN2018/079034 CN2018079034W WO2018171488A1 WO 2018171488 A1 WO2018171488 A1 WO 2018171488A1 CN 2018079034 W CN2018079034 W CN 2018079034W WO 2018171488 A1 WO2018171488 A1 WO 2018171488A1
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promoter
expression vector
seq
gene
plasmid
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PCT/CN2018/079034
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French (fr)
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邢盼盼
苏海霞
王炯
梅雪臣
万坤
宋盟军
李敬
刘爱福
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武汉远大弘元股份有限公司
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Priority claimed from CN201710169454.5A external-priority patent/CN107164369B/zh
Priority claimed from CN201710169693.0A external-priority patent/CN107164371B/zh
Priority claimed from CN201710169685.6A external-priority patent/CN107164370B/zh
Application filed by 武汉远大弘元股份有限公司 filed Critical 武汉远大弘元股份有限公司
Priority to EP18772338.2A priority Critical patent/EP3604530A4/en
Priority to US16/496,496 priority patent/US11639512B2/en
Publication of WO2018171488A1 publication Critical patent/WO2018171488A1/zh

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Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a Corynebacterium constitutive expression vector promoter and a screening method thereof and application thereof based on transcriptome sequencing screening, and to an expression vector and a recombinant strain containing the promoter.
  • Corynebacterium is a kind of Gram-positive bacteria.
  • the three main representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium glutamicum and Brevibacterium lactoferment have been widely used in production.
  • a variety of chemicals such as amino acids and nucleotides (Liebl et al., 1991).
  • the Corynebacterium mutant strain obtained by physical or chemical mutagenesis has a strong ability to synthesize useful substances of interest, and the transformation of wild strains or mutant strains of Corynebacterium by genetic engineering and metabolic engineering can obtain higher production intensity.
  • Strains. Metabolic engineering is to find key metabolic enzymes in various metabolic pathways of Corynebacterium, and to regulate the gene expression of key metabolic enzymes through genetic engineering.
  • genes are expressed under the control of a promoter whose expression intensity is dependent on the promoter element.
  • the promoters from Escherichia coli, Streptomyces and Bacillus subtilis can be expressed in Corynebacterium bacteria and can be applied to the construction of a vector system of Corynebacterium, such as the expression plasmid PXMJ19 constructed using Ptac and Ptrc promoters derived from Escherichia coli.
  • PEC-XK99E the gene is induced to express under the control of lacIq, but the expression level is low, and the inducer IPTG is needed in the production process, and IPTG is quite expensive, and is not suitable for gene expression inducer for large-scale production of the target product; Lactose can be used as an inducer for large-scale production, whereas for most coryneform strains, lactose cannot enter its cells (Brabetz et al., 1991), which also limits inducible expression systems. Application in coryneform bacteria.
  • the constitutive promoter can continuously express the foreign gene during the survival of the cell without special conditions such as induction, thereby simplifying the operation process and having relatively high safety, and thus is more suitable for application in actual production.
  • the constitutive genes with strong expression were screened and purified by PCR amplification of DNA probes in different housekeeping genes, and a constitutive strong promoter was selected for the construction of recombinant vector (Jensen P R et al. , Appl Environ Microbiol, 1998, 64(1): 82-87.). Yangliu et al. used the endogenous strong promoter Pgro of C.
  • glutamicum ATCC 13032 to effectively promote the expression of the exogenous gene xylA in Corynebacterium glutamicum ATCC 13032 (Yang Liu et al., New Energy Progress, 2014, 5(2): 353 -357), Wang Xiaoyuan and other tac-M promoters which have transcriptional initiation function by mutation, construct a constitutive expression vector PDXW-10, which is moderately expressed in Corynebacterium (CN: 200910210962.9).
  • the expression of the constitutive promoter has many advantages, the current screening method is complicated, and how to select a suitable promoter for regulation, to avoid the host cell growth stagnation or metabolic disorder due to excessive protein expression, or to deplete the host energy and the plasmid.
  • Loss is an urgent problem to be solved in industrial production.
  • Pgro as a promoter to construct an expression plasmid together with the gene of interest.
  • These plasmids also express the target gene in the logarithmic growth phase of the bacteria, resulting in a loss rate of more than 40% before the stable phase of the plasmid. The expression effect.
  • the technical problem to be solved by the present invention is that the current constitutive promoter screening method is complicated, and the expression plasmid constructed by the selected promoter is easily lost in the growth of the host, and also affects the growth and metabolism of the host. How to establish a rapid screening method for constitutive promoters, and screen out the endogenous promoter with high viability at the stationary phase, and the plasmid constructed by the promoter can ensure the stability of the plasmid during the stable growth period of the cells is what we need to solve. problem.
  • the object of the present invention is to establish a method for finding a promoter which is highly expressed in the logarithmic growth phase of bacteria and which is highly expressed during the stagnant period of bacterial growth, and uses it to construct a constitutive high-efficiency expression plasmid.
  • promoters are all regulatable promoters.
  • the expression vectors used green fluorescent protein (EGFP) as a marker gene to detect the activity of each promoter fragment and evaluate each probe plasmid. Passage stability, so as to screen out a series of promoters with low activity and stable start-up activity in the log phase promoter, and to evaluate the stability of plasmids containing each promoter, and further screen the promoters that stabilize the plasmid.
  • EGFP green fluorescent protein
  • a method for screening the promoter of baculobacter constitutive expression vector based on transcriptome sequencing, analyzing the transcriptional level of various genes in the log phase and stationary phase of Corynebacterium, and screening out the transcript abundance analysis of each gene in two periods A class of genes with low transcriptional levels in the log phase and high transcription levels in the stationary phase.
  • the promoter region of the gene is analyzed by promoter prediction software, and the promoter fragment of such a gene is amplified by PCR, and the DNA fragment of the promoter is ligated.
  • the expression vector Into the expression vector, insert the marker gene, construct the promoter detection vector, electrotransform the detection vector into the host cell, culture the host cell to the log phase and the stationary phase, and detect the OD and fluorescent protein expression of the bacterial growth by the multi-function microplate reader. At the same time, in the absence of resistance pressure, continuous passage is carried out, and the host cell with low fluorescence value of the log phase, high fluorescence value in the stationary phase and continuous passage for 50 generations without loss of the plasmid is selected, and the promoter contained therein is Corynebacterium constitutive expression vector promoter.
  • a Corynebacterium constitutive expression vector promoter selected based on the above method the nucleotide sequence of which is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • a Corynebacterium constitutive expression vector comprising the Corynebacterium constitutive expression vector promoter described above.
  • the coryneform constitutive expression vector is constructed by inserting the above-mentioned coryneform constitutive expression vector promoter and a target gene at a restriction site of the plasmid HY-P19, and the nucleus of the plasmid HY-P19
  • the nucleotide sequence is shown in SEQ ID NO: 4
  • the nucleotide sequence of the gene of interest is shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
  • the Corynebacterium constitutive expression vector promoter sequence is as shown in SEQ ID NO: 1
  • the nucleotide sequence of the inserted gene of interest is as shown in SEQ ID NO: 5
  • the constitutive expression vector of the bacillus constitutive expression vector is as shown in SEQ ID NO: 2
  • the nucleotide sequence of the inserted gene of interest is shown in SEQ ID NO: 6
  • the coryneform constitutive expression vector promoter is When the sequence is as shown in SEQ ID NO: 3
  • the nucleotide sequence of the inserted gene of interest is shown in SEQ ID NO: 7.
  • the expression vector electroporates a recombinant strain obtained by the host cell C. glutamicum, and the host cell C. glutamicum is preferably a strain having the preservation number CCTCC NO: M2016609, the strain is in November 2016. Deposited at the China Center for Type Culture Collection on the 01st.
  • the beneficial effect of the invention is that the expression vector constructed by such a promoter has a low expression level of the target gene protein in the log phase, but the expression level of the target protein in the stable phase is greatly increased relative to the log phase, and the growth of the host strain is low, and the plasmid is low. It has high stability and is suitable for vector construction of the target gene which does not require expression in the log phase and which needs high expression during the stationary phase. It is especially suitable for the construction of engineering strains for amino acid fermentation.
  • the Corynebacterium glutamicum H5 of the present invention has been deposited at the China Center for Type Culture Collection (CCTCC) on November 1, 2016, and was detected as a viable state on November 12, 2016.
  • the deposit address is: Wuhan University, Wuhan, China. , 430072
  • the accession number is: CCTCC NO: M2016609
  • the culture name is Corynebacterium glutamicum H5, Corynebacterium glutamicum H5, the classification is Corynebacterium glutamicum.
  • Figure 1 shows the original plasmid PXMJ19.
  • Figure 2 shows the synthetic DNA fragment hy-dna1.
  • Figure 3 shows plasmid HY-19 constructed after in vitro recombination.
  • Figure 4 shows the recombinant plasmid HY-P19-promoter-EGFP.
  • Bacterial culture Pick a ring of Corynebacterium glutamicum H5 strain producing isoleucine without plasmid, deposited at the China Center for Type Culture Collection on November 1, 2016, under the accession number CCTCC NO: M2016609), cultured in LB medium at 31 ° C overnight, centrifuged and centrifuged, resuspended in an equal volume of sterile water, and 5% inoculum into LBG freshly sterilized medium, 31 °C, 200 rpm culture to the middle of the log phase and the early stage of the stabilization period, take out the fermentation broth, quickly and equal volume Bacteria Reagent (QIAGEN) was mixed, centrifuged at 5000 rpm for 10 min, and 100 mg of wet cells were weighed and quickly stored in liquid nitrogen.
  • QIAGEN Bacteria Reagent
  • RNA extraction The sample stored in liquid nitrogen was placed in a mortar precooled with liquid nitrogen, and an appropriate amount of liquid nitrogen was added to ensure that the liquid nitrogen in the mortar was not dried, the sample was ground into a powder, and then according to QIAGEN RNeasy Plant Mini. Kit's experimental procedure for RNA extraction. The extracted sample was run on a 1% agarose gel and required no protein contamination, ie the agarose gel had no obvious bright bands. The concentration of the nucleic acid was measured by NanoDrop Spectrophotometer. The concentration of the sample was 421 ng/ul, and the concentration of the sample was 237 ng/ul. The sample was free of obvious protein contamination and the nucleic acid concentration was >60 ng/ul.
  • RNA samples were extracted and entrusted by Beijing Nuohe Zhiyuan Bioinformatics Co., Ltd. for sequencing.
  • the sequencing process is as follows: After the sample is tested, the rRNA is removed by the Ribo-zero kit to enrich the mRNA. Subsequently, the fragmentation buffer was used to break the mRNA into short fragments, and mRNA was used as a template to synthesize a strand cDNA with random hexamers. Then, buffer, dNTPs (dTTP in dNTP was replaced with dUTP) and DNA polymerase were added.
  • dNTPs dTTP in dNTP was replaced with dUTP
  • I and RNase H synthesize the double-stranded cDNA, and then purify the double-stranded cDNA with AMPure XP beads, and then degrade the second strand of the cDNA containing U with USER enzyme.
  • the purified double-stranded cDNA was firstly end-repaired, A-tailed and ligated to the sequencing linker, and the fragment size was selected using AMPure XP beads.
  • PCR amplification was performed and the PCR product was purified using AMPure XP beads to obtain the final library. After the library was constructed, preliminary quantification was performed using Qubit 2.0, and the library was diluted to 1 ng/ul. Then, the insert size of the library was detected using Agilent 2100.
  • the Q-PCR method was used.
  • the effective concentration of the library was accurately quantified (effective library concentration > 2 nM) to ensure library quality.
  • the different libraries were pooled according to the effective concentration and the target data volume, and then HiSeq/MiSeq was sequenced.
  • Bioinformatics analysis process The sequencing results were entrusted to Beijing Nuohe Zhiyuan Bioinformatics Co., Ltd. for information analysis. After obtaining the original sequenced sequence (Sequenced Reads), bioinformatics analysis is performed with the relevant species reference sequence or reference genome. The main processes of bioinformatics analysis are as follows:
  • Original sequence data Sequence data quality assessment ⁇ reference sequence alignment analysis ⁇ gene expression level analysis ⁇ RNA-seq overall quality assessment ⁇ gene differential expression analysis ⁇ differential gene GO enrichment analysis, differential gene KEGG enrichment analysis.
  • the bioinformatics reference species of the sample of the present invention is Corynebacterium glutamicum ATCC 13032 (purchased from Shanghai Fuxiang Biotechnology Co., Ltd.), and the reference genome is linked on ncbi as https://www.ncbi.nlm.nih.gov/ Nuccore/NC_006958.1.
  • the expression abundance of each gene in the log phase and the stationary phase was analyzed, and 10 genes and transcription analysis information of Table 1 were screened.
  • the consistent characteristics of the 10 genes were: logarithm The transcription level is low, and the transcription level is high in the stationary phase.
  • the genes CGTRNA_RS10085 and CGTRNA_RS10080 belong to the same operon, CGTRNA_RS07920, CGTRNA_RS07925, CGTRNA_RS07930 belong to the same operon, CGTRNA_RS00965, CGTRNA_RS00970 belong to the same operon, the same manipulation
  • the child shares a promoter.
  • Table 1 Corynebacterium glutamate transcriptome analysis S phase transcript abundance high L phase abundance low gene
  • Promoter region screening Through the NCBI blast function, the predicted gene fragments were compared with the genome of the C. glutamicum genome sequenced in the laboratory to find the corresponding gene sequence. The promoter region of each gene was predicted by consulting the literature or applying promoter prediction software, and the promoter region of each gene was selected for 2-3 fragments according to the predicted functional region for promoter activity detection.
  • Primer synthesis The primers were designed by snapgene software, and the primers were synthesized by Wuhan Tianyihuiyuan. The synthetic primer sequences are shown in Table 2 below.
  • Genomic extraction Pick a loop of Corynebacterium glutamicum H5, inoculate it in LB medium, incubate at 31 ° C overnight, centrifuge to remove the supernatant, and extract the bacterial genome using the procedure of the Tiangen Genome Extraction Kit.
  • the extracted genome was subjected to nucleic acid concentration detection using a NanoDrop Spectrophotometer, and the concentration of the nucleic acid subjected to PCR amplification was controlled to be between 100 and 200 ng/ul.
  • each synthetic primer was diluted with water to a final concentration of 10 uM, and amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: genomic template 1 ul, forward primer (10 uM) 1 ul, reverse primer (10 uM). 1ul, 5*TransStart FastPfu FlyBuffer 10ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1 ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 °C for 2 min, 95 °C for 20 s, 55 °C for 20 s, 72 °C for 10 s-1 min, 72 °C for 5 min, and the number of cycles was 32.
  • the amplified DNA fragments were respectively RS10085seq1, RS10085seq2, RS10085seq3 with recombinant linker.
  • Plasmid digestion and DNA synthesis using the original plasmid is PXMJ19, double digestion with NEB restriction endonuclease NarI and HindIII, removing the original lacIq and promoter Ptac, and the enzyme digestion system is 50ul system: plasmid 5- 10ul, NarI1ul, HindIII 1ul, cutsmart 5ul, ddH 2 O 33-38ul.
  • DNA agarose electrophoresis recovery PCR product and enzyme digestion product were added to 10*loading buffer, 50 ul was applied to 1.5% agarose gel, and takara 2000 DL DNA marker was used as a control, and electrophoresis was carried out at 70 V for 40 min. The PCR amplified bands were recovered according to the marker strips. The recovered DNA was recovered using the takara DNA recovery kit.
  • the DNA fragments were RS10085seq1 (167 bp), RS10085seq2 (180 bp), RS10085seq3 (240 bp), RS10840seq1 (107 bp), RS10840seq2 (282 bp), RS07910seq1 (110 bp), and RS07910seq2 (recombinant).
  • E. coli competent preparation E. coli competent DH5 ⁇ preparation was performed using the Biyuntian bacteria super-competitive kit, and the prepared state was stored in a refrigerator at -80 °C for use.
  • Transformation Slowly melt the competent cell DH5 ⁇ on ice, add the in vitro recombinant product to the competent cells, gently use the finger to force the centrifuge tube to mix the bacteria and recombinant products, and place in an ice bath or ice water bath for 30 minutes. , 42 ° C water bath, heat shock 2 minutes. Immediately after heat shock, place in an ice water bath for 2 minutes. 900 microliters of LB was added and incubated at 37 ° C for 200 hours at 200 rpm. The supernatant was centrifuged, and the cells were spread on a LB solid plate containing chloramphenicol 25 ug/ml, and cultured overnight at 37 °C.
  • HY-P19 was digested with NEB restriction endonuclease XbaI and EcoRI in a 37 ° C water bath for 1 h, the amount of plasmid was 1 ug, and the digestion system was 50 ul system: plasmid 5-10 ul, XbaI 1 ul, EcoRI1ul , cutsmart 5ul, ddH 2 O 33-38ul.
  • the digested product of HY-P19 was recovered, and the digested product and each promoter fragment and EGFP fragment were recombined in vitro using a Vazyme one Step Cloning Kit kit, and the other experimental procedures were the same as above (7)-(9).
  • HY-P19-promoter-EGFP A series of plasmids containing different promoters, HY-P19-promoter-EGFP, were constructed.
  • the original plasmid PXMJ19 in Figure 1 was digested with NarI and HindIII, and the digested product was recovered after digestion.
  • the digested product and the DNA fragment synthesized in Fig. 2 were recombined in vitro by one-step cloning. After transforming DH5 ⁇ , positive clones were picked, and after sequencing, the plasmid was extracted to obtain plasmid HY-P19 of Fig. 3.
  • HY-P19 was digested with XbaI and EcoRI, and the digested product was recovered.
  • the recombinant promoter fragment and the PCR-amplified EGFP fragment were recombined in vitro. After transforming DH5 ⁇ , positive clones were picked and verified by sequencing. Thereafter, the plasmid was extracted to obtain a probe vector of the series promoter of Fig. 4 and the marker gene EGFP.
  • the probe vectors containing the respective promoter fragments were designated as PRS10085seq1, PRS10085seq2, PRS10085seq3, PRS10840seq1, PRS10840seq2, PRS07910seq1, PRS07910seq2, PRS07930seq1, PRS07930seq2, PRS07930seq3, PRS00965seq1, PRS00965seq2, PRS04670seq1, and PRS04670seq2, respectively.
  • a single colony was picked from the fresh LB (about 12 h cultured) plate, and inserted into a test tube containing 5 mL of LBG liquid, and shake cultured at 30 ° C, 220 rpm overnight.
  • EPO yeast powder 5 g/L, peptone 10 g/L, Nacl 10 g/L, glycine 25 g/L, Tween 1 g/L. Incubate at 30 ° C with shaking until the OD is between 1.0 and 1.5 (approximately 4-5 h).
  • the competent cells were dispensed in 1.5 mL sterile EP tubes, 0.1 mL per tube. Store in an ultra-low temperature freezer at -80 °C.
  • the cell fluid was quickly inhaled into a pre-warmed EP tube containing 5 mL of LBHIS at 46 ° C, and the thermal shock was accurately timed for 6 minutes.
  • the 6EP tube was cultured at 30 ° C, shaking at 200 rpm, and the cells were resuscitated for 60 minutes.
  • the truffle transformants containing different promoter fragments were transferred to 5 ml LBG test medium and cultured at 30 ° C to an OD600 of 2.0 (log phase) and 3.2 (stability phase), respectively.
  • the cells were removed, centrifuged, and washed twice with PBS solution, resuspended in the same volume of PBS, and the cells were resuspended, and 200 ul of the cells were added to a black matrix 96-well plate using a multi-plate reader TECANM1000. Perform fluorescence value detection.
  • the fluorescence values of the detection plasmids in the log phase and the stationary phase of the host strain are shown in Table 3.
  • Promoter fragments RS10085seq2, RS10085seq3, RS10840seq2, RS07910seq2, RS07930seq3, RS00965seq2, RS04670seq2 showed our desired screening characteristics in the expression of the probe vector.
  • Table 3 Fluorescence detection and plasmid passage statistics of each probe plasmid in two periods of host bacteria
  • Passage stability means that more than 90% of the plasmids contain plasmids, 50 passages, 50 passages, and the plasmid is more than 90% stable.
  • the recombinant bacteria containing each promoter detection vector were inserted into LBG medium, and shaken at 30 ° C for 24 hours under the condition of no resistance pressure; the inoculation amount was 5%, and the passage was continued. Culture, each culture of bacteria is diluted and plated, and the same concentration is diluted and applied to the plate containing chloramphenicol and chloramphenicol.
  • the loss rate of the plasmid (the number of plate cells without chloramphenicol) - the number of plate cells containing chloramphenicol) / the number of plate cells without chloramphenicol ⁇ 100%, the plasmid loss rate ⁇ 10%, we defined this plasmid to be stable, when the plasmid loss rate is >10% This generation of plasmids is considered to be unstable.
  • the stable passage algebra of the cells containing each probe plasmid is shown in Table 3.
  • Example 2 Expression vector constructed using promoter fragment RS10085seq2 and application thereof
  • the key gene for isoleucine metabolism of Corynebacterium glutamicum is threonine dehydratase, the gene name is ilvA (the sequence is shown in SEQ ID NO: 5), and the selected promoter fragment RS10085seq2 is used as a promoter, and ilvA is The target gene was constructed to construct the expression vector HY-P19-RS10085seq2-ilvA.
  • DNA fragment amplification using plasmid PRS10085seq2 as a template, the upstream and downstream primers are:
  • the amplified DNA fragment RS10085seq2 was amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: 1 ul of PRS10085seq2 template, 1 ul of forward primer (10 uM), 1 ul of reverse primer (10 uM), 5 * TransStart FastPfu FlyBuffer 10 ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 ° C for 2 min, 95 ° C for 20 s, 55 ° C for 20 s, 72 ° C for 10 smin, 72 ° C for 5 min, and the number of cycles was 32.
  • the upstream and downstream primers are:
  • ilvARP caaaacagccaagctgaattcTTAGGTCAAGTATTCGTACTCAG
  • the amplified DNA fragment ilvA was amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: genomic template 1 ul, forward primer (10 uM) 1 ul, reverse primer (10 uM) 1 ul, 5* TransStart FastPfu FlyBuffer 10 ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 ° C for 2 min, 95 ° C for 20 s, 58 ° C for 20 s, 72 ° C for 30 s, 72 ° C for 5 min, and the number of cycles was 32.
  • Plasmid digestion The plasmid used was PXMJ19 minus the original lacIq and the promoter modified HY-P19.
  • HY-P19 was digested with NEB restriction endonuclease XbaI and EcoRI in a 37 ° C water bath for 1 h, the amount of plasmid was 1 ug, and the enzyme digestion system was 50 ul system: plasmid 5 ul, XbaI 1 ul, EcoRI1 ul, cutsmart 5ul, ddH 2 O 38ul.
  • DNA agarose electrophoresis recovery PCR product and enzyme digestion product were added to 10*loading buffer, 50ul was applied to 1.5% agarose gel, and takara 2000 DL DNA marker was used as control. Electrophoresis was carried out at 70V for 40min. The 160 bp and 1347 bp bands were amplified by PCR according to the marker strip recovery. Recycling is carried out using the takara DNA recovery kit.
  • In vitro recombination In vitro recombination using the Vazyme one Step Cloning Kit, the recombinant product was directly transformed into E. coli.
  • Competent preparation of E. coli construction of the same promoter promoter vector.
  • Transformation Slowly thaw competent bacteria on ice, add 10 ul of the in vitro recombinant product to 100 ul of competent cells, and gently use a finger to force the centrifuge tube to mix the bacteria and recombinant products. Leave in an ice bath or ice water bath for 30 minutes. 42 ° C water bath, heat shock 2 minutes. Immediately after heat shock, place in an ice water bath for 2 minutes. 900 microliters of LB was added and incubated at 37 ° C for 200 hours at 200 rpm. The supernatant was centrifuged, and the cells were spread on an LB solid plate containing chloramphenicol 25 ug/ml, and cultured at 37 ° C overnight.
  • Transformant verification and plasmid extraction Transformants that were grown on the resistant plates after transformation were sent to Wuhan Tianyihuiyuan for sequencing analysis, and the correct sequence of the transformants was picked and transferred to a 5 ml LB tube to a final concentration of 25 ug/ml. Chloramphenicol, shaken at 37 ° C, cultured at 200 rpm overnight, plasmid extraction using the Takara plasmid kit, expression plasmid HY-P19-RS10085seq2-ilvA concentration of 269 ng / ul.
  • Expression plasmid HY-P19-RS10085seq2-ilvA electrotransformation host cell Corynebacterium glutamicum H5: construction with the same promoter.
  • the electrolyzed bacterial liquid was centrifuged and concentrated, and coated on a LBHIS solid medium supplemented with 10 ug/mL chloramphenicol with a glass rod, and cultured in a 30 ° C incubator for 36 hours, and the grown transformant was plasmid HY-P19-
  • the strain of RS10085seq2-ilvA the strain was named H5-RS10085seq2-ilvA.
  • 5L fermenter fermentation medium corn slurry 15ml / L, glucose 140g / L (division, 0.075MPa moist heat sterilization 15min), ammonium sulfate 5g / L, potassium dihydrogen phosphate 0.4g / L, magnesium sulfate heptahydrate 0.6g /L, biotin 0.1mg / L, VB1 0.1mg / L, corn oil 1ml / L, Angel yeast powder 1g / L, defoamer 1ml / L, 121 ° C 0.01MPa moist heat sterilization 25min; added after sterilization For the initial sugar, adjust the pH to 7.0 with ammonia water.
  • Culture method the strain is connected to the seed culture medium (seed medium formula: glucose 17g / L, corn slurry 10ml / L, urea 1g / L, magnesium sulfate 0.5g / L, dipotassium hydrogen phosphate 1g / L, yeast paste 0.1g/L, biotin 0.1mg/L, vitamin B10.1mg/L, corn oil 0.1g/100ml, calcium carbonate 1g/100ml; pH 7.0 adjusted with NaOH, 31°C rotary shaker, 200rpm, cultured for 16h Then, 10% inoculation amount is connected to the 5L automatic control fermenter containing fermentation medium, and appropriate air is introduced to adjust the appropriate stirring speed.
  • seed medium seed medium formula: glucose 17g / L, corn slurry 10ml / L, urea 1g / L, magnesium sulfate 0.5g / L, dipotassium hydrogen phosphate 1g / L, yeast paste 0.1g/L, biotin 0.1m
  • the dissolved oxygen is controlled by the staged oxygen supply mode: 0-8h is 30%, 8- 24h is 25%, 24-56h is 12%, the pH is controlled at 7.0 by automatic flow of 15% ammonia water, the appropriate amount of foaming agent is defoamed by flow, and the residual sugar is controlled by adding glucose solution with a concentration of 800g/L. At about 3%, the fermentation ends at 56h.
  • the fermentation broth was detected by HPLC (Ilite C18 column, 5 ⁇ m, 4.6*250 mm, mobile phase of 0.015 mol/L of diammonium phosphate and 0.005 mol/L of a mixed aqueous solution of pH 7.20.
  • the flow rate was 0.5. Ml/min.
  • the column temperature is 23 ° C.
  • the detector is a UV detector with a wavelength of 199 nm.
  • the control strain of Corynebacterium glutamicum H5 produces 22.0 g/L of acid, 6.3 g/L of acid, and 12.83% of sugar acid.
  • the 56h cans were diluted and plated, and the gradient concentrations of 10 -6 , 10 -7 , 10 -8 , and 10 -9 were diluted .
  • 50 ul of bacterial solution was taken from each concentration gradient and coated on chloramphenicol and contained.
  • the chloramphenicol plate was placed in a 31 ° C incubator for 36 h, and the plasmid loss rate was calculated based on the number of colonies growing on the non-resistant plates and the resistant plates.
  • the plasmid loss rate (chloramphenicol-free plate bacteria) The number of cells - the number of plate cells containing chloramphenicol) / the number of plate cells without chloramphenicol ⁇ 100%.
  • Example 3 Expression vector constructed using promoter fragment RS07910seq2 and application thereof
  • the promoter fragment PRS07910seq2 was used as a promoter to lrp.
  • the exogenous expression plasmid HY-P19-RS07910seq2-lrp was constructed, and the expression plasmid was electrotransformed into the isoleucine-producing bacterium, Corynebacterium glutamicum H5, to construct a new strain, and the strain was evaluated by 5L fermentation. Acid production capacity.
  • an expression vector HY-P19-RS07910seq2-lrp was constructed.
  • DNA fragment amplification using plasmid PRS07910seq2 as a template, the upstream and downstream primers are:
  • RS07910seq2Fp tgcctgcaggtcgactctaga GCGATCACGTAGTCATCCAAG
  • the amplified DNA fragment RS07910seq2 was amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: P RS07910seq2 template 1 ul, forward primer (10 uM) 1 ul, reverse primer (10 uM) 1 ul, 5* TransStart FastPfu FlyBuffer 10 ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1 ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 ° C for 2 min, 95 ° C for 20 s, 55 ° C for 20 s, 72 ° C for 10 smin, 72 ° C for 5 min, and the number of cycles was 32.
  • the upstream and downstream primers are:
  • the amplified DNA fragment lrp was amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: 1 ul of genomic template, 1 ul of forward primer (10 uM), 1 ul of reverse primer (10 uM), 5 * TransStart FastPfu FlyBuffer 10 ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 ° C for 2 min, 95 ° C for 20 s, 56 ° C for 20 s, 72 ° C for 20 s, 72 ° C for 5 min, and the number of cycles was 32.
  • Plasmid digestion The plasmid used was PXMJ19 minus the original lacIq and the promoter modified HY-P19.
  • HY-P19 was digested with NEB restriction endonuclease XbaI and EcoRI in a 37 ° C water bath for 1 h, the amount of plasmid was 1 ug, and the enzyme digestion system was 50 ul system: plasmid 5 ul, XbaI 1 ul, EcoRI1 ul, cutsmart 5ul, ddH 2 O 38ul.
  • Expression plasmid HY-P19-RS07910seq2-lrp electrotransformed host cell Corynebacterium glutamicum construction of the same promoter promoter vector.
  • the electrolyzed bacterial liquid was centrifuged and concentrated, and coated on a LBHIS solid medium supplemented with 10 ug/mL chloramphenicol with a glass rod, and cultured in a 30 ° C incubator for 36 hours, and the grown transformant was plasmid HY-P19-
  • the strain of RS07910seq2-lrp, the strain was named H5-RS07910seq2-lrp.
  • Strain H5-RS07910seq2-lrp and Corynebacterium glutamicum H5 are fermented in 5L fermentor to produce acid.
  • the formulation of the fermentation medium of the 5L fermenter, the culture method of the strain and the detection method of the fermentation liquid during the canning were the same as those in the example 2, and the engineering bacteria H5-RS07910seq2-lrp was measured, and the fermentation yielded acid was 39.1 g/L, and the acid content was 6.9. g/L, the conversion rate of sugar acid reached 17.88%.
  • the 56h cans were diluted and plated, and the gradient concentrations of 10 -6 , 10 -7 , 10 -8 , and 10 -9 were diluted .
  • 50 ul of bacterial solution was taken from each concentration gradient and coated on chloramphenicol and contained.
  • the chloramphenicol plate was placed in a 31 ° C incubator for 36 h, and the plasmid loss rate was calculated based on the number of colonies growing on the non-resistant plates and the resistant plates.
  • the plasmid loss rate (chloramphenicol-free plate bacteria) The number of cells - the number of plate cells containing chloramphenicol) / the number of plate cells without chloramphenicol ⁇ 100%.
  • Example 4 Expression vector constructed using promoter fragment RS04670seq2 and application thereof
  • NADPH is mainly derived from the pentose phosphate pathway.
  • NADPH is supplied through this route, which consumes other metabolites and changes metabolism.
  • the carbon flux of the pathway may affect the accumulation of metabolites of interest, and by transcriptome analysis, the leucine-producing Corynebacterium glutamicum is down-regulated in the stationary phase, glucose-6-phosphate dehydrogenase in the pentose phosphate pathway.
  • an expression vector HY-P19-RS04670seq2-gnd was constructed.
  • DNA fragment amplification using plasmid PRS04670seq1 as a template, the upstream and downstream primers are:
  • RS04670seq2Fp tgcctgcaggtcgactctagaAGGCTGACAGAAACTCTAAAAAC
  • the amplified DNA fragment RS04670seq1 was amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: P RS04670seq1 template 1 ul, forward primer (10 uM) 1 ul, reverse primer (10 uM) 1 ul, 5* TransStart FastPfu FlyBuffer 10 ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1 ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 ° C for 2 min, 95 ° C for 20 s, 55 ° C for 20 s, 72 ° C for 10 smin, 72 ° C for 5 min, and the number of cycles was 32.
  • the upstream and downstream primers are:
  • Gnd-FP TTAACTTCGGAGGTATCCAtgaagctagattccattgattg
  • gnd-RP ccaaaacagccaagctgaattcTTAAGCTTCCACCTCGGAGCG
  • the amplified DNA fragment gnd was amplified using TransStart FastPfu Fly DNA Polymerase.
  • the PCR system was: genomic template 1 ul, forward primer (10 uM) 1 ul, reverse primer (10 uM) 1 ul, 5* TransStart FastPfu FlyBuffer 10 ul, 2.5 mM Dntps 4ul, TransStart FastPfu Fly DNA Polymerase 1ul, ddH 2 O 32ul.
  • the PCR amplification process was 95 ° C for 2 min, 95 ° C for 20 s, 56 ° C for 20 s, 72 ° C for 1 min, 72 ° C for 5 min, and the number of cycles was 32.
  • Plasmid digestion The plasmid used was PXMJ19 minus the original lacIq and the promoter modified HY-P19.
  • HY-P19 was digested with NEB restriction endonuclease XbaI and EcoRI in a 37 ° C water bath for 1 h, the amount of plasmid was 1 ug, and the enzyme digestion system was 50 ul system: plasmid 5 ul, XbaI 1 ul, EcoRI1 ul, cutsmart 5ul, ddH 2 O 38ul.
  • DNA agarose electrophoresis recovery PCR products and enzyme digestion products were added to 10*loading buffer, 50 ul was applied to 3%, 1% agarose gel, and takara 2000 DL DNA marker was used as a control, and electrophoresis was carried out at 70 V for 40 min.
  • the 77 bp and 1481 bp bands were amplified by PCR according to the marker strip recovery. Recycling is carried out using the takara DNA recovery kit.
  • Expression plasmid HY-P19-RS04670seq2-gnd electrotransformed host cell Corynebacterium glutamicum construction of the same promoter promoter vector.
  • the electrolyzed bacterial liquid was centrifuged and concentrated, and coated on a LBHIS solid medium supplemented with 10 ug/mL chloramphenicol with a glass rod, and cultured in a 30 ° C incubator for 36 hours, and the grown transformant was plasmid HY-P19-
  • the formulation of the fermentation medium of the 5L fermenter, the culture method of the strain and the detection method of the fermentation liquid during the canning were the same as those in the example 2, and the engineering bacteria H5-RS04670seq2-gnd was measured, and the acid production was 33.4 g/L, and the acid content was 7.08. g/L, the conversion rate of sugar acid reached 15.31%.
  • the 56h cans were diluted and plated, and the gradient concentrations of 10 -6 , 10 -7 , 10 -8 , and 10 -9 were diluted .
  • 50 ul of bacterial solution was taken from each concentration gradient and coated on chloramphenicol and contained.
  • the chloramphenicol plate was placed in a 31 ° C incubator for 36 h, and the plasmid loss rate was calculated based on the number of colonies growing on the non-resistant plates and the resistant plates.
  • the plasmid loss rate (chloramphenicol-free plate bacteria) The number of cells - the number of plate cells containing chloramphenicol) / the number of plate cells without chloramphenicol ⁇ 100%.

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Abstract

提供了一种基于转录组测序筛选棒杆菌组成型表达载体启动子的方法，还提供了基于转录组测序筛选出的棒杆菌组成型表达载体启动子、含有该启动子的表达载体以及该表达载体转化宿主细胞谷氨酸棒杆菌获得的重组菌株及其应用。

Description

一种基于转录组测序筛选的棒杆菌组成型表达载体启动子及其筛选方法和应用
本申请要求申请日为2017年03月21日的中国专利申请CN201710169454.5、CN201710169685.6以及CN201710169693.0的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于生物技术领域，具体涉及一种基于转录组测序筛选的棒杆菌组成型表达载体启动子及其筛选方法和应用，本发明还涉及含有所述启动子的表达载体及重组菌株。
背景技术
棒杆菌是一类革兰氏阳性菌，棒杆菌的三个主要代表：谷氨酸棒杆菌(谷氨酸棒状杆菌，Corynebacterium glutamicum)、黄色短杆菌和乳糖发酵短杆菌，已经被广泛用于生产氨基酸和核苷酸等多种化学物质(Liebl等，1991)。经过物理或化学方法诱变处理得到的棒杆菌突变株具有较强的合成目的有用物质的能力，而通过遗传工程和代谢工程对棒杆菌野生菌株或突变株进行改造，可以获得具有更高生产强度的菌株。代谢工程改造即是在棒杆菌的多种代谢途径中寻找关键的代谢酶，通过基因工程改造调控关键代谢酶的基因表达。通常基因都是在启动子的控制下表达，其表达强度依赖于启动子元件。来自大肠杆菌、链霉菌和枯草芽孢杆菌的启动子能够在棒杆菌属细菌中表达，可应用于棒杆菌的载体系统构建，如应用来源于大肠杆菌的Ptac、Ptrc启动子构建的表达质粒PXMJ19，PEC-XK99E，基因在lacIq的控制下进行诱导表达，但表达量低，且在生产过程中需要添加诱导剂IPTG，而IPTG相当昂贵，不适于用做大规模生产目的产品的基因表达诱导剂；乳糖能够用来替代IPTG作为诱导物应用于大规模的生产，而对于绝大多数的棒杆菌菌株而言，乳糖都不能进入其细胞中(Brabetz等,1991)，这也限制了诱导型表达系统在棒杆菌中的应用。
组成型启动子不需诱导等特殊条件即可在菌体存活期持续不断地表达外源基因，从而简化了操作过程、且具有相对较高的安全性，因此更适合在实际生产中应用。在不同细菌的管家基因中应用PCR扩增DNA探针等手段筛选并纯化具有强表达性的组成型基因，筛选出组成型的强启动子，用于重组载体的构建(Jensen P R et al.,Appl Environ Microbiol,1998,64(1):82-87.)。杨柳等利用谷氨酸棒杆菌ATCC 13032的内源强启动子Pgro有效促使外源基因xylA在谷氨酸棒杆菌ATCC 13032中的表达(杨柳等，新能源进展， 2014,5(2):353-357)，王小元等通过突变得到转录启动功能的tac-M启动子，构建组成型表达载体PDXW-10，在棒杆菌中，该载体表达外源蛋白的水平适中(CN：200910210962.9)。组成型启动子表达虽然具有很多优点，但目前筛选方法复杂，且如何选择合适的启动子进行调控，以避免因为过量的蛋白表达而导致宿主菌生长停滞或代谢障碍，或使宿主能量耗竭以及质粒丢失是工业化生产亟待解决的问题。目前存在使用如Pgro作为启动子，与目的基因一起构建组成表达型质粒，这类质粒在细菌对数生长期也进行目的基因的表达，导致质粒在稳定期之前丢失率达到40％以上，从而降低了表达效果。
发明内容
本发明要解决的技术问题是：目前的组成型启动子筛选方法复杂，而且筛选的启动子构建的表达质粒在宿主生长中容易丢失，还会影响宿主的生长与代谢。如何建立一个组成型启动子的快速筛选方法，且筛选出稳定期启动活力高的内源启动子，且启动子构建的质粒在菌体生长的稳定期能保证质粒的稳定性是我们需要解决的问题。
本发明的目的就是建立一种方法，寻找细菌对数生长期沉默，细菌生长停滞期高效表达的启动基因，用它来构建组成型高效表达质粒。在本发明中，我们应用转录组测序的分析数据，寻找一类能调控目的基因在对数期表达弱，而稳定期大量表达的启动子，一般这类启动子都是可调控型启动子，我们截取这些启动子不同的基因片段，构建了一系列棒杆菌组成型表达载体，该系列表达载体应用绿色荧光蛋白(EGFP)作为标记基因，探测各启动子片段活性强弱，且考核各探测质粒传代稳定性，从而筛选出一系列对数期启动子活力低、稳定期启动活力高的启动子，并考核含各启动子的质粒传代稳定性，进一步筛选出使质粒稳定的启动子。
本方面所采用的技术方案是：
一种基于转录组测序筛选棒杆菌组成型表达载体启动子的方法，分析棒杆菌在对数期与稳定期各基因的转录水平，通过对各基因在两个时期的转录丰度分析，筛选出一类对数期转录水平低，而稳定期转录水平高的基因，通过启动子预测软件分析基因的启动子区域，使用PCR扩增这类基因的启动子片段，并将启动子的DNA片段连入表达载体中，插入标记基因，构建启动子探测载体，电转化探测载体到宿主细胞中，培养宿主细胞到对数期和稳定期，多功能酶标仪检测细菌生长的OD与荧光蛋白表达情况，同时在无抗性压力的情况下，进行连续传代，选择探测载体对数期荧光值低、稳定期荧光值高且连续传代50代而质粒不丢失的宿主细胞，其含有的启动子即为棒杆菌组成型表达载体启动子。
一种基于上述方法筛选出的棒杆菌组成型表达载体启动子，其核苷酸序列如SEQ ID NO：1、SEQ ID NO：2或者SEQ ID NO：3所示。
一种棒杆菌组成型表达载体，含有以上所述的棒杆菌组成型表达载体启动子。
所述的棒杆菌组成型表达载体，它是在质粒HY-P19的酶切位点插入以上所述的棒杆菌组成型表达载体启动子和目的基因构建而成，所述质粒HY-P19的核苷酸序列如SEQ ID NO：4所示，所述目的基因的核苷酸序列如SEQ ID NO：5、SEQ ID NO：6或SEQ ID NO：7所示。其中，当所述的棒杆菌组成型表达载体启动子序列如SEQ ID NO：1所示时，插入的所述目的基因的核苷酸序列如SEQ ID NO：5所示；当所述的棒杆菌组成型表达载体启动子序列如SEQ ID NO：2所示时，插入的所述目的基因的核苷酸序列如SEQ ID NO：6所示；当所述的棒杆菌组成型表达载体启动子序列如SEQ ID NO：3所示时，插入的所述目的基因的核苷酸序列如SEQ ID NO：7所示。
所述的表达载体电转化宿主细胞谷氨酸棒杆菌获得的重组菌株，所述的宿主细胞谷氨酸棒杆菌较佳地为保藏编号为CCTCC NO：M2016609的菌株，该菌株于2016年11月01日保藏于中国典型培养物保藏中心。
所述的重组菌株在生产异亮氨酸中的应用。
本发明的有益效果：此类启动子构建的表达载体，对数期目的基因蛋白表达量低，但稳定期目的蛋白表达量相对于对数期大幅度提高，且对宿主菌生长影响低，质粒传代稳定性高，适合于对数期不需要表达，而稳定期需要高度表达的目的基因的载体构建。尤其适合于氨基酸发酵用工程菌株的构建。
本发明筛选出的启动子、表达载体和重组菌株在发酵生产异亮氨酸时，可提高产酸量和糖酸转化率(转化率＝发酵液中产目标氨基酸的总重量/发酵使用的葡萄糖重量×100％)，降低杂酸含量，具有很高的应用前景。
生物材料保藏信息
本发明的谷氨酸棒杆菌H5，已于2016年11月01日保藏在中国典型培养物保藏中心(CCTCC)，并于2016年11月12日检测为存活状态，保藏地址：中国武汉武汉大学，邮编430072，保藏编号为：CCTCC NO:M2016609，培养物名称是谷氨酸棒杆菌H5，Corynebacterium glutamicum H5，分类命名是谷氨酸棒杆菌Corynebacterium glutamicum。
附图说明
图1为原始质粒PXMJ19。
图2为合成的DNA片段hy-dna1。
图3为体外重组后构建的质粒HY-19。
图4为重组后的质粒HY-P19-启动子-EGFP。
具体实施方式
下面通过实施例对本发明进行详细地说明。
实施例1
1、转录组测序样本制备与转录信息分析：
细菌培养：挑一环不带质粒的生产异亮氨酸的谷氨酸棒杆菌(谷氨酸棒杆菌H5菌株，于2016年11月01日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M2016609)，在LB培养基中，31℃培养过夜，将菌液取出离心，用等体积的无菌水重悬，以5％的接种量接入LBG新鲜灭菌的培养基中，31℃，200rpm培养至对数期中期与稳定期前期，取出发酵液，迅速与等体积
Figure PCTCN2018079034-appb-000001
Bacteria Reagent(QIAGEN)混合，5000rpm离心10min，称量100mg湿菌体，迅速放入液氮中保存。
RNA提取：将液氮中保存的样品放入用液氮预冷的研钵中，加入适量液氮，保证研钵中的液氮不干，将样品研磨成粉末状，然后按照QIAGEN RNeasy Plant Mini Kit的实验步骤进行RNA提取。提取后的样品跑1％的琼脂糖凝胶检测，要求无蛋白污染，即琼脂糖胶的胶孔无明显亮带。用NanoDrop Spectrophotometer进行核酸浓度检测，对数期样品浓度421ng/ul，稳定期样品浓度237ng/ul，达到送样无明显蛋白污染、核酸浓度>60ng/ul测序要求。
2、转录组测序：提取RNA样品委托北京诺禾致源生物信息科技有限公司进行测序。测序流程如下：样品检测合格后，通过Ribo-zero试剂盒去除rRNA来富集mRNA。随后加入fragmentation buffer将mRNA打断成短片段，以mRNA为模板，用六碱基随机引物(random hexamers)合成一链cDNA，然后加入缓冲液、dNTPs(dNTP中的dTTP用dUTP取代)和DNA polymerase I和RNase H合成二链cDNA，再用AMPure XP beads纯化双链cDNA，之后用USER酶降解含有U的cDNA第二链。纯化的双链cDNA先进行末端修复、加A尾并连接测序接头，再用AMPure XP beads进行片段大小选择。最后进行PCR扩增，并用AMPure XP beads纯化PCR产物，得到最终的文库。文库构建完成后，先使用Qubit2.0进行初步定量，稀释文库至1ng/ul，随后使用Agilent 2100对文库的插入片段长度(insert size)进行检测，insert size符合预期后，使用Q-PCR方法对文库的有效浓度进行准确定量(文库有效浓度＞2nM)，以保证文库质量。库检合格后，把不同文库按照 有效浓度及目标下机数据量的需求pooling后进行HiSeq/MiSeq测序。
3、生物信息分析流程：测序结果委托北京诺禾致源生物信息科技有限公司进行信息分析。获得原始测序序列(Sequenced Reads)后，在有相关物种参考序列或参考基因组的情况下，进行生物信息分析。生物信息分析主要流程如下：
原始序列数据→测序数据质量评估→参考序列比对分析→基因表达水平分析→RNA-seq整体质量评估→基因差异表达分析→差异基因GO富集分析、差异基因KEGG富集分析。
本发明样本的生物信息分析参考物种为谷氨酸棒杆菌ATCC 13032(购自上海复祥生物科技有限公司)，参考基因组在ncbi上链接为https://www.ncbi.nlm.nih.gov/nuccore/NC_006958.1。根据对最终提供的信息分析数据表，对各基因在对数期与稳定期的表达丰度分析，筛选得到表一的10个基因与转录分析信息，这10个基因的一致特点为：对数期转录水平低，稳定期转录水平高，通过对操纵子预测，基因CGTRNA_RS10085、CGTRNA_RS10080属于同一个操纵子，CGTRNA_RS07920、CGTRNA_RS07925、CGTRNA_RS07930属于同一个操纵子、CGTRNA_RS00965、CGTRNA_RS00970属于同一个操纵子，同一个操纵子共用一个启动子。
表一：谷氨酸棒杆菌转录组分析S期转录丰度高L期丰度低基因
基因名称 Strand start end length S1_fpkm L1_fpkm
CGTRNA_RS10085 - 2142201 2143517 1317 27347.52 148.9929
CGTRNA_RS10080 - 2141798 2142136 339 24497.2 543.2887
CGTRNA_RS10840 + 2320501 2321934 1434 13058.69 838.0642
CGTRNA_RS07910 - 1672737 1673144 408 7453.836 37.12523
CGTRNA_RS07920 - 1674757 1675572 816 3383.739 15.18759
CGTRNA_RS07925 - 1675587 1676735 1149 2442.069 7.789865
CGTRNA_RS07930 - 1676732 1678090 1359 5493.98 22.03821
CGTRNA_RS00965 + 195241 199773 4533 6137.992 54.29964
CGTRNA_RS00970 + 199773 201293 1521 4023.859 33.94991
CGTRNA_RS04670 + 988209 989480 1272 2037.631 8.931068
4、启动子区域筛选：通过NCBI blast功能，将预测出来的基因片段与实验室测序出来的谷氨酸棒杆菌基因组进行比对，找到相应的基因序列。通过查阅文献或应用启动子预测软件，预测各基因的启动子区域，每个基因的启动子区域根据预测的功能区选择2-3个片段进行启动子活性检测。
(1)基因CGTRNA_RS10085的启动子区片段：
RS10085seq1:
Figure PCTCN2018079034-appb-000002
RS10085seq2:
Figure PCTCN2018079034-appb-000003
RS10085seq3:
Figure PCTCN2018079034-appb-000004
(2)基因CGTRNA_RS10840的启动子区片段
RS10840seq1:
Figure PCTCN2018079034-appb-000005
RS10840seq2:
Figure PCTCN2018079034-appb-000006
(3)基因CGTRNA_RS07910的启动子区片段
RS07910seq1:
Figure PCTCN2018079034-appb-000007
RS07910seq2:
Figure PCTCN2018079034-appb-000008
Figure PCTCN2018079034-appb-000009
(4)基因CGTRNA_RS07930的启动子区片段
RS07930seq1:
Figure PCTCN2018079034-appb-000010
RS07930seq2:
Figure PCTCN2018079034-appb-000011
RS07930seq3:
Figure PCTCN2018079034-appb-000012
(5)基因CGTRNA_RS00965的启动子区片段
RS00965seq1:
Figure PCTCN2018079034-appb-000013
RS00965seq2:
Figure PCTCN2018079034-appb-000014
(6)基因CGTRNA_RS04670的启动子区片段
RS04670seq1:
Figure PCTCN2018079034-appb-000015
RS04670seq2:
Figure PCTCN2018079034-appb-000016
5、启动子探测载体构建及荧光检测：
(1)引物合成：通过snapgene软件对引物进行设计，委托武汉天一辉远进行引物合成。各合成引物序列见下表二。
表二：构建探测载体的各引物序列
Figure PCTCN2018079034-appb-000017
(2)基因组提取：挑取一环谷氨酸棒杆菌H5，接种于LB培养基中，31℃培养过夜，离心去上清，菌体基因组提取使用天根基因组提取试剂盒的步骤进行提取，提取后 的基因组使用NanoDrop Spectrophotometer进行核酸浓度检测，控制进行PCR扩增的核酸浓度在100-200ng/ul之间。
(3)PCR扩增：将各合成引物加水稀释到终浓度10uM，，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：基因组模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。PCR扩增过程为95℃2min，95℃20s，55℃20s，72℃10s-1min，72℃5min，循环数为32个，得到扩增DNA片段分别为带重组接头的RS10085seq1、RS10085seq2、RS10085seq3、RS10840seq1、RS10840seq2、RS07910seq1、RS07910seq2、RS07930seq1、RS07930seq2、RS07930seq3、RS00965seq1、RS00965seq2、RS04670seq1、RS04670seq2、EGFP。
(4)质粒酶切与DNA合成：使用原始质粒是PXMJ19，用NEB限制性内切酶NarI与HindIII进行双酶切，去掉原始的lacIq和启动子Ptac，酶切体系为50ul体系：质粒5-10ul，NarI1ul，HindIII 1ul，cutsmart 5ul，ddH 2O 33-38ul。
委托武汉天一辉远合成DNA序列：hy-dna1
Figure PCTCN2018079034-appb-000018
(5)DNA琼脂糖电泳回收：将PCR产物与酶切产物加入10*loading buffer，点样50ul于1.5％琼脂糖凝胶，以takara 2000 DL DNA marker为对照，在70V电压下电泳40min。根据marker条带回收PCR扩增条带。回收使用takara DNA回收试剂盒进行回收，DNA片段分别为带重组接头的RS10085seq1(167bp)、RS10085seq2(180bp)、RS10085seq3(240bp)、RS10840seq1(107bp)、RS10840seq2(282bp)、RS07910seq1(110bp)、RS07910seq2(241bp)、RS07930seq1(93bp)、RS07930seq2(118bp)、RS07930seq3(229bp)、RS00965seq1(118bp)、RS00965seq2(241bp)、RS04670seq1(99bp)、RS04670seq2(247bp)、EGFP(760bp)。
(6)体外重组：使用Vazyme one Step Cloning Kit试剂盒将酶切产物与合成的DNA片段hydna1进行体外重组，重组产物直接转化大肠杆菌。
(7)大肠杆菌感受态制备：大肠杆菌感受态DH5α制备使用碧云天细菌超级感受态试剂盒进行感受态制备，制备后感受态保存于-80℃冰箱备用。
(8)转化：在冰上缓慢融解感受态细胞DH5α，将体外重组产物加入感受态细胞内，轻轻用手指弹动离心管，以混匀细菌和重组产物，冰浴或冰水浴放置30分钟，42℃水浴，热休克2分钟。热休克后立即置于冰水浴中，2分钟。加入900微升LB，37℃200rpm培养1小时。离心去上清，将菌体涂布于含氯霉素25ug/ml的LB固体平板中，37℃培养过 夜。
(9)转化子验证与质粒提取：将转化后在抗性平板上长出来的转化子，送武汉天一辉远进行测序分析，序列正确的转化子转接5ml LB试管中加入终浓度25ug/ml的氯霉素，37℃摇床，200rpm培养过夜，使用takara plasmid kit试剂盒进行质粒提取，使提取的质粒浓度在200-400ng/ul之间，构建的质粒命名为HY-P19，序列如SEQ ID NO：4所示。
将HY-P19使用NEB限制性内切酶XbaI和EcoRI，在37℃水浴锅内进行双酶切1h，酶切质粒量为1ug，酶切体系为50ul体系:质粒5-10ul,XbaI 1ul，EcoRI1ul，cutsmart 5ul，ddH 2O 33-38ul。回收HY-P19的酶切产物，使用Vazyme one Step Cloning Kit试剂盒将酶切产物与各启动子片段与EGFP片段进行体外重组，其他实验步骤同上(7)-(9)。
绿色荧光蛋白EGFP的片段DNA seq：720bp
Figure PCTCN2018079034-appb-000019
构建出一系列含有不同启动子的探测载体的质粒HY-P19-promoter-EGFP，构建流程为：将图1中的原始质粒PXMJ19用NarI和HindIII双酶切，酶切后回收酶切产物，将酶切产物与图2中合成的DNA片段用一步克隆的方法进行体外重组，转化DH5α后，挑取阳性克隆，测序验证后，提取质粒，得到图3的质粒HY-P19。将HY-P19用XbaI和EcoRI进行双酶切，回收酶切产物，与系列的合成启动子片段与PCR扩增的EGFP片段，进行一步克隆体外重组，转化DH5α后，挑取阳性克隆，测序验证后，提取质粒，得到 图4的系列启动子与标记基因EGFP的探测载体。含各启动子片段的探测载体分别命名为PRS10085seq1、PRS10085seq2、PRS10085seq3、PRS10840seq1、PRS10840seq2、PRS07910seq1、PRS07910seq2、PRS07930seq1、PRS07930seq2、PRS07930seq3、PRS00965seq1、PRS00965seq2、PRS04670seq1、PRS04670seq2。
(10)谷氨酸棒杆菌电转感受态细胞的制备：
①从新鲜的LB(大约培养12h)平板上面挑取一个单菌落，接入到装有5mL LBG液体的试管中，30℃，220rpm过夜振荡培养。
②取2mL菌体培养液接入到装有100mL EPO(酵母粉5g/L，蛋白胨10g/L，Nacl 10g/L，甘氨酸25g/L，吐温1g/L)培养基的1L三角瓶中，30℃振荡培养至OD为1.0-1.5之间(约为4-5h)。
③无菌条件下把培养液全部转移到4℃预冷的50mL离心管中，在冰上放置2分钟。
④4℃，7000rpm冷冻离心20分钟。去除上清液之后，倒置离心管于无菌的滤纸上面吸收残留的Epo培养基。
⑤用40mL 4℃预冷的10％甘油溶液重悬菌体并轻轻摇匀，7000rpm冷冻离心10分钟。倒去上清液。
⑥重复第5步2次。
⑦移液器吸取1mL预冷的10％甘油溶液，加入到离心管中并轻轻吹打重悬菌体。
⑧最后将感受态细胞用1.5mL的无菌EP管分装，每管0.1mL。放入超低温冰箱中-80℃保存。
(11)谷氨酸棒杆菌的电转化：
①取出一支-80℃保存的谷氨酸棒杆菌感受态细胞，插在冰上直到融化。
②加入样品质粒DNA到融化的感受态细胞中，用手指轻弹EP管，确保DNA和感受态细胞混合均匀。
③用移液器将含有DNA质粒的感受态细胞吸入到预冷的电击杯(孔径2mm中，用卫生纸迅速擦干。
④将电击杯放置到电转仪中，1800V电穿孔5毫秒。
⑤迅速将细胞液吸入到46℃预热的装有5mL LBHIS的EP管中，精确计时热击6分钟。
⑥EP管于30℃，200rpm振荡培养，细胞复苏60分钟。
⑦7000rpm室温离心2分钟，弃去大部分上清液，用剩余培养基重悬菌体，用玻璃棒涂布在添加10ug/mL氯霉素的LBHIS固体培养基上。
⑧室温放置1小时左右待表面液体被充分吸收后，倒置平板于30℃恒温培养箱培养36h，长出来的转化子即为含各探测载体的菌株。
(12)谷棒转化子荧光检测
将含有不同启动子片段的谷棒转化子，转接5ml LBG试管培养基中，30℃培养至OD600分别为2.0(对数期)、3.2(稳定期)。取出菌体，离心去上清后，用PBS溶液洗涤两次，用同体积的PBS进行重悬，重悬后的菌体，吸取200ul加入黑底96孔板中，使用多功能酶标仪TECANM1000进行荧光值检测。各探测质粒在宿主菌中对数期与稳定期检测荧光值见表三。从表中我们可以得出；同一个基因启动子，选取不同的启动子片段进行荧光值检测，得到不同的启动子活力数据，这与基因启动子区域的调控有关，我们需要筛选的即为对数期荧光值低、稳定期荧光值高的启动子片段。启动子片段RS10085seq2、RS10085seq3、RS10840seq2、RS07910seq2、RS07930seq3、RS00965seq2、RS04670seq2在探测载体的表达中，表现出我们所需的筛选特征。
表三：各探测质粒在宿主菌中两个时期荧光检测情况与质粒传代统计表
各探测质粒 对数期荧光值 稳定期荧光值 各质粒稳定传代代数
对照-无质粒 2871 8024 ——
PRS10085seq1 150352 231073 8
PRS10085seq2 6364 241445 50
PRS10085seq3 6191 225450 50
PRS10840seq1 102254 184533 6
PRS10840seq2 11022 177132 50
PRS07910seq1 9551 102310 6
PRS07910seq2 3564 98756 50
PRS07930seq1 12029 146021 5
PRS07930seq2 8211 133602 8
PRS07930seq3 3321 122564 50
PRS00965seq1 11083 85472 11
PRS00965seq2 5412 77450 50
PRS04670seq1 5441 35390 14
PRS04670seq2 3698 28428 50
注：传代稳定性指质粒有90％以上菌体含有质粒，传代50次，指传50次，质粒还稳定90％以上。
6、各启动子探测载体的质粒传代稳定性
将含有各启动子探测载体的重组菌，接入LBG培养基中，在无抗性压力的条件下，30℃旋转摇床，190rpm培养24h为一代；传代的接种量为5％，连续进行传代培养，每代培养的细菌进行稀释涂平板，稀释相同的浓度分别涂布于含氯霉素和不含氯霉素的平板中，质粒丢失率＝(不含氯霉素的平板菌体个数-含氯霉素的平板菌体个数)/不含氯霉素的平板菌体个数×100％,质粒丢失率≤10％，我们定义为此传代质粒稳定，当质粒丢失率>10％则认为此代质粒已不稳定。含各探测质粒的菌体的稳定传代代数见表三。
从表三我们可以得出质粒PRS10085seq2、PRS10085seq3、PRS10840seq2、PRS07910seq2、PRS07930seq3、PRS00965seq2、PRS04670seq2传到50代，质粒丢失率≤10％，表现出良好的稳定性。
实施例2 应用启动子片段RS10085seq2构建的表达载体及其应用
1、表达载体HY-P19-RS10085seq2-ilvA的构建
谷氨酸棒杆菌异亮氨酸代谢关键基因为苏氨酸脱水酶，其基因代号为ilvA(序列如SEQ ID NO：5所示)，使用筛选出来的启动子片段RS10085seq2作为启动子，ilvA为目的基因，构建表达载体HY-P19-RS10085seq2-ilvA。
DNA片段扩增：使用质粒PRS10085seq2为模板，上下游引物为：
RS10085seq2Fp:tgcctgcaggtcgactctagaCTATTCTATAGATCTATTG
RS10085seq2Rp:AGCGTGGATGACCTCCTTTGA
扩增DNA片段RS10085seq2，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：PRS10085seq2模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。PCR扩增过程为95℃2min，95℃20s，55℃20s，72℃10smin，72℃5min，循环数为32个。
以谷氨酸棒杆菌H5基因组为模板，上下游引物为：
ilvAFP：AAAGGAGGTCATCCACGCTATGAGTGAAACATACGTGTCTGAG
ilvARP：caaaacagccaagctgaattcTTAGGTCAAGTATTCGTACTCAG
扩增DNA片段ilvA，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：基因组模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。PCR扩增过程为95℃2min，95℃20s，58℃20s，72℃30s，72℃5min，循环数为32个。
质粒酶切：使用的质粒是PXMJ19去掉原始的lacIq和promoter改造后的HY-P19。 将HY-P19使用NEB限制性内切酶XbaI和EcoRI，在37℃水浴锅内进行双酶切1h，酶切质粒量为1ug，酶切体系为50ul体系：质粒5ul，XbaI 1ul,EcoRI1ul，cutsmart 5ul，ddH 2O 38ul。
DNA琼脂糖电泳回收：将PCR产物与酶切产物加入10*loading buffer，点样50ul于1.5％琼脂糖凝胶，以takara 2000 DL DNA marker为对照，在70V电压下电泳40min。根据marker条带回收PCR扩增160bp、1347bp条带。回收使用takara DNA回收试剂盒进行回收。
体外重组：使用Vazyme one Step Cloning Kit试剂盒进行体外重组，重组产物直接转化大肠杆菌。
大肠杆菌感受态制备：同启动子探测载体构建。
转化：在冰上缓慢融解感受态细菌，将10ul体外重组产物加入100ul感受态细胞内，轻轻用手指弹动离心管，以混匀细菌和重组产物。冰浴或冰水浴放置30分钟。42℃水浴，热休克2分钟。热休克后立即置于冰水浴中，2分钟。加入900微升LB，37℃200rpm培养1小时。离心去上清，将菌体涂布于含氯霉素25ug/ml的LB固体平板中，37℃培养过夜。
转化子验证与质粒提取：将转化后在抗性平板上长出来的转化子，送武汉天一辉远进行测序分析，挑取序列正确的转化子转接5ml LB试管中加入终浓度25ug/ml的氯霉素，37℃摇床，200rpm培养过夜，使用takara plasmid kit试剂盒进行质粒提取，表达质粒HY-P19-RS10085seq2-ilvA浓度为269ng/ul.
2、含表达载体HY-P19-RS10085seq2-ilvA的菌株的构建
谷氨酸棒杆菌电转感受态细胞的制备：同启动子探测载体构建。
表达质粒HY-P19-RS10085seq2-ilvA电转化宿主细胞谷氨酸棒杆菌H5：同启动子探测载体构建。
将电转后的菌液离心浓缩，用玻璃棒涂布在添加10ug/mL氯霉素的LBHIS固体培养基上，30℃培养箱中培养36h，长出来的转化子即为带质粒HY-P19-RS10085seq2-ilvA的菌株，菌株命名为H5-RS10085seq2-ilvA。
3、菌株H5-RS10085seq2-ilvA与谷氨酸棒杆菌H5在5L发酵罐发酵产酸
5L发酵罐发酵培养基：玉米浆15ml/L，葡萄糖140g/L(分消，0.075MPa湿热灭菌15min)，硫酸铵5g/L，磷酸二氢钾0.4g/L，七水硫酸镁0.6g/L，生物素0.1mg/L，VB1 0.1mg/L，玉米油1ml/L，安琪酵母粉1g/L，消泡剂1ml/L,121℃0.01MPa湿热灭菌25min；灭菌后加入初糖，用氨水调pH为7.0。
培养方法：将菌种接入种子培养基(种子培养基配方：葡萄糖17g/L，玉米浆10ml/L，尿素1g/L，硫酸镁0.5g/L，磷酸氢二钾1g/L，酵母膏0.1g/L，生物素0.1mg/L，维生素B10.1mg/L，玉米油0.1g/100ml，碳酸钙1g/100ml；用NaOH调pH7.0)，31℃旋转摇床，200rpm,培养16h后，以10％接种量接入含有发酵培养基的5L自动控制发酵罐中，通入适当空气，调节适当搅拌转速，采用分阶段供氧模式控制溶氧：0-8h为30％，8-24h为25％，24-56h为12％，通过自动流加15％的氨水控制pH在7.0，通过流加适量泡敌消泡，并通过流加浓度为800g/L的葡萄糖溶液将残糖控制在3％左右，发酵56h结束。
放罐时，发酵液通过HPLC检测(依力特C18色谱柱，5μm，4.6*250mm，流动相为0.015mol/L的磷酸氢二铵及0.005mol/L pH7.20的混合水溶液。流速为0.5ml/min。柱温为23℃。检测器为紫外检测器，波长199nm。对照菌谷氨酸棒杆菌H5发酵产酸22.0g/L，杂酸含量6.3g/L，糖酸转化率12.83％，工程菌H5-RS10085seq2-ilvA发酵产酸41.2g/L，杂酸含量6.8g/L，糖酸转化率18.79％。
4、菌株H5-RS10085seq2-ilvA 5L发酵罐发酵质粒稳定性
将56h放罐菌进行稀释涂平板，稀释10 -6、10 -7、10 -8、10 -9的梯度浓度，每个浓度梯度分别吸取50ul菌液，涂布于含氯霉素和不含氯霉素的平板中，放置于31℃培养箱中培养36h，根据无抗性平板和抗性平板上长出的菌落数计算质粒丢失率，质粒丢失率＝(不含氯霉素的平板菌体个数-含氯霉素的平板菌体个数)/不含氯霉素的平板菌体个数×100％。在10 -8稀释梯度的平板上，含氯霉素平板长出151个菌落，无氯霉素平板长出167个菌落，质粒丢失率仅为9.58％，质粒在发酵中稳定。
实施例3 应用启动子片段RS07910seq2构建的表达载体及其应用
在菌体代谢过程中，并不是所有的基因超强表达能增加代谢向目的产物流动，有很多基因表达以后产生的生物酶，在代谢中需要达到一个适度表达的平衡点时，反而能发挥作用。研究表明，谷氨酸棒杆菌内的广泛调节蛋白Lrp(其编码核苷酸为目的基因Lrp，核苷酸序列如SEQ ID NO：6所示)能提高谷氨酸棒杆菌L-异亮氨酸的合成和转运相关基因的转录水平，还能提高自身编码基因lrp转录水平，但Lrp的过量表达对谷氨酸棒杆菌的生长产生抑制作用，我们使用启动子片段PRS07910seq2作为启动子，以lrp为目的基因，构建外源表达质粒HY-P19-RS07910seq2-lrp，并将表达质粒电转化到异亮氨酸生产菌谷氨酸棒杆菌H5体内，构建新的菌株，并进行5L发酵考核菌株的产酸能力。
1、表达载体HY-P19-RS07910seq2-lrp的构建
使用筛选出来的启动子片段RS07910seq2作为启动子，lrp为目的基因，构建表达载体HY-P19-RS07910seq2-lrp。
DNA片段扩增：使用质粒PRS07910seq2为模板，上下游引物为：
RS07910seq2Fp:tgcctgcaggtcgactctaga GCGATCACGTAGTCATCCAAG
RS07910seq2Rp:CACGGTCACGGATGAAGCAT
扩增DNA片段RS07910seq2，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：P RS07910seq2模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。PCR扩增过程为95℃2min，95℃20s，55℃20s，72℃10smin，72℃5min，循环数为32个。以谷氨酸棒杆菌H5基因组为模板，上下游引物为：
lrpFP：GCTTCATCCGTGACCGTGAtgaagctagattccattgatt
lrpRP：caaaacagccaagctgaattctcacacctgggggcgagc
扩增DNA片段lrp，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：基因组模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。PCR扩增过程为95℃2min，95℃20s，56℃20s，72℃20s，72℃5min，循环数为32个。
质粒酶切：使用的质粒是PXMJ19去掉原始的lacIq和promoter改造后的HY-P19。将HY-P19使用NEB限制性内切酶XbaI和EcoRI，在37℃水浴锅内进行双酶切1h，酶切质粒量为1ug，酶切体系为50ul体系：质粒5ul，XbaI 1ul,EcoRI1ul，cutsmart 5ul，ddH 2O 38ul。
后续的体外重组、大肠杆菌感受态制备、转化以及转化子验证与质粒提取等步骤的操作均同实施例2，最终获得的表达质粒HY-P19-RS07910seq2-lrp浓度为321ng/ul。
2、含表达载体HY-P19-RS07910seq2-lrp的菌株的构建
谷氨酸棒杆菌电转感受态细胞的制备：同启动子探测载体构建。
表达质粒HY-P19-RS07910seq2-lrp电转化宿主细胞谷氨酸棒杆菌：同启动子探测载体构建。
将电转后的菌液离心浓缩，用玻璃棒涂布在添加10ug/mL氯霉素的LBHIS固体培养基上，30℃培养箱中培养36h，长出来的转化子即为带质粒HY-P19-RS07910seq2-lrp的菌株，菌株命名为H5-RS07910seq2-lrp。
3、菌株H5-RS07910seq2-lrp与谷氨酸棒杆菌H5在5L发酵罐发酵产酸
5L发酵罐发酵培养基的配方、菌株的培养方法以及放罐时发酵液的检测方法均同实施例2，测得工程菌H5-RS07910seq2-lrp，发酵产酸39.1g/L，杂酸含量6.9g/L，糖酸转化率达17.88％。
4、菌株H5-RS07910seq2-lrp 5L发酵罐发酵质粒稳定性
将56h放罐菌进行稀释涂平板，稀释10 -6、10 -7、10 -8、10 -9的梯度浓度，每个浓度梯度分别吸取50ul菌液，涂布于含氯霉素和不含氯霉素的平板中，放置于31℃培养箱中培养36h，根据无抗性平板和抗性平板上长出的菌落数计算质粒丢失率，质粒丢失率＝(不含氯霉素的平板菌体个数-含氯霉素的平板菌体个数)/不含氯霉素的平板菌体个数×100％。在10 -8稀释梯度的平板上，含氯霉素平板长出127个菌落，无氯霉素平板长出135个菌落，质粒丢失率仅为5.93％，质粒在发酵中稳定。
实施例4 应用启动子片段RS04670seq2构建的表达载体及其应用
在菌体代谢生产异亮氨酸的过程中，1mol异亮氨酸需要消耗4mol辅因子NADPH，NADPH主要来源于磷酸戊糖途径，生物体内NADPH通过此条途径供应需要消耗其他代谢产物，改变代谢途径的碳流量，可能会影响目的代谢物的积累，而通过转录组分析，产异亮氨酸的谷氨酸棒杆菌在稳定期，磷酸戊糖途径中的6-磷酸葡萄糖脱氢酶转录下调，所以为了保证稳定期代谢过程中辅因子NADPH的供应，又不对产物的积累造成影响，我们将6-磷酸葡萄糖脱氢酶的基因gnd(序列如SEQ ID NO：7所示)与一个启动子相连，启动此基因的表达。
1、表达载体HY-P19-RS04670seq2-gnd的构建
使用筛选出来的启动子片段RS04670seq2作为启动子，gnd为目的基因，构建表达载体HY-P19-RS04670seq2-gnd。
DNA片段扩增：使用质粒PRS04670seq1为模板，上下游引物为：
RS04670seq2Fp:tgcctgcaggtcgactctagaAGGCTGACAGAAACTCTAAAAAC
RS04670seq2Rp:GGATACCTCCGAAGTTAAGG
扩增DNA片段RS04670seq1，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：P RS04670seq1模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。PCR扩增过程为95℃2min,95℃20s，55℃20s，72℃10smin，72℃5min，循环数为32个。以谷氨酸棒杆菌H5基因组为模板，上下游引物为：
Gnd-FP：TTAACTTCGGAGGTATCCAtgaagctagattccattgattg
gnd-RP：ccaaaacagccaagctgaattcTTAAGCTTCCACCTCGGAGCG
扩增DNA片段gnd，使用TransStart FastPfu Fly DNA Polymerase进行扩增，PCR体系为：基因组模板1ul，正向引物(10uM)1ul，反向引物(10uM)1ul，5*TransStart FastPfu FlyBuffer 10ul，2.5mM Dntps 4ul，TransStart FastPfu Fly DNA Polymerase 1ul，ddH 2O 32ul。 PCR扩增过程为95℃2min，95℃20s，56℃20s，72℃1min，72℃5min，循环数为32个。
质粒酶切：使用的质粒是PXMJ19去掉原始的lacIq和promoter改造后的HY-P19。将HY-P19使用NEB限制性内切酶XbaI和EcoRI，在37℃水浴锅内进行双酶切1h，酶切质粒量为1ug，酶切体系为50ul体系：质粒5ul，XbaI 1ul,EcoRI1ul，cutsmart 5ul，ddH 2O 38ul。
DNA琼脂糖电泳回收：将PCR产物与酶切产物加入10*loading buffer，点样50ul于3％、1％琼脂糖凝胶，以takara 2000 DL DNA marker为对照，在70V电压下电泳40min。根据marker条带回收PCR扩增77bp、1481bp条带。回收使用takara DNA回收试剂盒进行回收。
后续的体外重组、大肠杆菌感受态制备、转化以及转化子验证与质粒提取等步骤的操作均同实施例2，最终获得的表达质粒HY-P19-RS04670seq2-gnd浓度为288ng/ul。
2、含表达载体HY-P19-RS04670seq2-gnd的菌株的构建
谷氨酸棒杆菌电转感受态细胞的制备：同启动子探测载体构建。
表达质粒HY-P19-RS04670seq2-gnd电转化宿主细胞谷氨酸棒杆菌：同启动子探测载体构建。
将电转后的菌液离心浓缩，用玻璃棒涂布在添加10ug/mL氯霉素的LBHIS固体培养基上，30℃培养箱中培养36h，长出来的转化子即为带质粒HY-P19-RS04670seq2-gnd的菌株，菌株命名为H5-RS04670seq2-gnd。
3、菌株H5-RS04670seq2-gnd与谷氨酸棒杆菌H5在5L发酵罐发酵产酸
5L发酵罐发酵培养基的配方、菌株的培养方法以及放罐时发酵液的检测方法均同实施例2，测得工程菌H5-RS04670seq2-gnd，发酵产酸33.4g/L，杂酸含量7.08g/L，糖酸转化率达15.31％。
4、菌株H5-RS04670seq2-gnd 5L发酵罐发酵质粒稳定性
将56h放罐菌进行稀释涂平板，稀释10 -6、10 -7、10 -8、10 -9的梯度浓度，每个浓度梯度分别吸取50ul菌液，涂布于含氯霉素和不含氯霉素的平板中，放置于31℃培养箱中培养36h，根据无抗性平板和抗性平板上长出的菌落数计算质粒丢失率，质粒丢失率＝(不含氯霉素的平板菌体个数-含氯霉素的平板菌体个数)/不含氯霉素的平板菌体个数×100％。在10 -8稀释梯度的平板上，含氯霉素平板长出118个菌落，无氯霉素平板长出121个菌落，质粒丢失率仅为2.48％，质粒在发酵中稳定。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅 是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (8)

  1. 一种基于转录组测序筛选棒杆菌组成型表达载体启动子的方法，其特征在于：分析棒杆菌在对数期与稳定期各基因的转录水平，通过对各基因在两个时期的转录丰度分析，筛选出一类对数期转录水平低，而稳定期转录水平高的基因，通过启动子预测软件分析基因的启动子区域，使用PCR扩增这类基因的启动子片段，并将启动子的DNA片段连入表达载体中，插入标记基因，构建启动子探测载体，电转化探测载体到宿主细胞中，培养宿主细胞到对数期和稳定期，多功能酶标仪检测细菌生长的OD与荧光蛋白表达情况，同时在无抗性压力的情况下，进行连续传代，选择探测载体对数期荧光值低、稳定期荧光值高且连续传代50代而质粒不丢失的宿主细胞，其含有的启动子即为棒杆菌组成型表达载体启动子。
  2. 一种基于权利要求1所述的方法筛选出的棒杆菌组成型表达载体启动子，其特征在于，其核苷酸序列如SEQ ID NO：1、SEQ ID NO：2或者SEQ ID NO：3所示。
  3. 一种棒杆菌组成型表达载体，其特征在于，其含有如权利要求2所述的棒杆菌组成型表达载体启动子。
  4. 如权利要求3所述的棒杆菌组成型表达载体，其特征在于，它是在质粒HY-P19的酶切位点插入目的基因和权利要求2所述的棒杆菌组成型表达载体启动子构建而成，所述质粒HY-P19的核苷酸序列如SEQ ID NO：4所示，所述目的基因的核苷酸序列如SEQ ID NO：5、SEQ ID NO：6或者SEQ ID NO：7所示。
  5. 如权利要求4所述的棒杆菌组成型表达载体，其特征在于，当权利要求2中所述的棒杆菌组成型表达载体启动子的核苷酸序列如SEQ ID NO：1所示时，所述目的基因的核苷酸序列如SEQ ID NO：5所示；
    当权利要求2中所述的棒杆菌组成型表达载体启动子的核苷酸序列如SEQ ID NO：2所示时，所述目的基因的核苷酸序列如SEQ ID NO：6所示；
    当权利要求2中所述的棒杆菌组成型表达载体启动子的核苷酸序列如SEQ ID NO：3所示时，所述目的基因的核苷酸序列如SEQ ID NO：7所示。
  6. 如权利要求3～5任一项所述的表达载体电转化宿主细胞谷氨酸棒杆菌获得的重组菌株。
  7. 如权利要求6所述的菌株，其特征在于，所述的宿主细胞谷氨酸棒杆菌的保藏编号为CCTCC NO：M2016609。
  8. 如权利要求6或7所述的重组菌株在生产异亮氨酸中的应用。
PCT/CN2018/079034 2017-03-21 2018-03-14 一种基于转录组测序筛选的棒杆菌组成型表达载体启动子及其筛选方法和应用 WO2018171488A1 (zh)

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