WO2022152125A1 - 具有l-脯氨酸外排功能的蛋白及其应用 - Google Patents

具有l-脯氨酸外排功能的蛋白及其应用 Download PDF

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WO2022152125A1
WO2022152125A1 PCT/CN2022/071346 CN2022071346W WO2022152125A1 WO 2022152125 A1 WO2022152125 A1 WO 2022152125A1 CN 2022071346 W CN2022071346 W CN 2022071346W WO 2022152125 A1 WO2022152125 A1 WO 2022152125A1
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proline
polypeptide
seq
efflux
corynebacterium glutamicum
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French (fr)
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孙际宾
刘娇
郑平
刘莫识
王钰
周文娟
孙冠男
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中国科学院天津工业生物技术研究所
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Priority to US18/260,861 priority Critical patent/US20240052386A1/en
Priority to EP22739004.4A priority patent/EP4279601A4/en
Publication of WO2022152125A1 publication Critical patent/WO2022152125A1/zh

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Definitions

  • the invention belongs to the fields of molecular biology and bioengineering, in particular to a new use of the threonine efflux protein ThrE of Corynebacterium glutamicum as an L-proline efflux protein, and the production of L-proline by using the protein ThrE Methods of Acids and Derivatives thereof.
  • L-proline a naturally occurring non-essential amino acid in the human body, has a wide range of applications in clinical, biological materials and industry.
  • the production methods of L-proline mainly include chemical method and fermentation method. Due to the serious pollution and high cost of chemical extraction method, it has gradually lost the market. Microbial fermentation method has low production cost, high production intensity, high specificity and environmental pollution. It has become the most widely used method in industry today.
  • the commonly used industrial fermentation strains are Corynebacterium and Escherichia, commonly used Escherichia such as Escherichia coli, commonly used Corynebacterium such as Corynebacterium glutamicum, Brevibacterium such as yellow short bacterium Brevibacterium flavum, Brevibacterium lactofermentus, and certain species of Arthrobacter and certain species of Microbacterium. Due to the physiological superiority of Corynebacterium glutamicum, it has become the most important production strain in the industry to produce amino acids and other products.
  • glutamic acid is mainly used as a substrate through ⁇ -glutamyl kinase (Glutamate-5-kinase, ProB), glutamate-semialdehyde dehydrogenase (Glutamate-semialdehyde dehydrogenase, ProA), pyrroline- 5-carboxylic acid reductase (Pyrroline-5-carboxylic acid reductase, ProC) catalyzes the production of L-proline.
  • the prior art mainly produces L-proline through the genetic transformation of the key enzymes ProA and ProB of the L-proline synthesis pathway. For example, CN101084312A reported that the mutation at position 149 of the ProB protein derived from Corynebacterium glutamicum can relieve L-proline. Feedback inhibition of proline improves the production of L-proline in engineered strains.
  • the amino acids synthesized in the microorganisms are transported to the extracellular in time through the transport pathway, which is also very important for promoting the biosynthesis of amino acids.
  • Expression can significantly increase the lysine production of the engineered strain (CN1283797C).
  • L-proline efflux proteins there has been no report of utilizing L-proline efflux proteins to enhance L-proline production. Therefore, there is an urgent need in the art to excavate effective L-proline efflux proteins, so that L-proline produced in the cell can be efficiently excreted to the outside of the cell to obtain high-yielding L-proline engineered bacteria.
  • the present invention screened the L-proline efflux protein of Corynebacterium glutamicum through the whole genome scale membrane transporter inhibition library. Based on this screening, the present invention screened the thrE (Cgl2622, which has been confirmed in the literature as the encoding gene of threonine efflux protein) gene inhibition, and the L-proline specific yield decreased significantly.
  • SZCgP1 the G149D mutation of the ProB protein introduced by Corynebacterium glutamicum ATCC13869, the codon was mutated from GGT to GAT, see CN101084312A for details
  • ThrE was overexpressed in extracellular proline throughout the fermentation process.
  • ThrE is an L-proline efflux protein.
  • One of the objects of the present invention is to provide the use of proline efflux protein in the production of L-proline or hydroxyproline.
  • Another object of the present invention is to provide a L-proline or hydroxyproline producing strain.
  • Another object of the present invention is to provide a method for producing L-proline or hydroxyproline.
  • Another object of the present invention is to provide a method for constructing L-proline or hydroxyproline producing strains.
  • polypeptide as a proline efflux protein in the production of L-proline and hydroxyproline, the polypeptide is:
  • the polypeptide is a polypeptide derived from the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 or 2, and the amino acid sequence shown in SEQ ID NO: 1 or 2 passes through either or both ends of the two ends.
  • One or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residues are added to form, and have L-proline Acid efflux function.
  • amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2.
  • a L-proline production strain wherein the strain expresses the following polypeptide:
  • the polypeptide is a polypeptide derived from the polypeptide of the amino acid sequence shown in SEQ ID NO: 1 or 2, and the amino acid sequence shown in SEQ ID NO: 1 or 2 passes through either or both ends of the two ends.
  • One or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residues are added to form, and have L-proline Acid efflux function.
  • amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2.
  • the production strains are bacteria.
  • the production strain is selected from Escherichia or Corynebacterium, preferably Escherichia coli or Corynebacterium glutamicum, more preferably Corynebacterium glutamicum, more specifically Corynebacterium glutamicum Bacillus ATCC 13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067.
  • the glutamate kinase in the producing bacteria is not subject to feedback inhibition by L-proline or is weakened by feedback inhibition by L-proline.
  • the activity of glutamate kinase and/or glutamate semialdehyde dehydrogenase and/or pyrroline-5-carboxylic acid reductase in the producing bacteria is enhanced.
  • a method for producing L-proline comprising: culturing the aforementioned production strain to produce L-proline.
  • the method further includes the step of separating L-proline from the fermentation broth.
  • a method for constructing an L-proline-producing strain comprising enhancing the activity of the following polypeptides with L-proline efflux function in the strain:
  • the polypeptide is a polypeptide derived from the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 or 2, and the amino acid sequence shown in SEQ ID NO: 1 or 2 passes through a or several, preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, most preferably 1 amino acid residues are added to form, and have L-proline Ejection function.
  • amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or SEQ ID NO:2.
  • the production strains are bacteria.
  • the production strain is selected from Escherichia or Corynebacterium, preferably Escherichia coli or Corynebacterium glutamicum, more preferably Corynebacterium glutamicum, more specifically Corynebacterium glutamicum Bacillus ATCC 13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067.
  • the glutamate kinase in the producing bacteria is not subject to feedback inhibition by L-proline or is weakened by feedback inhibition by L-proline.
  • the activity of glutamate kinase and/or glutamate semialdehyde dehydrogenase and/or pyrroline-5-carboxylic acid reductase in the production bacterium is enhanced.
  • a host cell in which the activity of the polypeptide with L-proline efflux function is weakened, and the polypeptide with L-proline efflux function activity is:
  • the host cell is selected from the genus Escherichia or Corynebacterium, preferably Escherichia coli or Corynebacterium glutamicum, more preferably Corynebacterium glutamicum, more specifically Corynebacterium glutamicum Bacillus ATCC 13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067.
  • the glutamate kinase in the host cell is not subject to feedback inhibition by L-proline or is weakened by feedback inhibition by L-proline.
  • the activity of proline hydroxylase in the host cell is enhanced.
  • the proline hydroxylase is trans-proline-4-hydroxylase, and its nucleotide sequence is shown in SEQ ID NO: 5.
  • the activity of glutamate kinase and/or glutamate semialdehyde dehydrogenase and/or pyrroline-5-carboxylic acid reductase in the production bacterium is enhanced.
  • a method for producing hydroxyprolines such as trans-4-hydroxy-L-proline comprising: culturing the above-mentioned host cells to produce trans-4- Hydroxyproline such as hydroxylated-L-proline.
  • the method further includes the step of separating hydroxyproline such as trans-4-hydroxy-L-proline from the fermentation broth.
  • hydroxyproline producing strains such as trans-4-hydroxy-L-proline, wherein the method comprises:
  • the activity of glutamate kinase and/or glutamate semialdehyde dehydrogenase and/or pyrroline-5-carboxylic acid reductase in the production bacterium is enhanced.
  • the proline hydroxylase is trans-proline-4-hydroxylase, and its nucleotide sequence is shown in SEQ ID NO:5.
  • the present invention finds that the threonine efflux protein ThrE is an L-proline efflux protein of Corynebacterium glutamicum through screening.
  • L-proline-producing strains SZCgP1 and SZCgP3 were constructed. Overexpression of ThrE in these two strains could increase L-proline production by 1.57 times, respectively. , 2.56 times. Therefore, in practice, it can be used for bacterial fermentation to produce L-proline, which is convenient for popularization and application, and has important industrial application value.
  • ThrE is also used in the production of hydroxyproline such as trans-4-hydroxy-L-proline, and knocking out the thrE gene will significantly reduce the output of extracellular L-proline by-products and increase the production of hydroxyproline.
  • the ratio of hydroxyproline such as trans-4-hydroxy-L-proline to L-proline helps to obtain hydroxyproline such as trans-4-hydroxy-L-proline with higher purity acid.
  • Knockout of the thrE gene is important for the production of hydroxyproline products such as trans-4-hydroxy-L-proline.
  • the present invention provides a new idea for constructing a hydroxyproline producing strain, namely knocking out the L-proline efflux protein of the strain, so as to increase the concentration of intracellular L-proline and increase the intracellular L-proline.
  • Proline can be used as a substrate of hydroxyproline, so that under the action of proline hydroxylase, intracellular L-proline is converted into hydroxyproline, and a higher-yield hydroxyproline is constructed for the technology in the art.
  • Amino acid producing strains have reference significance.
  • the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods.
  • the experimental method of unreceipted specific conditions in the following examples usually according to conventional conditions such as Sambrook et al., molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer the proposed conditions.
  • polypeptide with L-proline efflux function refers to a protein or polypeptide that can efflux intracellular L-proline to extracellular, which can be the protein described in SEQ ID NO: 1 or SEQ ID NO: 2 , or it can be a protein or polypeptide with L-proline efflux function obtained by mutating sequence 1 or sequence 2.
  • protein protein
  • polypeptide and “peptide” are used interchangeably and have the meanings commonly understood by one of ordinary skill in the art. Used interchangeably herein and is an amino acid polymer of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).
  • fusion protein is a protein obtained by fusing the mutant protein to a protein tag.
  • the protein tag can be located at the N-terminus of the mutant protein or at the C-terminus of the mutant protein. There may also be spacer amino acid residues between the mutant protein and the protein tag, specifically, there may be less than 10 spacer amino acid residues.
  • host cell as used herein means any cell type that is amenable to transformation, transfection, transduction, etc. comprising a polypeptide having L-proline efflux functional activity, a polynucleotide encoding a polypeptide, or an expression vector of the present disclosure .
  • recombinant microorganism encompasses host cells that differ from the parental cells after introduction of transcription initiation elements or recombinant expression vectors, in particular by transformation.
  • host cells suitable for use in the present invention include, but are not limited to, Escherichia, Corynebacterium, preferably Escherichia coli or Corynebacterium glutamicum, more preferably Corynebacterium glutamicum, more particularly Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC13869, Corynebacterium glutamicum B253, Corynebacterium glutamicum ATCC 14067.
  • transformation has the meaning commonly understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host.
  • the method of transformation includes any method of introducing nucleic acid into cells, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.
  • the culture of the host cells herein can be carried out according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and can be appropriately adjusted according to actual conditions. Culture conditions such as temperature, time, and pH of the medium.
  • a selected/alternative/preferred “numerical range” includes both the numerical endpoints at both ends of the range and, with respect to the aforementioned numerical endpoints, all natural numbers subsumed between the numerical endpoints.
  • wild-type and “naturally occurring” refer to objects that can be found in nature.
  • a polypeptide or polynucleotide sequence that exists in an organism can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring.
  • amino acid mutation or “nucleotide mutation” includes “substitution, duplication, deletion or addition of one or more amino acids or nucleotides”.
  • mutation refers to changes in nucleotide sequence or amino acid sequence. In a specific embodiment, the term “mutation” refers to "substitution”.
  • mutant state refers to the activity of a polypeptide in a microorganism in an unmodified state, ie, the activity in its natural state.
  • L-proline efflux functional activity has the same or similar meaning as those commonly understood by those skilled in the art, and both refer to the fact that the amino acid sequence of a fragment is a part of the amino acid sequence of a complete protein or polypeptide, and has The same or similar function or activity as the intact protein or polypeptide. Specifically, in the present invention, it refers to any amino acid fragment having an L-proline efflux function obtained from the ThrE protein of the present invention.
  • the L-proline efflux protein of the present invention it is obvious to further mutate the L-proline efflux protein of the present invention to obtain further mutants still possessing corresponding functions and activities.
  • Preferably 1 amino acid residue does not affect the function of the resulting mutant.
  • technicians often add a 6 ⁇ His tag on either end of the obtained protein, and this protein has the same function as a protein without a 6 ⁇ His tag. Therefore, the present invention shall include conservative mutations obtained on the basis of the present invention.
  • conservative mutation refers to a mutation that normally maintains the function of a protein. Representative examples of conservative mutations are conservative substitutions.
  • the term "conservative substitution” relates to the replacement of amino acid residues with amino acid residues having similar side chains.
  • Families of amino acid residues with similar side chains have been defined in the art and include those with basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid and glutamic acid) ), non-polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (eg, alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (eg tyrosine, phenylalanine, tryptophan and histidine).
  • basic side chains eg, lysine
  • substitutions typically exchange one amino acid at one or more sites in a protein. Such substitutions can be conservative. Examples of substitutions considered conservative substitutions include substitution of Ala to Ser or Thr, substitution of Arg to Gln, His, or Lys, substitution of Asn to Glu, Gln, Lys, His, or Asp, substitution of Asp to Glu, Gin, Lys, His, or Asp.
  • the term “corresponds to” has the meaning commonly understood by one of ordinary skill in the art. Specifically, “corresponds to” means the position at which one sequence corresponds to a specified position in the other sequence after the two sequences are aligned for homology or sequence identity. Therefore, for example, with regard to “the amino acid residue corresponding to the 40th position of the amino acid sequence shown in SEQ ID NO: 1", if a 6 ⁇ His tag is added to one end of the amino acid sequence shown in SEQ ID NO: 1, the resulting mutant corresponds to The 40th position of the amino acid sequence shown in SEQ ID NO: 1 may be the 46th position.
  • the homology or sequence identity may be more than 90%, preferably more than 95%, more preferably 96%, 97%, 98%, 99% homology.
  • the preferred method of determining identity is to obtain the greatest match between the sequences tested.
  • Methods for determining identity are codified in publicly available computer programs.
  • Preferred computer program methods for determining the identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al, 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al, 1990).
  • the BLASTX program is publicly available from NCBI and other sources (BLAST Handbook, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990).
  • the well-known Smith Waterman algorithm can also be used to determine identity.
  • Example 1 Screening of L-proline efflux protein of Corynebacterium glutamicum in Corynebacterium glutamicum ATCC13869
  • the inventors screened the L-proline efflux proteins of Corynebacterium glutamicum through a genome-scale membrane transporter inhibition library.
  • the cas9 gene of pCas9 (LIU, Jiao, et al. Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microbial cell factories, 2017, 16.1:205.) was mutated by D10A and H840A, and the The Bsa I restriction site was removed to obtain the pdCas9 plasmid; then from pnCas9(D10A) ⁇ AID ⁇ gRNA ⁇ ccdB TS (WANG, Yu, et al.
  • the gRNA-ccdB expression cassette was amplified on the plasmid and cloned into the same position of pdCas9 to obtain a CRISPRi plasmid pdCas9 gRNA-ccdB that can be efficiently constructed, the sequence of which is shown in SEQ ID NO: 3 , the suppression efficiency of the system can reach 96%.
  • the primers for constructing the CRISPRi plasmid of the thrE gene were thrE-F and thrE-R (Table 1).
  • the control strain was SZCgP1 (pdCas9gRNA-control)
  • the suppressor plasmid of thrE gene was pdCas9gRNA-thrE
  • the suppressor strain of thrE gene was SZCgP1 (pdCas9gRNA-thrE).
  • the composition of seed medium is (g/L): glucose, 5g/L; yeast powder, 1g/L; soy peptone, 3g/L; urea, 3g/L; succinic acid, 0.5g/L; K 2 HPO 4 ⁇ 3H 2 O, 1 g/L; MgSO 4 ⁇ 7H 2 O, 0.1 g/L; Biotin, 0.01 mg/L; Vitamin B1, 0.1 mg/L; MOPS, 20 g/L.
  • the composition of fermentation medium is: glucose, 80g/L; yeast powder, 1g/L; soy peptone, 1g/L; NaCl, 1g/L; ammonium sulfate, 1g/L; urea, 6g/L; K 2 HPO 4 ⁇ 3H 2 O, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.45g/L; FeSO 4 ⁇ 7H 2 O, 0.05g/L; Biotin, 0.4mg/L; Vitamin B1, 0.1mg/L; MOPS, 40g/L; initial pH7.2.
  • the strains were inoculated into a seed medium containing 15 ⁇ g/ml chloramphenicol for 8 h, and the culture was inoculated as a seed into a 24-well plate containing 15 ⁇ g/ml chloramphenicol and 0.03 mM IPTG fermentation medium, and the medium was filled with liquid
  • the amount was 800 ⁇ l, and the inoculum was controlled at an initial OD 600 of 0.03 (detected by microplate reader), cultured at 30°C for 21 h, the plate shaker speed was 800 rpm, and each strain was 3 parallel. Amino acid production.
  • Detection method of L-proline Dilute with 3% (W/V) sulfosalicylic acid to an appropriate concentration; take 1 mL of diluent, add 1 mL of ninhydrin (1.25 g of ninhydrin dissolved in 30 mL of glacial acetic acid) and 20 mL of 6M H 3 PO 4 , heated at 70° C. to dissolve) and 1 mL of glacial acetic acid, reacted in a boiling water bath at 100° C. for 45 min; measured OD 520 after cooling.
  • Draw a standard curve using L-proline at a concentration of 0-100 mg/L and calculate the concentration of the sample to be tested according to the standard curve. The results are shown in Table 2, the L-proline production and specific yield of the strain decreased by 63% and 49%, respectively, after the thrE gene expression was inhibited.
  • the inventors constructed knockout mutants based on CRISPR/Cas9 genome editing technology. Using pCas9 (LIU, Jiao, et al. Development of a CRISPR/Cas9 genome editing toolbox for Corynebacteriu m glutamicum.
  • gRNA-1/gRNA -2 is the primer to amplify the plasmid backbone fragment containing the gRNA ⁇ ccdB expression cassette and the temperature-sensitive origin of replication.
  • the above two fragments were cloned and connected by Novozan's one-step recombination kit to obtain a pCas9gRNA-ccdB plasmid that can be efficiently constructed.
  • the plasmid map is shown in Figure 1, and the sequence is shown in SEQ ID NO: 4.
  • the ATCC13869 genome was used as a template, and thrE-1/thrE-2 and thrE-3/thrE-4 were used as primers to amplify the upstream and downstream homology arms, respectively;
  • pCas9gRNA- The c cdB plasmid was used as the template, and cas9-3/cas9-4, cas9-5/cas9-6, and gRNA-3/gRNA-4 were used as primers to amplify three plasmid backbone fragments, and 20bp gRNA target DNA was introduced at the same time.
  • SZCgP1 strain was made competent, and about 2 ⁇ g of pCas9gRNA-thrE plasmid was electroporated, and 1 mL of pre-warmed TSB medium at 46°C was added, heat-shocked at 46°C for 6 min, incubated at 30°C for 3 h, and added 15 ⁇ g/mL chloramphenicol and 0.05mM IPTG for coating.
  • the TSB plate was cultured for 2 days, and the correct mutant was named SZCgP2 strain by PCR and sequencing.
  • the overexpression plasmid was constructed as follows: thrE was overexpressed on the pEC-ccdB plasmid, a derivative of the pEC-XK99E plasmid, and the pEC-ccdB plasmid was digested with Bsa I; according to the reported genome sequence of Corynebacterium glutamicum ATCC13869, the ATCC13869 genome was used as The template is used to amplify the thrE1 gene fragment with thrE-5/thrE-6. The amino acid sequence of the protein encoded by the thrE1 gene is shown in SEQ ID NO: 1.
  • the pEC-thrE1 plasmid was transformed into the SZCgP2 strain to obtain the complementing strain SZCgP2 (pEC-thrE1); the pEC-thrE1 and pEC-control plasmids were transformed into SZCgP1, respectively, to obtain the overexpression strain and the control strains SZCgP1 (pEC-thrE1) and SZCgP1 (pEC-control).
  • the composition of seed medium is (g/L): glucose, 5g/L; yeast powder, 1g/L; soy peptone, 3g/L; urea, 3g/L; succinic acid, 0.5g/L; K 2 HPO 4 ⁇ 3H 2 O, 1 g/L; MgSO 4 ⁇ 7H 2 O, 0.1 g/L; Biotin, 0.01 mg/L; Vitamin B1, 0.1 mg/L; MOPS, 20 g/L.
  • the fermentation medium composition is: glucose, 80g/L; yeast powder, 1g/L; soy peptone, 1g/L; NaCl, 1g/L; ammonium sulfate, 1g/L; urea, 6g/L; K 2 HPO 4 ⁇ 3H 2 O, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.45g/L; FeSO 4 ⁇ 7H 2 O, 0.05g/L; Biotin, 0.4mg/L; Vitamin B1, 0.1mg/L; MOPS, 40g/L; initial pH7.2.
  • the strain was inoculated into the seed medium for 8 hours, and the culture was inoculated into a 250 mL conical flask of the fermentation medium as a seed.
  • the medium filling volume was 25 mL.
  • the inoculum amount was controlled at an initial OD 600 of 0.1 (determined by a spectrophotometer), cultured at 30 °C for 21 h, and the shaker speed was 2200 rpm, with 3 parallels for each strain.
  • the fermentation process was sampled to determine OD 600 , intracellular L-proline concentration and Extracellular L-proline concentration.
  • the method for measuring the concentration of L-proline inside and outside the cell is as follows: add 400 ⁇ L of silicone oil to a 1.5 mL centrifuge tube in advance, quickly aspirate 400 ⁇ L of the fermentation broth and slowly add it, centrifuge at 4°C and 12000 rpm for 10 min, and the supernatant is the extracellular sample.
  • the bacterial pellet at the bottom of the centrifuge tube was cut off, placed in a 1.5 mL centrifuge tube, an appropriate amount of 3% sulfosalicylic acid was added according to the amount of bacterial cells, boiled at 100°C for 20 min, and centrifuged at 12,000 rpm for 10 min.
  • the supernatant was the intracellular sample.
  • the L-proline detection method is the same as that shown in Example 1, and the intracellular L-proline concentration is converted according to the volume of Corynebacterium glutamicum reported in the literature.
  • the measurement results of intracellular and extracellular L-proline concentration of each strain during the fermentation process are shown in Table 4 and Table 5.
  • the extracellular L-proline concentration of the thrE gene knockout strain SZCgP2 during the entire fermentation process was significantly lower than
  • the starting strain SZCgP1 had a slightly higher intracellular L-proline concentration than the starting strain SZCgP1 in the early stage of fermentation (3-9h);
  • the extracellular L-proline concentration of the overexpressing strain SZCgP1 (pEC-thrE1) was significantly higher than that of the control strain SZCgP1 (pEC-control) during the whole fermentation process, and the intracellular concentration of the whole fermentation process (especially the rapid acid production period) was higher.
  • the L-proline concentrations were significantly lower than those of the control strain SZCgP1 (pEC-control). Taken together, the above results indicate that ThrE is a proline efflux protein. Overexpression of ThrE in SZCgP1 strain can increase the production of L-proline.
  • the overexpression strain SZCgP1 (pEC-thrE1) increased the extracellular L-proline production at 21 h 1.57 times, indicating that overexpression of L-proline efflux protein gene is of great significance in the transformation of L-proline production strains.
  • an L-proline-producing strain SZCgP3 was constructed on the basis of Corynebacterium glutamicum ATCC13032 strain, that is, the G149K mutation of proB gene was introduced, and the codon was mutated from GGT to AAG (see CN101084312A).
  • the construction of ThrE overexpression strain is similar to that of Corynebacterium glutamicum 13869 strain.
  • the thrE was overexpressed on the pEC-ccdB plasmid derived from the pEC-XK99E plasmid, the pEC-ccdB plasmid was digested with Bsa I, and the thrE2 gene was amplified with thrE-5/thrE-6 using the genome of Corynebacterium glutamicum ATCC13032 as a template Fragment, the amino acid sequence of the protein encoded by the thrE2 gene is shown in SEQ ID NO: 2, and the above two fragments were cloned and connected by Novozan's one-step recombination kit to obtain the pEC-thrE2 plasmid.
  • the plasmid map is shown in Figure 3.
  • the pEC-thrE2 and pEC-control plasmids were transformed into SZCgP3, respectively, to obtain overexpression strains and control strains SZCgP3 (pEC-thrE2) and SZCgP3 (pEC-control).
  • the extracellular L-proline production of the above strains was evaluated by shake flask fermentation. The fermentation method was the same as that shown in Example 2. The results were shown in Table 6. Overexpression of ThrE in Corynebacterium glutamicum SZCgP3 can increase L-proline.
  • the pEC-mip4h and pEC-XK99E control plasmids were transformed into SZCgP1 and SZCgP2 strains, respectively, to obtain overexpression strains and control strains SZCgP1 (pEC-mip4h) and SZCgP1 (pEC-XK99E), and SZCgP2 (pEC-mip4h) and SZCgP2 (pEC -XK99E).
  • composition of seed medium is (g/L): glucose, 5g/L; yeast powder, 1g/L; soy peptone, 3g/L; urea, 3g/L; succinic acid, 0.5g/L; K 2 HPO 4 ⁇ 3H 2 O, 1 g/L; MgSO 4 ⁇ 7H 2 O, 0.1 g/L; Biotin, 0.01 mg/L; Vitamin B1, 0.1 mg/L; MOPS, 20 g/L.
  • the composition of fermentation medium is: glucose, 80g/L; yeast powder, 1g/L; soy peptone, 1g/L; NaCl, 1g/L; ammonium sulfate, 1g/L; urea, 6g/L; K 2 HPO 4 ⁇ 3H 2 O, 1g/L; MgSO 4 ⁇ 7H 2 O, 0.45g/L; FeSO 4 ⁇ 7H 2 O, 0.2g/L; Biotin, 0.4mg/L; Vitamin B1, 0.1mg/L; MOPS, 40g/L; initial pH7.2.
  • the strains were inoculated into the seed medium and cultivated for 8 hours, and the culture was inoculated into a 24-well plate containing 800 ⁇ l of fermentation medium as seeds.
  • the rotating speed of the orifice shaker was 800 rpm, and each strain had 3 parallels.
  • OD 600 After the fermentation, OD 600 , trans-4-hydroxy-L-proline and L-proline production were detected.
  • the detection method of trans-4-hydroxy-L-proline refers to the national standard GB/T 9695.23-2008, and the detection method of L-proline is the same as that shown in Example 1. The results are shown in Table 7.
  • the strains that do not express trans-proline-4-hydroxylase cannot produce trans-4-hydroxy-L-proline, and the L-proline efflux protein thrE gene is knocked out. Will significantly reduce the production of extracellular L-proline by-product, increase the ratio of trans-4-hydroxy-L-proline to L-proline, and help to obtain higher purity trans-4- Hydroxy-L-proline production. It is expected to further strengthen the activity of trans-proline-4-hydroxylase, so that the L-proline synthesized in the strain can be rapidly converted into trans-4-hydroxy-L-proline, and the L-proline can be knocked out.
  • the proline efflux protein thrE gene also reduces the efflux of L-proline to the outside of the cell, and can also increase the production of trans-4-hydroxy-L-proline. Therefore, knockout of the L-proline efflux protein thrE is important for trans-4-hydroxy-L-proline production.
  • the present invention first constructs a plasmid expressing trans-proline-3-hydroxylase, based on the pEC-XK99E plasmid
  • the expression plasmid pEC-Ubp4h expressing trans-proline-3-hydroxylase Ubp4h as shown in patent CN110804596A was artificially synthesized.
  • the pEC-Ubp4h and pEC-XK99E control plasmids were transformed into SZCgP1 and SZCgP2 strains, respectively, to obtain overexpression strains and control strains SZCgP1 (pEC-Ubp4h) and SZCgP1 (pEC-XK99E), and SZCgP2 (pEC-Ubp4h) and SZCgP2 (pEC -XK99E).
  • SZCgP1 pEC-Ubp4h
  • SZCgP1 pEC-XK99E
  • SZCgP2 pEC-Ubp4h
  • SZCgP2 pEC-XK99E
  • Hydroxyproline exists in four natural stereoisomers, namely trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, and trans-3-hydroxy-L -proline, cis-3-hydroxy-L-proline, those skilled in the art all know that these four different hydroxyproline are produced by the corresponding trans-proline-4-hydroxylase, cis-proline-4-hydroxylase, trans-proline-3-hydroxylase, cis-proline-3-hydroxylase directly catalyze L-proline to obtain, existing
  • the technical literature has also reported that different hydroxyproline can be produced by overexpressing different proline hydroxylases on the basis of L-proline producing strains (Zhang F, Liu H, Zhang T, Pijning T, Yu L).

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Abstract

本发明公开了一种具有L-脯氨酸外排功能的蛋白及其应用。利用蛋白ThrE生产L-脯氨酸或羟基脯氨酸的方法，是通过在产L-脯氨酸的菌株中增强具有L-脯氨酸外排功能的多肽的活性生产L-脯氨酸。或者，使产L-脯氨酸的宿主细胞中具有L-脯氨酸外排功能的多肽的活性减弱并使脯氨酸羟化酶的活性增强生产羟基脯氨酸。

Description

具有L-脯氨酸外排功能的蛋白及其应用
本申请要求2021年01月13日向中国国家知识产权局提交的专利申请号为2021100451754，发明名称为“具有L-脯氨酸外排功能的蛋白及其应用”的在先申请的优先权。该件在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于分子生物学和生物工程领域，具体涉及一种谷氨酸棒杆菌的苏氨酸外排蛋白ThrE作为L-脯氨酸外排蛋白的新用途，以及利用蛋白ThrE生产L-脯氨酸及其衍生物的方法。
背景技术
L-脯氨酸，是天然存在的一种人体的非必需氨基酸，在临床、生物材料和工业等方面有广泛的应用。L-脯氨酸的生产方法主要有化学法和发酵法，由于化学提取法污染严重成本高，已逐渐失去市场，微生物发酵法由于具有生产成本低、生产强度高、高特异性和对环境污染小等优点而成为当今工业应用最广泛的方法。目前，常用的工业发酵菌株有棒杆菌和埃希氏菌，常用的埃希氏菌如大肠杆菌(Escherichia coli)，常用的棒杆菌如谷氨酸棒杆菌(Corynebacterium glutamicum)，短杆菌如黄色短杆菌(Brevibacterium flavum)、乳酸发酵短杆菌(Brevibacterium lactofermentus)，以及节杆菌属的某些种和微杆菌属的某些种。而由于谷氨酸棒杆菌的生理优越性，已成为工业中最重要的生产菌株用来生产氨基酸等产品。
在棒杆菌中，主要以谷氨酸为底物经过γ-谷氨酰激酶(Glutamate-5-kinase，ProB)、谷氨酸半醛脱氢酶(Glutamate-semialdehyde dehydrogenase，ProA)、吡咯啉-5-羧酸还原酶(Pyrroline-5-carboxylic acid reductase，ProC)催化后生产L-脯氨酸。现有技术主要通过对L-脯氨酸合成途径的关键酶ProA、ProB的遗传改造生产L-脯氨酸，如CN101084312A报道了谷氨酸棒杆菌来源的ProB蛋白的149位突变可以解除L-脯氨酸的反馈抑制，提高工程菌株L-脯氨酸的产量。
在发酵过程中，通过转运途径将微生物体内合成的氨基酸及时转运到胞外，对于促进氨基酸的生物合成也十分重要，比如在赖氨酸的生产过程中，通过赖氨酸外排蛋白LysE的过表达，可以将工程菌株的赖氨酸产量显著提高(CN1283797C)。然而，在L-脯氨酸生产过程中，尚未有利用L-脯氨酸外排蛋白来提高L-脯氨酸产量的报道。因此，本领域急需挖掘有效的L-脯氨酸外排蛋白，以便将胞内生产的L-脯氨酸高效地排到胞外，获得高产的L-脯氨酸工程菌。
发明内容
为克服现有技术中的问题，本发明通过全基因组规模膜转运蛋白抑制库筛选谷氨酸棒杆菌的L-脯氨酸外排蛋白。基于此筛选，本发明筛选到thrE(Cgl2622，已有文献证实为苏氨酸外排蛋白的编码基因)基因抑制后，L-脯氨酸比产率下降明显。在一个L-脯氨酸生产菌SZCgP1(谷氨酸棒杆菌ATCC13869引入ProB蛋白的G149D突变，密码子从GGT突变为GAT，具体参见CN101084312A)中过表达ThrE，在整个发酵过程的胞外脯氨酸浓度都明显高于对照菌株SZCgP1(pEC-control)，在整个发酵过程(特别是快速产酸期)的胞内脯氨酸浓度都明显低于对照菌株SZCgP1(pEC-control)。上述结果表明ThrE是一个L-脯氨酸外排蛋白。
本发明的目的之一是提供脯氨酸外排蛋白在生产L-脯氨酸或羟脯氨酸中的用途。
本发明的又一目的是提供一种L-脯氨酸或羟脯氨酸的生产菌株。
本发明的另一目的是提供一种生产L-脯氨酸或羟脯氨酸的方法。
本发明的再一目的是提供一种L-脯氨酸或羟脯氨酸生产菌株的构建方法。
在本发明的第一方面，提供了一种多肽作为脯氨酸外排蛋白在生产L-脯氨酸、羟脯氨酸中的用途，所述多肽是：
A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽。
在另一优选例中，所述多肽是从SEQ ID NO:1或2所示氨基酸序列的多肽衍生的多肽，由SEQ ID NO:1或2所示氨基酸序列在两端的任意一端或两端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基 的添加而形成，并且具有L-脯氨酸外排功能。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
在本发明的第二方面，提供了一种L-脯氨酸的生产菌株，其特征在于，所述菌株表达以下多肽：
A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽。
在另一优选例中，所述多肽是从SEQ ID NO:1或2所示氨基酸序列的多肽衍生的多肽，由SEQ ID NO:1或2所示氨基酸序列在两端的任意一端或两端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成，并且具有L-脯氨酸外排功能。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
在另一优选例中，所述生产菌株是细菌。
在另一优选例中，所述生产菌株选自埃希氏菌属或棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，进一步优选为谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067。
在另一优选例中，所述生产细菌中的谷氨酸激酶不受L-脯氨酸的反馈抑制或L-脯氨酸的反馈抑制减弱。
在另一优选例中，所述生产细菌中的谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
在本发明的第三方面，提供一种生产L-脯氨酸的方法，所述方法包括：培养前面所述的生产菌株，使之生产L-脯氨酸。
在另一优选例中，所述方法还包括从发酵液中分离L-脯氨酸的步骤。
在本发明的第四方面，提供一种L-脯氨酸生产菌株的构建方法，所述方法包括增强所述菌株中以下具有L-脯氨酸外排功能的多肽的活性：
A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽。
在另一优选例中，所述多肽是从SEQ ID NO:1或2所示氨基酸序列多肽衍生的多肽，由SEQ ID NO:1或2所示氨基酸序列在两端的任意一端或两端经过一个或几个，优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基的添加而形成，并且具有L-脯氨酸外排功能。
在另一优选例中，所述多肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
在另一优选例中，所述生产菌株是细菌。
在另一优选例中，所述生产菌株选自埃希氏菌属或棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，进一步优选为谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067。
在另一优选例中，所述生产细菌中的谷氨酸激酶不受L-脯氨酸的反馈抑制或L-脯氨酸的反馈抑制减弱。
在另一优选例中，所述生产细菌中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
在本发明的第五方面，提供一种宿主细胞，所述宿主细胞中具有L-脯氨酸外排功能的多肽的活性减弱，所述具有L-脯氨酸外排功能活性的多肽为：
A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-
脯氨酸外排功能活性的多肽；或
C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽。
在另一优选例中，所述宿主细胞选自埃希氏菌属或棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，进一步优选为谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067。
在另一优选例中，所述宿主细胞中的谷氨酸激酶不受L-脯氨酸的反馈抑制或L-脯氨酸的反馈抑制减弱。
在另一优选例中，所述宿主细胞中的脯氨酸羟化酶的活性增强。
在另一优选例中，所述脯氨酸羟化酶为反式-脯氨酸-4-羟基酶，其核苷酸序列如SEQ ID NO:5所示。
在另一优选例中，所述生产细菌中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
在本发明的第六方面，提供一种生产反式-4-羟基-L-脯氨酸等羟脯氨酸的方法，所述方法包括：培养上述宿主细胞，使之生产反式-4-羟化-L-脯氨酸等羟脯氨酸。
在另一优选例中，所述方法还包括从发酵液中分离反式-4-羟基-L-脯氨酸等羟脯氨酸的步骤。
在本发明的第七方面，提供一种反式-4-羟基-L-脯氨酸等羟脯氨酸生产菌株的构建方法，其特征在于，所述方法包括：
(1)弱化所述菌株中以下具有L-脯氨酸外排功能的多肽的活性，所述具有L-脯氨酸外排功能活性的多肽为：
A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽；
(2)解除L-脯氨酸对谷氨酸激酶的反馈抑制或L-脯氨酸的反馈抑制减弱。
(3)将脯氨酸羟化酶的编码基因导入到步骤(2)获得的菌株中。
在另一优选例中，增强所述生产细菌中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性。在另一优选例中，所述脯氨酸羟化酶为反式-脯氨酸-4-羟化酶，其核苷酸序列如SEQ ID NO:5所示。
本发明的有益效果：
1、本发明经过筛选发现苏氨酸外排蛋白ThrE是一个谷氨酸棒杆菌的L-脯氨酸外排蛋白。在谷氨酸棒杆菌ATCC13869、ATCC13032菌株基础上构建L-脯氨酸生产菌SZCgP1、SZCgP3，使这两种菌株中过表达ThrE，均可以提高L-脯氨酸产量，提高幅度分别为1.57倍、2.56倍。因此，在实践上可用于细菌发酵生产L-脯氨酸，便于推广应用，具备重要的工业应用价值。
2、本发明还将ThrE用于反式-4-羟基-L-脯氨酸等羟脯氨酸的生产中，敲除thrE基因会显著降低胞外L-脯氨酸副产物的产量，提高反式-4-羟基-L-脯氨酸等羟脯氨酸与L-脯氨酸的比例，有助于获得更高纯度的反式-4-羟基-L-脯氨酸等羟脯氨酸。敲除thrE 基因对反式-4-羟基-L-脯氨酸等羟脯氨酸产品的生产具有重要意义。
3、本发明为构建羟脯氨酸生产菌株提供了新的思路，即敲除菌株的L-脯氨酸外排蛋白，以便增加胞内L-脯氨酸的浓度，增加的胞内L-脯氨酸可以作为羟脯氨酸的底物，从而在脯氨酸羟化酶的作用下，将胞内L-脯氨酸转化为羟脯氨酸，为本领域技术构建更高产的羟脯氨酸生产菌株具有借鉴意义。
附图说明
图1.pCas9gRNA-ccdB质粒图谱；
图2.pEC-thrE1质粒图谱；
图3.pEC-thrE2质粒图谱。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
定义与说明：
本文所用的术语“具有L-脯氨酸外排功能的多肽”是指可以将胞内L-脯氨酸外排到胞外的蛋白或者多肽，其可以是序列1或序列2所述的蛋白，也可以是对序列1或序列2进行突变而获得的仍然具有L-脯氨酸外排功能的蛋白或多肽。
本文所用的术语“蛋白”、“多肽”和“肽”可互换使用，并具有本领域普通技术人员通常理解的含义。在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。
本文所用的术语“融合蛋白”为将所述突变体蛋白与蛋白标签融合得到的蛋白质。蛋白标签可位于突变体蛋白的N端，也可位于突变体蛋白的C端。突变体蛋白和蛋白标签之间还可以具有间隔氨基酸残基，具体可具有10个以下间隔氨基酸残基。
本文中的术语“宿主细胞”意指易于用包含本公开的具有L-脯氨酸外排功能活性的多肽、编码多肽的多核苷酸或表达载体转化、转染、转导等的任何细胞类型。术语“重组微生物”涵盖导入转录起始元件或重组表达载体后不同于亲本细胞的宿主细胞，重组宿主细胞具体通过转化来实现。例如，适用于本发明的宿主细胞包括但不限于埃希氏菌属、棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，更优选谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067。
本文中的术语“转化”具有本领域技术人员普遍理解的意思，即将外源性的DNA导入宿主的过程。所述转化的方法包括任何将核酸导入细胞的方法，这些方法包括但不限于电穿孔法、磷酸钙沉淀法、氯化钙(CaCl 2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
本文的宿主细胞的培养可以根据本领域的常规方法进行，包括但不限于孔板培养、摇瓶培养、批次培养、连续培养和分批补料培养等，并可以根据实际情况适当地调整各种培养条件如温度、时间和培养基的pH值等。
本文所用的术语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
本文所用的“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
本文所用的“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
本文所用的选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
本文所用的术语“野生型的”、“天然存在的”指在自然界中可以找到的对象。例如，一种存在于生物体中，可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽或多核苷酸序列是天然存在的。
本文所用的术语“氨基酸突变”或“核苷酸突变”，包括“取代、重复、缺失或添加一个或多个氨基酸或核苷酸”。在本发明中，术语“突变”是指核苷酸序列或者氨基酸序列的改变。在一个具体的实施方式中，术语“突变”是指“取代”。
本文所用的术语“自然状态”是指微生物中多肽处于未修饰状态的活性，即自然状态下的活性。
本文所用的术语“具有L-脯氨酸外排功能活性”与本领域技术人员常规理解的含义相同或相似，均是指某一片段的氨基酸序列是完整蛋白或多肽的氨基酸序列的一部分，具备与完整蛋白或多肽相同或相似的功能或活性。具体地说，在本发明中，表示从本发明的ThrE蛋白获得的具有L-脯氨酸外排功能的任意氨基酸片段。
本领域技术人员知晓，为提升活性而对野生型多肽进行突变，找到能实现所需目的的位点更为重要。因此，基于本发明的教导，本领域技术人员会对序列1或2所示氨基酸序列进行突变，并检测突变体的相关活性。此外，本领域普通技术人员也不难知晓，在多肽的某些区域，例如非重要区域改变少数氨基酸残基基本上不会改变生物活性，例如，适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等，Molecular Biology of The Gene，第四版，1987，The Benjamin/Cummings Pub.Co.P224)。因此，本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。
因此，对本发明的L-脯氨酸外排蛋白作进一步突变而得到仍具备相应功能和活性的进一步突变体是显而易见的。例如，本领域技术人员公知在多肽的任一端增加或减少数个氨基酸残基，例如优选1-20个、更优选1-15个、更优选1-10个、更优选1-3个、最优选1个氨基酸残基不会影响得到的突变体的功能。例如，为便于纯化，技术人员往往在得到的蛋白的任一端带上6×His标签，而这种蛋白与不具备6×His标签的蛋白具有相同的功能。因此，本发明应包括在本发明基础上得到的保守性突变。
术语“保守突变”是指可正常维持蛋白质的功能的突变。保守突变的代表性例子为保守置换。
如本公开所使用的，术语“保守置换”涉及用具有类似侧链的氨基酸残基替换氨基酸残基。本领域已经定义了具有类似侧链的氨基酸残基家族，并且包括具有碱性侧链(例如赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸和谷氨酸)、不带电极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、和半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸和色氨酸)、β-支链(例如苏氨酸、缬氨酸和异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸和组氨酸)。
如本公开所使用的，“保守置换”通常在蛋白质的一个或多个位点上交换一种氨基酸。这种取代可以是保守的。作为被视作保守置换的置换，示例性的，可以举出Ala向Ser或Thr的置换、Arg向Gln、His或Lys的置换、Asn向Glu、Gln、Lys、His或Asp的置换、Asp向Asn、Glu或Gln的置换、Cys向Ser或Ala的置换、Gln向Asn、 Glu、Lys、His、Asp或Arg的置换、Glu向Gly、Asn、Gln、Lys或Asp的置换、Gly向Pro的置换、His向Asn、Lys、Gln、Arg或Tyr的置换、Ile向Leu、Met、Val或Phe的置换、Leu向Ile、Met、Val或Phe的置换、Lys向Asn、Glu、Gln、His或Arg的置换、Met向Ile、Leu、Val或Phe的置换、Phe向Trp、Tyr、Met、Ile或Leu的置换、Ser向Thr或Ala的置换、Thr向Ser或Ala的置换、Trp向Phe或Tyr的置换、Tyr向His、Phe或Trp的置换、及Val向Met、Ile或Leu的置换。此外，保守突变还包括起因于基因所来源的个体差异、株、种的差异等天然产生的突变。
本文所用的术语“对应于”具有本领域普通技术人员通常理解的意义。具体地说，“对应于”表示两条序列经同源性或序列相同性比对后，一条序列与另一条序列中的指定位置相对应的位置。因此，例如，就“对应于序列1所示氨基酸序列的第40位的氨基酸残基”而言，如果在序列1所示氨基酸序列的一端加上6×His标签，那么所得突变体中对应于序列1所示氨基酸序列的第40位就可能是第46位。
在具体的实施方式中，所述同源性或序列相同性可以是90％以上，优选95％以上，更优选96％、97％、98％、99％的同源性。
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。
除非另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
实施例1：在谷氨酸棒杆菌ATCC13869中筛选谷氨酸棒杆菌的L-脯氨酸外排蛋白
发明人通过全基因组规模膜转运蛋白抑制库筛选谷氨酸棒杆菌的L-脯氨酸外排蛋白。将pCas9(LIU,Jiao,et al.Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum.Microbial cell factories,2017,16.1:205.)质粒的cas9基因进行D10A和H840A突变，同时将质粒骨架中的Bsa I酶切位点去除，获得pdCas9质粒；再从pnCas9(D10A)‐AID‐gRNA‐ccdB TS(WANG,Yu,et al.Expanding targeting scope,editing window,and base transition capability of base editing in Corynebacterium glutamicum.Biotechnology and bioengineering,2019，116：3016-3029)质粒上扩增gRNA‐ccdB表达盒克隆至pdCas9的相同位置，获得可以高效构建的CRISPRi质粒pdCas9gRNA-ccdB，其序列如SEQ ID NO：3所示，该系统的抑制效率可以达到96％。基于以上巧妙设计，抑制目标基因时只需要设计20bp的sgRNA靶DNA结合区，再合成2条24bp互补引物，退火成双链并通过Golden Gate克隆构建目标基因的抑制质粒。发明人同时构建了没有20bp定位序列的对照质粒pdCas9gRNA-control。将谷氨酸棒杆菌ATCC13869全基因组规模的蛋白质序列在TransportDB数据库进行膜转运蛋白预测，获得全基因组规模的397个膜转运蛋白；再基于CRISPRi系统构建397个膜转运蛋白基因的抑制质粒，将抑制库质粒和对照质粒分别导入一个L-脯氨酸生产菌SZCgP1(谷氨酸棒杆菌ATCC13869引入proB基因的G149D突变，密码子从GGT突变为GAT，具体参见CN101084312A)，获得抑制库菌株；最后通过96孔板发酵评价所有菌株的L-脯氨酸比产率，抑制后L-脯氨酸比产率下降的基因即为可能的L-脯氨酸外排蛋白基因。基于以上筛选，发明人筛选到thrE(Cgl2622，已有文献证实为L-苏氨酸外排蛋白)基因抑制后，L-脯氨酸比产率下降明显。构建thrE基因的CRISPRi质粒的引物为thrE-F和thrE-R(表1)。对照菌株为SZCgP1(pdCas9gRNA-control)，thrE基因的抑制质粒为pdCas9gRNA-thrE，thrE基因的抑制菌株为SZCgP1(pdCas9gRNA-thrE)。
表1
引物 核苷酸序列 SEQ ID NO.
thrE-F TTCACACTGCTCGAACTTGTACCT 6
thrE-R AAACAGGTACAAGTTCGAGCAGTG 7
为了进一步确认thrE基因的抑制对L-脯氨酸生产的影响，采用24孔深孔板进行发 酵评价。种子培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，1g/L；大豆蛋白胨，3g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。发酵培养基成份为：葡萄糖，80g/L；酵母粉，1g/L；大豆蛋白胨，1g/L；NaCl，1g/L；硫酸铵，1g/L；尿素，6g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.45g/L；FeSO 4·7H 2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。首先将菌株接种到含有15μg/ml氯霉素的种子培养基中培养8h，培养物作为种子接种到含有15μg/ml氯霉素和0.03mM IPTG发酵培养基的24孔板中，培养基装液量为800μl，接种量控制在初始OD 600为0.03(酶标仪检测)，30℃培养21h，孔板摇床转速为800rpm，每个菌株3个平行，发酵结束后检测OD 600和L-脯氨酸产量。L-脯氨酸的检测方法：用3％(W/V)磺基水杨酸稀释到合适浓度；取1mL稀释液，加入1mL酸合茚三酮(1.25g茚三酮溶于30mL冰醋酸和20mL 6M H 3PO 4中，70℃加热溶解)和1mL冰醋酸，100℃沸水浴反应45min；冷却后测定OD 520。采用0-100mg/L浓度的L-脯氨酸绘制标准曲线，根据标准曲线计算待测样品的浓度。结果如表2所示，thrE基因表达抑制后菌株的L-脯氨酸产量和比产率分别下降63％和49％。
表2 thrE基因表达抑制对L-脯氨酸产量和比产率的影响
Figure PCTCN2022071346-appb-000001
实施例2.在谷氨酸棒杆菌ATCC13869中鉴定谷氨酸棒杆菌的L-脯氨酸外排蛋白
(1)在SZCgP1菌株中构建thrE基因的敲除、回补和过表达菌株
发明人基于CRISPR/Cas9基因组编辑技术构建敲除突变体。以pCas9(LIU,Jiao,et al.Development of a CRISPR/Cas9 genome editing toolbox for Corynebacteriu m glutamicum.Microbial cell factories,2017,16.1:205.)质粒为模板，以cas9-1/cas9-2为引物，在引物上引入操纵元件lacO突变(TGTGTGGAATTGTGAGCG GATAACAATTTCACACA突变为TGTGTGGAATTGTGAGCG CTCACAATTTCACACA)和RBS突变(AAAGG AGTTGAGA突变为AAAGG CACCCGAT)，扩增包含cas9的片 段；再以pnCas9(D10A)‐AID‐gRNA‐ccdB TS(WANG,Yu,et al.Expanding targeti ng scope,editing window,and base transition capability of base editing in Coryne bacterium glutamicum.Biotechnology and bioengineering,2019，116：3016-3029)质粒为模板，以gRNA-1/gRNA-2为引物，扩增包含gRNA‐ccdB表达盒和温敏复制原点的质粒骨架片段。以上2个片段通过诺唯赞的一步重组试剂盒克隆连接，获得可以高效构建的pCas9gRNA-ccdB质粒，质粒图谱如图1所示，序列如SEQ ID NO：4所示。根据已报道的谷氨酸棒杆菌ATCC13869基因组序列，以ATCC13869基因组为模板，以thrE-1/thrE-2和thrE-3/thrE-4为引物分别扩增上游和下游同源臂；以pCas9gRNA-c cdB质粒为模板，分别以cas9-3/cas9-4、cas9-5/cas9-6、gRNA-3/gRNA-4为引物，扩增3个质粒骨架片段，同时引入20bp的gRNA靶DNA结合区；以上5个片段通过诺唯赞的一步重组试剂盒克隆连接，获得pCas9gRNA-thrE质粒。SZCgP1菌株制备感受态，电转化约2μg pCas9gRNA-thrE质粒，加入1mL 46℃预热的TSB培养基，46℃热击6min，30℃孵育3h，涂布添加15μg/mL氯霉素和0.05mM IPTG的TSB平板，培养2天，PCR及测序验证正确的突变体命名为SZCgP2菌株。
过表达质粒构建如下：在pEC-XK99E质粒的衍生质粒pEC-ccdB质粒上过表达thrE，采用Bsa I酶切pEC-ccdB质粒；根据已报道的谷氨酸棒杆菌ATCC13869基因组序列，以ATCC13869基因组为模板，以thrE-5/thrE-6扩增thrE1基因片段，thrE1基因所编码的蛋白质的氨基酸序列如SEQ ID NO:1所示，以上2个片段通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-thrE1质粒，质粒图谱如图2所示。同时构建一个质粒上没有克隆任何基因的对照质粒pEC-control。将pEC-thrE1质粒转化至SZCgP2菌株，获得回补菌株SZCgP2(pEC-thrE1)；分别将pEC-thrE1和pEC-control质粒转化至SZCgP1，获得过表达菌株及对照菌株SZCgP1(pEC-thrE1)和SZCgP1(pEC-control)。
表3
Figure PCTCN2022071346-appb-000002
Figure PCTCN2022071346-appb-000003
(2)摇瓶发酵测定thrE基因的敲除、回补和过表达菌株的胞内和胞外L-脯氨酸浓度
为了进一步确认thrE基因是否为L-脯氨酸外排蛋白，采用摇瓶发酵测定敲除、回补和过表达菌株的胞内和胞外L-脯氨酸浓度。种子培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，1g/L；大豆蛋白胨，3g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。发酵培养基成份为：葡萄糖，80g/L；酵母粉，1g/L；大豆蛋白胨，1g/L；NaCl， 1g/L；硫酸铵，1g/L；尿素，6g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.45g/L；FeSO 4·7H 2O，0.05g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。首先将菌株接种到种子培养基中培养8h，培养物作为种子接种到发酵培养基的250mL三角瓶中，培养基装液量为25mL，根据需要加入25μg/ml卡那霉素和0.05mM IPTG，接种量控制在初始OD 600为0.1(分光光度计测定)，30℃培养21h，摇床转速为2200rpm，每个菌株3个平行，发酵过程取样测定OD 600、胞内L_脯氨酸浓度和胞外L-脯氨酸浓度。
胞内外L-脯氨酸浓度测定方法如下：预先在1.5mL离心管中加入400μL硅酮油，快速吸取发酵菌液400μL缓慢加入，4℃，12000rpm离心10min，上清即胞外样品。将离心管底部的菌体沉淀切割下来，置于1.5mL离心管中，根据菌体量加入适量3％磺基水杨酸，100℃煮沸20min，12000rpm离心10min，上清即为胞内样品。L-脯氨酸检测方法同实施例1所示，胞内L-脯氨酸浓度根据文献报道的谷氨酸棒杆菌体积进行换算。
发酵过程各个菌株的胞内和胞外L-脯氨酸浓度测定结果如表4和表5所示，thrE基因敲除菌株SZCgP2在整个发酵过程的胞外L-脯氨酸浓度都明显低于出发菌株SZCgP1，在发酵前期(3-9h)胞内的L-脯氨酸浓度较出发菌株SZCgP1略高；回补菌株SZCgP2(pEC-thrE1)在整个发酵过程的胞外L-脯氨酸浓度都明显高于出发菌株SZCgP1，在快速产酸期(3-15h)胞内L-脯氨酸浓度都明显低于出发菌株。过表达菌株SZCgP1(pEC-thrE1)在整个发酵过程的胞外L-脯氨酸浓度都明显高于对照菌株SZCgP1(pEC-control)，在整个发酵过程(特别是快速产酸期)的胞内L-脯氨酸浓度都明显低于对照菌株SZCgP1(pEC-control)。综合以上结果，表明ThrE是一个脯氨酸外排蛋白。在SZCgP1菌株中过表达ThrE可以提高L-脯氨酸产量，过表达菌株SZCgP1(pEC-thrE1)相对导入空质粒的对照菌SZCgP1(pEC-control)在21h胞外L-脯氨酸产量提高幅度达1.57倍，表明过表达L-脯氨酸外排蛋白基因在L-脯氨酸生产菌株改造中具有重要意义。
表4 谷氨酸棒杆菌ATCC13869中thrE基因敲除、回补和过表达菌株的胞内L-脯氨酸浓度
Figure PCTCN2022071346-appb-000004
Figure PCTCN2022071346-appb-000005
表5 谷氨酸棒杆菌ATCC13869中thrE基因敲除、回补和过表达菌株的胞外L-脯氨酸浓度
Figure PCTCN2022071346-appb-000006
实施例3.L-脯氨酸外排蛋白在谷氨酸棒杆菌ATCC13032菌株中应用于L脯氨酸生产
首先在谷氨酸棒杆菌ATCC13032菌株基础上构建一个L-脯氨酸生产菌SZCgP3，即引入proB基因的G149K突变，密码子从GGT突变为AAG(参见CN101084312A)。ThrE过表达菌株构建与谷氨酸棒杆菌13869菌株类似。在pEC-XK99E质粒的衍生质粒pEC-ccdB质粒上过表达thrE，采用Bsa I酶切pEC-ccdB质粒，以谷氨酸棒杆菌ATCC13032基因组为模板，以thrE-5/thrE-6扩增thrE2基因片段，thrE2基因所编码的蛋白质的氨基酸序列如SEQ ID NO:2所示，以上2个片段通过诺唯赞的一步重组试剂盒克隆连接，获得pEC-thrE2质粒，质粒图谱如图3所示。分别将pEC-thrE2和pEC-control质粒转化至SZCgP3，获得过表达菌株及对照菌株SZCgP3(pEC-thrE2)和SZCgP3(pEC-control)。摇瓶发酵评价以上菌株的胞外L-脯氨酸产量，发酵方法同实施例2所示，结果如表6所示，在谷氨酸棒杆菌SZCgP3中过表达ThrE可以提高L-脯氨酸产量，过表达菌株SZCgP3(pEC-thrE2)相对导入空质粒的对照菌SZCgP3(pEC-control)在21h提高幅度达2.56倍，进一步表明在谷氨酸棒杆菌中过表达L-脯氨酸外排蛋白基因在L-脯氨酸生产中具有重要意义。
表6 谷氨酸棒杆菌ATCC13032中thrE基因过表达菌株的胞外L-脯氨酸产量
Figure PCTCN2022071346-appb-000007
实施例4.L-脯氨酸外排蛋白在羟脯氨酸生产中的应用
首先构建一个表达反式-脯氨酸-4-羟化酶的质粒，在pEC-XK99E质粒基础上人工合成表达如专利CN201710661945.1所示的反式-脯氨酸-4-羟化酶的表达质粒pEC-mip4h，其中，反式-脯氨酸-4-羟化酶的序列如SEQ ID NO：5所示。分别将pEC-mip4h和pEC-XK99E对照质粒转化至SZCgP1和SZCgP2菌株，获得过表达菌株及对照菌株SZCgP1(pEC-mip4h)和SZCgP1(pEC-XK99E)，以及SZCgP2(pEC-mip4h)和SZCgP2(pEC-XK99E)。
种子培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，1g/L；大豆蛋白胨，3g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。发酵培养基成份为：葡萄糖，80g/L；酵母粉，1g/L；大豆蛋白胨，1g/L；NaCl，1g/L；硫酸铵，1g/L；尿素，6g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.45g/L；FeSO 4·7H 2O，0.2g/L；生物素，0.4mg/L；维生素B1，0.1mg/L；MOPS，40g/L；初始pH7.2。首先将菌株接种种子培养基中培养8h，培养物作为种子接种到装有800μl发酵培养基的24孔板中，接种量控制在初始OD 600为0.1(酶标仪检测)，30℃培养18h，孔板摇床转速为800rpm，每个菌株3个平行，发酵结束后检测OD 600、反式-4-羟基-L-脯氨酸和L-脯氨酸产量。反式-4-羟基-L-脯氨酸的检测方法参考国标GB/T 9695.23-2008，L-脯氨酸检测方法同实施例1所示。结果如表7所示，没有表达反式-脯氨酸-4-羟化酶的菌株不能产生反式-4-羟基-L-脯氨酸，敲除L-脯氨酸外排蛋白thrE基因会显著降低胞外L-脯氨酸副产物的产量，提高反式-4-羟基-L-脯氨酸与L-脯氨酸的比例，有助于获得更高纯度的反式-4-羟基-L-脯氨酸 产量。预计进一步强化反式-脯氨酸-4-羟化酶的活性，使得菌株胞内合成的L-脯氨酸可以快速转化为反式-4-羟基-L-脯氨酸，敲除L-脯氨酸外排蛋白thrE基因也会减少L-脯氨酸外排至胞外，同时还可以提高反式-4-羟基-L-脯氨酸产量。因此，敲除L-脯氨酸外排蛋白thrE对反式-4-羟基-L-脯氨酸生产具有重要意义。
表7 thrE基因敲除对胞外反式-4-羟化-L-脯氨酸和L-脯氨酸产量的影响
Figure PCTCN2022071346-appb-000008
实施例5.L-脯氨酸外排蛋白在羟脯氨酸生产中的应用
为了进一步验证L-脯氨酸外排蛋白在生产其他羟脯氨酸中的应用，本发明首先构建一个表达反式-脯氨酸-3-羟化酶的质粒，在pEC-XK99E质粒基础上人工合成表达如专利CN110804596A所示的反式-脯氨酸-3-羟化酶Ubp4h的表达质粒pEC-Ubp4h。分别将pEC-Ubp4h和pEC-XK99E对照质粒转化至SZCgP1和SZCgP2菌株，获得过表达菌株及对照菌株SZCgP1(pEC-Ubp4h)和SZCgP1(pEC-XK99E)，以及SZCgP2(pEC-Ubp4h)和SZCgP2(pEC-XK99E)。培养基成份和菌株的培养参照实施例4。反式-3-羟基-L-脯氨酸的检测方法参考专利CN110804596A，L-脯氨酸检测方法同实施例1所示。结果显示，没有表达反式-脯氨酸-3-羟化酶的菌株不能产生反式-3-羟基-L-脯氨酸，敲除L-脯氨酸外排蛋白thrE基因会显著降低胞外L-脯氨酸副产物的产量，提高反式-3-羟基-L-脯氨酸与L-脯氨酸的比例，有助于获得更高纯度的反式-3-羟基-L-脯氨酸产量。也就是说，敲除L-脯氨酸外排蛋白thrE对反式-3-羟基-L-脯氨酸生产具有重要意义。
羟脯氨酸存在四种天然的立体异构体，分别是反式-4-羟基-L-脯氨酸、顺式-4-羟基-L-脯氨酸、反式-3-羟基-L-脯氨酸、顺式-3-羟基-L-脯氨酸，本领域技术人员均知晓这四种不同的羟脯氨酸是由对应的反式-脯氨酸-4-羟化酶、顺式-脯氨酸-4-羟化酶、反式-脯氨酸-3-羟化酶、顺式-脯氨酸-3-羟化酶直接催化L-脯氨酸得到的，现有技术文献也已报道了在L-脯氨酸生产菌株基础上过表达不同的脯氨酸羟化酶便可生产不同的羟脯氨酸(Zhang F,Liu H,Zhang T,Pijning T,Yu L,Zhang W,Liu W,Meng X.Biochemical and genetic characterization of fungal proline hydroxylase in echinocandin biosynthesis.Appl Microbiol  Biotechnol.2018 Sep；102(18):7877-7890.Chen K,Pang Y,Zhang B,Feng J,Xu S,Wang X,Ouyang P.Process optimization for enhancing production of cis-4-hydroxy-L-proline by engineered Escherichia coli.Microb Cell Fact.2017 Nov 22；16(1):210.Mori H,Shibasaki T,Yano K,Ozaki A.Purification and cloning of a proline 3-hydroxylase,a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline.J Bacteriol.1997Sep；179(18):5677-83.)。因此，对于本领域技术人员而言，在本发明实施例4、5证明了敲除L-脯氨酸外排蛋白ThrE可以增加胞内L-脯氨酸浓度，并在该菌株基础上过表达反式-脯氨酸-4-羟化酶可以增加反式-4-羟基-L-脯氨酸产量、过表达反式-脯氨酸-3-羟化酶可以增加反式-3-羟基-L-脯氨酸产量的情况下，本领域技术人员可显而易见地知晓，在敲除L-脯氨酸外排蛋白ThrE的基础上过表达不同的脯氨酸羟化酶均可降低胞外L-脯氨酸副产物的产量，提高相应的羟脯氨酸与L-脯氨酸的比例，即敲除L-脯氨酸外排蛋白对以上4种羟脯氨酸的生产均具有重要意义。以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种多肽在生产L-脯氨酸或羟脯氨酸中的用途，其特征在于，所述多肽是：
    A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
    B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
    C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽；
    优选地，所述多肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
  2. 一种L-脯氨酸的生产菌株，其特征在于，所述菌株表达以下多肽：
    A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
    B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
    C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽；
    优选地，所述多肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
    优选地，所述的生产菌株是细菌；
    优选地，所述的生产菌株选自埃希氏菌属或棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，进一步优选为谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067；
    进一步优选地，所述的生产细菌中的谷氨酸激酶不受L-脯氨酸的反馈抑制或L-脯氨酸的反馈抑制减弱；
    更进一步优选地，所述的生产细菌中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
  3. 一种生产L-脯氨酸的方法，其特征在于，所述方法包括：培养权利要求2所述的生产菌株，使之生产L-脯氨酸；
    优选地，所述方法还包括从发酵液中分离L-脯氨酸的步骤。
  4. 一种L-脯氨酸生产菌株的构建方法，其特征在于，所述方法包括增强所述菌株中以下多肽的活性：
    A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
    B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
    C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽；
    优选地，所述多肽的氨基酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
    优选地，所述的生产菌株是细菌；
    优选地，所述的生产菌株选自埃希氏菌属或棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，进一步优选为谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067；
    进一步优选地，所述生产细菌中的谷氨酸激酶不受L-脯氨酸的反馈抑制或L-脯氨酸的反馈抑制减弱；
    更进一步优选地，所述生产细菌中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
  5. 一种宿主细胞，其特征在于，所述宿主细胞中具有L-脯氨酸外排功能的多肽的活性减弱，所述具有L-脯氨酸外排功能活性的多肽为：
    A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽；或
    B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
    C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽；
    优选地，所述的生产菌株是细菌；
    优选地，所述宿主细胞选自埃希氏菌属或棒杆菌属，优选大肠杆菌或谷氨酸棒杆菌，进一步优选为谷氨酸棒杆菌，更具体的是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌B253、谷氨酸棒杆菌ATCC 14067；
    进一步优选地，所述宿主细胞中的谷氨酸激酶不受L-脯氨酸的反馈抑制或L-脯氨酸的反馈抑制减弱；
    更进一步优选地，所述宿主细胞中脯氨酸羟化酶的活性增强；
    更进一步优选地，所述宿主细胞中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
  6. 如权利要求5中所述的宿主细胞，其特征在于，所述脯氨酸羟化酶为反式-脯氨酸-4-羟化酶，其核苷酸序列如SEQ ID NO:5所示。
  7. 一种生产羟脯氨酸的方法，其特征在于，所述方法包括：培养权利要求5或6所述的宿主细胞，使之生产羟脯氨酸；
    优选地，所述方法还包括从发酵液中分离羟脯氨酸的步骤。
  8. 一种羟脯氨酸生产菌株的构建方法，其特征在于，所述方法包括：
    (1)弱化所述菌株中具有L-脯氨酸外排功能的多肽的活性，所述具有L-脯氨酸外排功能活性的多肽为：
    A)SEQ ID NO:1或SEQ ID NO:2所示氨基酸序列的多肽，并且具有L-脯氨酸外排功能活性；或
    B)在A)所述多肽的两端的任意一端或两端添加、缺失部分氨基酸而仍然具有L-脯氨酸外排功能活性的多肽；或
    C)与A)所述多肽同源性高于90％、95％、96％、97％、98％或99％，且来源于棒杆菌属的具有L-脯氨酸外排功能活性的多肽；
    (2)解除L-脯氨酸对谷氨酸激酶的反馈抑制或L-脯氨酸的反馈抑制减弱。
    (3)将脯氨酸羟化酶的编码基因导入到步骤(2)获得的菌株中；
    优选地，所述宿主细胞中谷氨酸激酶和/或谷氨酸半醛脱氢酶和/或吡咯啉-5-羧酸还原酶的活性增强。
  9. 如权利要求8所述的羟脯氨酸生产菌株的构建方法，其特征在于，所述脯氨酸羟化酶为反式-脯氨酸-4-羟化酶，其核苷酸序列如SEQ ID NO:5所示。
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