WO2016127469A1 - 用于生产β-丙氨酸的工程菌及生产β-丙氨酸的方法 - Google Patents

用于生产β-丙氨酸的工程菌及生产β-丙氨酸的方法 Download PDF

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WO2016127469A1
WO2016127469A1 PCT/CN2015/074683 CN2015074683W WO2016127469A1 WO 2016127469 A1 WO2016127469 A1 WO 2016127469A1 CN 2015074683 W CN2015074683 W CN 2015074683W WO 2016127469 A1 WO2016127469 A1 WO 2016127469A1
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gene
pand
escherichia coli
carboxylase
recombinant
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蔡真
张君丽
李寅
张冬竹
郭恒华
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中国科学院微生物研究所
安徽华恒生物科技股份有限公司
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01011—Aspartate 1-decarboxylase (4.1.1.11)
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to the field of biocatalysis, in particular to the construction and application of genetic engineering bacteria for producing ⁇ -alanine.
  • ⁇ -alanine is the only ⁇ -alanine naturally occurring in nature and has a wide range of applications in the medical, food, and chemical industries. For example, it is used as a chemical raw material to synthesize calcium pantothenate, carnosine, and sodium pamidium phosphate for synthesizing tumor hypercalcemia and the cost-specific anti-inflammatory drug balsalazide for treating ulcerative enteritis.
  • ⁇ -alanine can also be used as a precipitant in the preparation of a medicament, a water purification flocculant, a plating corrosion inhibitor, a lead poisoning antidote, and a synthetic sweetener.
  • the ⁇ -alanine chemical production includes an acrylonitrile method, an acrylic acid method, a succinimide degradation method, and a ⁇ -aminopropionitrile method.
  • the acrylonitrile method is the main method for producing ⁇ -alanine in China.
  • the method ammoxidizes acrylonitrile and ammonia to form ⁇ -aminopropionitrile, and then hydrolyzes under acidic or basic conditions.
  • the method has easy availability of raw materials and low cost, but has side reactions, and a large amount of inorganic salts are formed during the hydrolysis process, and the products are difficult to be purified.
  • the biotransformation method mainly utilizes L-aspartate ⁇ -carboxylase to specifically remove the ⁇ -carboxyl group of L-aspartic acid to form ⁇ -alanine.
  • L-aspartate ⁇ -carboxylase which is self-expressed in E. coli, has an enzyme activity of only 0.22 U/mg, and is expressed in Escherichia coli from Corynebacterium glutamicum.
  • L-aspartate alpha carboxylase with an enzyme activity of 3.42 U/mg (Nicole, 1999, Applied and Environmental Microbiology).
  • Chopra et al. found that the enzyme activity of L-aspartate alpha carboxylase derived from M. tuberculosis was 35 ⁇ mol/g ⁇ h (Chopra, 2002, Protein Expression and Purification).
  • Chinese Patent Application Publication No. CN 101210230A discloses an Escherichia coli BL21 (DE3) overexpressing its own source of L-aspartate alpha carboxylase. The ⁇ -alanine obtained by catalyzing L-aspartic acid using this strain was only 2.94 g/L.
  • an object of the present invention is to provide a recombinant Escherichia coli engineered bacterium capable of efficiently expressing L-aspartate ⁇ -carboxylase having high activity, and to produce ⁇ by whole cell transformation of L-aspartic acid.
  • - A method of alanine.
  • a recombinant Escherichia coli engineered bacterium capable of overexpressing L-aspartate alpha carboxylase derived from Bacillus subtilis represented by SEQ ID NO: 6 or A polypeptide that overexpresses at least 60%, preferably 80%, more preferably 90%, further preferably 95% and most preferably 98%, or even 99% homology to the enzyme, and which has the enzymatic activity.
  • the Escherichia coli is not particularly limited, but BL21 (DE3) is preferred.
  • the engineered bacteria is introduced into a recombinant plasmid in Escherichia coli, which is inserted into the cloning site of the vector and inserted into the Bacillus subtilis L-aspartate alpha carboxylation shown in SEQ ID NO: 5.
  • the carrier is not particularly limited, but pET-30a(+) is preferred.
  • the engineered bacterium is a recombinant bacterium having a DNA sequence integrated at one or more sites (for example, 2 to 4) on the genome of the Escherichia coli, the DNA sequence comprising the coding sequence shown in SEQ ID NO: 5
  • the gene and encoding of B. subtilis L-aspartate alpha carboxylase are at least 60%, preferably 80%, more preferably 90%, further preferably 95% and most preferably 98%, or even 99% homologous to the enzyme.
  • the insertion site is not particularly limited, but a site where the frdB gene and the tktB gene are located in the genome is preferred.
  • the DNA sequence further comprises a promoter sequence.
  • the promoter sequence is not particularly limited as long as any promoter capable of initiating a downstream L-aspartate ⁇ -carboxylase-encoding gene is feasible.
  • a promoter of the B. subtilis L-aspartate alpha carboxylase gene itself a promoter carried in the plasmid used in the construction of the engineered strain of the present invention, for example, a promoter in PUC19, pET-30a (+ The promoter in the ) can also be a commonly used strong promoter such as Lac, T7 and the like.
  • a T7 promoter (sequence 8) is preferred.
  • the 1 ⁇ 2 ⁇ 3 ⁇ 4 fragment consisting of four DNA fragments is integrated into the frdB gene in the E. coli BL21 (DE3) genome by homologous recombination, wherein the 1 ⁇ 2 ⁇ 3 ⁇ In the 4 fragment, fragment 1 is a DNA fragment of nucleotides 4287847-4287308 of Escherichia coli BL21 (DE3) genome (GenBank: AM946981.2), and fragment 2 is plasmid 68-998 of plasmid pET30-panD B (sequence 7)
  • a DNA fragment of nucleotides fragment 3 is a DNA fragment of nucleotides 31-1507 of plasmid PKD4 (GenBank: AY048743.1), and fragment 4 is a genome of Escherichia coli BL21 (DE3) (GenBank: AM946981.2) a DNA fragment of nucleotides 4287127-4286588;
  • fragment 2-1 is a DNA fragment of nucleotides 2444239-2444839 of Escherichia coli BL21 (DE3) genome (GenBank: AM946981.2), and fragment 2-2 is plasmid pET30.
  • DNA fragment of nucleotides 236-735 is a DNA fragment of nucleotides 31-1508 of plasmid PKD4 (GenBank: AY048743.1), and fragment 4 is the large intestine a DNA fragment of the nucleotide number 2445379-2445978 of the Bacillus BL21 (DE3) genome (GenBank: AM946981.2);
  • the kan resistance was discarded, and a recombinant strain in which two copies of the L-aspartate ⁇ -carboxylase gene were inserted into the frdB gene and the tktB gene was obtained.
  • the engineered bacteria of the present invention can be used for biotransformation of L-aspartic acid to produce ⁇ -alanine.
  • a method for whole-cell biotransformation of L-aspartic acid to produce ⁇ -alanine comprising the step of fermenting the above-mentioned engineered bacteria. Also included is the use of L-aspartic acid as a substrate for biotransformation with the engineered bacteria to produce beta-alanine.
  • the present invention screens for a highly active Bacillus subtilis L-aspartate ⁇ -carboxylase, and enhances the expression of the enzyme in Escherichia coli by genetic recombination, thereby enabling efficient conversion of L-aspartic acid to obtain ⁇ -alanine. acid.
  • Bacillus subtilis L-aspartate alpha carboxylase is integrated into the E. coli genome to further increase the activity of L-aspartate alpha carboxylase in the fermentation broth.
  • the engineering bacteria of the invention avoids the problem of plasmid loss during the large-scale cultivation of the bacteria, and ensures the stability of the enzyme activity in the industrial application process.
  • the engineering bacteria constructed by the invention does not need to add antibiotics in the cell culture process, which saves the cultivation cost and the subsequent sewage treatment cost, and is beneficial to the biological production of ⁇ -alanine in the industry.
  • Figure 1 is an HPLC chromatogram of a standard solution.
  • Fig. 2 is a column diagram showing the enzyme activity of the supernatant obtained by crushing the engineering bacteria A, engineering bacteria B, engineering bacteria C and engineering bacteria fermentation liquid.
  • Figure 3 is a graph showing the concentration of ⁇ -alanine produced in the conversion system over time during biotransformation.
  • A is an electropherogram of the fragment FrdB-panD B- kan-FrdB when constructing the PAND-1 strain according to the method of the present invention
  • B is to verify whether the constructed PAND-1-1 strain has the FrdB-panD B- kan
  • C is an electropherogram showing that kanamycin resistance has been lost in the constructed PAND1 strain.
  • A is an electrophoretogram of the fragment tktB-panD B- kan-tktB when the PAND-2 strain is constructed according to the method of the present invention
  • B is to verify whether the constructed PAND-2-1 strain has tktB-panD B- kan
  • C is an electropherogram showing the loss of kanamycin resistance in the constructed PAND2 strain.
  • Figure 6 shows the results of detection of L-aspartate ⁇ -carboxylase activity in culture medium after incubation of recombinant strains PAND-1 and PAND-2 for 12 hours at different temperatures.
  • Figure 7 is a schematic diagram showing a homologous recombination method for inserting a gene encoding Bacillus subtilis L-aspartate alpha carboxylase into Escherichia coli BL21 (DE3) according to an embodiment of the present invention, wherein
  • A is a wild type (BL21 (DE3)) E. coli genome using primers frdB-for and frdB-rev to obtain a 1735 bp DNA fragment by PCR;
  • C was the 2583 bp DNA fragment obtained by PCR using the primers FrdB-For and FrdB-rev after removing the kan gene.
  • Vector pET-30a (+) purchased from Novagen.
  • E. coli BL21 (DE3) purchased from Novagen.
  • L-aspartic acid standard purchased from Bioengineering Biotechnology (Shanghai) Co., Ltd., product number AB0091CAS (56-84-8).
  • Beta-alanine standard product purchased from Bioengineering Biotechnology (Shanghai) Co., Ltd., product number A6168CAS (107-95-9).
  • yeast extract 0.5%), peptone (1%), sodium chloride (1%)
  • LB solid medium composition yeast extract (0.5%), peptone (1%), sodium chloride (1%), 1.5% agar.
  • homology refers to the degree of similarity between the nucleotide sequence of a DNA or the amino acid sequence of a protein, and the DNA having a (some degree) homology as described herein is encoded.
  • the protein has the same activity at least for the function of the present invention, and the same protein having (a certain degree of homology) has the same activity at least for the function of the present invention.
  • the engineered bacteria of the present invention is a recombinant strain in which a DNA sequence is integrated at one or more sites on the genome of the Escherichia coli, and the DNA sequence comprises a gene selected from the group consisting of L-aspartate ⁇ -carboxylase and a coding and preparation thereof.
  • a DNA sequence is integrated at one or more sites on the genome of the Escherichia coli, and the DNA sequence comprises a gene selected from the group consisting of L-aspartate ⁇ -carboxylase and a coding and preparation thereof.
  • One of the genes of the polypeptide having at least 60%, preferably 80%, more preferably 90%, further preferably 95% and most preferably 98%, or even 99% homology, and having said enzymatic activity.
  • the highly active L-aspartate alpha carboxylase obtained by the following examples was Bacillus subtilis L-aspartate alpha carboxylase (SEQ ID NO: 6). Of course, the higher the activity, the more advantageous the L-aspartate alpha carboxylase.
  • the present invention integrates the gene of the highly active L-aspartate ⁇ -carboxylase into the genome of Escherichia coli by a method of homologous recombination, thereby obtaining an engineered bacteria capable of stably proliferating and overexpressing the enzyme.
  • the copy number of the integrated gene is not particularly limited, but is preferably 2 to 4 copies, more preferably 2 copies.
  • the integration site should be those sites that do not adversely affect the proliferation and growth of the bacteria.
  • a site in the frdB gene and/or the tktB gene in the E. coli genome is preferred.
  • Escherichia coli there is no particular limitation on Escherichia coli, and it is preferably BL21 (DE3).
  • Example 1 Construction of an engineered strain expressing L-aspartate alpha carboxylase on a plasmid
  • L-aspartate ⁇ -carboxylase from four hosts, Escherichia coli, Corynebacterium glutamicum, Mycobacterium tuberculosis, and Bacillus subtilis.
  • the coding gene was used to construct four engineering bacteria. Among them, the DNA sequence shown in SEQ ID NO: 1 of the Sequence Listing is the coding gene panD E of L-aspartate ⁇ -carboxylase in Escherichia coli BL21(DE)3.
  • the DNA sequence shown in SEQ ID NO: 2 of the Sequence Listing is the coding gene panD C of L-aspartate ⁇ -carboxylase in Corynebacterium glutamicum ATCC13032.
  • the DNA sequence shown in SEQ ID NO: 3 of the Sequence Listing is the coding gene panD M of L-aspartate ⁇ -carboxylase in Mycobacterium tuberculosis H37Rv.
  • the DNA sequence shown in SEQ ID NO: 4 of the Sequence Listing is the coding gene panD B of L-aspartate ⁇ -carboxylase in Bacillus subtilis 168.
  • PCR amplification was carried out using a primer pair consisting of Ec-pand-for and Ec-pand-rev to obtain a PCR amplification product.
  • Ec-pand-rev 5'-TTACGG GGTACC TCAAGCAACCTGTACCGGAA-3' (SEQ ID NO: 10).
  • the vector pET-30a(+) was digested with restriction endonucleases Nde I and Kpn I to recover a vector backbone of about 5300 bp.
  • the recombinant plasmid pET30-panD E was structurally described as follows: Sequence 1 of the sequence listing was inserted between the Nde I and Kpn I restriction sites of the vector pET-30a(+) from the 5' end 4th to 381 A double-stranded DNA molecule as shown.
  • the recombinant plasmid pET30-panD E was introduced into Escherichia coli BL21 (DE3), and the obtained recombinant strain was an engineering strain A, also known as an engineering strain E. coli BL21(DE3)/pET30-panD E .
  • PCR amplification was carried out using a primer pair consisting of cg-pand-for and cg-pand-rev to obtain a PCR amplification product.
  • Cg-pand-rev 5'-TTACGG CTCGAG TCAAATACTACGGCTCGTCAGC-3' (SEQ ID NO: 12).
  • the vector pET-30a(+) was digested with restriction endonucleases Nde I and Xho I to recover a vector backbone of about 5300 bp.
  • the recombinant plasmid pET30-panD C was structurally described as follows: Sequence 2 of the sequence listing was inserted between the Nde I and Xho I cleavage sites of the vector pET-30a(+) from the 5' end 4th to 411 The DNA fragment shown.
  • the recombinant plasmid pET30-panD C was introduced into Escherichia coli BL21 (DE3), and the obtained recombinant strain was engineered bacteria B, also known as engineering bacteria E. coli BL21 (DE3) / pET30-panD C .
  • PCR amplification was carried out using a primer pair consisting of mt-pand-for and mt-pand-rev to obtain a PCR amplification product.
  • Mt-pand-rev 5'-TTACGG GGTACC CTATCCCACACCGAGCCG-3' (SEQ ID NO: 14).
  • the vector pET-30a(+) was digested with restriction endonucleases Nde I and Kpn I to recover a vector backbone of about 5300 bp.
  • the recombinant plasmid pET30-panD M was structurally described as follows: Sequence 3 of the sequence listing was inserted between the Nde I and Kpn I restriction sites of the vector pET-30a (+) from the 5' end 4 to 420 The DNA fragment shown.
  • the recombinant plasmid pET30-panD M was introduced into Escherichia coli BL21 (DE3), and the obtained recombinant strain was an engineering bacteria C, also known as an engineering strain E. coli BL21(DE3)/pET30-panD M .
  • the DNA fragment synthesized in the step (1) is used as a template, and a primer pair consisting of bs-pand-for and bs-217-rev is subjected to PCR amplification to obtain a PCR amplification product.
  • PCR amplification was carried out using a primer pair consisting of bs-217-for and bs-pand-rev to obtain a PCR amplification product.
  • Bs-pand-rev 5'-TTACGG GGTACC CTACAAAATTGTACGGGCTGGT-3' (SEQ ID NO: 18).
  • the PCR amplification product of the step (4) was double-digested with restriction endonucleases Nde I and Kpn I, and the digested product was recovered.
  • the vector pET-30a(+) was digested with the restriction enzymes Nde I and Kpn I to recover a vector skeleton of about 5300 bp.
  • step (7) and the digestion products of step (6) of step (5) the vector backbone connection, to obtain recombinant plasmid pET30-panD B.
  • sequence 5 of the sequence listing was inserted between the Nde I and Kpn I restriction sites of the vector pET-30a (+) from the 5' end 4 to 384
  • the DNA fragment shown differs from sequence 4 only in that sequence 4 is mutated from A at position 207 of the 5' end from A to C to remove the Nde I restriction recognition sequence in SEQ ID NO:4; Both Sequence 4 and Sequence 5 encode the protein shown in SEQ ID NO: 6 of the Sequence Listing).
  • the DNA sequence of the recombinant plasmid pET30-panD B is sequence 7 in the sequence listing.
  • the recombinant plasmid pET30-panD B was introduced into Escherichia coli BL21 (DE3), and the obtained recombinant strain was an engineering bacterium, also known as an engineering strain E. coli BL21(DE3)/pET30-panD B .
  • the engineering bacteria A, engineering bacteria B, engineering bacteria C and engineering bacteria constructed in Example 1 were respectively subjected to the following operations:
  • the whole culture system obtained in the step 1 was inoculated into 100 mL of LB liquid medium containing 50 ⁇ g/ml kanamycin and 0.2 mM IPTG, and cultured at 30 ° C, shaking at 200 rpm for 12 hours.
  • take 2mL of culture solution add 1mL of phosphate buffer with pH 8.0 and 0.01mol/L, mix and then ultrasonically crush (power 200W, work 3 Stop for 3 seconds in seconds, total time is 4 min), centrifuge at 10,000 rpm for 1 min, and take the supernatant.
  • step 4 Take 500 ⁇ L of the supernatant obtained in step 3, and add 536 ⁇ L of 60 g/L L-aspartic acid solution (preparation method of L-aspartic acid solution: take L-aspartic acid, add water, add NaOH The pH was adjusted to 7.0 to promote the dissolution of L-aspartic acid. The reaction was allowed to stand at 37 ° C for 2 h, centrifuged at 12,000 rpm for 1 min, and the supernatant was taken.
  • the supernatant obtained in the step 4 was diluted with distilled water to 50 volumes to obtain a diluted solution, and the diluted solution was derivatized and subjected to HPLC detection.
  • Derivatization method Take 300 ⁇ L of the dilution solution, add 360 ⁇ L of pH 9.5, 0.05 mol/L sodium borate buffer, then add 240 ⁇ L of the derivatizing agent, mix, react at room temperature for 2 min, and perform HPLC detection.
  • the derivatizing agent consisted of 1.3 g of o-phthalaldehyde, 0.59 g of N-acetylcysteine, 20 mL of absolute ethanol, and 78.11 mL of a pH 9.5, 0.05 mol/L borate buffer.
  • a standard solution (standard solution containing 0.4 g/L L-aspartic acid and 0.3 g/L ⁇ -alanine; preparation method of standard solution: taking L-aspartic acid standard and ⁇ - Alanine standard, adding water, adding NaOH to adjust pH to 7.0 to promote L-aspartic acid and ⁇ -alanine dissolution)
  • HPLC chart is shown in Figure 1 ( In Figure 1, the unit of the ordinate is mAU).
  • the peak time of the L-aspartic acid standard is 1.409min
  • the peak time of the ⁇ -alanine standard was 2.910min.
  • the amount of enzyme required to produce 1 ⁇ mol of product ( ⁇ -alanine) per minute is defined as an enzyme unit.
  • the enzyme activity histogram of the supernatant obtained from the cell suspension of the bacterial cells of engineering bacteria A, engineering bacteria B, engineering bacteria C and engineering bacteria is shown in Fig. 2. It can be seen that the supernatant obtained from the engineered bacterium has the highest enzyme activity of 2.09 U/mL. That is, the engineered fungus expressing L-aspartate ⁇ -carboxylase of Bacillus subtilis on the plasmid has the highest activity.
  • Example 1 A single colony of the engineered bacterium prepared in Example 1 was picked, inoculated into 100 mL of LB liquid medium, and cultured at 37 ° C, shaking at 200 rpm for 12 h.
  • the whole culture system obtained in the step 1 was inoculated into 1000 mL of LB liquid medium containing 50 ⁇ g/ml kanamycin and 0.2 mM IPTG, and cultured at 30 ° C, shaking at 200 rpm for 12 h.
  • the culture solution obtained in the step 2 was centrifuged at 9000 rpm for 10 minutes to collect the cells.
  • step 4 Take all the cells obtained in step 3, suspend with 300 mL of distilled water, add L-aspartic acid (90 g), biotransformation at 37 ° C, 450 rpm for 15 h, during the conversion, sample once every hour, using the example 2 Methods The concentration of ⁇ -alanine was determined by HPLC.
  • the present invention integrates the gene encoding pan- B of L-aspartate alpha carboxylase of Bacillus subtilis by selecting an appropriate integration site on the E. coli genome, namely, the frdB gene and the tktB gene locus. On the E. coli genome, an engineered strain containing two copies of the panD B gene and containing no plasmid was obtained.
  • Example 4 Construction of an engineered bacterium on the genome expressing L-aspartate alpha carboxylase of Bacillus subtilis
  • the frdB gene (encoding fumarate reductase, converting succinate to fumaric acid, Gene ID: 948666) and the tktB gene (encoding transketolase, D-sedoheptulose) were selected in the E. coli BL21 (DE3) genome. Conversion of 7-phosphoric acid and D-glyceraldehyde-3-phosphate to D-ribose-5-phosphate and D-xylulose-5-phosphate, Gene ID: 12932322) as L-aspartate alpha carboxylate of Bacillus subtilis The integration site of the gene encoding the gene panD B.
  • the panD B gene (sequence 5, in which the 207th nucleotide at the 5' end of the sequence has been mutated from A to C to remove the Nde I restriction sequence in the sequence) is integrated into the frdB of the E. coli BL21 (DE3) genome.
  • an engineered PAND-1 that integrates a copy of the panD B gene was constructed.
  • the engineering strain PAND-2 integrating the two copies of the panD B gene was constructed with tktB as the integration site.
  • the specific construction method is as follows:
  • DNA fragment of nucleotides 68-998 (containing the T7 promoter sequence (sequence 8)), obtained fragment 2 (991 bp); plasmid PKD4 (purchased from Changsha Winrun Biotechnology Co., Ltd., sequence GenBank: AY048743.
  • a DNA fragment of nucleotides 31-1507 was amplified with primer f-1450-for and primer f-2978-rev to obtain fragment 3 (1519 bp); Escherichia coli BL21 (DE3) genome was used as a template
  • a DNA fragment of nucleotides 4287127-4286588 on the genome was amplified with primer f-2822-for and primer f-3488-rev to obtain fragment 4 (567 bp).
  • Fragments 1, 2, 3, 4 were fused in equimolar to obtain the fragment FrdB-panD B- kan-FrdB (PCR conditions: 94 ° C, 5 min; 94 ° C, 30 s, 55 ° C, 30 s, 72 ° C, 3 min 30 s, 30 cycles; 72 ° C, 10 min.).
  • a full length 3488 bp fragment was obtained (see Figure 4A). Treat with Dpn 1 and store at -20 °C until use.
  • PCP20 was then transferred to the strain, and kan resistance was discarded.
  • Colony PCR was performed using the primer frbB-for and the primer frdB-rev, at which time a 2583 bp fragment was obtained by PCR (Fig. 4C, Fig. 7), demonstrating that the kan gene integrated into the frdB gene locus in the previous step was successfully eliminated.
  • the correct strain was cultured at 37 ° C to lose the plasmid PCP20, and an engineering strain PAND-1 integrating a copy of the panD B gene was obtained.
  • FIG. 1 A schematic diagram of the above method is shown in FIG. An engineered strain integrating the two copies of the panD B gene was then constructed in the same manner.
  • fragment 2-4 (628 bp). Fragments 2-1, 2-2, 2-3, 2-4 were fused in equimolar to obtain the fragment tktB-panD B- kan-tktB, which was 3176 bp in length (Fig. 5A). Treat with Dpn 1 and store at -20 °C until use.
  • PCP20 was transferred into the strain, kanamycin resistance was discarded, and colony PCR was performed using primer Tktb-for and primer Tktb-rev to obtain a DNA fragment of about 2060 bp (Fig. 5C), which proved that it was successfully eliminated.
  • the previous step was integrated into the kan gene in the tktB gene locus.
  • the correct strain was cultured at 37 ° C to lose the plasmid PCP20, and an engineered strain PAND-2 integrating the two copies of the panD B gene was obtained.
  • the engineering strain PAND-2 constructed by the present invention is obtained by replacing the frdB gene and the tktB gene in the genome of Escherichia coli BL21 (DE3) with the coding gene panD B of L-aspartate ⁇ -carboxylase of Bacillus subtilis.
  • An engineered strain containing no plasmid, high L-aspartate alpha carboxylase activity It can be seen from the above examples that the use of PAND-2 not only obtains higher L-aspartate alpha carboxylase activity, but also eliminates the problem of plasmid loss during large-scale cultivation, ensuring industrial application.
  • the enzyme activity is stable.
  • the engineered bacteria constructed by the invention does not need to add antibiotics in the cell culture process, reduces the cost of cell culture and the cost of subsequent sewage treatment, and is beneficial to the biological production of ⁇ -alanine in the industry.

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Abstract

提供了用于生产β-丙氨酸的工程菌及生产β-丙氨酸的方法。该工程菌相对于原始菌能够过表达序列6所示的枯草芽孢杆菌L-天冬氨酸a羧化酶，或者过表达与该酶具有至少60%、优选80%、更优选90%、进一步优选95%和最优选98%、甚至99%同源性且具有所述酶活性的多肽。

Description

用于生产β-丙氨酸的工程菌及生产β-丙氨酸的方法 技术领域
本发明涉及生物催化领域，尤其涉及用于生产β-丙氨酸的基因工程菌的构建及应用。
背景技术
β-丙氨酸是自然界天然存在的唯一一个β型丙氨酸，在医疗，食品，化工领域有广泛的应用。如作为化工原料合成泛酸钙、肌肽以及用于合成治疗肿瘤高血钙的帕米磷酸钠和治疗溃疡性肠炎的费特异性抗炎药物巴柳氮。另外，β-丙氨酸还可以作为药剂制备中的沉淀剂、水的净化絮凝剂、电镀缓蚀剂、铅中毒解毒剂以及用于合成甜味剂等。
β-丙氨酸化学法生产包括丙烯腈法、丙烯酸法、琥珀酰亚胺降解法及β-氨基丙腈法。其中，丙烯腈法是国内生产β-丙氨酸的主要方法。该方法将丙烯腈和氨水氨化反应生成β-氨基丙腈，再在酸性或碱性条件下水解即得。该方法原料易得，成本较低，但有副反应，且水解过程中生成大量无机盐，产物较难纯化。
近年来，通过生物转化法来生产β-丙氨酸因其反应条件温和、专一性强、环境友好等特点受到越来越多的关注。生物转化法主要利用L-天冬氨酸α羧化酶特异性地脱去L-天冬氨酸的α羧基，生成β-丙氨酸。1999年，Nicole等报道了在大肠杆菌中过表达其自身来源的L-天冬氨酸α羧化酶，酶活仅为0.22U/mg，而在大肠杆菌中表达来源于谷氨酸棒状杆菌的L-天冬氨酸α羧化酶，酶活为3.42U/mg(Nicole,1999,Applied and Environmental Microbiology)。2002年，Chopra等发现来源于结核杆菌(M.tuberculosis)的L-天冬氨酸α羧化酶的酶活为35μmol/g·h(Chopra,2002,Protein Expression and Purification)。2006年，中国专利申请公开No.CN 101210230A公开了一株过表达其自身来源的L-天冬氨酸α羧化酶的大肠杆菌BL21(DE3)。使用该菌株催化L-天冬氨酸得到的β-丙氨酸仅为2.94g/L。由此可见，这些早期研究中由于所选择的L-天冬氨酸α羧化酶活性较低，从而导致生物转化得到的β-丙氨酸产量极低，无法达到工业化生产的需要。因此，仍需要进一步开发能高效转化L-天冬氨酸生产β-丙氨酸的工程菌。
发明内容
针对上述问题，本发明的目的在于提供一种能高效表达具有较高活性的L-天冬氨酸α羧化酶的重组大肠杆菌工程菌，以及利用全细胞转化L-天冬氨酸生产β-丙氨酸的方法。
为此，根据本发明，提供一种重组大肠杆菌工程菌，所述工程菌相对于原始菌能够过表达序列6所示的来源于枯草芽孢杆菌的L-天冬氨酸α羧化酶，或者过表达与该酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性，且具有所述酶活性的多肽。所述大肠杆菌没有特别限制，但是优选BL21(DE3)。
根据一种实施方式，所述工程菌是在大肠杆菌中导入重组质粒，所述重组质粒是在载体的多克隆位点插入序列5所示的编码枯草芽孢杆菌L-天冬氨酸α羧化酶的基因或编码与该酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性且具有所述酶活性的多肽的基因。所述载体没有特别限制，但是优选pET-30a(+)。
根据另一种实施方式，所述工程菌是在大肠杆菌基因组上的一个或多个位点(例如2～4个)整合有DNA序列的重组菌，所述DNA序列包含序列5所示的编码枯草芽孢杆菌L-天冬氨酸α羧化酶的基因和编码与所述酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性且具有所述酶活性的多肽的基因中的一个。
所述插入位点没有特别限制，但是优选基因组中frdB基因和tktB基因所在位点。
所述DNA序列进一步包含启动子序列。
对所述启动子序列没有特别的限制，只要能够启动下游的L-天冬氨酸α羧化酶编码基因的任何启动子都是可行的。例如但不限于：枯草芽孢杆菌L-天冬氨酸α羧化酶基因自身的启动子，构建本发明工程菌所用质粒中携带的启动子，例如可以是PUC19中启动子、pET-30a(+)中的启动子，还可以是常用的强启动子，如Lac、T7等。优选T7启动子(序列8)。
根据更为具体的实施方式，通过同源重组将由四个DNA片段组成的1·2·3·4片段整合到大肠杆菌BL21(DE3)基因组中frdB基因中，其中所述1·2·3·4片段中，片段1为大肠杆菌BL21(DE3)基因组(GenBank:AM946981.2)第4287847-4287308位核苷酸的DNA片段，片段2为质粒pET30-panDB(序列7)第68-998位核苷酸的DNA片段，片段3为质粒PKD4(GenBank:AY048743.1)第31-1507位核苷酸的DNA片段，和片段4为大肠杆菌BL21(DE3)基因组(GenBank:AM946981.2)第4287127-4286588位核苷酸的DNA片段；
丢掉kan抗性，获得在frdB基因中插入一拷贝枯草芽孢杆菌L-天冬氨酸α羧化酶基因的重组菌；
通过同源重组将由四个DNA片段组成的2-1·2-2·2-3·2-4片段整合到大肠杆菌BL21(DE3)基因组中tktB基因中，其中所述2-1·2-2·2-3·2-4片段中，片段2-1为大肠杆菌 BL21(DE3)基因组(GenBank:AM946981.2)第2444239-2444839位核苷酸的DNA片段，片段2-2为质粒pET30-panDB(序列7)第236-735位核苷酸的DNA片段，片段2-3为质粒PKD4(GenBank:AY048743.1)第31-1508位核苷酸的DNA片段，和片段4为大肠杆菌BL21(DE3)基因组(GenBank:AM946981.2)第2445379-2445978位核苷酸的DNA片段；
丢掉kan抗性，获得在frdB基因和tktB基因中插入两拷贝L-天冬氨酸α羧化酶基因的重组菌。
本发明的工程菌可用于生物转化L-天冬氨酸生产β-丙氨酸。
根据本发明的第二方面，提供一种全细胞生物转化L-天冬氨酸生产β-丙氨酸的方法，包括发酵培养上述工程菌的步骤。还包括以L-天冬氨酸为底物，用所述工程菌进行生物转化，生产β-丙氨酸。
本发明筛选得到高活性的枯草芽孢杆菌L-天冬氨酸α羧化酶，并通过基因重组在大肠杆菌中增强该酶的表达，从而能够高效转化L-天冬氨酸获得β-丙氨酸。
而且根据本发明优选的实施方式，将枯草芽孢杆菌L-天冬氨酸α羧化酶整合到大肠杆菌基因组中，进一步提高了发酵液中L-天冬氨酸α羧化酶的活性。本发明的工程菌避免了菌体在大规模培养过程中的质粒丢失问题，保证了工业化应用过程中酶活的稳定。另外，本发明所构建的工程菌在菌体培养过程中无需添加抗生素，节约了培养成本和后续污水处理成本，有利于在工业上实现β-丙氨酸的生物法生产。
附图说明
通过以下附图可以更好地理解本发明。
图1为标准品溶液的HPLC图谱。
图2为工程菌甲、工程菌乙、工程菌丙和工程菌丁发酵液经细胞破碎后得到的上清液的酶活柱形图。
图3为生物转化过程中转化体系中生成的β-丙氨酸浓度随时间变化的曲线图。
图4中A为根据本发明的方法构建PAND-1菌株时片段FrdB-panDB-kan-FrdB的电泳图；B为验证构建的PAND-1-1菌株中是否带有FrdB-panDB-kan-FrdB片段的电泳图；C为验证构建的PAND1菌株中已丢掉卡那霉素抗性的电泳图。
图5中A为根据本发明的方法构建PAND-2菌株时片段tktB-panDB-kan-tktB的电泳图；B为验证构建的PAND-2-1菌株中是否带有tktB-panDB-kan-tktB片段的电泳图；C为验证构建的PAND2菌株中已丢掉卡那霉素抗性的电泳图。
图6为重组菌株PAND-1和PAND-2在不同温度下培养12小时后培养液中L-天冬氨酸α羧化酶活性检测结果。
图7为根据本发明实施例将编码枯草芽孢杆菌L-天冬氨酸α羧化酶的基因插入大肠杆菌BL21(DE3)的同源重组方法示意图，其中
A为野生型(BL21(DE3))大肠杆菌基因组中用引物frdB-for和frdB-rev通过PCR获得1735bp DNA片段；
B为将片段FrdB-panDB-kan-FrdB插入到基因组后，用引物FrdB-For和FrdB-rev通过PCR验证获得的3963bp DNA片段；和
C为去掉kan基因后，用引物FrdB-For和FrdB-rev通过PCR验证获得的2583bp DNA片段。
具体实施方式
以下结合附图通过具体实施例来进一步详细地说明本发明。本领域技术人员应理解，以下具体实施例仅用于说明本发明，而非对本发明的限制。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下通过实施例进一步说明本发明的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，可参见《分子克隆实验指南(第3版)》(科学出版社)、《微生物学实验(第4版)》(高等教育出版社)以及相应仪器和试剂的厂商说明书等。实施例中所用仪器设备和试剂为市售的常用仪器、试剂。以下实施例中的定量试验，均设置三次重复实验，每次重复实验中含有10个重复处理，结果取平均值。
载体pET-30a(+)：购自Novagen公司。大肠杆菌BL21(DE3)：购自Novagen公司。L-天冬氨酸标准品：购自生工生物工程(上海)股份有限公司，产品编号AB0091CAS(56-84-8)。β-丙氨酸标准品：购自生工生物工程(上海)股份有限公司，产品编号A6168CAS(107-95-9)。
LB液体培养基组成：酵母膏(0.5％)，蛋白胨(1％)，氯化钠(1％)
LB固体培养基组成：酵母膏(0.5％)，蛋白胨(1％)，氯化钠(1％)，1.5％琼脂。
本文提及的“同源性(homology)”是指DNA的核苷酸序列或蛋白质的氨基酸序列之间的相似程度，同时本文所说的具有(一定程度)同源性的DNA其所编码的蛋白至少在用于本发明的功能方面具有相同的活性，同样的具有(一定程度)同源性的蛋白质至少在用于本发明的功能方面具有相同的活性。
本文提及的方法中各步骤的执行顺序并不限于本文的文字所体现出来的顺序，也就是说，各个步骤的执行顺序是可以改变的，而且两个步骤之间根据需要可以插入其他步骤。
本发明的工程菌是在大肠杆菌基因组上的一个或多个位点整合有DNA序列的重组菌，所述DNA序列包含选自编码L-天冬氨酸α羧化酶的基因和编码与所述酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性且具有所述酶活性的多肽的基因中的一个。
经以下实施例筛选得到的高活性L-天冬氨酸α羧化酶是枯草芽孢杆菌L-天冬氨酸α羧化酶(序列6)。当然，活性越高的L-天冬氨酸α羧化酶是越有利的。
本发明通过同源重组的方法将高活性L-天冬氨酸α羧化酶的基因整合到大肠杆菌的基因组中，从而获得能够稳定增殖且过表达该酶的工程菌。
对于整合基因的拷贝数没有特别限制，但是优选为2～4拷贝，更优选为2拷贝。
整合位点应当是不会对菌的增殖和生长造成不良影响的那些位点。优选大肠杆菌基因组中的frdB基因和/或tktB基因中的位点。
对大肠杆菌也没有特别的限制，优选为BL21(DE3)。
实施例1、构建质粒上表达L-天冬氨酸α羧化酶的工程菌
我们分别选择来源于大肠杆菌、谷氨酸棒状杆菌(Corynebacterium glutamicum)、结核分枝杆菌(Mycobacterium tuberculosis)和枯草芽孢杆菌(Bacillus subtilis)这4种宿主的L-天冬氨酸α-羧化酶的编码基因，进行4种工程菌的构建。其中，序列表的序列1所示的DNA序列是大肠杆菌BL21(DE)3中L-天冬氨酸α-羧化酶的编码基因panDE。序列表的序列2所示的DNA序列是谷氨酸棒状杆菌ATCC13032中L-天冬氨酸α-羧化酶的编码基因panDC。序列表的序列3所示的DNA序列是结核分枝杆菌H37Rv中L-天冬氨酸α-羧化酶的编码基因panDM。序列表的序列4所示的DNA序列是枯草芽孢杆菌168中L-天冬氨酸α-羧化酶的编码基因panDB。
一、质粒上表达大肠杆菌L-天冬氨酸α-羧化酶的工程菌甲的构建
1、重组质粒pET30-panDE的构建
(1)合成序列表的序列1所示的DNA片段。
(2)以步骤(1)合成的DNA片段为模板，用Ec-pand-for和Ec-pand-rev组成的引物对进行PCR扩增，得到PCR扩增产物。
Ec-pand-for：5’-GGCTTCCATATGATTCGCACGATGCTGC-3’(序列9)；
Ec-pand-rev：5’-TTACGGGGTACCTCAAGCAACCTGTACCGGAA-3’(序列10)。
(3)用限制性内切酶Nde Ⅰ和Kpn Ⅰ双酶切步骤(2)获得的PCR扩增产物，回收酶切产物。
(4)用限制性内切酶Nde Ⅰ和Kpn Ⅰ双酶切载体pET-30a(+)，回收约5300bp的载体骨架。
(5)将步骤(3)的酶切产物和步骤(4)的载体骨架用T4连接酶，16℃过夜连接，得到重组质粒pET30-panDE。
根据测序结果对重组质粒pET30-panDE进行结构描述如下：在载体pET-30a(+)的Nde Ⅰ和Kpn Ⅰ酶切位点之间插入了序列表的序列1自5’末端第4至381所示的双链DNA分子。
2、工程菌甲的构建
将重组质粒pET30-panDE导入大肠杆菌BL21(DE3)，得到的重组菌即为工程菌甲，又称工程菌E.coli BL21(DE3)/pET30-panDE。
二、质粒上表达谷氨酸棒状杆菌L-天冬氨酸α-羧化酶的工程菌乙的构建
1、重组质粒pET30-panDC的构建
(1)合成序列表的序列2所示的DNA片段。
(2)以步骤(1)合成的DNA片段为模板，用cg-pand-for和cg-pand-rev组成的引物对进行PCR扩增，得到PCR扩增产物。
cg-pand-for：5’-GGCTTCCATATGCTGCGTACCATCCTG-3’(序列11)；
cg-pand-rev：5’-TTACGGCTCGAGTCAAATACTACGGCTCGTCAGC-3’(序列12)。
(3)用限制性内切酶Nde Ⅰ和Xho Ⅰ双酶切步骤(2)的PCR扩增产物，回收酶切产物。
(4)用限制性内切酶Nde Ⅰ和Xho Ⅰ双酶切载体pET-30a(+)，回收约5300bp的载体骨架。
(5)将步骤(3)的酶切产物和步骤(4)的载体骨架连接，得到重组质粒pET30-panDC。
根据测序结果对重组质粒pET30-panDC进行结构描述如下：在载体pET-30a(+)的Nde Ⅰ和Xho Ⅰ酶切位点之间插入了序列表的序列2自5’末端第4至411所示的DNA片段。
2、工程菌乙的构建
将重组质粒pET30-panDC导入大肠杆菌BL21(DE3)，得到的重组菌即为工程菌乙，又称工程菌E.coli BL21(DE3)/pET30-panDC。
三、质粒上表达结核分枝杆菌L-天冬氨酸α-羧化酶的工程菌丙的构建
1、重组质粒pET30-panDM的构建
(1)合成序列表的序列3所示的DNA片段。
(2)以步骤(1)合成的DNA片段为模板，用mt-pand-for和mt-pand-rev组成的引物对进行PCR扩增，得到PCR扩增产物。
mt-pand-for：5’-GGCTTCCATATGTTACGGACGATGCTG-3’(序列13)；
mt-pand-rev：5’-TTACGGGGTACCCTATCCCACACCGAGCCG-3’(序列14)。
(3)用限制性内切酶Nde Ⅰ和Kpn Ⅰ双酶切步骤(2)的PCR扩增产物，回收酶切产物。
(4)用限制性内切酶Nde Ⅰ和Kpn Ⅰ双酶切载体pET-30a(+)，回收约5300bp的载体骨架。
(5)将步骤(3)的酶切产物和步骤(4)的载体骨架连接，得到重组质粒pET30-panDM。
根据测序结果对重组质粒pET30-panDM进行结构描述如下：在载体pET-30a(+)的Nde Ⅰ和Kpn Ⅰ酶切位点之间插入了序列表的序列3自5’末端第4至420所示的DNA片段。
2、工程菌丙的构建
将重组质粒pET30-panDM导入大肠杆菌BL21(DE3)，得到的重组菌即为工程菌丙，又称工程菌E.coli BL21(DE3)/pET30-panDM。
四、质粒上表达枯草芽孢杆菌L-天冬氨酸α-羧化酶的工程菌丁的构建
1、重组质粒pET30-panDB的构建
(1)合成序列表的序列4所示的DNA片段。
(2)以步骤(1)合成的DNA片段为模板，用bs-pand-for和bs-217-rev组成的引物对进行PCR扩增，得到PCR扩增产物。
bs-pand-for：5’-GGCTTCCATATGTATCGAACAATGATGAGCG-3’(序列15)；
bs-217-rev：5’-TGCACCGTTTAAGCA
Figure PCTCN2015074683-appb-000001
ATGACGCCGCTTCCC-3’(序列16)。
(3)以步骤(1)合成的DNA片段为模板，用bs-217-for和bs-pand-rev组成的引物对进行PCR扩增，得到PCR扩增产物。
bs-217-for：5’-GGGAAGCGGCGTCAT
Figure PCTCN2015074683-appb-000002
TGCTTAAACGGTGCA-3’(序列17)；
bs-pand-rev：5’-TTACGGGGTACCCTACAAAATTGTACGGGCTGGT-3’(序列18)。
(4)同时将步骤(2)得到的PCR扩增产物和步骤(3)得到的PCR扩增产物作为模板，用bs-pand-for和bs-pand-rev组成的引物对进行PCR扩增，得到PCR扩增产物。
(5)用限制性内切酶Nde Ⅰ和Kpn Ⅰ双酶切步骤(4)的PCR扩增产物，回收酶切产物。
(6)用限制性内切酶Nde Ⅰ和Kpn Ⅰ双酶切载体pET-30a(+)，回收约5300bp的载体骨架。
(7)将步骤(5)的酶切产物和步骤(6)的载体骨架连接，得到重组质粒pET30-panDB。
根据测序结果对重组质粒pET30-panDB进行结构描述如下：在载体pET-30a(+)的Nde Ⅰ和Kpn Ⅰ酶切位点之间插入了序列表的序列5自5’末端第4至384所示的DNA片段(序列 5与序列4的差别仅在于将序列4自5’末端第207位核苷酸由A突变为了C，以去除序列4中的Nde Ⅰ酶切识别序列；序列表的序列4和序列5均编码序列表的序列6所示的蛋白质)。该重组质粒pET30-panDB的DNA序列为序列表中的序列7。
2、工程菌丁的构建
将重组质粒pET30-panDB导入大肠杆菌BL21(DE3)，得到的重组菌即为工程菌丁，又称工程菌E.coli BL21(DE3)/pET30-panDB。
实施例2、检测质粒上表达L-天冬氨酸α羧化酶的工程菌的L-天冬氨酸α羧化酶活性
分别将实施例1构建的工程菌甲、工程菌乙、工程菌丙和工程菌丁进行如下操作：
1、挑取工程菌的单菌落，接种于10mL含50μg/ml卡那霉素的LB液体培养基，37℃、200rpm振荡培养12h。
2、取步骤1得到的整个培养体系，接种于100mL含50μg/ml卡那霉素和0.2mM IPTG的LB液体培养基，30℃、200rpm振荡培养12h。
3、步骤2的终止时刻(即OD600nm＝3的菌液)，取2mL培养液，加入1mL pH 8.0、0.01mol/L的磷酸缓冲液，混匀，然后进行超声波破碎(功率200W，工作3秒停3秒，总时间为4min)，10000rpm离心1min，取上清液。
4、取500μL步骤3得到的上清液，加入536μL 60g/L的L-天冬氨酸溶液(L-天冬氨酸溶液的制备方法：取L-天冬氨酸，加入水，加入NaOH调整pH为7.0以促进L-天冬氨酸溶解)，37℃静置反应2h，12000rpm离心1min，取上清液。
5、HPLC检测
取步骤4得到的上清液，用蒸馏水稀释至50倍体积，得到稀释液，稀释液衍生化后进行HPLC检测。
衍生化的方法：取300μL稀释液，加入360μL pH 9.5、0.05mol/L的硼酸钠缓冲液，然后再加入240μL衍生剂，混匀，室温反应2min后进行HPLC检测。
衍生剂由1.3g邻苯二甲醛、0.59g N-乙酰半胱氨酸、20mL无水乙醇和78.11mL pH 9.5、0.05mol/L的硼酸缓冲液组成。
HPLC检测采用Agilent色谱柱(Eclipse XDB-C18，5μm，4.6×150mm)，流动相为2.871g/L的乙酸钠水溶液，流速为1mL/min，在334nm(紫外)进行实时监测。
将标准品溶液(标准品溶液即含有0.4g/L L-天冬氨酸和0.3g/Lβ-丙氨酸的溶液；标准品溶液的制备方法：取L-天冬氨酸标准品和β-丙氨酸标准品，加入水，加入NaOH调整pH为7.0以促进L-天冬氨酸和β-丙氨酸溶解)按相同的方法进行衍生化后进行HPLC检测，HPLC图谱见图1(图1中，纵坐标的单位是mAU)。L-天冬氨酸标准品的出峰时间为 1.409min，β-丙氨酸标准品的出峰时间为2.910min。
β-丙氨酸的标准曲线方程为y＝10437x，y代表β-丙氨酸的峰面积，x代表β-丙氨酸的浓度(g/L)。L-天冬氨酸的标准曲线方程为y＝9884x，y代表L-天冬氨酸的峰面积，x代表L-天冬氨酸的浓度(g/L)。
每分钟生成1μmol产物(β-丙氨酸)所需要的酶量定义为一个酶活单位。
工程菌甲、工程菌乙、工程菌丙和工程菌丁的发酵液菌体经细胞破碎后得到的上清液的酶活柱形图见图2。可见，工程菌丁得到的上清液的酶活最高，为2.09U/mL。即质粒上表达枯草芽孢杆菌的L-天冬氨酸α羧化酶的工程菌丁的活性最高。
实施例3、利用工程菌丁转化L-天冬氨酸生产β-丙氨酸
1、挑取实施例1制备的工程菌丁的单菌落，接种于100mL LB液体培养基，37℃、200rpm振荡培养12h。
2、取步骤1得到的整个培养体系，接种于1000mL含50μg/ml卡那霉素和0.2mM IPTG的LB液体培养基，30℃、200rpm振荡培养12h。
3、取步骤2得到的培养液，9000rpm离心10min，收集菌体。
4、取全部步骤3得到的菌体，用300mL蒸馏水悬浮，加入L-天冬氨酸(90g)，37℃、450rpm生物转化15h，转化过程中，每小时取样一次，采用实施例2中的方法通过HPLC检测β-丙氨酸的浓度。
检测结果见图3。转化15h时转化体系中β-丙氨酸的浓度达178g/L，转化率达99％。
由以上实施例1～3的研究，比较了来源于大肠杆菌、谷氨酸棒状杆菌、结合杆菌及枯草芽孢杆菌的L-天冬氨酸α羧化酶的活性。当将这些酶的基因克隆到质粒中并在大肠杆菌中过表达时，我们发现过表达枯草芽孢杆菌的L-天冬氨酸α羧化酶的工程菌丁的酶活高达2.09U/mL。使用该工程菌转化L-天冬氨酸15h后β-丙氨酸产量即达178g/L，转化率高达99％，是目前报道的最高水平，具有工业化应用的潜力。
然而，在中式规模(300L)时发现，即便在添加抗生素的情况下，工程菌丁仍然存在严重的质粒丢失现象。培养结束后几乎一半的菌体细胞已经不含有质粒。这大大降低了发酵液的总酶活，导致后续转化中β-丙氨酸的产量低下。另外，菌体培养时抗生素的使用不但增加了成本，也给后续污水处理带来负担。
针对这些问题，本发明通过在大肠杆菌基因组上选择合适的整合位点，即，frdB基因和tktB基因位点，将枯草芽孢杆菌的L-天冬氨酸α羧化酶的编码基因panDB整合到大肠杆菌基因组上，获得包含两个拷贝的panDB基因、且不包含质粒的工程菌。
实施例4：构建基因组上表达枯草芽孢杆菌的L-天冬氨酸α羧化酶的工程菌
选择大肠杆菌BL21(DE3)基因组中frdB基因(编码富马酸还原酶，将琥珀酸转化成延胡索酸，Gene ID:948666)和tktB基因(编码转酮醇酶，将D-景天庚酮糖-7-磷酸和D-甘油醛-3-磷酸转化成D-核糖-5-磷酸和D-木酮糖-5-磷酸,Gene ID:12933222)作为枯草芽孢杆菌的L-天冬氨酸α羧化酶的编码基因panDB的整合位点。
将panDB基因(序列5，其中序列5’末端第207位核苷酸已由A突变为了C，以去除序列中的Nde Ⅰ酶切识别序列)整合到大肠杆菌BL21(DE3)基因组上的frdB基因所在位点，构建整合一拷贝panDB基因的工程菌PAND-1。在此菌株的基础上，以tktB为整合位点再构建整合两拷贝panDB基因的工程菌PAND-2。具体构建方法如下：
(1)以frdB作为一拷贝整合位点，以大肠杆菌BL21(DE3)基因组(GenBank:AM946981.2)为模板，用引物f-1-for和引物f-558-rev扩增基因组上第4287847-4287308位核苷酸的DNA片段，获得FrdB部分片段1(558bp)；以以上实施例1构建的质粒pET30-panDB为模板，用引物f-511-for和引物f-1501-rev扩增第68-998位核苷酸的DNA片段(其中含有T7启动子序列(序列8))，获得片段2(991bp)；以质粒PKD4(购自长沙赢润生物技术有限公司，序列GenBank:AY048743.1)为模板，用引物f-1450-for和引物f-2978-rev扩增第31-1507位核苷酸的DNA片段，获得片段3(1519bp)；以大肠杆菌BL21(DE3)基因组为模板，用引物f-2822-for和引物f-3488-rev扩增基因组上第4287127-4286588位核苷酸的DNA片段，获得片段4(567bp)。将片段1，2，3，4以等摩尔进行融合，获得片段FrdB-panDB-kan-FrdB(PCR条件：94℃，5min；94℃，30s，55℃，30s，72℃，3min 30s,30cycle；72℃，10min。)。经验证，获得全长3488bp的片段(参见图4A)。用Dpn 1处理，-20℃保存待用。
(2)将PKD46质粒转入到BL21(DE3)菌株获得BL21(DE3)/PKD46菌株。
(3)从平板上挑取BL21(DE3)/PKD46菌株单克隆，接入LB液体培养基，37℃过夜培养。1:100转接新的含有10mM阿拉伯糖的LB液体培养基，待OD600nm＝0.6，制备感受态，将1μg的以上制备的DNA片段FrdB-panDB-kan-FrdB转入感受态细胞中，30℃，摇床震荡复苏12h。将菌液全部涂在含50μg/mL卡那霉素的LB/kan平板，37℃培养12h。
用验证引物frdB-for和frdB-rev对菌落进行PCR验证，获得约3963bp的片段(图4B，附野生型对照为1735bp)，证明panDB以及卡那霉素抗性基因kan已经成功整合到frdB基因位点，将此工程菌命名为PAND-1-kan。
(4)然后在该菌株中转入PCP20，丢掉kan抗性。用引物frbB-for和引物frdB-rev做菌落PCR验证，此时PCR获得约2583bp的片段(图4C，图7)，证明已经成功消除上一步整合到frdB基因位点中的kan基因。验证正确的菌株37℃培养，使质粒PCP20丢失，获得整合一拷贝panDB基因的工程菌PAND-1。
以上方法的示意图见图7。然后以同样的方法构建整合两拷贝panDB基因的工程菌。
(5)以大肠杆菌BL21(DE3)基因组为模板，以引物t-1-for和t-627-rev对第2444239-2444839位核苷酸的DNA片段进行PCR扩增，获得片段2-1(627bp)；以质粒pET30-panDB为模板，用引物t-570-for和引物t-1129-rev对序列7的第236-735位核苷酸的DNA片段，进行扩增，获得片段2-2(560bp)，其中含有T7启动子序列(序列8)；以质粒PKD4为模板，以引物t-1072-for和引物t-2606-rev对第31-1508位核苷酸的DNA片段，进行PCR扩增，获得片段2-3(1535bp)；以大肠杆菌BL21(DE3)基因组为模板，用引物t-2549-for和引物t-3176-rev对第2445379-2445978位核苷酸的DNA片段，进行PCR扩增，获得片段2-4(628bp)。将片段2-1，2-2，2-3，2-4以等摩尔进行融合，获得片段tktB-panDB-kan-tktB，全长3176bp(图5A)。用Dpn 1处理，-20℃保存待用。
(6)将PKD46质粒转入到PAND-1菌株获得PAND-1/PKD46菌株
(7)从平板上挑取PAND-1/PKD46菌株单克隆，LB培养基过夜培养。1:100转接新的含有10mM阿拉伯糖的LB液体培养基中，待OD600nm＝0.6，制备感受态细胞，将1μg的DNA片段tktB-panDB-kan-tktB转入感受态细胞，30℃，复苏12h，将菌液离心全部涂在含50μg/mL卡那霉素的LB/kan平板。用引物Tktb-for和引物Tktb-rev做菌落PCR验证，获得约3440bp的DNA片段(图5B，附野生型对照为2004bp)，证明第二个拷贝的panDB基因以及卡那霉素抗性基因kan已经成功整合到tktB基因位点，将此菌株命名为PAND-2-kan。
(8)然后在该菌株中转入PCP20，丢掉卡那霉素抗性，用引物Tktb-for和引物Tktb-rev做菌落PCR验证，得到约2060bp的DNA片段(图5C)，证明已经成功消除上一步整合到tktB基因位点中的kan基因。将验证正确的菌株在37℃培养，使质粒PCP20丢失，获得整合两拷贝panDB基因的工程菌PAND-2。
表1.实施例1中菌株构建所涉及的引物及序列
Figure PCTCN2015074683-appb-000003
Figure PCTCN2015074683-appb-000004
实施例5检测基因组上表达L-天冬氨酸α羧化酶的工程菌的L-天冬氨酸α羧化酶活性
从平板上分别挑取PAND-1和PAND-2单菌落接种于10mL的LB培养基37℃过夜培养。取上述培养液1:10转接于100mL的含有0.2mM的IPTG的LB培养基，分别在25℃，30℃和37℃培养12h。分别收集2mL菌体，用磷酸缓冲液(pH8.0)悬浮，然后超声波破碎菌体细胞，离心取上清500μL，加入536μL L-天冬氨酸(60g/L)，37℃，反应120min，12000r/min离心取上清，稀释50倍，衍生化后HPLC(Agilent Technologies 1200series)检测(具体衍生化步骤见实施例2)。
结果如图6所示，工程菌PAND-2在25℃、30℃和37℃温度下培养12h后，其所表达的 L-天冬氨酸α羧化酶的活性比PAND-1菌株的酶活分别提高了33％，50％和59％，证明工程菌PAND-2优于PAND-1。其中，工程菌PAND-2在37℃培养12h的酶活达到2.27U/mL，为目前所构建工程菌中产生L-天冬氨酸α羧化酶活性的最高水平。
本发明所构建的工程菌PAND-2是将大肠杆菌BL21(DE3)基因组中的frdB基因和tktB基因替换为枯草芽孢杆菌的L-天冬氨酸α羧化酶的编码基因panDB，从而获得不含质粒的、高L-天冬氨酸α羧化酶活性的工程菌。通过以上实施例可以看出，使用PAND-2不但获得了更高的L-天冬氨酸α羧化酶活性，而且还杜绝了大规模培养过程中的质粒丢失问题，保证了工业应用过程中酶活的稳定。另外，本发明所构建的工程菌在菌体培养过程中无需添加抗生素，降低了菌体培养成本和后续污水处理成本，有利于在工业上实现β-丙氨酸的生物法生产。

Claims (10)

  1. 一种重组大肠杆菌工程菌，所述工程菌相对于原始菌能够过表达序列6所示的枯草芽孢杆菌L-天冬氨酸α羧化酶，或者过表达该酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性且具有所述酶活性的多肽。
  2. 根据权利要求1所述的重组大肠杆菌工程菌，其中所述工程菌是在大肠杆菌中导入重组质粒，所述重组质粒是在载体的多克隆位点插入有编码所述枯草芽孢杆菌L-天冬氨酸α羧化酶的基因或编码与该酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性且具有所述酶活性的多肽的基因。
  3. 根据权利要求1所述的重组大肠杆菌工程菌，其中所述工程菌是在大肠杆菌基因组上的一个或多个位点整合有DNA序列的重组菌，其中所述DNA序列为编码所述枯草芽孢杆菌L-天冬氨酸α羧化酶的基因和编码与该酶具有至少60％、优选80％、更优选90％、进一步优选95％和最优选98％、甚至99％同源性且具有所述酶活性的多肽的基因。
  4. 根据权利要求2或3所述的重组大肠杆菌工程菌，其中所述编码枯草芽孢杆菌L-天冬氨酸α羧化酶的基因为序列5所示的核苷酸序列。
  5. 根据权利要求3所述的重组大肠杆菌工程菌，其中所述DNA序列进一步包含启动子序列。
  6. 根据权利要求5所述的重组大肠杆菌工程菌，其中所述启动子序列为序列8所示的T7启动子序列。
  7. 根据权利要求3所述的重组大肠杆菌工程菌，其中所述整合位点是大肠杆菌基因组中的frdB基因和tktB基因中的位点。
  8. 根据权利要求2所述的重组大肠杆菌工程菌，其中所述载体为pET-30a(+)。
  9. 根据权利要求1所述的重组大肠杆菌工程菌，其中所述大肠杆菌为BL21(DE3)。
  10. 一种生产β-丙氨酸的方法，包括收集权利要求1所述的工程菌，加入底物L-天冬氨酸，生产β-丙氨酸。
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