RECOMBINANT STRAIN FOR PRODUCING SHIKIMIC ACID, AND CONSTRUCTION METHOD AND USE THEREOF

Abstract

The present invention relates to a recombinant strain for producing shikimic acid, in which a target gene that regulates the asymmetric cell division and target genes that regulate the shikimic acid production are expressed The target gene that regulates the asymmetric cell division includes cytoskeletal protein PopZ coding gene popZ, and the target genes that regulate the shikimic acid production include DAHP synthase coding gene aroG, 3-dehydroquinate synthase coding gene aroB, and transketolase coding gene tktA. The recombinant strain of the present invention realizes the de novo synthesis of shikimic acid using glucose as a substrate, with a low cost. After fermentation with the strain in a 7.5 L fermentor, the highest production of shikimic acid is 88.1 g/L, the yield is 0.33 g/g, and the production intensity of shikimic acid is 1.1 g/L/h.

Claims

1. A recombinant strain for producing shikimic acid, wherein in the recombinant strain a shikimate kinase I coding gene aroK and a shikimate kinase II coding gene aroL are knocked out, and a target gene that regulates the asymmetric cell division and target genes that regulate the shikimic acid production are expressed, the target gene that regulates the asymmetric cell division comprises a cytoskeletal protein PopZ coding gene popZ, and the target genes that regulate the shikimic acid production are 3-dehydroquinate synthase coding gene aroB, DAHP synthase coding gene aroG, and transketolase coding gene tktA.

2. The recombinant strain according to claim 1, wherein the recombinant strain is constructed with strain GL0002 as a host.

3. The recombinant strain according to claim 1, wherein the recombinant strain is constructed by using pETac-PopZ and PJ01-GAB as expression vectors, wherein pETac-PopZ is constructed by ligating the gene popZ to the vector pETac, and PJ01-GAB is constructed by ligating the gene aroB, the gene aroG, and the gene tktA sequentially to the vector PJ01.

4. The recombinant strain according to claim 3, wherein the expression vectors are constructed by inserting the gene popZ containing B0034RBS into the vector pETac to obtain pETac-PopZ; and inserting the genes aroB, aroG and tkta containing B0034RBS sequentially into the vector PJO1 to obtain PJ01-GAB.

5. The recombinant strain according to claim 1, wherein the cytoskeletal protein PopZ coding gene popZ has a nucleotide sequence as shown in SEQ ID NO: 1.

6. A method for constructing the recombinant strain according to claim 1, comprising steps of: inserting a popZ fragment into the plasmid pETac, to obtain a recombinant plasmid pETac-PopZ; sequentially ligating a aroB, aroG, and tktA fragment in tandem to the plasmid PJO1 to construct a recombinant plasmid PJO1-GAB; and transforming the recombinant plasmid pETac-PopZ and the recombinant plasmid PJO1-GAB into a strain GL0002 to obtain the recombinant strain.

7. A method for producing shikimic acid, comprising producing shikimic acid by fermentation using the recombinant strain according to claim 1.

8. The method according to claim 7, wherein fermentation conditions comprise a temperature of 35-38° C., a revolving speed of 480-530 rpm, an inoculation amount of 5-10%, a liquid load of 30-50%, a pH of 6.0-7.0, an initial OD.sub.600 of 0.04-0.3 for fermentation, an air flow rate of 1-2 vvm, and fermentation time of 72-100 h.

9. The method according to claim 7, wherein the fermentation conditions comprise a temperature of 35-38° C., a revolving speed of 200-220 rpm, an initial OD.sub.600 of 0.04-0.1 for fermentation, and fermentation time of 70-90 h.

10. A method, comprising: using the recombinant strain according to claim 1 to produce shikimic acid, a product containing shikimic acid, or a target protein.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To make the disclosure of the present invention more comprehensible, the present invention will be further described in detail by way of specific embodiments of the present invention with reference the accompanying drawings, in which:

[0031] FIG. 1 shows the regulation of parameters in asymmetric cell division;

[0032] FIG. 2 shows the production path of shikimic acid;

[0033] FIG. 3 shows the amount of shikimic acid produced in the control group CT and in the experimental group GS within 75 h of shake flask fermentation; and

[0034] FIG. 4 shows the amount of shikimic acid produced in the fermentor after 80 h of fermentation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention will be further described below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

[0036] Materials and methods involved in the examples:

[0037] The plasmid is constructed by a typical molecular biology method.

[0038] The cell morphological parameters are measured using a fluorescence microscope (Nikon microscop, 80i) at an ambient temperature controlled to 30° C.

[0039] Seed medium: LB medium, containing peptone 10 g/L, yeast powder 5 g/L, and sodium chloride 10 g/L.

[0040] Fermentation medium: including a standard fermentation medium (NBS medium, 1 L), K.sub.2HPO.sub.4 (7.5 g), ferric ammonium (III) citrate (0.3 g), citric acid monohydrate (2.1 g), L-phenylalanine (0.7 g), L-tyrosine (0.7 g), L-Tryptophan (0.35 g), and concentrated H.sub.2SO.sub.4 (1.2 mL). The fermentation medium is adjusted to pH 7.0 by adding concentrated aqueous ammonia before reaching a high pressure. The following supplements are added immediately before fermentation: glucose, MgSO.sub.4 (0.24 g), p-hydroxybenzoic acid (0.010 g), potassium p-aminobenzoate (0.010 g), 2,3-dihydroxybenzoic acid (0.010 g) and trace minerals (NH.sub.4).sub.6(Mo.sub.7O.sub.24).Math.4H.sub.2O (0.0037 g), ZnSO.sub.4.Math. 7H.sub.2O (0.0029 g), H.sub.3BO.sub.3 (0.0247 g), CuSO.sub.4 .Math. 5H.sub.2O (0.0025 g), and MnCl.sub.2.Math.4H.sub.2O (0.0158 g), and methyl-α-d-glucopyranoside of a final concentration of 1 mM. The glucose, MgSO.sub.4, and methyl-α-d-glucopyranoside solutions are respectively autoclaved, and the aromatic vitamin and trace mineral solutions are sterilized with a 0.22 .Math.M membrane. Before adding the fermentation medium, pH is adjusted to 7 with KOH, and the system is sterilized with a 0.22 .Math.m membrane. An antifoaming agent (Sigma 204) is added as needed.

[0041] Preparation of fermentation sample: A fermentation broth sample is centrifuged at 12000 rpm for 5 min, the supernatant is collected, diluted, and filtered through a 0.22 .Math.m aqueous-system filter membrane. The filtrate is analyzed by liquid chromatography analysis.

[0042] Determination of polylactic acid content: Dionex High Performance Liquid Chromatograph (with UV-Vis detector), Bio-Rad Aminex HPX-87H (300 × 7.8 mm, 9 .Math.m) chromatographic column; mobile phase: 0.005 M H.sub.2SO.sub.4 filtered through 0.22 .Math.m filter and ultrasonically degassed; flow rate: 0.6 mL/min; column temperature: 35° C.; and UV detection wavelength: 210 nm.

Example 1: Screening of Asymmetric Division Gene

[0043] The popZ gene encoding asymmetric cell division and the green fluorescent protein gfp gene (having a nucleotide sequence as shown in SEQ ID NO: 5) were fused, to obtain the GFP-PopZ protein. B0034RBS was inserted at the upstream ATG position of the fused gene by fusion PCR. The PCR product was recovered, and ligated, by homologous recombination, to the vector pETac plasmid digested at BamH1 and Sal1 restriction sites, to obtain the recombinant plasmid pETac-GFP-PopZ. The pETac-GFP plasmid was constructed similarly.

[0044] The obtained recombinant plasmids pETac-GFP-PopZ and pETac-GFP were introduced into competent cells E. coli JM109, respectively. Strains carrying the asymmetric cell division gene pETac-GFP-PopZ and the pETac-GFP plasmid were obtained, respectively.

[0045] In LB medium, the above strains were evaluated for parameters of asymmetric cell division by fluorescence microscopy. The results are shown in FIG. 1. It can be seen from FIGS. 1A-D, the GFP-PopZ fusion protein is expressed, and this allows GFP to accumulate at a pole of the cells. The data was analyzed by software matching with the fluorescence microscopy. The results show that the relative fluorescence distribution of GFP in the strain expressing GFP-PopZ fusion protein is higher at one end, and the control group shows a uniform GFP fluorescence distribution. Further, the relative pole number (1E) and the asymmetry (1F) of the strain were analyzed. The relative pole number and asymmetry of the strain expressing GFP-PopZ fusion protein reach 92% and 3.8, respectively. The results show that asymmetric cell division can be achieved effectively by expression of PopZ.

Example 2. Production of Shikimic Acid by Shake-flask Fermentation

[0046] The constructed vectors PJ01-GAB and pETac-PopZ expressing related genes in the shikimic acid synthesis pathway (FIG. 2) were introduced into the competent cells of S1 (strain GL0002), and a recombinant GS strain for producing shikimic acid and carrying PJ01-GAB and pETac-PopZ was obtained. The E. coli GL0002 is obtained by knocking out the gene coding alcohol dehydrogenase (adhE, SEQ ID NO:6) and the gene coding acetate kinase (ackA, SEQ ID NO:7) from the starting strain E. coli ATCC 8739.

[0047] The fermentation conditions for shikimic acid production were: 37° C., 220 rpm, initial OD.sub.600 of 0.1 for fermentation, and fermentation time of 75 h.

[0048] The shikimic acid-producing strain was cultured in NBS medium, and the content was determined. The results are shown in FIG. 3. With the extension of the bacterial culture time, the shikimic acid content in the control group CT is increased to 18.2 g/L, and the yield reaches 0.29 g/g. The shikimic acid content of the GS strain in the experimental group is increased to 28.2 g/L, and the yield reaches 0.33 g/g, which are respectively 54.9% and 13.8% higher than the control group.

Example 3: Production of Shikimic Acid in a Fermentor

[0049] The fermentation performance with the GS strain was detected in a 7.5 L fermentor. The fermentation conditions for shikimic acid production were 38° C., 480 rpm, inoculation amount of 5%, liquid load of 46%, pH of 7.0, initial OD.sub.600 of 0.3 for fermentation, air flow rate of 1 vvm, and fermentation time of 80 h.

[0050] As shown in FIG. 4, at the end of fermentation, the production and yield of shikimic acid reach 88.1 g/L, 0.33 g/g, respectively.

[0051] Apparently, the above-described embodiments are merely examples provided for clarity of description, and are not intended to limit the implementations of the present invention. Other variations or changes can be made by those skilled in the art based on the above description. The embodiments are not exhaustive herein. Obvious variations or changes derived therefrom also fall within the protection scope of the present invention.