Method for biosynthesizing D-glucaric acid by using D-xylose and methanol

Abstract

The invention provides a method for biosynthesizing D-glucaric acid by using D-xylose and methanol. At present, most researchers use D-glucose hexose as a substrate to prepare D-glucaric acid. According to the invention, mdh, hps, phi, ino1, suhb, miox and udh genes are over-expressed in escherichia coli, and a new way for biologically synthesizing D-glucaric acid from D-xylose and methanol is constructed; the genes frmRAB, rpiA, pfkA and pfkB are knocked out, so that a carbon source is reasonably controlled; and combining an adaptive laboratory evolution method, fusing a dissolution promoting label and an inositol biosensor to improve the efficiency of a biosynthetic pathway, and finally realizing efficient synthesis of D-glucaric acid.

Description

Technical Field

[0001] The present application relates to a method for biosynthesizing D-glucaric acid from D-xylose and methanol, and belongs to the field of metabolic engineering. Background Art

[0002] D-glucaric acid is a dicarboxylic acid derivative that occurs naturally in fruits, vegetables, and mammalian tissues. It has a wide range of applications in the chemical, pharmaceutical, food, and therapeutic industries. In 2004, it was identified as one of the "12 high value-added chemicals from biomass." It serves as a precursor for synthetic nylon, biodegradable plastics, preservatives, detergents, and food additives. As research continues, D-glucaric acid and its derivatives are being explored for their potential in adjuvant therapy, such as lowering cholesterol, improving the efficacy of cancer chemotherapy, and regulating human hormone levels.

[0003] Traditional methods for the synthesis of D-glucaric acid include nitric acid oxidation and TEMPO electrochemical oxidation. However, these methods bring challenges such as environmental pollution and resource limitations. In contrast, advances in metabolic engineering have made the biosynthetic production of GA more cost-effective, environmentally friendly and efficient. Existing studies have explored the use of glucose, sucrose, lignocellulose and hemicellulose as substrates for the synthesis of D-glucaric acid, but there are no reports on the synthesis of D-glucaric acid from D-xylose and methanol. Xylose is the second most abundant sugar in lignocellulose and is expected to be a renewable resource for biofuels and bio-based chemicals. In Escherichia coli, the xylose utilization pathway includes the isomerization of xylose to xylulose, the phosphorylation of xylulose to xylulose-5-phosphate, and finally entering the pentose phosphate pathway for cellular metabolism. Methanol is considered to be a promising co-substrate or single substrate due to its abundant reserves, strong reducing properties and low cost. The conversion of D-xylose and methanol to D-glucaric acid by genetically engineering Escherichia coli has laid the foundation for the research on the production of D-glucaric acid using one-carbon and five-carbon resources. Summary of the invention

[0004] In view of the above problems, the present invention aims to provide a method for biosynthesizing D-glucaric acid from D-xylose and methanol. The present invention overexpresses mdh, hps, phi, ino1, suhb, miox and udh genes in Escherichia coli to construct a new pathway for biosynthesizing D-glucaric acid from D-xylose and methanol; by knocking out frmRAB, rpiA, pfkA and pfkB genes, the rational control of carbon source is achieved; the efficiency of the biosynthetic pathway is improved by combining adaptive laboratory evolution method, fusion solubilization tag and inositol biosensor, and finally the efficient synthesis of D-glucaric acid is achieved.

[0005] The specific technical solutions of the present invention are as follows: A method for biosynthesizing D-glucaric acid by using D-xylose and methanol, comprising: co-expressing inositol-1-phosphate synthase gene ino1, inositol oxygenase gene miox, methanol dehydrogenase gene mdh, 3-hexose-6-phosphate synthase gene hps, 6-phospho-3-hexose isomerase gene phi, endogenous phosphatase gene suhb and uronate dehydrogenase gene udh in Escherichia coli, constructing a biosynthetic pathway for preparing D-glucaric acid by using D-xylose and methanol as carbon sources; knocking out frmRAB gene, ribose-5-phosphate isomerase A gene rpiA, 6-phosphofructokinase 1 gene pfkA and The 6-phosphofructokinase 2 gene pfkB regulates the carbon flux in the biosynthetic pathway; the adaptive laboratory evolution method is used to enhance the growth ability of the cells; the solubility-promoting tag NusA is fused with the inositol oxygenase Miox to enhance the catalytic ability of the key rate-limiting enzyme in the biosynthetic pathway, the inositol oxygenase Miox; an inositol biosensor is constructed, and the reactive transcription factor IpsA is used to sense the exact amount of inositol in the cell. The accumulation of inositol eliminates the transcriptional inhibition mediated by the transcription factor IpsA and activates the expression of inositol oxygenase Miox, thereby improving the efficiency of the biosynthetic pathway, and finally obtaining an engineered bacterium that efficiently synthesizes D-glucaric acid; Among them, the NCBI sequence number of the ino1 gene is NP_012382.2; the NCBI sequence number of the miox gene is AAF25202.1; the NCBI sequence number of the mdh gene is WP_013953014.1; the NCBI sequence number of the hps gene is WP_014707664; the NCBI sequence number of the phi gene is WP_064496767; the NCBI gene number of the suhb gene is 947285; the NCBI sequence number of the udh gene is WP_011103399.1; the frmRAB gene includes the transcriptional repressor gene The three genes are frmR, S-(hydroxymethyl)glutathione dehydrogenase gene frmA and S-formylglutathione hydrolase gene frmB, the NCBI gene number of the frmR gene is 944986, the NCBI gene number of the frmA gene is 944988, and the NCBI gene number of the frmB gene is 944991; the NCBI gene number of the rpiA gene is 947407; the NCBI gene number of the pfkA gene is 948412; the NCBI gene number of the pfkB gene is 946230; the nucleotide sequence of the gene encoding the transcription factor IpsA is shown in SEQ ID NO.5.

[0006] The above method for biosynthesizing D-glucaric acid using D-xylose and methanol comprises the following steps: S1: Based on E. coli JM109 (DE3), the frmRAB gene, rpiA gene, pfkA gene and pfkB gene were sequentially cloned using the λ-red homologous recombination method to obtain an engineered bacterium; S2: using the engineered bacteria obtained in step S1 as the starting strain to perform adaptive laboratory evolution, thereby improving the growth ability of the strain in a medium containing D-xylose, and obtaining domesticated engineered bacteria; S3: Based on the engineered bacteria domesticated in step S2, the mdh gene, hps gene, Phi gene, suhb gene, udh gene, nusA-miox fusion gene, transcription factor gene ipsA, and inositol-1-phosphate synthase gene ino1 are overexpressed to obtain an engineered bacteria that can utilize D-xylose and methanol to biosynthesize D-glucaric acid.

[0007] The method for the above adaptive laboratory evolution is: 1) The engineered bacteria obtained in step S1 were washed with M9 liquid medium and then resuspended, inoculated into M9 liquid medium containing 12 g / L D-xylose and 1 wt% casamino acids, and cultured at 37° C. and 250 rpm until the exponential growth phase to obtain the first domesticated bacterial solution; 2) The first domestication bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / LD-xylose and 0.8 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the second domestication bacterial solution; 3) The second domestication bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / LD-xylose and 0.6 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the third domestication bacterial solution; 4) The third domestication bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / LD-xylose and 0.4 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the fourth domestication bacterial solution; 5) The fourth acclimation bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / LD-xylose and 0.2 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase, to obtain the fifth acclimation bacterial solution; 6) The fifth acclimation bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / LD-xylose, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the sixth acclimation bacterial solution; 7) The sixth culture solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, resuspended, and inoculated into M9 liquid medium containing 6 g / LD-xylose, and cultured at 37°C and 250 rpm until OD 600 The value is 0.6, which is the seventh domestication solution; 8) After continuous subculturing 20 times according to the method of step 7), the mixture was spread on M9 solid medium containing 6 g / L D-xylose and cultured at 37° C. until colonies grew to obtain domesticated engineered bacteria; The formula of M9 liquid culture medium is as follows: 3g / L KH2PO4, 1g / L NH4Cl, 0.5g / L NaCl, 6.78g / LNa2HPO4, 0.246g / L MgSO4·7H2O, 0.1mg / L H3BO4, 0.84mg / L ZnCl2, 8.3mg / L FeCl3·6H2O, 44.1mg / L CaCl2·2H2O, 0.016mg / L MnCl2·4H2O, 50mg / L EDTA, 0.13mg / L CuCl2·H2O, 1.15g / L L-proline, 1mg / L thiamine, and 1mg / L biotin; the formula of M9 solid culture medium is as follows: 1.5% agar powder is added to M9 liquid culture medium.

[0008] The above step S3 is specifically as follows: using the pRSFDuet vector as a backbone vector, inserting the mdh gene between the Nco I and HindIII restriction sites, and sequentially inserting the hps gene and the Phi gene between the Nde I and xho I restriction sites, to obtain the recombinant vector pRSFDuet-mdh-hps-phi; using the pCDFDuet vector as a backbone vector, inserting the suhb gene between the Nco I and BamHI restriction sites, and inserting the udh gene between the NdeI and KpnI restriction sites, to obtain the recombinant vector pCDFDuet-suhb-udh; using the pETDuet vector as a backbone vector, inserting the P cg44 -nusA-miox-T T7Te -P2-ipsA-T rrnB The ino1 gene was inserted between the Nde I and Kpn I restriction sites to obtain the recombinant vector pETDuet-P cg44 -nusA-miox-T T7Te -P2-ipsA-TrrnB -ino1; the recombinant vector pRSFDuet-mdh-hps-phi, the recombinant vector pCDFDuet-suhb-udh and the recombinant vector pETDuet-P cg44 -nusA-miox-T T7Te -P2-ipsA-T rrnB -ino1 is simultaneously transformed into the engineered bacteria domesticated in step S2, thereby obtaining an engineered bacteria capable of utilizing D-xylose and methanol to biosynthesize D-glucaric acid; Among them, P cg44 The nucleotide sequence of the promoter is shown in SEQ ID NO.1; the nucleotide sequence of the nusA-miox fusion gene is shown in SEQ ID NO.2; T7Te The nucleotide sequence of the terminator is shown in SEQ ID NO.3; the nucleotide sequence of the P2 promoter is shown in SEQ ID NO.4; the nucleotide sequence of the transcription factor gene ipsA is shown in SEQ ID NO.5; rrnB The nucleotide sequence of the terminator is shown in SEQ ID NO.6.

[0009] The method further comprises: inoculating the engineered bacteria obtained in step S3 into a fermenter for fed-batch fermentation, wherein the engineered bacteria obtained in step S3 are activated in LB liquid culture medium and then transferred to improved TB liquid culture medium, and cultured to an exponential growth phase at a temperature of 37° C. and a rotation speed of 250 rpm to obtain a seed solution; 3 L of improved TB liquid culture medium containing 22.8 g / L D-xylose and 8.2 g / L methanol is loaded into a 5 L fermenter, and the seed solution is inoculated into the fermenter at an inoculation amount of 10% for fermentation, wherein the temperature of the fermentation process is maintained at 30° C., the pH of the culture medium is stabilized at 7, and the dissolved oxygen level is maintained at 30% to 35%; at the 24th hour of fermentation, D-xylose is added to a final concentration of 22.8 g / L, and methanol is added to a final concentration of 7.7 g / L; at the 48th hour of fermentation, D-xylose is added to a final concentration of 22.3 g / L, and methanol is added to a final concentration of 8.3 g / L; and the fermentation is terminated after 64 hours; Among them, the formula of the improved TB liquid culture medium is: 12g / L soy peptone, 24g / L yeast extract, 5g / L glycerol, 2.31g / L KH2PO4, 12.54g / L K2HPO4, 1.491g / L KCl, 1.169g / L NaCl, 2.517g / L MnCl2, 1.904g / L MgCl2, and 0.2g / L thiamine.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The ability of Escherichia coli to biosynthesize D-glucaric acid using xylose and methanol as substrates was demonstrated. In order to overcome the challenges of slow cell growth, uneven carbon distribution, instability of key enzymes, and low yields, a multifaceted approach was adopted. This included carbon flux redirection, adaptive evolution, protein labeling, inositol (MI) biosensors, and fermentation in fermenters, which ultimately resulted in a significant increase in the yield of D-glucaric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Maps of recombinant vector pETDuet-miox-ino1, recombinant vector pRSFDuet-mdh-hps-phi, and pCDFDuet-suhb-udh.

[0012] Figure 2 : Map of the recombinant vector pETDuet-nusA-miox-ino1.

[0013] Figure 3 : Recombinant vector pETDuet-P cg44 -nusA-miox-T T7Te -P2-ipsA-T rrnB -map of ino1.

[0014] Figure 4 : Metabolic flow chart.

[0015] Figure 5 : Diagram of D-glucaric acid synthesis via D-xylose and methanol utilization pathways; (A) Fermentation results in Example 1; (B) LC-MS confirmed that the product in Figure A is D-glucaric acid.

[0016] Figure 6 : Fermentation results in Example 2; (A) E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB); (B) E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA); (C) E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA; ΔpfkA).

[0017] Figure 7 : Fermentation results of EERC-6 and EERC-9 in Example 3.

[0018] Figure 8: Fermentation results of EERC-10 and EERC-22 in Example 3.

[0019] Fig. 9 : Fermentation results of EERC-22 fermenter fed batch fermentation in Example 4. DETAILED DESCRIPTION

[0020] The present application is described in detail below with reference to embodiments, but the present invention is not limited to these embodiments.

[0021] The methanol dehydrogenase gene mdh involved in the embodiment of the present invention is from hookworm, and the NCBI sequence number is WP_013953014.1; the 3-hexose-6-phosphate synthase gene hps is from methanolic Bacillus, and the NCBI sequence number is WP_014707664; the 6-phospho-3-hexose isomerase gene Phi is from Methylococcus capsulatus, and the NCBI sequence number is WP_064496767; the inositol-1-phosphate synthase gene ino1 is from Saccharomyces cerevisiae, and the NCBI sequence number is NP_012382.2; the endogenous phosphatase gene suhb is from Escherichia coli, and the NCBI gene number is 947285; the inositol oxygenase gene miox is from mouse, and the NCBI sequence number is AAF25202.1; the uronic acid dehydrogenase gene udh is from Pseudomonas syringae, and the NCBI sequence number is WP_011103399.1; the frmRAB gene includes the transcription repressor gene Since the three genes frmR, S-(hydroxymethyl)glutathione dehydrogenase gene frmA and S-formylglutathione hydrolase gene frmB have NCBI gene numbers of frmR gene 944986, frmA gene 944988 and frmB gene 944991, and frmR gene, frmA gene and frmB gene are arranged continuously in the Escherichia coli genome, the frmR gene, frmA gene and frmB gene can be knocked out at one time by using the λ-red homologous recombination method, without considering the influence of "knockout order"; the NCBI gene number of ribose-5-phosphate isomerase A gene rpiA is 947407; the NCBI gene number of 6-phosphofructokinase 1 gene pfkA is 948412; the NCBI gene number of 6-phosphofructokinase 2 gene pfkB is 946230; cg44 The nucleotide sequence of the promoter is shown in SEQ ID NO.1; the nucleotide sequence of the nusA-miox fusion gene is shown in SEQ ID NO.2; T7Te The nucleotide sequence of the terminator is shown in SEQ ID NO.3; the nucleotide sequence of the P2 promoter is shown in SEQ ID NO.4; the nucleotide sequence of the responsive transcription factor gene ipsA is shown in SEQ ID NO.5;rrnB The nucleotide sequence of the terminator is shown in SEQ ID NO.6.

[0022] The recombinant vector pETDuet-miox-ino1 involved in the embodiment of the present invention is obtained by using the pETDuet vector as a backbone vector, inserting the miox gene between the EcoRI and HindIII restriction sites, and inserting the ino1 gene between the NdeI and KpnI restriction sites; the recombinant vector pCDFDuet-suhb-udh is obtained by using the pCDFDuet vector as a backbone vector, inserting the suhb gene between the NcoI and BamHI restriction sites, and inserting the udh gene between the NdeI and KpnI restriction sites; the recombinant vector pRSFDuet-mdh-hps-phi is obtained by using the pRSFDuet vector as a backbone vector, inserting the mdh gene between the Nco I and HindIII restriction sites, and sequentially inserting the hps gene and the Phi gene between the Nde I and xho I restriction sites; the recombinant vector pETDuet-nusA-miox-ino1 is obtained by using the pETDuet vector as a backbone vector, inserting the nusA-miox fusion gene between the EcoR I and HindIII restriction sites, and inserting the udh gene between the NdeI and KpnI restriction sites. I and KpnI restriction sites to insert the ino1 gene; the recombinant vector pETDuet-P cg44 -nusA-miox-T T7Te -P2-ipsA-T rrnB -ino1 is based on the pETDuet vector as the backbone vector, and a sequence of P cg44 Promoter, nusA-miox fusion gene, T T7Te terminator, P2 promoter, responsive transcription factor gene ipsA and T rrnB The terminator P cg44 -nusA-miox-T T7Te -P2-ipsA-T rrnB The element was obtained by inserting the ino1 gene between the NdeI and KpnI restriction sites.

[0023] The formula of the M9 liquid culture medium involved in the embodiment of the present invention is: 3g / L KH2PO4, 1g / L NH4Cl, 0.5g / L NaCl, 6.78g / L Na2HPO4, 0.246g / L MgSO4·7H2O, 0.1mg / L H3BO4, 0.84mg / L ZnCl2, 8.3mg / L FeCl3·6H2O, 44.1mg / L CaCl2·2H2O, 0.016mg / L MnCl2·4H2O, 50mg / L EDTA, 0.13mg / L CuCl2·H2O, 1.15g / L L-proline, 1mg / L thiamine, and 1mg / L biotin.

[0024] The M9 solid culture medium involved in the embodiment of the present invention is M9 liquid culture medium with 1.5% agar powder added thereto.

[0025] The formula of the improved TB liquid culture medium involved in the embodiment of the present invention is: 12g / L soy peptone, 24g / L yeast extract, 5g / L glycerol, 2.31g / L KH2PO4, 12.54g / L K2HPO4, 1.491g / L KCl, 1.169g / L NaCl, 2.517g / L MnCl2, 1.904g / LMgCl2, and 0.2g / L thiamine.

[0026] Embodiment 1: The recombinant vector pRSFDuet-mdh-hps-phi, the recombinant vector pETDuet-ino1-miox, and the recombinant vector pCDFDuet-suhb-udh were simultaneously transformed into E. coli JM109 (DE3) to obtain the engineered bacteria E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh).

[0027] E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh) was fermented and cultured. The specific method was as follows: 0.1% of the glycerol-preserved bacteria of E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh) was inoculated into 4 mL of LB liquid culture medium, activated and cultured overnight at a temperature of 37° C. and a rotation speed of 250 rpm, and then transferred to 50 mL of 9 g / L LB liquid culture medium at an inoculum of 1%; The invention discloses a method for preparing a fermentation broth comprising the steps of: adding an M9 liquid medium containing D-xylose, adding ampicillin with a final concentration of 100 mg / L, kanamycin with a final concentration of 30 mg / L and spectinomycin with a final concentration of 50 mg / L, culturing at a temperature of 37° C. and a rotation speed of 250 rpm until an exponential growth phase, and obtaining a seed solution; transferring the seed solution to 50 mL of a fermentation medium according to an inoculation amount of 2%, and fermenting for 60 hours at a liquid volume ratio of 20%, a temperature of 30° C. and a rotation speed of 250 rpm, and measuring the yield of D-glucaric acid in the fermentation broth to be 69.9 mg / L; wherein the fermentation medium is based on an M9 liquid medium, adding D-xylose with a final concentration of 9 g / L and methanol with a final concentration of 8 g / L, and adding ampicillin with a final concentration of 100 mg / L, kanamycin with a final concentration of 30 mg / L, spectinomycin with a final concentration of 50 mg / L and IPTG with a final concentration of 0.1 mM.

[0028] Embodiment 2: Based on E. coli JM109 (DE3), the frmRAB gene was knocked out by the λ-red homologous recombination method to obtain the engineered bacteria E. coli JM109 (DE3) (ΔfrmRAB); the recombinant vectors pRSFDuet-mdh-hps-phi, pETDuet-ino1-miox and pCDFDuet-suhb-udh were simultaneously transformed into E. coli JM109 (DE3) (ΔfrmRAB) to obtain the engineered bacteria E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB); in E. coli On the basis of JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB), the λ-red homologous recombination method was used to knock out the rpiA gene to obtain the engineered bacteria E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA); on the basis of E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA), the λ-red homologous recombination method was used to knock out the pfkA gene to obtain the engineered bacteria E. coli JM109(DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA; ΔpfkA).

[0029] The engineered bacteria were fermented and cultured for a certain period of time according to the method in Example 1, and the yield of D-glucaric acid in the fermentation broth of E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB) was measured to be 103.1 mg / L, the yield of D-glucaric acid in the fermentation broth of E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA) was measured to be 127.9 mg / L, and the yield of D-glucaric acid in the fermentation broth of E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA; ΔpfkA) was measured to be 197.7 mg / L.

[0030] Embodiment 3: Based on E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA; ΔpfkA), the pfkB gene was knocked out using the λ-red homologous recombination method to obtain the engineered bacteria E. coli JM109 (DE3) (pET-miox-ino1; pRSF-mdh-hps-phi; pCDF-suhb-udh; ΔfrmRAB; ΔrpiA; ΔpfkA; ΔpfkB), which was recorded as EERC-6. EERC-6 was fermented and cultured according to the method in Example 1. The results showed that it could not grow in a medium containing D-xylose, nor could it use D-xylose and methanol in the fermentation medium to biosynthesize D-glucaric acid, and the yield of D-glucaric acid was 0.

[0031] Based on E. coli JM109 (DE3), the frmRAB gene, rpiA gene, pfkA gene and pfkB gene were knocked out in sequence using the λ-red homologous recombination method to obtain the engineered bacteria E. coli JM109 (DE3) (ΔfrmRAB; ΔrpiA; ΔpfkA; ΔpfkB), which was recorded as EERC-7. EERC-7 was subjected to adaptive laboratory evolution, and the specific steps are as follows: 1) Wash the EERC-7 cells with M9 liquid medium and resuspend them to OD 600 The value was 0.1, inoculated into M9 liquid medium containing 12g / LD-xylose and 1wt% casamino acid, and cultured at 37°C and 250rpm until the exponential growth phase to obtain the first domestication bacterial solution; 2) Centrifuge the first cultured bacterial solution, collect the bacterial precipitate, wash it with M9 liquid medium, and resuspend it to OD 600 The value was 0.1, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.8 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the second domestication bacterial solution; 3) Centrifuge the second cultured bacterial solution, collect the bacterial precipitate, wash it with M9 liquid medium, and resuspend it to OD 600 The value was 0.1, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.6 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the third domestication bacterial solution; 4) Centrifuge the third culture solution, collect the bacterial precipitate, wash it with M9 liquid medium, and resuspend it to OD 600The value was 0.1, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.4 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the fourth domesticated bacterial solution; 5) Centrifuge the fourth culture solution, collect the bacterial precipitate, wash it with M9 liquid medium, and resuspend it to OD 600 The value was 0.1, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.2 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the fifth domesticated bacterial solution; 6) Centrifuge the fifth culture solution, collect the bacterial precipitate, wash it with M9 liquid medium, and resuspend it to OD 600 The value was 0.1, inoculated into M9 liquid medium containing 12 g / L D-xylose, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the sixth domestication bacterial solution; 7) Centrifuge the sixth culture solution, collect the bacterial precipitate, wash it with M9 liquid medium, and resuspend it to OD 600 The value was 0.1, and the culture was inoculated into M9 liquid medium containing 6 g / L D-xylose, and cultured at 37 °C and 250 rpm until OD 600 The value is 0.6, which is the seventh domestication solution; 8) After continuous subculturing for 20 times according to the method in step 7), the mixture was spread on M9 solid culture medium containing 6 g / L D-xylose and cultured at 37° C. for 12 hours until colonies grew. Then, each colony was fermented and cultured according to the method in Example 1. The results showed that all colonies were able to grow in the fermentation medium, and the colony that grew the fastest in the fermentation medium was recorded as EERC-8.

[0032] The recombinant vector pRSFDuet-mdh-hps-phi, the recombinant vector pETDuet-ino1-miox, and the recombinant vector pCDFDuet-suhb-udh were simultaneously transformed into EERC-8 to obtain the engineered bacteria EERC-9.

[0033] The recombinant vector pETDuet-nusA-miox-ino1, the recombinant vector pRSFDuet-mdh-hps-phi and the recombinant vector pCDFDuet-suhb-udh were simultaneously transformed into EERC-8 to obtain the engineered bacteria EERC-10.

[0034] The recombinant vectors pRSFDuet-mdh-hps-phi, pCDFDuet-suhb-udh and pETDuet-P cg44 -nusA-miox-TT7Te -P2-ipsA-T rrnB -ino1 was simultaneously transformed into EERC-8 to obtain the engineered bacterium EERC-22.

[0035] The engineered bacteria were fermented and cultured according to the method in Example 1, and it was found that EERC-9, EERC-10 and EERC-22 were all able to grow in the fermentation medium. The yield of D-glucaric acid in the fermentation broth of EERC-9 was measured to be 217.1 mg / L, the yield of D-glucaric acid in the fermentation broth of EERC-10 was 286.5 mg / L, and the yield of D-glucaric acid in the fermentation broth of EERC-22 was 301.4 mg / L.

[0036] Embodiment 4: The glycerol-preserved bacteria of EERC-22 were inoculated into 4 mL LB liquid culture medium at an inoculation rate of 0.1%, and activated and cultured overnight at a temperature of 37°C and a rotation speed of 250 rpm. Then, the inoculation rate was transferred to 50 mL modified TB liquid culture medium at an inoculation rate of 2%, and cultured at a temperature of 37°C and a rotation speed of 250 rpm until the exponential growth phase to obtain seed liquid. A 5 L fermenter was filled with 3 L of modified TB liquid culture medium containing 22.8 g / L D-xylose and 8.2 g / L methanol, and the seed liquid was inoculated into the fermenter at an inoculation rate of 10%. The temperature during the fermentation process was maintained at 30°C and the culture medium was maintained at 250 rpm. The pH of the culture medium was stabilized at 7, and the dissolved oxygen (DO) level was maintained at 30% to 35%; D-xylose and methanol were added at the 24th hour of fermentation to make the concentration of D-xylose reach 22.8 g / L, and the concentration of methanol reach 7.7 g / L; D-xylose and methanol were added at the 48th hour of fermentation to make the concentration of D-xylose reach 22.3 g / L, and the concentration of methanol reach 8.3 g / L; the fermentation was terminated after 64 hours, and the production of D-glucaric acid in the fermentation broth was measured to be about 3 g / L.

[0037] The above are only several embodiments of the present application and are not intended to limit the present application in any form. Although the present application is disclosed as above with preferred embodiments, they are not intended to limit the present application. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for biosynthesizing D-glucaric acid using D-xylose and methanol, characterized in that: Overexpression of inositol-1-phosphate synthase gene in Escherichia coli ino1, Inositol oxygenase gene Miox, Methanol dehydrogenase gene mdh, 3-Hexose-6-phosphate synthase gene hps, 6-Phospho-3-hexose isomerase gene phi, Endogenous phosphatase gene suhb and uronate dehydrogenase gene udh , construct a biosynthetic pathway for preparing D-glucaric acid using D-xylose and methanol as carbon sources; knock out frmRAB Gene 、 Ribose-5-phosphate isomerase A gene rpiA, 6-phosphofructokinase 1 gene pkq and 6-phosphofructokinase 2 gene pkq , regulating the carbon flux in the biosynthetic pathway; using adaptive laboratory evolution to enhance the growth ability of cells; fusing the solubility-promoting tag NusA with the inositol oxygenase Miox to enhance the catalytic ability of the key rate-limiting enzyme in the biosynthetic pathway, the inositol oxygenase Miox; constructing an inositol biosensor, using the reactive transcription factor IpsA to sense the exact amount of inositol in the cell, eliminating the transcriptional inhibition mediated by the transcription factor IpsA and activating the expression of inositol oxygenase Miox through the accumulation of inositol, thereby improving the efficiency of the biosynthetic pathway, and finally obtaining an engineered bacterium that efficiently synthesizes D-glucaric acid.

2. The method according to claim 1, characterized in that: ino1 The NCBI sequence number of the gene is NP_012382.2; miox The NCBI sequence number of the gene is AAF25202.1; md The NCBI sequence number of the gene is WP_013953014.1; hps The NCBI sequence number of the gene is WP_014707664; phi The NCBI sequence number of the gene is WP_064496767; suhb The NCBI gene number of the gene is 947285; udh The NCBI sequence number of the gene is WP_011103399.1; frmRAB transcriptional repressor gene fR , S-(hydroxymethyl)glutathione dehydrogenase gene frmA and S-formylglutathione hydrolase gene fB Three genes, fR The NCBI gene number of the gene is 944986. frmA The NCBI gene number of the gene is 944988. fB The NCBI gene number of the gene is 944991; rpI The NCBI gene number of the gene is 947407; pkq The NCBI gene number of the gene is 948412; pkq The NCBI gene number of the gene is 946230; the nucleotide sequence of the gene encoding the transcription factor IpsA is shown in SEQ ID NO.

5.

3. The method according to claim 2, characterized in that: The following steps are involved: S1: E. coli Based on JM109 (DE3), the λ-red homologous recombination method was used to knock out frmRAB Gene 、 rpI Gene , pfkA Genes and pkq Gene, get engineered bacteria; S2: using the engineered bacteria obtained in step S1 as the starting strain to perform adaptive laboratory evolution, thereby improving the growth ability of the strain in a medium containing D-xylose, and obtaining domesticated engineered bacteria; S3: Based on the engineered bacteria domesticated in step S2, overexpress md Gene, hps Gene, Phi Gene, suhb Gene, udh Gene, nusA - miox Fusion gene, transcription factor gene iPS , inositol-1-phosphate synthase gene ino1 , and obtained an engineered bacterium that can utilize D-xylose and methanol to biosynthesize D-glucaric acid.

4. The method according to claim 3, characterized in that: The methods of adaptive laboratory evolution are: 1) The engineered bacteria obtained in step S1 were washed with M9 liquid medium and then resuspended, and inoculated into M9 liquid medium containing 12 g / L D-xylose and 1 wt% casamino acids, and cultured at 37° C. and 250 rpm until the exponential growth phase to obtain the first domesticated bacterial solution; 2) The first domestication bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.8 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the second domestication bacterial solution; 3) The second domestication bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.6 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the third domestication bacterial solution; 4) The third domestication bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.4 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the fourth domestication bacterial solution; 5) The fourth acclimation bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / L D-xylose and 0.2 wt% casamino acids, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the fifth acclimation bacterial solution; 6) The fifth acclimation bacterial solution was centrifuged, the bacterial precipitate was collected, washed with M9 liquid medium, and then resuspended, inoculated into M9 liquid medium containing 12 g / L D-xylose, and cultured at 37°C and 250 rpm until the exponential growth phase to obtain the sixth acclimation bacterial solution; 7) Centrifuge the sixth culture solution, collect the bacterial precipitate, wash it with M9 liquid medium, resuspend it, and inoculate it into M9 liquid medium containing 6g / L D-xylose. Cultivate it at 37℃ and 250rpm until OD 600 The value is 0.6, which is the seventh domestication solution; 8) After continuous subculturing 20 times according to the method in step 7), spread it on M9 solid culture medium containing 6 g / L D-xylose, and culture it at 37°C for 12 hours until colonies grow to obtain domesticated engineered bacteria.

5. The method according to claim 4, characterized in that: The formula of M9 liquid culture medium is: 3g / L KH2PO4, 1g / L NH4Cl, 0.5g / L NaCl, 6.78g / L Na2HPO4, 0.246g / L MgSO4·7H2O, 0.1mg / L H3BO4, 0.84mg / LZnCl2, 8.3mg / L FeCl3·6H2O, 44.1mg / L CaCl2·2H2O, 0.016mg / L MnCl2·4H2O, 50mg / LEDTA, 0.13mg / L CuCl2·H2O, 1.15g / L L-proline, 1mg / L thiamine, 1mg / L biotin; M9 solid culture medium is M9 liquid culture medium with 1.5% agar powder added.

6. The method according to claim 3, characterized in that: Step S3 specifically includes: using pRSFDuet vector as the backbone vector, NcoI and Hind III Insertion between restriction sites md Genes, and N I and xh Insertion between restriction sites hps Genes and Phi Gene, recombinant vector pRSFDuet-mdh-hps-phi ; Using pCDFDuet as the backbone vector, NcoI and BamH Insertion between restriction sites suhb gene and inserted between the Nde I and Kpn I restriction sites udh Gene, recombinant vector pCDFDuet- suhb-udh ; Using pETDuet vector as backbone vector, EcoR I and Hind III Insert P between restriction sites cg44 - nusa-miox -T T7Te -P2- iPS -T rrnB components and in N and KqI Insertion between restriction sites ino1 Gene, recombinant vector pETDuet-P cg44 - nusA-miox -T T7Te -P2- iPS -T rrnB - ino1 ; The recombinant vector pRSFDuet- mdh-hps-phi , recombinant vector pCDFDuet- suhb-udh and the recombinant vector pETDuet-P cg44 - nusA-miox -T T7Te -P2- iPS -T rrnB - ino1 At the same time, the engineered bacteria domesticated in step S2 are transformed to obtain engineered bacteria capable of utilizing D-xylose and methanol to biosynthesize D-glucaric acid.

7. The method according to claim 6, characterized in that: P cg44 The nucleotide sequence of the promoter is shown in SEQ ID NO.1; nusA - miox The nucleotide sequence of the fusion gene is shown in SEQ ID NO.2; T7Te The nucleotide sequence of the terminator is shown in SEQ ID NO.3; the nucleotide sequence of the P2 promoter is shown in SEQ ID NO.4; the responsive transcription factor gene iPS The nucleotide sequence is shown in SEQ ID NO.5; rrnB The nucleotide sequence of the terminator is shown in SEQ ID NO.

6.

8. The method according to claim 3, characterized in that: The method further comprises: inoculating the engineered bacteria obtained in step S3 into a fermentation tank for fed-batch fermentation.

9. The method according to claim 8, characterized in that: The method of fed-batch fermentation is as follows: the engineered bacteria obtained in step S3 are activated in LB liquid culture medium and then transferred to modified TB liquid culture medium, and cultured at a temperature of 37° C. and a rotation speed of 250 rpm until the exponential growth phase to obtain seed liquid; 3L of modified TB liquid culture medium containing 22.8g / L D-xylose and 8.2g / L methanol is loaded into a 5L fermenter, and the seed liquid is inoculated into the fermenter at an inoculum of 10% for fermentation, and the temperature of the fermentation process is maintained at 30° C., the pH of the culture medium is stabilized at 7, and the dissolved oxygen level is maintained at 30%-35%; at the 24th hour of fermentation, D-xylose is added to a final concentration of 22.8g / L, and methanol is added to a final concentration of 7.7g / L; at the 48th hour of fermentation, D-xylose is added to a final concentration of 22.3g / L, and methanol is added to a final concentration of 8.3g / L; and the fermentation is terminated after 64 hours.

10. The method according to claim 9, characterized in that: The formula of the improved TB liquid culture medium is: 12 g / L soy peptone, 24 g / L yeast extract, 5 g / L glycerol, 2.31 g / L KH2PO4, 12.54 g / L K2HPO4, 1.491 g / L KCl, 1.169 g / L NaCl, 2.517 g / L MnCl2, 1.904 g / L MgCl2, and 0.2 g / L thiamine.

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