A method for whole-cell biosynthesis of hexanediamines

Abstract

The application discloses a kind of whole-cell biosynthesis methods of hexamethylenediamine, belong to the field of bioengineering.The method of the application utilizes carboxylic acid reductase (CAR) and transaminase (TA) to construct the biological conversion pathway from adipic acid to hexamethylenediamine using Escherichia coli as host strain.The adaptability between key enzymes CAR and TA is optimized by TA isozyme screening, and the whole-cell biosynthesis of hexamethylenediamine is realized;Further introduce CAR-TA-TA system to optimize the coordination between each step of hexamethylenediamine synthesis pathway, effectively improve the accumulation of hexamethylenediamine.The application provides a method for synthesizing hexamethylenediamine by microbial cell factory, with the advantages of simple operation, less pollution, high product quality, etc., and lays a foundation for the development of future hexamethylenediamine synthesis technology.

Description

Technical Field

[0001] This invention relates to a whole-cell biosynthesis method for hexamethylenediamine, belonging to the field of bioengineering. Background Technology

[0002] Hexamethylenediamine (HDMA), an important chemical raw material, plays a crucial role in the industrial production of nylon 66. Currently, its synthesis primarily relies on the hydrogenation of adiponitrile. The main preparation technologies for adiponitrile include butadiene hydrocyanation, acrylonitrile electrolysis, and adipic acid amination dehydration, with butadiene hydrocyanation accounting for the largest share in industrial applications. However, this technology requires the use of the highly volatile and toxic hydrogen cyanide, and is accompanied by the consumption of non-renewable fossil fuels. The harsh reaction conditions, complex separation processes, and high degree of technological monopoly limit global adiponitrile production capacity, consequently limiting HDMA production capacity. With the decline in adipic acid market prices, the synthesis of adiponitrile via adipic acid amination dehydration has gradually gained some market competitiveness, but some technical drawbacks remain, such as difficulties in catalyst recovery and regeneration, and numerous side reactions.

[0003] In recent years, the depletion of fossil fuels has led to increasing environmental problems, and international emphasis on controlling CO2 emissions has resulted in significant changes in the acquisition methods of various chemical raw materials. Recent reports indicate that hexamethylenediamine can be synthesized from substrates such as succinyl-CoA, 4-aminobutyryl-CoA, 4-aminobutyraldehyde, glutamic acid, or 2,5-(hydroxymethyl)furfural via a multi-stage enzyme-linked reaction. With the development of bio-based adipic acid, pathways for synthesizing hexamethylenediamine from adipic acid have been developed. The bioconversion of adipic acid to hexamethylenediamine involves two branches. One route involves the reduction of adipic acid to hexanoic acid semialdehyde by carboxylic acid. Hexanoic acid semialdehyde is then converted to 6-aminohexanoic acid by transaminase. 6-aminohexanoic acid is further reduced by carboxylic acid to 6-aminohexanol, and finally, 6-aminohexanol undergoes transamination to synthesize hexamethylenediamine. The other route involves adipic acid undergoing two consecutive carboxylic acid reduction reactions to synthesize hexamethylenedialdehyde, which then undergoes two consecutive transamination reactions to synthesize hexamethylenediamine. Fedorchuk's team constructed an in vitro synthesis system for adipic acid to hexamethylenediamine using the carboxyl reductase MAB4714 from *Mycobacterium chelonae* and the transaminase SAV2585 from *Streptomyces avermitilis*. They further optimized the reaction system using a mutant of MAB4714 (L342E) and the butyramine transaminase PatA from *E. coli*, reducing the accumulation of the intermediate 6-aminohexanoic acid and achieving a one-pot biocatalytic synthesis of adipic acid to hexamethylenediamine. However, this system is based in vitro, requiring the external addition of adipic acid substrate and multiple amino acid donors, resulting in high raw material costs. The preparation of multiple pure enzymes further increases operating costs. Microbial cell factories can prepare abundant enzyme systems and convert low-cost raw materials (such as glucose) into various amino acids and cofactors. Establishing a whole-cell biosynthesis system for hexamethylenediamine will be a major direction for overcoming the challenges of industrializing bio-based hexamethylenediamine in the future. Summary of the Invention

[0004] The purpose of this invention is to overcome the current technological bottlenecks in the synthesis of hexamethylenediamine and to provide a method for the microbial synthesis of hexamethylenediamine. This method utilizes carboxylic acid reductase (CAR) and transaminase (TA) heterologously constructed in engineered strains of *Escherichia coli* to establish a hexamethylenediamine biosynthetic pathway. Figure 1 This study achieved the complete biotransformation of adipic acid to hexamethylenediamine, including the construction of the initial CAR-TA hexamethylenediamine synthesis system and the construction of a further optimized CAR-TA-TA hexamethylenediamine synthesis system, which improved the conversion efficiency of adipic acid to hexamethylenediamine.

[0005] The first objective of this invention is to provide a genetically engineered bacterium that produces hexamethylenediamine, wherein the genetically engineered bacterium overexpresses carboxylic acid reductase and transaminase.

[0006] In one embodiment of the present invention, the genetically engineered bacteria overexpress one or more transaminases derived from different sources.

[0007] In one embodiment of the present invention, the genetically engineered bacteria overexpress one or two transaminases derived from different sources.

[0008] In one embodiment of the present invention, the carboxylic acid reductase includes the carboxylic acid reductase MAB CAR derived from Mycobacteroides abscessus.

[0009] In one embodiment of the present invention, the transaminase includes aspartate transaminase CVTA derived from Chromobacterium violaceum ATCC12472, succinyldiamine transaminase PatA derived from E. coli MG1655, 4-aminobutyric acid transaminase GabT derived from Streptomyces avermitilis, succinyldiamine transaminase HATA derived from Hafnia alvei, 4-aminobutyric acid transaminase STTA derived from Solanum tuberosum, aspartate transaminase SPTA derived from Silicibacter pomeroyi, pyruvate transaminase VFTA derived from Vibrio fluvialis, or transaminase PDTA derived from Paracoccus denitrificans.

[0010] In one embodiment of the present invention, the amino acid sequence of the carboxylic acid reductase MAB CAR is shown in SEQ ID NO.1, the amino acid sequence of the transaminase CVTA is shown in SEQ ID NO.2, the amino acid sequence of the transaminase PatA is shown in SEQ ID NO.3, the amino acid sequence of the transaminase GabT is shown in SEQ ID NO.4, the amino acid sequence of the transaminase HATA is shown in SEQ ID NO.5, the amino acid sequence of the transaminase STTA is shown in SEQ ID NO.6, the amino acid sequence of the transaminase SPTA is shown in SEQ ID NO.7, the amino acid sequence of the transaminase VFTA is shown in SEQ ID NO.8, and the amino acid sequence of the transaminase PDTA is shown in SEQ ID NO.9.

[0011] In one embodiment of the present invention, the nucleotide sequence of the carboxylic acid reductase MAB CAR is shown in SEQ ID NO. 10, the nucleotide sequence of the transaminase CVTA is shown in SEQ ID NO. 11, the nucleotide sequence of the transaminase PatA is shown in SEQ ID NO. 12, the nucleotide sequence of the transaminase GabT is shown in SEQ ID NO. 13, the nucleotide sequence of the transaminase HATA is shown in SEQ ID NO. 14, the nucleotide sequence of the transaminase STTA is shown in SEQ ID NO. 15, the nucleotide sequence of the transaminase SPTA is shown in SEQ ID NO. 16, the nucleotide sequence of the transaminase VFTA is shown in SEQ ID NO. 17, and the nucleotide sequence of the transaminase PDTA is shown in SEQ ID NO. 18.

[0012] In one embodiment of the present invention, the genetically engineered bacteria overexpress carboxylic acid reductase MAB CAR with an amino acid sequence as shown in SEQ ID NO.1 and transaminase PatA with an amino acid sequence as shown in SEQ ID NO.3 or transaminase HATA with an amino acid sequence as shown in SEQ ID NO.5;

[0013] Alternatively, the genetically engineered bacteria may overexpress carboxylic acid reductase MAB CAR with the amino acid sequence shown in SEQ ID NO.1 and transaminase PatA with the amino acid sequence shown in SEQ ID NO.3 or transaminase HATA with the amino acid sequence shown in SEQ ID NO.5.

[0014] In one embodiment of the present invention, the genetically engineered bacteria express genes encoding carboxylic acid reductase and transaminase using pRSFDuet-1, pETDuet-1 and / or pACYCDuet-1 plasmids.

[0015] In one embodiment of the present invention, the genetically engineered bacteria express a gene encoding carboxylic acid reductase using the pRSFDuet-1 plasmid.

[0016] In one embodiment of the present invention, the genetically engineered bacteria express a gene encoding a transaminase using pETDuet-1 and / or pACYCDuet-1 plasmids.

[0017] The second objective of this invention is to provide a method for synthesizing hexamethylenediamine, wherein the method uses adipic acid as a reaction substrate and the above-mentioned genetically engineered bacteria to carry out the reaction to obtain hexamethylenediamine.

[0018] In one embodiment of the present invention, the method involves inoculating the above-mentioned genetically engineered bacteria into a solution containing 4-8 g·L⁻¹ of... -1In glucose culture medium, at 35–38°C, 225–275 rpm. -1 After shaking flask fermentation for 3–5 hours under the specified conditions, add [a solution] to a final concentration of 0.1 mmol·L⁻¹. -1 IPTG induction and addition of 2-5 g·L -1 Adipic acid substrate.

[0019] In one embodiment of the present invention, the components of the culture medium comprise 10–20 g·L⁻¹. -1 Tryptone, 5-10 g / L -1 Yeast extract, 0.5-10 g·L -1 Sodium chloride, 0.95 g·L -1 MgCl2 and 0.186 g·L -1 KCl.

[0020] The present invention also provides the application of the above-described genetically engineered bacteria and the above-described method in the preparation of products containing hexamethylenediamine.

[0021] Beneficial effects:

[0022] The method of this invention utilizes *Escherichia coli* as the host strain to heterologously construct a biotransformation pathway from adipic acid to hexamethylenediamine using carboxylic acid reductase (CAR) and transaminase (TA). By introducing the CAR-TA-TA system, the coordination between the steps of the hexamethylenediamine synthesis pathway is optimized, effectively increasing the accumulation of hexamethylenediamine without further accumulating the byproduct 6-aminohexanoic acid, thus significantly increasing the hexamethylenediamine synthesis yield to 22 mg·L⁻¹. -1 This invention provides a method for synthesizing hexamethylenediamine through a microbial cell factory, which has the advantages of simple operation, low pollution, and high product quality, laying the foundation for the future development of hexamethylenediamine synthesis technology. Attached Figure Description

[0023] Figure 1 A schematic diagram of the biotransformation pathway from adipic acid to hexamethylenediamine;

[0024] Figure 2 Figure showing the results of shake-flask fermentation for the synthesis of hexamethylenediamine and 6-aminohexanoic acid in engineered strains;

[0025] Figure 3 Figure 1. Results of shake-flask fermentation for the synthesis of hexamethylenediamine and 6-aminohexanoic acid in the CAR-TA-TA system engineered strain. Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments. The embodiments described below are illustrative and not limiting, and should not be used to limit the scope of protection of the present invention.

[0027] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0028] The technical means employed in this invention:

[0029] The plasmids pRSFDuet-1, pETDuet-1, and pACYCDuet-1, the molecular manipulation techniques, the microbial culture techniques, and the detection of hexamethylenediamine involved in this invention are all well known to those skilled in the art.

[0030] The culture media involved in the following examples:

[0031] LB solid culture: 10 g·L -1 Tryptone, 5 g / L -1 Yeast extract, 10 g·L -1 Sodium chloride and 2 g·L -1 Agar powder.

[0032] LB liquid medium: 10 g·L -1 Tryptone, 5 g / L -1 Yeast extract and 10 g·L -1 Sodium chloride.

[0033] SOB culture: 5 g·L -1 Yeast extract, 20 g·L -1 Peptone, 0.5 g / L -1 NaCl, 0.95 g·L -1 MgCl2 and 0.186 g·L -1 KCl.

[0034] Example 1: Construction of genetically engineered bacteria expressing carboxylic acid reductase

[0035] The synthesized MAB CAR gene and pRSFDuet-1 plasmid were double-digested with Nco I and BamHI. After purification, the digested MAB CAR gene and pRSFDuet-1 plasmid were ligated using T4 DNA ligase. The ligation product was introduced into *E. coli* JM109. The transformed *E. coli* JM109 were streaked onto LB agar and cultured overnight at 37°C. Colony PCR was performed for identification. Positive transformants were picked and inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 12–14 h. The bacterial culture was verified by Sanger sequencing. After successful verification, the bacterial cells were collected, and the plasmid pRSF-MABCAR was extracted. The recombinant plasmid pRSF-MABCAR was transformed into *E. coli* BL21(DE3) to obtain the recombinant strain BL21 / pRSF-MABCAR.

[0036] Example 2: Construction of an engineered strain for the complete synthesis of hexamethylenediamine using carboxylic acid reductase and transaminase.

[0037] The nucleotide sequences of transaminases with restriction endonuclease sites at both ends were synthesized by Shanghai Sangon Biotech Co., Ltd. The genes encoding eight transaminases with chemically synthesized amino acid sequences (SEQ ID NO.2–SEQ ID NO.9) were digested with Nco I and BamHI and then inserted into the pETDuet-1 expression plasmid to construct recombinant expression plasmids for the eight transaminases: pET-CVTA, pET-PatA, pET-GabT, pET-HATA, pET-STTA, pET-SPTA, pET-VFTA, and pET-PDTA.

[0038] The eight recombinant plasmids obtained were transformed into the recombinant strain BL21 / pRSF-MAB CAR constructed in Example 1 to obtain hexamethylenediamine-producing recombinant strains DAH1 to DAH8, which were stored in a -80°C freezer.

[0039] The specific carboxyl reductase and transaminase expression plasmids contained in the hexamethylenediamine-producing engineered strains DAH1 to DAH8 are as follows: DAH1: pRSF-MAB CAR and pET-CVTA plasmids; DAH2: pRSF-MAB CAR and pET-PatA plasmids; DAH3: pRSF-MAB CAR and pET-GabT plasmids; DAH4: pRSF-MAB CAR and pET-HATA plasmids; DAH5: pRSF-MAB CAR and pET-STTA plasmids; DAH6: pRSF-MAB CAR and pET-SPTA plasmids; DAH7: pRSF-MAB CAR and pET-VFTA plasmids; DAH8: pRSF-MAB CAR and pET-PDTA plasmids.

[0040] Example 3: Shake-flask fermentation of recombinant strain and determination of hexamethylenediamine yield

[0041] 1. Shake flask fermentation

[0042] Take 50 μL of the bacterial culture of the hexamethylenediamine engineered strains DAH1-DAH8 constructed in Example 2 and inoculate them into 20 mL of LB liquid medium, respectively, and incubate at 37 °C and 250 r·min. -1 The culture was incubated overnight on a shaker to obtain the seed culture; 1 mL of the seed culture was transferred to a 250 mL flask containing 50 mL of SOB medium, and 6 g·L⁻¹ was added. -1 Glucose, at 37℃, 250 r·min -1 Shake-flask fermentation was conducted under the specified conditions. After culturing for 4 hours, a final concentration of 0.1 mmol·L⁻¹ was added.-1 Isopropylβ-D-1-thiogalactopyranoside (IPTG) was used to induce the reaction, and a final concentration of 2.5 g·L⁻¹ was added. -1 adipic acid substrate, transferred to 30℃, 250 r·min -1 Fermentation broth was obtained by culturing in the environment for 72 hours.

[0043] 2. HPLC detection of hexamethylenediamine

[0044] 1) Sample derivatization and extraction

[0045] The pre-column derivatization procedure for dansyl chloride is as follows: the fermentation broth is subjected to 12000 r·min -1 After centrifugation for 10 min, take 500 μL of the supernatant as the sample, add 500 μL of saturated NaHCO3 solution and internal standard (5 μL with a concentration of 10 g·L⁻¹) -1 After mixing thoroughly with heptanediamine, the pH was adjusted to 10 with saturated NaOH solution, followed by the addition of 1 mL of the derivatization reagent dansyl chloride (5 g·L⁻¹). -1 (Soluble in acetone). The mixture was incubated in a light-protected water bath at 60°C for 30 min. After extraction with 2 mL of anhydrous diethyl ether for 10 min, the upper organic phase was collected. This extraction process was repeated twice. The two collected organic phases were mixed and dried using a nitrogen evaporator to remove the diethyl ether. The derivatized compound was dissolved in 500 μL of acetonitrile solution, filtered through a 0.22 μm filter membrane, and then analyzed by HPLC.

[0046] 2) HPLC chromatographic determination

[0047] Chromatographic conditions:

[0048] High-performance liquid chromatography (HPLC) separation of diamine dansyl chloride derivatives was performed on a C18 column at a separation temperature of 30 °C and a UV detection wavelength of 254 nm. The injection volume was 10 μL. Mobile phase A was ultrapure water, and mobile phase B was HPLC-grade acetonitrile. Both mobile phases were filtered through a 0.22 μm filter before use. The gradient elution program was set as follows: 0–4 min, 55%–70% B; 4–6 min, 70% B; 6–11 min, 70% B; 11–12 min, 95% B; 12–13 min, 95% B; 13–16 min, 55% B. The total flow rate was set to 0.7 mL / min. -1 .

[0049] The fermentation results of hexamethylenediamine engineered strains DAH1-DAH8 are as follows: Figure 2 As shown, DAH2 and DAH4 had relatively high hexamethylenediamine yields, at 13 mg·L⁻¹. -1The whole-cell biosynthesis of hexamethylenediamine was achieved; the final product of hexamethylenediamine engineered strains DAH1, DAH3 and DAH8 was 6-aminohexanoic acid, which could not effectively produce hexamethylenediamine; the hexamethylenediamine engineered strain DAH5 could not achieve the synthesis of hexamethylenediamine or 6-aminohexanoic acid; the final product concentration of hexamethylenediamine engineered strains DAH6 and DAH7 was low, and they also contained the byproduct 6-aminohexanoic acid.

[0050] Example 4: Hexamethylenediamine engineered strain expressing dual transaminase

[0051] Seven transaminase genes (CVTA, GabT, HATA, STTA, SPTA, VFTA, PDTA) other than PatA were digested with Nco I and BamHI and then ligated into the pACYCDuet-1 expression plasmid to construct seven recombinant transaminase plasmids: pACYC-CVTA, pACYC-GabT, pACYC-HATA, pACYC-STTA, pACYC-SPTA, pACYC-VFTA, and pACYC-PDTA. These seven recombinant plasmids were then transformed into the hexamethylenediamine engineered strain DAH2 constructed in Example 2 to obtain hexamethylenediamine engineered strains DAH9–DAH15.

[0052] The specific carboxyl reductase and transaminase expression plasmids contained in the hexamethylenediamine engineered strains DAH9–DAH15 are as follows:

[0053] DAH9: pRSF-MAB CAR, pET-PatA, and pACYC-CVTA plasmids; DAH10: pRSF-MAB CAR, pET-PatA, and pACYC-GabT plasmids; DAH11: pRSF-MAB CAR, pET-PatA, and pACYC-HATA plasmids; DAH12: pRSF-MAB CAR, pET-PatA, and pACYC-STTA plasmids; DAH13: pRSF-MAB CAR, pET-PatA, and pACYC-SPTA plasmids; DAH14: pRSF-MAB CAR, pET-PatA, and pACYC-VFTA plasmids; DAH15: pRSF-MAB CAR, pET-PatA, and pACYC-PDTA plasmids.

[0054] After whole-cell shake-flask fermentation of hexamethylenediamine engineered strains DAH9-DAH15 according to the method described in Example 3, the hexamethylenediamine synthesis results are as follows: Figure 3 As shown, the hexamethylenediamine synthesis yield of the engineered strain DAH10 was significantly increased, reaching 22 mg·L⁻¹. -1 Compared to the hexamethylenediamine engineered strain DAH2, the level of 6-aminocaproic acid was increased by 1.7 times, while the level of 6-aminocaproic acid in the hexamethylenediamine engineered strain DAH10 did not increase significantly.

[0055] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered bacterium that produces hexamethylenediamine, characterized in that, The genetically engineered bacteria overexpress carboxylic acid reductase MABCAR, transaminase PatA, and transaminase GabT. The carboxylic acid reductase MAB CAR is derived from Mycobacteroides abscessus The transaminase PatA is derived from... E. coli MG1655, the transaminase GabT is derived from Streptomyces avermitilis ; The amino acid sequence of the carboxylic acid reductase MAB CAR is shown in SEQ ID NO.1, the amino acid sequence of the transaminase PatA is shown in SEQ ID NO.3, and the amino acid sequence of the transaminase GabT is shown in SEQ ID NO.

4. The nucleotide sequence of the carboxylic acid reductase is shown in SEQ ID NO.10, the nucleotide sequence of the transaminase PatA is shown in SEQ ID NO.12, and the nucleotide sequence of the transaminase GabT is shown in SEQ ID NO.

13. The genetically engineered bacteria express the gene encoding carboxylic acid reductase using the pRSFDuet-1 plasmid, and express the gene encoding transaminase using the pETDuet-1 and / or pACYCDuet-1 plasmids.

2. A method for synthesizing hexamethylenediamine, characterized in that, The method involves using adipic acid as a reaction substrate and reacting it with the genetically engineered bacteria described in claim 1 to obtain hexamethylenediamine.

3. The method according to claim 2, characterized in that, The method involves inoculating the above-mentioned genetically engineered bacteria into a solution containing 4-8 g·L⁻¹ of... -1 In glucose culture medium, at 35–38°C and 225–275 r·min -1 After shaking flask fermentation for 3–5 h under the specified conditions, add 0.1 mmol·L⁻¹. -1 IPTG induction and addition of 2-5 g·L -1 Adipic acid substrate.

4. The use of the genetically engineered bacteria of claim 1 or the method of claim 2 or 3 in the preparation of products containing hexamethylenediamine.

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