Application of thermococcus-sourced aldolase and mutant thereof in glycol synthesis

Abstract

The invention relates to the field of biochemical engineering, and provides application of thermococcus-derived aldolase and a mutant thereof in glycol synthesis, and the aldolase mutant, a coding gene, a recombinant plasmid and a recombinant bacterium thereof. Wherein the amino acid sequence of the thermococcus-derived aldolase is as shown in SEQ ID NO. 1; the mutant is obtained by mutating the aldolase. The aldolase and the mutant thereof are high in catalytic efficiency, efficient biosynthesis of 1, 3-propylene glycol and 1, 3-butanediol is achieved, and the aldolase and the mutant thereof have good industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of biochemical engineering, and more particularly to a thermococcus (…). 嗜热栖热球菌 Applications of aldolase and its mutants in the synthesis of diols (1,3-propanediol, 1,3-butanediol). Background Technology

[0002] 1,3-Propanediol (1,3-PDO) and 1,3-Butanediol (1,3-BDO), as key C3 and C4 linear glycol platform chemicals, have irreplaceable application value in the chemical, materials, and daily chemical industries. 1,3-Propanediol is a core raw material for the production of polypropylene terephthalate (PTT). The superior performance of PTT fibers in high-end textiles and carpets is driving the continuous growth of global demand for 1,3-PDO. my country's demand is projected to reach 65,000 tons in 2025, with a market size of approximately US$264 million, and is expected to further increase to US$518 million by 2031. 1,3-Butanediol, as a multifunctional green solvent and fine chemical intermediate, is widely used in cosmetic moisturizers, fragrances, and the synthesis of biodegradable polyesters. Its global market size is projected to reach approximately US$350 million by 2030, indicating huge market potential.

[0003] Currently, the industrial production of 1,3-propanediol has gradually shifted towards biological methods. However, existing biosynthetic technologies still have significant shortcomings, making it difficult to meet the demands of large-scale applications. Mainstream processes often use glucose or glycerol as substrates. Glucose, in particular, relies on grain resources, raising concerns about food security due to competition with human resources for food, and its cost is significantly affected by fluctuations in grain prices. Glycerol-based routes face challenges such as volatile glycerol market prices and unstable supply, making production economics difficult to guarantee. Furthermore, existing microbial fermentation methods (such as using Klebsiella pneumoniae to produce 1,3-propanediol) suffer from complex metabolic pathways, numerous byproducts, and difficulties in separating and purifying the target product, further increasing production costs. Meanwhile, the biosynthetic technology for 1,3-butanediol is still immature. Existing pathways also rely on high-cost substrates or complex fermentation systems and are incompatible with the 1,3-propanediol production process, failing to achieve efficient synthesis of multiple products on the same platform.

[0004] Aldolase-catalyzed carbon-carbon bond formation reactions offer a promising green biocatalytic pathway for the one-step, highly selective synthesis of chiral diols from inexpensive substrates such as formaldehyde, acetaldehyde, and pyruvate. This reaction is mild, highly atom-economical, and fully meets the requirements of sustainable development, making it an ideal direction for addressing the shortcomings of existing biological methods.

[0005] However, the application of currently reported natural aldolases in the industrial synthesis of 1,3-propanediol and 1,3-butanediol still faces significant limitations: most natural aldolases exhibit low catalytic activity towards non-natural substrates such as formaldehyde and acetaldehyde, making it difficult to meet the yield requirements of industrial production; some enzymes suffer from insufficient regio / stereoselectivity and significant product inhibition effects, resulting in limited purity and yield of the target product; furthermore, the currently reported aldolase synthesis routes use formaldehyde and acetaldehyde as substrates, raising concerns about raw material safety. These limitations collectively restrict their large-scale application. Although researchers have attempted to screen aldolases from different sources or mutate them, a highly active natural aldolase capable of efficiently synthesizing 1,3-propanediol in a one-pot process using methanol, ethanol, or other similar substrates, while simultaneously possessing the ability to synthesize 1,3-butanediol, has not yet been discovered. The catalytic efficiency of existing enzyme preparations falls far short of the yield and cost requirements of industrial production.

[0006] Therefore, exploring highly active natural aldolase resources and further optimizing their catalytic performance through targeted mutagenesis to construct an efficient and economical biocatalytic process for the green and large-scale production of 1,3-propanediol and 1,3-butanediol has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an application of thermococcus-derived aldolase and its mutant in the synthesis of diols (1,3-propanediol, 1,3-butanediol).

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: The application of a thermococcus-derived aldolase B6YUB3 in the synthesis of 1,3-propanediol and / or 1,3-butanediol, wherein the amino acid sequence of the aldolase is shown in SEQ ID NO.1, methanol and ethanol are used as common substrates in the synthesis of 1,3-propanediol, and ethanol is used as substrate in the synthesis of 1,3-butanediol, and the products are obtained through multi-enzyme catalytic reaction.

[0009] An aldolase mutant, said mutant being obtained by mutating the above-mentioned thermococcal-derived aldolase, wherein the mutation site is selected from one or more of positions 23, 64, 172, 213, and 214.

[0010] As one of the preferred embodiments of the present invention, the mutation sites of the mutant are selected from one or more of positions 64, 213, and 214, and the mutation directions are: position 64 is mutated to cysteine ​​(C), position 213 is mutated to aspartic acid (D), and position 214 is mutated to aspartic acid (D).

[0011] As one of the preferred embodiments of the present invention, the mutant is a triple mutant with mutation sites at positions 64, 213, and 214. Position 64 is mutated to cysteine ​​(C), position 213 is mutated to aspartic acid (D), and position 214 is mutated to aspartic acid (D). Its amino acid sequence is shown in SEQ ID NO.8.

[0012] The encoding gene of one of the above-mentioned aldolase mutants.

[0013] A recombinant plasmid containing the above-mentioned encoding gene.

[0014] A recombinant bacterium containing the above-mentioned encoding gene.

[0015] An application of the above-mentioned aldolase mutant or encoding gene in the synthesis of 1,3-propanediol and / or 1,3-butanediol, wherein methanol and ethanol are used as common substrates in the synthesis of 1,3-propanediol, and ethanol is used as substrate in the synthesis of 1,3-butanediol, and the products are obtained through multi-enzyme catalytic reaction.

[0016] As one of the preferred embodiments of the present invention, when the coding gene is used as the application object, the method further includes the following approach: constructing a recombinant bacterium containing the coding gene; using the wet bacterial cells obtained by fermentation culture of the recombinant bacterium, or the pure enzyme obtained by crushing and purifying the bacterial cells as a catalyst to carry out the multi-enzyme catalytic reaction.

[0017] As one of the preferred embodiments of the present invention, in the application of the above-mentioned thermococcus-derived aldolase, aldolase mutant, or encoding gene, the multi-enzyme catalytic reaction system includes a coenzyme cycling enzyme and an auxiliary enzyme; wherein, the coenzyme cycling enzyme is selected from NADH-dependent formate dehydrogenase (catalyzing the regeneration of NAD). + The enzymes are selected from one or more of the following: NAD(P)H-dependent formate dehydrogenase (catalyzes the regeneration of NAD(P)H), 1,3-propanediol dehydrogenase (NADH-dependent, catalyzes the reduction of 3-hydroxypropanal to 1,3-propanediol), and aldehyde-ketone reductase (NAD(P)H-dependent, reduces 3-hydroxybutyral to 1,3-butanediol); the coenzyme is selected from one or more of the following: catalase (decomposes H2O2 and catalyzes the oxidation of ethanol to acetaldehyde) and alcohol oxidase (catalyzes the oxidation of methanol to formaldehyde); the above enzymes maintain the dynamic balance of coenzyme NADH / NAD(P)H through synergistic action, improve the conversion efficiency of aldolase to substrate, and reduce the accumulation of byproducts.

[0018] As one of the preferred embodiments of the present invention, the one-pot reaction system for synthesizing 1,3-propanediol specifically contains catalase, alcohol oxidase, aldolase (B6YUB3 or its mutant), 1,3-propanediol dehydrogenase, and NADH-dependent formate dehydrogenase; wherein the protein concentration of alcohol oxidase is 1 mg / mL to 10 mg / mL (more preferably 4 mg / mL), the protein concentration of catalase is 0.1 mg / mL to 4 mg / mL (more preferably 0.5 mg / mL), and the protein concentration of aldolase is 1 mg / mL to 20 mg / mL. (More preferably 10 mg / mL), the protein concentration of NADH-dependent formate dehydrogenase is 0.1 mg / mL to 4 mg / mL (more preferably 2 mg / mL), the protein concentration of 1,3-propanediol dehydrogenase is 0.1 mg / mL to 1 mg / mL (more preferably 0.5 mg / mL); the amount of methanol is 100 mmol / L to 1000 mmol / L (more preferably 500 mmol / L), the amount of ethanol is 100 mmol / L to 500 mmol / L (more preferably 300 mmol / L), NAD + The dosage is 0.1~10 mmol / L (more preferably 1 mmol / L), the dosage of sodium formate is 100~500 mmol / L (more preferably 300 mmol / L), and the catalytic process is controlled at pH 5.0~9.0 (more preferably 7.0) and temperature 20℃~60℃ (more preferably 35℃).

[0019] As one of the preferred embodiments of the present invention, the one-pot reaction system for synthesizing 1,3-butanediol specifically contains catalase, alcohol oxidase, aldolase (B6YUB3 or its mutant), aldehyde reductase, and NAD(P)H-dependent formate dehydrogenase; wherein the protein concentration of alcohol oxidase is 1 mg / mL to 10 mg / mL (more preferably 4 mg / mL), the protein concentration of catalase is 0.1 mg / mL to 4 mg / mL (more preferably 0.5 mg / mL), the protein concentration of aldolase (B6YUB3 or its mutant) is 1 mg / mL to 20 mg / mL (more preferably 10 mg / mL), and the NAD(P)H-dependent formate dehydrogenase... The protein concentration of H-dependent formate dehydrogenase is 0.1 mg / mL to 4 mg / mL (more preferably 2 mg / mL), and the protein concentration of aldehyde reductase is 2 mg / mL to 10 mg / mL (more preferably 4 mg / mL); the amount of ethanol used is 200 mmol / L to 1000 mmol / L (more preferably 800 mmol / L), and NADP... +The dosage is 0.1~10 mmol / L (more preferably 1 mmol / L), the dosage of sodium formate is 100~500 mmol / L (more preferably 300 mmol / L), and the catalytic process is controlled at pH 5.0~9.0 (more preferably 7.0) and temperature 20℃~60℃ (more preferably 35℃).

[0020] In the above system, NAD(P) + The NAD(P)H coenzyme cycle system is a commonly used coenzyme cycle system, including alcohol dehydrogenase / isopropanol, formate dehydrogenase / formate, glucose dehydrogenase / glucose, etc., with Aquabacterium tumefaciens being the preferred choice. 水生杆菌属 Formate dehydrogenase and sodium formate (CN5-332) are derived from this source.

[0021] NAD + The NADH coenzyme cycle is a commonly used coenzyme cycle system, including alcohol dehydrogenase / isopropanol, formate dehydrogenase / formate, glucose dehydrogenase / glucose, etc., preferably *Bacillus wallichiana* (…). 壁栖类芽孢杆菌 ), Simple Bacillus ( 简单类芽孢杆菌 Formate dehydrogenase and sodium formate are derived from ( ).

[0022] Alcohol oxidase is preferably found in *Platycodon grandiflorus* ( 无花果拟盘多毛孢 Alcohol oxidase from ( ).

[0023] 1,3-Propanediol dehydrogenase is preferably found in Clostridium pasteurellii ( 巴氏梭菌 1,3-propanediol dehydrogenase from ).

[0024] Aldehyde reductase is preferably found in Pseudomonas aeruginosa ( 铜绿假单胞菌 Aldol reductase from )

[0025] Reaction principle: This invention establishes a method for the synthesis of 1,3-propanediol starting from methanol / ethanol: as follows Figure 1 As shown, methanol is first oxidized by alcohol oxidase to formaldehyde and ethanol to acetaldehyde; then, carbon-carbon bond linkage is catalyzed by aldolase to generate the intermediate 3-hydroxypropane; finally, 1,3-propanediol is obtained by reduction catalyzed by 1,3-propanediol dehydrogenase. The experiment modified the rate-limiting enzyme aldolase by mutating alanine at position 64 to cysteine ​​to form a disulfide bond, reducing the inhibition of enzyme activity by the intermediate; simultaneously, serine at positions 213 and 214 was mutated to aspartic acid, enhancing the enzyme's binding ability to the substrate and significantly improving aldolase activity. This successfully removed the inhibition of the rate-limiting step, resulting in a substantial increase in the yield of 1,3-propanediol.

[0026] The advantages of this invention compared to the prior art are: 1. The inventors systematically screened and compared the catalytic activities of 21 aldolases from different sources, and found that Thermococcus (…嗜热栖热球菌 The aldolase derived from this enzyme (Uniprot accession number: B6YUB3) exhibits significantly superior catalytic activity compared to previously reported natural aldolases. It can be directly adapted for the synthesis of 1,3-propanediol and 1,3-butanediol, and the yield of 1,3-propanediol using methanol and ethanol as substrates can reach 27.09 mM, which is the best level among known natural aldolases to date, demonstrating excellent carbon-carbon linkage potential.

[0027] 2. This invention utilizes semi-rational design (combining PDB ID: 1JCJ crystal modeling, molecular docking, and enzyme catalytic mechanisms) to perform site-directed and combinatorial mutagenesis on B6YUB3 aldolase, screening for mutants with significantly improved catalytic performance. This is comparable to existing enzymatic methods for preparing 1,3-propanediol (e.g., J. Am. Chem. Soc. 2025, 147, 39827). Compared to 39837), the mutants optimized in this invention can significantly increase the yield of 1,3-propanediol. Among them, the optimal triple mutant A64C / S213D / S214D increases the yield by 1.8 times compared to the wild type, effectively solving the problems of blind modification of existing mutants and insignificant improvement in enzyme activity and yield.

[0028] 3. The highly active aldolase mutant obtained by screening in this invention can be used not only efficiently in the synthesis of 1,3-propanediol, but also in the synthesis of 1,3-butanediol. The optimized mutant can catalyze a 129 mM (11.6 g / L) yield of 1,3-butanediol, showing good industrial application performance. Compared with existing single-product synthesis technologies, it has a wider range of applications and is suitable for different industrial needs.

[0029] 4. This invention uses inexpensive and readily available methanol and ethanol as substrates, combined with a highly efficient multi-enzyme synergistic system. The reaction conditions are mild and the implementation is convenient. It does not require high temperature, high pressure or precious metal catalysts, which effectively reduces the cost of industrial applications. It lays the foundation for the efficient and green synthesis of two high-valent diols and has both practicality and value for large-scale promotion. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the reaction principle of the one-pot enzymatic catalytic synthesis of 1,3-propanediol and 1,3-butanediol according to the present invention. Figure 2 This is an HPLC chromatogram of the 1,3-propanediol standard and catalytic process of the present invention; Figure 3 This is an HPLC chromatogram of the 1,3-butanediol standard and catalytic process of the present invention; Figure 4 This is a schematic diagram of the active cavity mutation site of the aldolase B6YUB3 of the present invention; Figure 5This is a yield diagram of the process of synthesizing 1,3-propanediol catalyzed by the wild-type aldolase B6YUB3 and the triple mutant enzymes A64C / S213D / S214D of the present invention. Figure 6 This is a yield diagram of the process of synthesizing 1,3-butanediol catalyzed by the triple mutant enzymes A64C / S213D / S214D of this invention. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the culture media, carriers, plasmids, and reagents used in the following embodiments are conventional culture media, carriers, plasmids, and reagents in this technical field; and unless otherwise specified, the methods and equipment used in the following embodiments are conventional methods and equipment in this technical field.

[0032] The culture medium formulations involved in the following examples are as follows: LB medium: yeast extract 5.0 g / L, peptone 10.0 g / L, NaCl 10.0 g / L.

[0033] Example 1: Construction of recombinant expression systems for aldolases from different sources 1. Twenty-one candidate aldolases (containing Thermococcus) were screened from the Uniprot database. 嗜热栖热球菌属 嗜热栖热球菌 The aldolase B6YUB3 from which the catalytic synthesis of 1,3-propanediol was derived was analyzed, and its Uniprot accession number is shown in Table 1.

[0034] Table 1. Information on aldolases from different sources and comparison of their catalytic activity in the synthesis of 1,3-propanediol.

[0035] 2. Based on the amino acid sequences of these enzymes in Table 1, gene optimization and synthesis were commissioned to a gene synthesis company (Beijing Qingke Biotechnology Co., Ltd.). The gene optimization standard was: based on the host cell *Escherichia coli*. 大肠杆菌 The codons of BL21 (DE3) are optimized using a preference-based synonymous codon approach. During the optimization process, the amino acid sequences of each enzyme are kept unchanged. At the same time, it is ensured that the optimized gene contains restriction endonuclease recognition sequences (specifically BamHI and NdeI) that match the multiple cloning site of the commercially available expression vector pET21a at both ends, and that the reading frame is consistent with the His6 tag coding sequence on the vector, thus avoiding frameshift mutations.

[0036] 3. Twenty-one artificially synthesized aldolase genes were double-digested with BamHI and NdeI and then directionally ligated into the multiple cloning site (MCS) of the pET21a expression vector, which was also double-digested. The target gene sequence was located between the T7 promoter and T7 terminator of the pET21a vector and immediately adjacent to the His6 tag coding sequence on the vector (located downstream of the MCS), thus constructing 21 recombinant expression plasmids (the recombinant plasmid containing the Thermococcus-derived B6YUB3 gene was named pET21a-B6YUB3). Each recombinant plasmid was transformed into *E. coli*. 大肠杆菌 BL21 (DE3) competent cells were plated on LB agar plates containing ampicillin. Single colonies were picked and sequenced to verify the gene sequence, restriction sites, and reading frames. Finally, 21 recombinant engineered bacteria were obtained (including recombinant engineered bacteria expressing thermococcal aldolase B6YUB3, the amino acid sequence of the expressed B6YUB3 is shown in SEQ ID NO.1, and the corresponding nucleotide sequence encoding the protein is shown in SEQ ID NO.2).

[0037] Example 2: Construction of recombinant Escherichia coli with other required enzymes This embodiment describes the construction of recombinant *E. coli* containing other coenzymes and coenzyme cycling enzymes required for the catalytic reaction system. The required enzymes include alcohol oxidase, catalase, 1,3-propanediol dehydrogenase, NADH-dependent formate dehydrogenase, NAD(P)H-dependent formate dehydrogenase, and aldehyde-ketone reductase. The specific sources, amino acid sequences, and coenzyme cycling system design for each enzyme are as follows: 1. Coenzyme Cycle System Design Application of coenzyme NAD(P) + / NAD(P)H and NAD + The design employs a NADH-dependent coenzyme cycle, where the NADH-dependent cycle corresponds to the synthesis of 1,3-propanediol (1,3-PDO) and the NAD(P)H-dependent cycle corresponds to the synthesis of 1,3-butanediol (1,3-BDO). This design avoids the use of expensive coenzyme raw materials, instead using the relatively inexpensive NAD(P)H. + or NAD + It serves as a raw material and can significantly reduce the amount of coenzyme used, effectively controlling production costs.

[0038] 2. Specific sources and amino acid sequences of each enzyme NAD(P) + / NAD(P)H coenzyme cycle system, selected by Aquabacterium ( 水生杆菌属 Formate dehydrogenase derived from CN5-332, the amino acid sequence of which is as described in SEQ ID NO.3; NAD + / NADH coenzyme cycle system, selected from simple Bacillus ( 简单类芽孢杆菌 Formate dehydrogenase derived from ), the amino acid sequence of which is as described in SEQ ID NO.4; Alcohol oxidase, selected from *Plasmodium spp.* ( 无花果拟盘多毛孢 An alcohol oxidase derived from [source missing], the amino acid sequence of which is as described in SEQ ID NO. 5; 1,3-Propanediol dehydrogenase, selected from Clostridium pasteurellii ( 巴氏梭菌 1,3-Propanediol dehydrogenase from [source missing], the amino acid sequence of which is as described in SEQ ID NO. 6; Aldehyde reductase, selected from Pseudomonas aeruginosa ( 铜绿假单胞菌 The aldehyde-ketone reductase is derived from the following source, and the amino acid sequence of the aldehyde-ketone reductase is as described in SEQ ID NO.7.

[0039] The recombinant E. coli construction methods for the above enzymes are as follows, referring to the construction process of the aldolase recombinant expression system in Example 1: 1. Beijing Qingke Biotechnology Co., Ltd. was commissioned to optimize and artificially synthesize the enzymes according to the amino acid sequences of the above enzymes and the gene optimization standards of Example 1 (based on the codon preference optimization of E. coli BL21(DE3), retaining the amino acid sequence unchanged, adding BamHI and NdeI restriction sites at both ends, consistent with the His6 tag reading frame of the pET21a vector).

[0040] 2. After double digestion of the synthesis genes of each enzyme, they were ligated into the pET21a expression vector to construct the recombinant expression plasmids corresponding to each enzyme. These plasmids were then transformed into Escherichia coli BL21(DE3) competent cells. Single colonies were picked and sequenced to verify the construction and ensure that the recombinant engineered bacteria corresponding to each enzyme were obtained.

[0041] Example 3: Induction and expression of recombinant engineered bacteria and protein purification In this embodiment, the 21 recombinant engineered bacteria with aldolase obtained in Example 1 and the recombinant engineered bacteria with each auxiliary enzyme and coenzyme cycling enzyme obtained in Example 2 were induced to express and their proteins were purified.

[0042] 1. Take the 21 recombinant engineered aldolases obtained in Example 1 and the recombinant engineered coenzymes and coenzyme cycling enzymes obtained in Example 2, respectively, and inoculate them into LB medium containing 50 μg / mL ampicillin. Culture at 37℃ and 200 rpm until the mid-log growth phase. Add IPTG to a final concentration of 1.0 mmol / L and induce expression at 24℃ for 6 h to obtain the induced expression fermentation broth for later use.

[0043] 2. Centrifuge the fermentation broth, resuspend it in pH 7.0, 50 mmol / L PBS buffer, and then homogenize the cells using a high-pressure cell homogenizer to obtain the crude enzyme solution, which needs to be purified as soon as possible. 3. Take 3.0~4.0 mL of nickel chelate agarose gel packing material into the chromatography column, let it stand, and wash it with water for 10 column volumes before use. Equilibrate the adsorption column with 10 column volumes of equilibration buffer (50 mmol / L sodium phosphate, 300 mmol / L NaCl, 20 mmol / L imidazole, pH 7.4). After equilibration, centrifuge the crude enzyme solution obtained in the above operation at 12000 rpm for 10 min at 4℃. Take an appropriate amount of supernatant for loading. Seal both ends of the chromatography column after loading and place it on ice with gentle shaking for 40~60 min. Reposition the chromatography column vertically and discard the loading waste solution. Wash off non-specifically adsorbed contaminating proteins with 10 column volumes of washing buffer (50 mmol / L sodium phosphate, 300 mmol / L NaCl, 40 mmol / L imidazole, pH 7.4). Then elute the target protein with 10 column volumes of elution buffer (50 mmol / L sodium phosphate, 300 mmol / L NaCl, 300 mmol / L imidazole, pH 7.4). Collect the eluent in separate tubes and store on ice.

[0044] 4. SDS-PAGE verification of the target protein in the collected solution: The collected solution containing the target protein was transferred to a dialysis bag and placed in a low-temperature environment. Desalination was performed using 50 mmol / L, pH 7.0 phosphate buffer solution as the dialysis buffer. The dialysis buffer was changed every 4 hours. After dialysis, PEG 20000 was added to the outside of the dialysis bag to concentrate the enzyme solution. After concentration, the protein concentration of the purified enzyme solution was determined according to the Bradford kit manual.

[0045] Example 4: Comparison of the activities of aldolases from various sources in the multi-enzyme catalytic synthesis of 1,3-propanediol using ethanol and methanol as initial substrates. In this embodiment, 21 different aldolases purified from different sources obtained in Example 3 were used, along with purified coenzymes and coenzyme cycling enzymes. A catalytic reaction system was constructed using ethanol and methanol as initial substrates. By detecting the yield of 1,3-propanediol, the catalytic activity of aldolases from different sources was compared, and the aldolase with the best catalytic activity was screened.

[0046] 1. Construct a one-pot catalytic reaction system as follows: alcohol oxidase 4 mg / mL, catalase (purchased from Solarbio) 0.5 mg / mL, aldolase 10 mg / mL, NADH-dependent formate dehydrogenase protein 2 mg / mL, 1,3-propanediol dehydrogenase 0.5 mg / mL, ethanol 500 mmol / L, methanol 300 mmol / L, NAD... +1 mmol / L, sodium formate 300 mmol / L, phosphate buffer 50 mM (pH 7.0).

[0047] 2. Place the above reaction solution in a 1.5 mL EP tube, shake in a metal bath, and react at 35°C for 1 h.

[0048] 3. Add 10% v / v 6mM hydrochloric acid solution to terminate the reaction. Centrifuge at 12000×g for 5 min, and pass the supernatant through a 0.22μm membrane for liquid chromatography analysis.

[0049] 4. High-performance liquid chromatography (HPLC) analysis method. A high-performance liquid chromatography system equipped with an Aminex HPX-87H column (300x7.8mm, Bio-Red) was used, with 5mM sulfuric acid solution as the mobile phase and a flow rate of 0.6 mL / min. -1 Quantitative analysis of diols (1,3-propanediol and 1,3-butanediol) was performed. The column temperature was 60℃, and the RID detector temperature was 35℃.

[0050] The test results are shown in Table 1. Figure 2 , Figure 3 The HPLC chromatograms are for 1,3-propanediol, 1,3-butanediol standards, and the catalytic process, respectively.

[0051] The results above indicate that there are significant differences in the activities of aldolases from different sources in the multi-enzyme catalytic synthesis of 1,3-propanediol from ethanol and methanol. Among these, those from *Thermococcus* show the most significant differences. 嗜热栖热球菌 The B6YUB3 aldolase from Thermococcus showed the best catalytic activity, yielding 27.09 mM of 1,3-propanediol, significantly higher than aldolases from other sources. Some aldolases, however, exhibited no or very low catalytic activity. These results indicate that the B6YUB3 aldolase from Thermococcus is the optimal candidate enzyme for the multi-enzyme catalytic synthesis of 1,3-propanediol from ethanol and methanol, and can serve as a core enzyme for the construction of efficient catalytic synthesis systems for subsequent 1,3-propanediol and 1,3-butanediol.

[0052] Example 5: Site-directed mutagenesis and screening of key amino acids in the active pocket of aldolase B6YUB3. This embodiment is based on the optimal aldolase B6YUB3 (amino acid sequence shown in SEQ ID NO.1) screened in Example 4. By locating the key amino acid sites in its active pocket through bioinformatics analysis, site-directed mutagenesis was performed to construct a mutant recombinant engineered bacterium. After induction expression and protein purification, mutants with catalytic activity superior to wild-type B6YUB3 were screened to provide high-quality single-point mutation targets for subsequent combined mutagenesis.

[0053] 1. To improve the yield of 1,3-propanediol by aldolase B6YUB3, site-directed mutation sites for B6YUB3 were selected using computer-aided design. Based on the reported aldolase crystal structure (PDB ID: 1JCJ), homology modeling of aldolase B6YUB3 was performed. Simulation analysis was conducted using protein 3D structure analysis software and molecular docking software. Considering the aldolase-substrate docking results, the characteristics of the enzyme's substrate binding pocket, and the enzyme's catalytic mechanism, the mutation site distribution is as follows: Figure 4 As shown, the final amino acid sequence SEQ ID NO. 2 was modified by mutating the following mutations: threonine at position 16 was mutated to serine; threonine at position 23 was mutated to leucine and glycine; alanine at position 64 was mutated to cysteine; phenylalanine at position 69 was mutated to tyrosine; glutamic acid at position 172 was mutated to glycine; aspartic acid at position 173 was mutated to alanine; and serine at positions 213 and 214 was mutated to aspartic acid.

[0054] 2. Nine site-directed mutagenesis primers were designed, as shown in Table 2. Using the vector pET21a-B6YUB3 (constructed in Example 1) as a template, the entire plasmid was amplified by PCR using the site-directed mutagenesis primers. The amplified bands of the correct size were obtained by 1% agarose gel electrophoresis. The PCR product was digested with the restriction endonuclease DpnI for 1 hour to digest the methylated plasmid template. The digested PCR product was ligated using a one-step cloning method, and then transformed into *E. coli* BL21(DE3) cells. Colony PCR and sequencing verification confirmed the presence of an aldolase mutant with the target site mutation.

[0055] Table 2. Site-directed mutagenesis primers for B6YUB3

[0056] 3. Following the induction expression and protein purification steps in Example 3, the above-mentioned aldolase mutant was induced to express, purified by Ni-NTA affinity chromatography, desalted by dialysis, and concentrated to obtain a pure enzyme preparation.

[0057] 4. Referring to the catalytic reaction system and detection method in Example 4, catalytic reaction systems were constructed using wild-type aldolase B6YUB3 and pure enzyme preparations of 9 mutants, respectively. The yield of 1,3-propanediol was detected, and the catalytic activity of each mutant was compared. The results are shown in Table 3.

[0058] Table 3. Comparison of enzyme activities between wild-type B6YUB3 and nine B6YUB3 mutants

[0059] The results showed that T23L, A64C, E172G, S213D, and S214D had increased aldolase activity compared to wild-type aldolase, with A64C and S214D exhibiting the highest catalytic activity.

[0060] Example 6: Further combined multi-site mutation modification of the A64C site of aldolase B6YUB3. This embodiment is based on the single-point mutant A64C screened in Example 5. It combines several other key sites (T23, T16, E172, S213, S214) selected for multi-site mutation modification to construct two-point and three-point combined mutants, further enhancing the catalytic activity of aldolase and obtaining a highly efficient mutant aldolase.

[0061] 1. For the construction of a multi-point mutant using aldolase B6YUB3-A64C as a template, five double-point mutants and one triple-point mutant were designed, with the mutation primers shown in Table 4. Specifically, using the vector pET21a-B6YUB3-A64C as a template (constructed in Example 5), the entire plasmid was amplified by PCR. The correctly sized amplified band was obtained by 1% agarose gel electrophoresis. The PCR product was digested with the restriction endonuclease DpnI for 1 hour to digest the methylated plasmid template. The digested PCR product was ligated using a one-step cloning method, and then transformed into *E. coli* BL21(DE3) cells. Colony PCR and sequencing verification confirmed the aldolase mutant with the target mutation site.

[0062] Table 4. Primers for multiple mutations of B6YUB3

[0063] 2. Following the induction expression and protein purification steps in Example 3, the above-mentioned aldolase mutant was induced to express, purified by Ni-NTA affinity chromatography, desalted by dialysis, and concentrated to obtain a pure enzyme preparation.

[0064] 3. Referring to the catalytic reaction system and detection method in Example 4, catalytic reaction systems were constructed using wild-type aldolase B6YUB3, A64C single-point mutant, and the pure enzyme preparations of the six multi-point mutants in this example. The yield of 1,3-propanediol was detected, and the catalytic activity of each mutant was compared. The results are shown in Table 5.

[0065] Table 5. Comparison of enzyme activities between wild-type B6YUB3 and seven B6YUB3 mutants

[0066] The results showed that A64C / S213D, A64C / S214D, and A64C / S213D / S214D exhibited increased enzyme activity compared to aldolase A64C. Among them, A64C / S213D / S214D (amino acid sequence shown in SEQ ID NO. 8, and the corresponding nucleotide sequence encoding the enzyme shown in SEQ ID NO. 9) showed the highest catalytic activity, which was 321% higher than that of the wild type.

[0067] Example 7: One-pot enzymatic catalytic preparation of 1,3-propanediol This embodiment uses the highly efficient mutant aldolase A64C / S213D / S214D screened in Example 6, combined with the purified coenzyme and coenzyme cycle enzyme from Example 3, and uses ethanol and methanol as initial substrates. A one-pot enzymatic catalytic reaction system and conditions are used to achieve the efficient preparation of 1,3-propanediol.

[0068] 1. Reaction system: alcohol oxidase 4 mg / mL, catalase (purchased from Solarbio) 0.5 mg / mL, aldolase mutant A64C / S213D / S214D protein 10 mg / mL, NADH-dependent formate dehydrogenase 2 mg / mL, 1,3-propanediol dehydrogenase 0.5 mg / mL, ethanol 500 mmol / L, methanol 300 mmol / L, NAD + 1 mmol / L, sodium formate 300 mmol / L, phosphate buffer 50 mM (pH 7.0).

[0069] Reaction conditions: Place the above reaction solution in a 1.5 mL EP tube, shake in a metal bath, and react at 35 °C.

[0070] 2. Take a sample every two hours and add 10% v / v 6mM hydrochloric acid solution to terminate the reaction.

[0071] 3. Centrifuge at 12000×g for 5 min, take the supernatant and pass it through a 0.22μm membrane for liquid chromatography analysis, and calculate the yield.

[0072] The results are as follows Figure 5 As shown, the triple mutant enzyme A64C / S213D / S214D has a significantly faster catalytic rate than the wild type, and the final yield of 1,3-propanediol reaches a final concentration of 256 mM (19.5 g / L), which is 1.8 times higher than that of the wild type.

[0073] Example 8: One-pot enzymatic catalytic preparation of 1,3-butanediol This embodiment uses the highly efficient mutant aldolase A64C / S213D / S214D screened in Example 6, combined with the purified coenzyme and coenzyme cycling enzyme from Example 3, and employs a one-pot enzymatic catalytic reaction system and conditions with ethanol as the substrate to achieve the efficient preparation of 1,3-butanediol.

[0074] 1. Reaction system: alcohol oxidase 4 mg / mL, catalase (purchased from Solarbio) 0.5 mg / mL, aldolase mutant A64C / S213D / S214D protein 10 mg / mL, NAD(P)H-dependent formate dehydrogenase 2 mg / mL, aldehyde reductase 4 mg / mL, ethanol 800 mmol / L, NAD + 1 mmol / L, sodium formate 300 mmol / L, phosphate buffer 50 mM (pH 7.0).

[0075] Reaction conditions: Place the above reaction solution in a 1.5 mL EP tube, shake in a metal bath, and react at 35 °C.

[0076] 2. Take a sample every two hours and add 10% v / v 6mM hydrochloric acid solution to terminate the reaction.

[0077] 3. Centrifuge at 12000×g for 5 min, take the supernatant and pass it through a 0.22 μm membrane for liquid chromatography analysis, and calculate the yield.

[0078] The results are as follows Figure 6 As shown, the triple mutant enzymes A64C / S213D / S214D can catalyze the synthesis of 1,3-butanediol, with a final yield of 129 mM (11.6 g / L).

[0079] In summary, this invention obtains Thermococcus through screening. 嗜热栖热球菌 The derived aldolase B6YUB3 is the optimal candidate enzyme for the synthesis of 1,3-propanediol from ethanol and methanol. Through site-directed mutagenesis and combined multi-position mutagenesis of key amino acids in its active pocket, the mutant with the highest catalytic activity, A64C / S213D / S214D, was successfully obtained. This mutant exhibits significantly increased enzyme activity and catalytic efficiency compared to the wild type. Using this highly efficient mutant aldolase as the core, a one-pot enzymatic catalytic system was constructed, enabling the efficient catalytic preparation of 1,3-propanediol and 1,3-butanediol. The final yield of 1,3-propanediol reached 256 mM (19.5 g / L), a 1.8-fold increase compared to the wild type, and the final yield of 1,3-butanediol reached 129 mM (11.6 g / L), achieving highly efficient synthesis of both diol products. The recombinant aldolase mutant constructed in this invention exhibits excellent catalytic activity. The one-pot enzymatic catalysis system has mild reaction conditions and is easy to operate. Furthermore, the production cost is significantly reduced by utilizing a coenzyme recycling system. All enzyme preparations used can be prepared on a large scale through recombinant expression in Escherichia coli. At the same time, the yield of the target product is greatly increased, demonstrating promising prospects for industrial application.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a thermococcus-derived aldolase in the synthesis of 1,3-propanediol and / or 1,3-butanediol, characterized in that, The amino acid sequence of the aldolase is shown in SEQ ID NO.

1. Methanol and ethanol are used as common substrates for the synthesis of 1,3-propanediol, and ethanol is used as substrate for the synthesis of 1,3-butanediol. The products are obtained through multi-enzyme catalytic reaction.

2. The application according to claim 1, characterized in that, The multi-enzyme catalytic reaction system includes a coenzyme cycling enzyme and an auxiliary enzyme; the coenzyme cycling enzyme is selected from one or more of NADH-dependent formate dehydrogenase, NAD(P)H-dependent formate dehydrogenase, 1,3-propanediol dehydrogenase, and aldehyde-ketone reductase; the auxiliary enzyme is selected from one or more of catalase and alcohol oxidase; the reaction conditions are: pH 5.0~9.0, temperature 20℃~60℃.

3. An aldolase mutant, characterized in that, The mutant is obtained by mutating the thermococcal-derived aldolase of claim 1, wherein the mutation site is selected from one or more of positions 23, 64, 172, 213, and 214.

4. The aldolase mutant according to claim 3, characterized in that, The mutation sites of the mutant are selected from one or more of positions 64, 213, and 214, and the mutation directions are: position 64 is mutated to cysteine, position 213 is mutated to aspartic acid, and position 214 is mutated to aspartic acid.

5. The aldolase mutant according to claim 4, characterized in that, The mutant is a triple mutant with mutation sites at positions 64, 213, and 214. Position 64 is mutated to cysteine, position 213 to aspartic acid, and position 214 to aspartic acid. Its amino acid sequence is shown in SEQ ID NO.

8.

6. A gene encoding an aldolase mutant as described in any one of claims 3 to 5.

7. A recombinant plasmid containing the encoding gene of claim 6, and a recombinant bacterium.

8. The use of an aldolase mutant according to any one of claims 3 to 5, or the encoding gene according to claim 6, in the synthesis of 1,3-propanediol and / or 1,3-butanediol, characterized in that, Methanol and ethanol were used as common substrates for the synthesis of 1,3-propanediol, while ethanol was used as substrate for the synthesis of 1,3-butanediol. The products were obtained through multi-enzyme catalytic reactions.

9. The application according to claim 8, characterized in that, When the coding gene is used as the application object, the following approach is also included: constructing recombinant bacteria containing the coding gene; using wet bacterial cells obtained by fermentation culture of the recombinant bacteria, or pure enzymes obtained by breaking and purifying the bacterial cells as catalysts to carry out the multi-enzyme catalytic reaction.

10. The application according to claim 8, characterized in that, The multi-enzyme catalytic reaction system includes a coenzyme cycling enzyme and an auxiliary enzyme; the coenzyme cycling enzyme is selected from one or more of NADH-dependent formate dehydrogenase, NAD(P)H-dependent formate dehydrogenase, 1,3-propanediol dehydrogenase, and aldehyde-ketone reductase; the auxiliary enzyme is selected from one or more of catalase and alcohol oxidase; the reaction conditions are: pH 5.0~9.0, temperature 20℃~60℃.

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