Geranial dehydrogenase as well as preparation method and application thereof

Abstract

The invention provides geranial dehydrogenase as well as a preparation method and application thereof, and relates to the technical field of biology, and the amino acid sequence of the geranial dehydrogenase is as shown in SEQ ID NO.1. Citral can be used as a substrate to catalytically synthesize the aromatic folic acid, and the conversion rate reaches 90%. The geranial dehydrogenase can be used for efficiently synthesizing the aromatic folic acid and can be used for biocatalytically converting the aromatic folic acid. Meanwhile, NAD < + > is used as a coenzyme to improve the enzymatic reaction efficiency, so that efficient catalytic synthesis of the aromatic folic acid is realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to geranaldehyde dehydrogenase and a preparation method and application thereof. Background Art

[0002] Geranic acid, also known as geranic acid and 3,7-dimethyl-2,6-octadienoic acid, is an acyclic monoterpene compound with stable properties. It is often used in food flavors, cosmetics, and pharmaceutical manufacturing. In the field of skin care, it can be used as an antioxidant and anti-inflammatory substance, and has a therapeutic effect on acne, eczema and other problems; in the food industry, it can act as a preservative and antioxidant to help extend the shelf life of food and increase its nutritional value; in addition, this substance can also be used in drug research and development, and is converted into a compound with anti-cancer activity through chemical reactions. Its molecular formula is as follows:

[0003]

[0004] In recent years, the main production methods of geranic acid include plant extraction, chemical synthesis and biocatalytic conversion. However, plant extraction is hindered by low content and limited natural resources, which hinders large-scale production; chemical synthesis has many disadvantages such as cumbersome steps, low yield, complex by-products, difficult post-processing, and environmental pollution. In comparison, the enzyme synthesis method is not restricted by raw materials and climate planting risks. The use of enzyme engineering technology platform to produce terpenoids is expected to achieve large-scale, economical, efficient and environmentally friendly industrial production. However, the enzymes used to synthesize geranic acid in the existing technology are of low efficiency and difficult to use in large-scale industrial production.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a geranaldehyde dehydrogenase to solve the technical problem in the prior art that there is a lack of an enzyme that can be used for the efficient synthesis of geranic acid in a biocatalytic pathway.

[0007] A second object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned geranaldehyde dehydrogenase.

[0008] The third object of the present invention is to provide a vector containing the above-mentioned nucleic acid molecule.

[0009] A fourth object of the present invention is to provide a recombinant cell expressing geranaldehyde dehydrogenase.

[0010] A fifth object of the present invention is to provide a method for constructing the above-mentioned recombinant cell.

[0011] The sixth object of the present invention is to provide the use of the above-mentioned recombinant cell or the recombinant cell constructed by the above-mentioned method in producing geranyl aldehyde dehydrogenase, increasing the yield of geranyl aldehyde dehydrogenase or producing geranic acid.

[0012] A seventh object of the present invention is to provide a method for producing geranaldehyde dehydrogenase.

[0013] An eighth object of the present invention is to provide a method for preparing geranic acid.

[0014] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0015] In a first aspect, the present invention provides a geranaldehyde dehydrogenase, the amino acid sequence of the geranaldehyde dehydrogenase is shown in SEQ ID NO.1.

[0016] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned geranaldehyde dehydrogenase;

[0017] Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2 or has more than 80% identity with the nucleotide sequence shown in SEQ ID NO.2 and has the function of increasing the amino acid production of microorganisms.

[0018] In a third aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

[0019] In a fourth aspect, the present invention provides a recombinant cell expressing geranaldehyde dehydrogenase, comprising the above-mentioned nucleic acid molecule, or comprising the above-mentioned vector, or expressing the above-mentioned geranaldehyde dehydrogenase.

[0020] In a fifth aspect, the present invention provides a method for constructing the above-mentioned recombinant cell, comprising introducing the above-mentioned vector into a biological cell;

[0021] Preferably, the biological cells include prokaryotic cells;

[0022] Preferably, the prokaryotic cells include Escherichia coli BL21 cells.

[0023] In a sixth aspect, the present invention provides the use of the above-mentioned recombinant cell or the recombinant cell constructed by the above-mentioned method in any of the following:

[0024] A. Produce geranaldehyde dehydrogenase;

[0025] B. Increase the production of geranaldehyde dehydrogenase;

[0026] C. Produce geranic acid.

[0027] In a seventh aspect, the present invention provides a method for producing geranaldehyde dehydrogenase, comprising culturing the above-mentioned recombinant cell or the recombinant cell prepared by the above-mentioned method to obtain a fermentation product, and obtaining geranaldehyde dehydrogenase from the fermentation product;

[0028] Preferably, the culturing of the above-mentioned recombinant cells or the recombinant cells prepared by the above-mentioned method comprises subjecting the activated recombinant cells to seed culture to obtain seed liquid, and subjecting the seed liquid to fermentation culture;

[0029] Preferably, the seed culture conditions include shaking culture at 37°C and 200 rpm for 8 to 12 hours, preferably 10 hours;

[0030] Preferably, the fermentation culture comprises inoculating the seed liquid into the fermentation medium, shaking the culture for 15 hours, and continuously adding nutrients until the OD 600 The temperature is 30 to 40, an inducer is added, and the fermentation product is obtained by inducing the culture;

[0031] Preferably, the nutrients include glucose at a concentration of 195 g / L, peptone at a concentration of 32 g / L, yeast powder at a concentration of 32 g / L, and MgSO4 at a final concentration of 60 mM;

[0032] Preferably, the shaking culture conditions in the fermentation culture include shaking culture at 500-900 rpm at 37°C, preferably 600 rpm;

[0033] Preferably, after adding the inducer, the culture temperature is adjusted to 28-35°C, preferably 30°C;

[0034] Preferably, the induction culture time is 4 to 8 hours, preferably 6 hours;

[0035] Preferably, the inducing agent is IPTG or Lactose, preferably IPTG;

[0036] Preferably, obtaining geranyl aldehyde dehydrogenase from the fermentation product comprises collecting bacterial cells and crushing the cells to obtain geranyl aldehyde dehydrogenase;

[0037] Preferably, the cell disruption comprises mixing the cells and the disruption buffer in a mass volume ratio of 1:1 to 2 and then homogenizing and disrupting the cells, preferably in a mass volume ratio of 1:1.5;

[0038] Preferably, the crushing buffer is Na2HPO4-NaH2PO4 buffer.

[0039] In an eighth aspect, the present invention provides a method for preparing geranic acid, comprising using a cell disrupted liquid containing geranal dehydrogenase prepared by the above method as a catalyst, and carrying out a dehydrogenation reaction with citral as a substrate to prepare geranic acid.

[0040] Furthermore, the dehydrogenation reaction is carried out in NAD + / NADH coenzyme circulation system works in coordination;

[0041] Preferably, the enzyme activity of the cell disruption solution containing geranyl aldehyde dehydrogenase is ≥2.21 U / mL;

[0042] Preferably, the concentration of citral in the dehydrogenation reaction system is 10-20 g / L, preferably 20 g / L;

[0043] Preferably, the amount of cell disrupted liquid containing geranyl aldehyde dehydrogenase added to the dehydrogenation reaction system is 70% to 90% (v / v), preferably 80% to 82% (v / v);

[0044] Preferably, the concentration of nicotinamide adenine dinucleotide in the dehydrogenation reaction system is 1 to 5 mM / mL, preferably 2 mM / mL;

[0045] Preferably, in the dehydrogenation reaction system, the volume ratio of the cell disrupted liquid containing NADH oxidase to the cell disrupted liquid containing geranaldehyde dehydrogenase is 1:1 to 1:8, preferably 1:5.

[0046] Further, the preparation of geranic acid comprises reacting at 150-250 r / min until the citric acid is consumed, preferably at 200 r / min;

[0047] Preferably, the reaction temperature is 25-35°C, preferably 30°C;

[0048] Preferably, the reaction time is 10 to 15 hours, preferably 12 hours.

[0049] The invention provides a geranaldehyde dehydrogenase, which can catalyze the synthesis of geranic acid using citral as a substrate, and the conversion rate reaches 90%. The invention provides a geranaldehyde dehydrogenase capable of efficiently synthesizing geranic acid, which can be used for biocatalytic conversion of geranic acid.

[0050] On the other hand, a method for preparing geranic acid is provided, which uses citral as a substrate and converts it into geranic acid under the catalytic action of geranaldehyde dehydrogenase; at the same time, nicotinamide adenine dinucleotide is used as a coenzyme to improve the efficiency of the enzymatic reaction and achieve efficient catalytic synthesis of geranic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0052] Figure 1 A schematic diagram of the catalytic system pathway for preparing geranic acid by geranaldehyde dehydrogenase provided by the present invention;

[0053] Figure 2 An alignment diagram of different geranaldehyde dehydrogenase amino acid sequences provided in Example 1 of the present invention;

[0054] Figure 3 The SDS-PAGE electrophoretogram of different geranaldehyde dehydrogenases provided in Example 3 of the present invention;

[0055] Figure 4 Gas chromatograms of catalytic reactions of three high enzyme activity proteins provided in Example 4 of the present invention;

[0056] Figure 5 This is a gas chromatogram of citral and geranic acid in the detection catalytic system provided in Example 6 of the present invention. DETAILED DESCRIPTION

[0057] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0058] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0059] In one aspect, the present invention provides a geranaldehyde dehydrogenase, the amino acid sequence of the geranaldehyde dehydrogenase is shown as SEQ ID NO.1.

[0060] The geranaldehyde dehydrogenase provided by the invention is derived from Pseudomonadales bacterium, can catalyze the synthesis of geranic acid using citral as a substrate, and the conversion rate reaches 90%. A geranaldehyde dehydrogenase capable of efficiently synthesizing geranic acid is provided, which can be used for biocatalytic conversion of geranic acid.

[0061] According to another aspect of the present invention, a nucleic acid molecule encoding the above-mentioned geranaldehyde dehydrogenase is also provided.

[0062] Nucleic acid molecules refer to polymeric forms of nucleotides of any length, including ribonucleotides and / or deoxyribonucleic acids. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases.

[0063] In some specific embodiments, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2 or has more than 80% identity with the nucleotide sequence shown in SEQ ID NO.2 and has the function of increasing the amino acid production of a microorganism.

[0064] According to another aspect of the present invention, a vector containing the above-mentioned nucleic acid molecule is also provided.

[0065] Wherein, the vector is well known to those skilled in the art, including but not limited to: plasmid, phagemid, cosmid or artificial chromosome, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); bacteriophage such as λ phage or M13 phage and animal virus, etc. Animal viruses that can be used as vectors include but are not limited to retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (such as herpes simplex virus), poxvirus, baculovirus, papillomavirus, papillomavirus. In some embodiments, the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0066] In some specific embodiments, the vector is pET28a.

[0067] According to another aspect of the present invention, a recombinant cell expressing geranaldehyde dehydrogenase is provided, which contains the above-mentioned nucleic acid molecule, or contains the above-mentioned vector, or expresses the above-mentioned geranaldehyde dehydrogenase.

[0068] Wherein, the cell may refer to a single cell, a cell line, or a cell culture. Cells herein include their offspring, which may not necessarily be identical to the primary cell due to natural, accidental or deliberate mutations, and may differ from the primary cell in morphology and / or in genomic DNA. The cell may be a natural cell or a transformant.

[0069] According to another aspect of the present invention, there is also provided a method for constructing the above-mentioned recombinant cell, comprising introducing the above-mentioned vector into a biological cell;

[0070] In some specific embodiments, the biological cell comprises a prokaryotic cell;

[0071] In some specific embodiments, the prokaryotic cells include Escherichia coli BL21 cells.

[0072] According to another aspect of the present invention, there is also provided the use of the above-mentioned recombinant cell or the recombinant cell constructed by the above-mentioned method in any of the following:

[0073] A. Produce geranaldehyde dehydrogenase;

[0074] B. Increase the production of geranaldehyde dehydrogenase;

[0075] C. Produce geranic acid.

[0076] According to another aspect of the present invention, a method for producing geranaldehyde dehydrogenase is also provided, comprising culturing the above-mentioned recombinant cell or the recombinant cell prepared by the above-mentioned method to obtain a fermentation product, and obtaining geranaldehyde dehydrogenase from the fermentation product.

[0077] In some specific embodiments, the culturing of the recombinant cells or the recombinant cells prepared by the above method comprises subjecting the activated recombinant cells to seed culture to obtain seed liquid, and fermenting the seed liquid.

[0078] In some specific embodiments, the seed culture conditions include shaking culture at 200 rpm at 37° C. for 8 to 12 hours. The shaking culture time in the seed culture may be, but is not limited to, 8 hours, 8.5 hours, 9 hours, 9.5 hours, 10 hours, 10.5 hours, 11 hours, 11.5 hours or 12 hours, or any value between 8 and 12 hours, preferably 10 hours.

[0079] In some specific embodiments, the fermentation culture comprises inoculating the seed liquid into the fermentation medium, shaking the culture for 15 hours, and continuously adding nutrients until the OD is 600 The pH value is 30-40, an inducer is added, and the fermentation product is obtained by inducing the culture.

[0080] In some specific embodiments, the nutrients include glucose at a concentration of 195 g / L, peptone at a concentration of 32 g / L, yeast powder at a concentration of 32 g / L, and MgSO4 at a final concentration of 60 mM.

[0081] In some specific embodiments, the shaking culture conditions in the fermentation culture include shaking culture at 500-900 rpm at 37° C. The shaking culture speed in the fermentation culture may be, but is not limited to, 500 rpm, 540 rpm, 580 rpm, 600 rpm, 640 rpm, 680 rpm, 700 rpm, 740 rpm, 780 rpm, 800 rpm, 850 rpm or 900 rpm, or any value between 500 and 900 rpm, preferably 600 rpm.

[0082] In some specific embodiments, the addition of the inducer further includes adjusting the culture temperature to 28-35° C. Wherein, the culture temperature can be adjusted to, but not limited to, 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C. or 35° C., or any temperature between 28 and 35° C., preferably 30° C. after the addition of the inducer.

[0083] In some specific embodiments, the induction culture time is 4 to 8 hours. The induction culture time may be, but is not limited to, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours or 8 hours, or any value between 4 and 8 hours, preferably 6 hours.

[0084] In some specific embodiments, the inducer is IPTG or Lactose, preferably IPTG.

[0085] Among them, IPTG is isopropyl-β-D-thiogalactoside and Lactose is lactose.

[0086] In some specific embodiments, obtaining geranyl aldehyde dehydrogenase from the fermentation product includes collecting bacterial cells and obtaining geranyl aldehyde dehydrogenase after cell disruption.

[0087] In order to ensure that the obtained cell disruption solution contains sufficient geranaldehyde dehydrogenase, in some specific embodiments, the cell disruption includes mixing the bacteria and the disruption buffer in a mass volume ratio of 1:1 to 2 and then homogenizing and disrupting. The mass volume ratio of the bacteria and the disruption buffer can be, but is not limited to, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2, and can also be any ratio between 1:1 and 2, preferably a mass volume ratio of 1:1.5.

[0088] In some specific embodiments, the fragmentation buffer is a Na2HPO4-NaH2PO4 buffer, wherein the pH of the Na2HPO4-NaH2PO4 buffer is 8.0 and the concentration is 100 mM.

[0089] According to another aspect of the present invention, a method for preparing geranic acid is also provided, comprising using the cell disrupted liquid containing geranal dehydrogenase prepared by the above method as a catalyst, and carrying out a dehydrogenation reaction with citral as a substrate to prepare geranic acid.

[0090] With citral as substrate, it is converted into geranic acid under the catalysis of geranial dehydrogenase. Citral includes trans-citral (α-citral) and cis-citral (β-citral).

[0091] α-Citral, also known as geranial and geranyl aldehyde, is the E-isomer with a double bond at the 2nd position. Its structural formula is shown below:

[0092]

[0093] β-Citral: Also known as orange aldehyde, it is the Z-isomer with a double bond at position 2. Its structural formula is shown below:

[0094]

[0095] In order to improve the efficiency of the enzymatic reaction, in some specific embodiments, the dehydrogenation reaction is carried out in NAD + / NADH coenzyme circulation system.

[0096] Nicotinamide adenine dinucleotide (NAD + ) is used as a coenzyme to improve the efficiency of the enzymatic reaction and achieve efficient catalytic synthesis of geranic acid. The specific enzyme catalytic system pathway is as follows Figure 1 In some specific embodiments, the enzyme activity of the cell disruption solution containing geranyl aldehyde dehydrogenase is ≥ 2.21 U / mL.

[0097] In some specific embodiments, the concentration of citral in the dehydrogenation reaction system is 10 to 20 g / L. The concentration of citral in the dehydrogenation reaction system may be, but is not limited to, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L or 20 g / L, or any value between 10 and 20 g / L, preferably 20 g / L.

[0098] In some specific embodiments, the amount of cell lysate containing geranyl aldehyde dehydrogenase added to the dehydrogenation reaction system is 70% to 90% (v / v). Wherein, the amount of cell lysate containing geranyl aldehyde dehydrogenase added to the dehydrogenation reaction system can be, but not limited to, 70%, 73%, 75%, 78%, 80%, 82%, 85%, 88% or 90%, and can also be any value between 70% and 90% (v / v), preferably 80% to 82% (v / v).

[0099] In some specific embodiments, the concentration of nicotinamide adenine dinucleotide in the dehydrogenation reaction system is 1 to 5 mM / mL. The concentration of nicotinamide adenine dinucleotide in the dehydrogenation reaction system may be, but is not limited to, 1 mM / mL, 1.5 mM / mL, 2 mM / mL, 2.5 mM / mL, 3 mM / mL, 3.5 mM / mL, 4 mM / mL, 4.5 mM / mL or 5 mM / mL, and may be any value between 1 and 5 mM / mL, preferably 2 mM / mL.

[0100] In some specific embodiments, the volume ratio of the cell disrupted liquid containing NADH oxidase to the cell disrupted liquid containing geranaldehyde dehydrogenase in the dehydrogenation reaction system is 1:1 to 1:8.

[0101] The volume ratio of the cell disrupted liquid containing NADH oxidase to the cell disrupted liquid containing geranaldehyde dehydrogenase in the dehydrogenation reaction system may be, but is not limited to, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 or 1:8, or any value between 1:1 and 1:8, preferably 1:5.

[0102] In some specific embodiments, the preparation of geranic acid comprises reacting at 150-250 r / min until the citric acid is consumed. Specifically, it can be but not limited to 150 r / min, 160 r / min, 170 r / min, 180 r / min, 190 r / min, 200 r / min, 210 r / min, 220 r / min, 230 r / min, 240 r / min, or 250 r / min, and can also be any speed between 150 and 250 r / min, preferably 200 r / min.

[0103] In some specific embodiments, the reaction temperature is 25-35°C. The reaction temperature may be, but is not limited to, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C, or any temperature between 25 and 35°C, preferably 30°C;

[0104] In some specific embodiments, the reaction time is 10 to 15 hours. The reaction time may be, but is not limited to, 10 hours, 10.5 hours, 11 hours, 11.5 hours, 12 hours, 12.5 hours, 13 hours, 13.5 hours, 14 hours, 14.5 hours or 15 hours, or any value between 10 and 15 hours, preferably 12 hours.

[0105] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0106] Example 1

[0107] The basic information of the geranaldehyde dehydrogenase (KDI24_07135) provided by the present invention is shown in Table 1. The geranaldehyde dehydrogenase is derived from Pseudomonadales bacterium, and its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.2.

[0108] Table 1 Basic information of geranyl aldehyde dehydrogenase

[0109]

[0110] Geranaldehyde dehydrogenase (KDI24_07135) was used as the target gene, and the obtained target gene was codon optimized using E. coli BL21 (DE3) as the expression host to make it more suitable for expression in Escherichia coli. The optimized nucleotide sequence is shown in SEQ ID NO.3 and cloned into the pET28a expression plasmid to obtain the pET28a-KDI24_07135 recombinant plasmid.

[0111] It should be noted that in this embodiment, only E. coli BL21 (DE3) is used as an expression host as an example. Other expression systems can also be used to select codons that are more in line with the biological preferences of the expression system for optimization, and replace synonymous codons in the original sequence with preferred codons.

[0112] At the same time, other aldehyde dehydrogenase encoding genes were selected for comparison, including WP_043683932, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; Br6_04489, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; AldA_Rv0768, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively; CFAEC_02300, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively; JSS57_06825, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively; AAGA71_06380, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively; SMF913_12953, whose amino acid sequence and nucleotide sequence are shown in SEQ ID NO.16 and SEQ ID NO.17, respectively. NTW01_04870, the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.18 and SEQ ID NO.19, respectively; AAF724_03170, the amino acid sequence and nucleotide sequence are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively. Among them, WP_043683932 is an aldehyde dehydrogenase known from Castellaniella defragrans. The secondary structure of WP_043683932 is used as a reference to compare the amino acid sequences of different proteins. Figure 2 As shown, the secondary structure of WP_043683932 is presented at the top of the comparison text, and the amino acid sequence with 100% similarity is presented in red, and the amino acid sequence with 50% to 70% similarity is presented in yellow. It can be seen that KDI24_07135, Br6_04489, AldA_Rv0768, CFAEC_02300, JSS57_06825, AAGA71_06380, SMF913_12953, NTW01_04870 and AAF724_03170 all have homology with WP_043683932, and all belong to aldehyde dehydrogenase, which has the ability to catalyze dehydrogenation reactions.

[0113] The above target genes were cloned into the pET28a expression plasmid to obtain the recombinant plasmids pET28a-WP_043683932, pET28a-Br6_04489, pET28a-AldA_Rv0768, pET28a-CFAEC_02300, pET28a-JSS57_06825, pET28a-AAGA71_06380, pET28a-SMF913_12953, pET28a-NTW01_04870 and pET28a-AAF724_03170, respectively.

[0114] Example 2 Transformation of recombinant plasmid (introduction into Escherichia coli)

[0115] This embodiment uses the recombinant plasmid provided in the embodiment to introduce Escherichia coli, and the operation is specifically carried out according to the following steps: First, take out the E.coli BL21 (DE3) competent cells from the -80°C refrigerator and melt them on ice. A certain concentration of recombinant plasmid is added to the competent cells and left to stand on ice for 30 minutes. It is then heat-shocked at 42°C for 90 seconds, and then quickly ice-bathed for 2 minutes after the heat shock. Next, 900 μL of LB liquid culture medium is added and cultured at 37°C and 200rpm for 1 hour. The cultured cell fluid is centrifuged at 1500×g for 5 minutes, and a certain volume of supernatant is discarded and the bacteria are resuspended. The bacterial liquid is then spread on an LB plate containing 50 mg / L kanamycin, inverted and cultured at 37°C for 12 hours, and finally a single colony is picked for PCR and DNA sequencing verification, and the correctly sequenced recombinant strain is inoculated in 50mL of LB culture medium containing 50 mg / L kanamycin, and cultured at 37°C and 200rpm. The culture solution was centrifuged at 5000 rpm and 4°C, and then resuspended in 5 mL of LB medium and 5 mL of 50% glycerol, and stored at -80°C.

[0116] Example 3 Cultivation and expression of recombinant strains

[0117] In this example, the recombinant strain prepared in Example 2 was selected for culture and expression of the corresponding protein. The operation was specifically carried out in the following steps: the stored recombinant protein was selected and inoculated into 5 mL of LB liquid culture medium containing 50 mg / L kanamycin, and cultured at 37°C and 200 rpm for 8 to 12 h, and then transferred to 50 mL of LB liquid culture medium containing 50 mg / L kanamycin at a 2% inoculum, and cultured at 37°C and 200 rpm with shaking until the OD 600 At 0.6-0.8, the concentration of isopropyl-β-D-thiogalactoside (IPTG) added was 0.1 mM, the induction temperature was set to 20°C, and the induction culture was carried out at 200 rpm for 12 hours. The fermentation broth obtained by the induced culture was centrifuged at 6000 r / min for 5 minutes, and the supernatant was discarded to retain the bacteria.

[0118] Take 0.1g of wet cells and suspend them in 1mL of 100mM Na2HPO4-NaH2PO4 cell disruption buffer at pH 8.0. Perform ultrasonic disruption in an ice bath at a frequency of 20kHz and a power of 150W. Repeat the disruption three times for 60s each and 60s apart. The disruption solution prepared in this way contains the recombinant protein. Centrifuge the disruption solution at 13000r / min for 5min to obtain a supernatant containing the recombinant protein.

[0119] After preparing the electrophoresis samples, the protein soluble expression was tested by polyacrylamide gel electrophoresis (SDS-PAGE). Figure 3 As shown, M is a protein marker of 18-66 kDa; C is a protein fragment and supernatant of pET28a expressed in BL21; 1 is a protein fragment and supernatant of pET28a-WP_043683932 expressed in BL21; 2 is a protein fragment and supernatant of pET28a-Br6_04489 expressed in BL21; 3 is a protein fragment and supernatant of pET28a-KD124_07135 expressed in BL21; 4 is a protein fragment and supernatant of pET28a-AldA_Rv0768 expressed in BL21; 5 is a protein fragment and supernatant of pET28a-KD124_07135 expressed in BL21; a is the protein crushed liquid and supernatant of pET28a-CFAEC_02300; 6 is the protein crushed liquid and supernatant of pET28a-JSS57_06825 expressed in BL21; 7 is the protein crushed liquid and supernatant of pET28a-AAGA71_06380 expressed in BL21; 8 is the protein crushed liquid and supernatant of pET28a-SMF913_12953 expressed in BL21; 9 is the protein crushed liquid and supernatant of pET28a-NTW01_04870 expressed in BL21; 10 is the protein crushed liquid and supernatant of pET28a-AAF724_03170 expressed in BL21. It can be seen that the expression of empty pET28a (C) was used as a control to verify the soluble expression of the bacterial cell fragments and supernatants of the ten proteins (referred to as protein 1 to protein 10). The results of SDS-PAGE analysis showed that proteins 1 to 8 all showed soluble expression. Among them, protein 1, protein 3, protein 4 and protein 6 had better soluble expression effects.

[0120] Example 4 Analysis of Geranial Dehydrogenase Activity

[0121] In this example, the supernatant of the broken liquid of protein 1 to protein 10 was selected as the enzyme solution to be tested, and the enzyme activity of geranyl aldehyde dehydrogenase was analyzed in a customized system, which specifically included 5 μL of the enzyme solution to be tested, a substrate with a final concentration of 1 mM, and a final concentration of 1 mM NAD + , the reaction system was filled to 200 μL with Na2HPO4-NaH2PO4 buffer. The substrates were citral and geranyl aldehyde. The reaction was carried out at 30°C, and the change in absorbance at 340 nm was detected by a multifunctional microplate reader. The results are shown in Table 2. The enzyme activity unit (U) is defined as the catalytic reduction of 1 μmol NAD per minute. + The amount of enzyme consumed. The enzyme activity was calculated according to the following formula (1):

[0122]

[0123] In the formula: EW is the change in absorbance within 1 min; 6220 is the extinction coefficient, unit is L / (mol·cm); V is the reaction volume, unit is mL; v is the amount of enzyme added, unit is mL; L is the optical path distance, unit is cm.

[0124] Table 2 Geranyl aldehyde dehydrogenase enzyme activity detection

[0125]

[0126] Where a means that the substrate is citral, and the protein is incubated at 100 mM, pH 8.0

[0127] The detection was carried out in Na2HPO4-NaH2PO4 buffer; b means that the substrate is geranaldehyde, and the protein was incubated in 100mM, pH8.0 Na2HPO4-NaH2PO4 buffer for detection; c means that the substrate is citral, and the protein was incubated in 100mM, pH9.4 Glycine-NaOH buffer for detection; d means that the substrate is geranaldehyde, and the protein was incubated in 100mM, pH9.4 Glycine-NaOH buffer for detection; e means ND means the detection is inactive.

[0128] As shown in Table 2, protein 1, protein 3, protein 4 and protein 6 with good soluble expression effects were detected to be active when citral and geranial were used as substrates. The enzyme activities of different proteins were detected in 100mM, pH 8.0 Na2HPO4-NaH2PO4 and 100mM, 9.4 Glycine-NaOH buffer systems. Incubation in 100mM, pH 8.0 Na2HPO4-NaH2PO4 buffer was more likely to stimulate the activity of geranial dehydrogenase. Among them, protein 3 performed best, which was 0.80U / mL (substrate was citral) and 1.46U / mL (substrate was geranial). Protein 1, protein 3 and protein 6 had good enzyme activities and were screened for the next step of catalytic reaction verification.

[0129] Example 5 High-density fermentation of engineered strains

[0130] The engineered strain of protein 3 with the highest enzyme activity in this example was subjected to high-density fermentation, specifically according to the following steps:

[0131] First, take 100μL of bacterial solution from the glycerol tube and spread it evenly on the LB plate, and culture it at 37℃ for 12h. Then scrape the bacterial moss from the plate and inoculate it into a 250mL shaking flask with baffles containing 100mL LB seed culture medium, and culture it at 37℃ and 200rpm for 10h. Inoculate 200μL of seed culture solution into 2.6L upper tank fermentation medium, which contains 1.73g / L (NH4)2SO4, 6.72g / L KH2PO4, 9.43g / L K2HPO4, 9g / L glucose, 1.48g / L peptone and a drop of defoamer. Use ammonia solution to maintain the pH of the culture solution at about 7.0, and filter air at 220L / h for oxygen supply. The stirring speed is adjusted in the range of 500-900rpm. After culturing at 37℃ and 600rpm for 15h, start continuous feeding. The feed solution contained 195 g / L glucose, 32 g / L peptone, 32 g / L yeast powder, and a final concentration of 60 mM MgSO4. The culture was continuously fed until OD 600 The fermentation temperature was 30-40 °C, and a final concentration of 0.1 mM IPTG was added. The mixture was induced at 30 °C for about 6 h until the feeding was completed. Samples were taken regularly to monitor the changes in various parameters during the fermentation process in real time. After the cells were collected, the cells were broken using a high-pressure homogenizer.

[0132] The bacterial cells and disruption buffer (100 mM, pH 8.0 Na2HPO4-NaH2PO4 buffer) were mixed at a ratio of 1:1.5 (g:mL) and then homogenized to prepare a cell disruption solution containing geranyl aldehyde dehydrogenase.

[0133] Example 6 Construction of 300 mL Catalytic System (Preparation of Geranic Acid)

[0134] Geranic acid was prepared by using the cell disrupted liquid containing geranaldehyde dehydrogenase of the engineered strain for preparing protein 3 in Example 5. The operation was specifically carried out according to the following steps.

[0135] Based on the previous optimization results, the 300mL catalytic reaction system was constructed as follows: final concentration 2mM NAD + , 20 g / L citral (6 mL added), 244 mL of cell lysate containing geranyl aldehyde dehydrogenase, 50 mL of cell lysate containing NOX, reacted at 30°C, 200 r / min for 12 h. Both enzymes were resuspended in a 100 mM, pH 8.0 Na2HPO4-NaH2PO4 buffer system. Among them, the cell lysate of NOX was prepared by BL21 recombinant cells containing pET28a-NOX recombinant plasmid (the amino acid sequence and nucleotide sequence of NOX expressed are shown in SEQ ID NO.22 and SEQ ID NO.23, respectively) according to the method of Example 5.

[0136] Samples were taken at 0 h and 12 h, respectively, and extracted with an equal volume of ethyl acetate for 1 h. The samples were used for GC to detect the changes of citral and geranic acid in the upper solution.

[0137] Example 7 Gas Chromatography Detection Method

[0138] All samples of Example 6 were subjected to GC detection using a capillary column Kromat KB-624 according to the following operating steps:

[0139] The column length is 50m, the inner diameter is 0.3mm, and the film thickness is 1μm. The instrument uses Furley 9790II, equipped with a flame ionization detector, and N2 as the carrier gas. The sample is injected 2μL, the detector and injector temperatures are both 300℃, and the split ratio is 1:50. The sample is analyzed isobarically at a column box temperature of 220℃, and the analysis is maintained for 15min.

[0140] The gas chromatograms of the catalytic reactions of three high enzyme activity proteins are shown in Figure 2. Figure 4As shown, Standard is a standard, citral (a mixture of neral and geranial) and geranic acid (a mixture of geranic acid and neral) solutions are used as standard; Protein1 is a sample with code No. 1 without adding NADH oxidase; Protein3 is a sample with code No. 3 without adding NADH oxidase; Protein6 is a sample with code No. 6 without adding NADH oxidase; Protein1+LPNOX is a sample with code No. 1 with NADH oxidase added; Protein1+LPNOX is a sample with code No. 3 with NADH oxidase added; Protein6+LPNOX is a sample with code No. 6 with NADH oxidase added; Geranic acid 1 is cis-geranic acid, also known as neral, Geranic acid 2 is trans-geranic acid, and Geranic acid 2 is the target product. The catalytic reaction system without adding NADH oxidase uses 100mM, pH 8.0 Na2HPO4-NaH2PO4 buffer instead of NADH oxidase. It can be seen that there are unconverted substrates in Protein1 and Protein6, while Protein3 is completely converted, and the geranaldehyde dehydrogenase conversion efficiency of Protein3 is the best; after adding LPNOX to Protein1+LPNOX, Protein3+LPNOX and Protein6+LPNOX, the amount of trans-geranic acid (i.e., geranic acid 2) generated is significantly increased, and the substrate conversion rate is improved, among which Protein3+LPNOX has the highest output and the highest conversion efficiency.

[0141] Figure 5 The test results of citral and geranic acid in 300mL catalytic system, where samples 1 to 3 are the results of three parallel experiments of sample No. 3, and the control is to use 100mM, pH8.0 Na2HPO4-NaH2PO4 buffer instead of enzyme solution. It can be seen that the substrate is catalytically converted to synthesize the target product geranic acid 2 in 12h. The experimental results show that in the 300mL reaction system with 20g / L citral as the substrate, the substrate converted three times has been completely converted, and the conversion rate of citral to (trans) geranic acid reaches more than 90%.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A geranaldehyde dehydrogenase, characterized in that The amino acid sequence of the geranaldehyde dehydrogenase is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the geranaldehyde dehydrogenase according to claim 1; Preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2 or has more than 80% identity with the nucleotide sequence shown in SEQ ID NO.2 and has the function of increasing the amino acid production of microorganisms.

3. A vector comprising the nucleic acid molecule according to claim 2.

4. A recombinant cell expressing geranaldehyde dehydrogenase, characterized in that Contains the nucleic acid molecule according to claim 2, or contains the vector according to claim 3, or expresses the geranaldehyde dehydrogenase according to claim 1.

5. A method for constructing the recombinant cell according to claim 4, characterized in that: Comprising: introducing the vector of claim 3 into a biological cell; Preferably, the biological cells include prokaryotic cells; Preferably, the prokaryotic cells include Escherichia coli BL21 cells.

6. Use of the recombinant cell according to claim 4 or the recombinant cell constructed by the method according to claim 5 in any of the following: A. Produce geranaldehyde dehydrogenase; B. Increase the production of geranaldehyde dehydrogenase; C. Produce geranic acid.

7. A method for producing geranaldehyde dehydrogenase, characterized in that: The method comprises culturing the recombinant cell according to claim 4 or the recombinant cell prepared by the method according to claim 5 to obtain a fermentation product, and obtaining geranaldehyde dehydrogenase from the fermentation product; Preferably, the culturing of the recombinant cell according to claim 4 or the recombinant cell prepared by the method according to claim 5 comprises subjecting the activated recombinant cell to seed culture to obtain a seed solution, and subjecting the seed solution to fermentation culture; Preferably, the seed culture conditions include shaking culture at 37°C and 200 rpm for 8 to 12 hours, preferably 10 hours; Preferably, the fermentation culture comprises inoculating the seed liquid into the fermentation medium, shaking the culture for 15 hours, and continuously adding nutrients until the OD 600 The temperature is 30 to 40, an inducer is added, and the fermentation product is obtained by inducing the culture; Preferably, the nutrients include glucose at a concentration of 195 g / L, peptone at a concentration of 32 g / L, yeast powder at a concentration of 32 g / L, and MgSO4 at a final concentration of 60 mM; Preferably, the shaking culture conditions in the fermentation culture include shaking culture at 500-900 rpm at 37°C, preferably 600 rpm; Preferably, after adding the inducer, the culture temperature is adjusted to 28-35°C, preferably 30°C; Preferably, the induction culture time is 4 to 8 hours, preferably 6 hours; Preferably, the inducing agent is IPTG or Lactose, preferably IPTG; Preferably, obtaining geranyl aldehyde dehydrogenase from the fermentation product comprises collecting bacterial cells and crushing the cells to obtain geranyl aldehyde dehydrogenase; Preferably, the cell disruption comprises mixing the cells and the disruption buffer in a mass volume ratio of 1:1 to 2 and then homogenizing and disrupting the cells, preferably in a mass volume ratio of 1:1.5; Preferably, the crushing buffer is Na2HPO4-NaH2PO4 buffer.

8. A method for preparing geranic acid, characterized in that: The method comprises using the cell disrupted liquid containing geranyl aldehyde dehydrogenase prepared by the method according to claim 7 as a catalyst and carrying out a dehydrogenation reaction with citral as a substrate to prepare geranic acid.

9. The method according to claim 8, characterized in that The dehydrogenation reaction is in NAD + / NADH coenzyme circulation system works in coordination; Preferably, the enzyme activity of the cell disruption solution containing geranyl aldehyde dehydrogenase is ≥2.21 U / mL; Preferably, the concentration of citral in the dehydrogenation reaction system is 10-20 g / L, preferably 20 g / L; Preferably, the amount of cell disrupted liquid containing geranyl aldehyde dehydrogenase added to the dehydrogenation reaction system is 70% to 90% (v / v), preferably 80% to 82% (v / v); Preferably, the concentration of nicotinamide adenine dinucleotide in the dehydrogenation reaction system is 1 to 5 mM / mL, preferably 2 mM / mL; Preferably, in the dehydrogenation reaction system, the volume ratio of the cell disrupted liquid containing NADH oxidase to the cell disrupted liquid containing geranaldehyde dehydrogenase is 1:1 to 1:8, preferably 1:

5.

10. The method according to claim 8 or 9, characterized in that: The preparation of geranic acid comprises reacting at 150-250 r / min until the citric acid is consumed, preferably at 200 r / min; Preferably, the reaction temperature is 25-35°C, preferably 30°C; Preferably, the reaction time is 10 to 15 hours, preferably 12 hours.

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