Part:BBa_K4929004
pAO815-AocI-FAD
Composite Part - BBa_K4929004(pAO815-AocI-FAD)
Construction Design
After preparing the PCR templates for the target genes AocI(BBa_K4929002) and FAD(BBa_K4929000) and the linear pAO815(BBa_K4929001) vector, we performed separate PCR amplifications for these two fragments. The amplified fragment was then used to synthesize pAO815-AocI-FAD plasmid. Then, the groups of plasmids were transformed into DH5α cells and lysed, followed by agarose gel electrophoresis to confirm the correctness of the resulting products. Finally, these plasmids were introduced into our yeast strain for further protein synthesis and functional verification.
Engineering Principle
This gene encodes a metal-binding membrane glycoprotein that oxidatively deaminates putrescine, histamine, and related compounds. The encoded protein is inhibited by amiloride, a diuretic that acts by closing epithelial sodium ion channels. Alternatively, spliced transcript variants encoding multiple isoforms have been observed for this gene. [provided by RefSeq, Jan 2013]
Amine oxidases (AOs) catalyze the oxidation of biogenic amines to form aldehydes, ammonia, and hydrogen peroxide. AOs were first discovered by Yamada et al [1], where fungi formed AOs in mycelium when grown with mono- or diamines as nitrogen sources, whereas other nitrogen sources did not have any effect on enzyme formation. Certain microorganisms can secrete amine oxidase, which reduces the biogenic amine content in fermented foods; meanwhile, biogenic amine oxidase, which can degrade biogenic amines, also exists in the human gut and is used to regulate intracellular amine content, maintain intra- and extracellular acid-base balance, and safeguard human health [2]. The classification of AOs mainly depends on their molecular structures, amino acid sequences, and cofactor structures. Currently, amine oxidases of microbial origin are found to be mainly flavin-containing amine oxidases (EC 1.4.3.4) and copper-containing amine oxidases (EC 1.4.3.6).
Copper amine oxidases (CuAOs) are a class of copper-containing reductases, and such enzymes contain two types of cofactors: 2,4,5-dihydroxyphenylalanine quinone (TPQ) and lysine tyrosine quinone (LTQ) that are produced post-translationally from specific tyrosine residues [3]. CuAOs are present in Escherichia coli, Arthrobacter globigii, Hansenula polymorphica Arthrobacter sphaericus, Hansenula polymorphica, and Picrosporum, and the amine oxidase from Arthrobacter sphaericus KAIT-B-007 was purified twice on a dextran gel S-200 [4]. The specific activity of amine oxidase from Arthrobacter sphaericus KAIT-B-007 was 14.3 U/mg. The enzyme has good heat resistance, and the enzyme activity remained stable at 65°C, and the residual enzyme activity was 91% after 10 min at 70°C [5]. The copper-containing oxidase 2 from Arthrobacter taurus TC-1 showed high catalytic efficiency for phenylethylamine, tyramine and histamine, and the relative value of Kcat/Km was 100:49.6:7.6 [6]; Hansenula polymorpha H525 contains a copper amine oxidase gene, and the crude enzyme solution was added to grape juice containing biogenic amines for 7 d, the content of phenylethylamine and tyramine was reduced to almost zero [4].
Amine Dehydrogenase (AmDH), most of which use Tryptophan Tryptophylquinone (TTQ) as a cofactor, can oxidize amines to dehydrogenate to produce the corresponding aldehydes and ammonia, and the electrons generated by its oxidation of primary amines are passed from TTQ through Cu2+ on the copper-containing protein cofactor to the final electron receptor [7]. Methylamine Dehydrogenase (MADH) was the first enzyme discovered to use TTQ as a cofactor, and the electrons generated from the oxidation of methylamine are passed from TTQ to the electron acceptor cytochrome c via amicyanin [8]. In addition to amine dehydrogenases with TTQ as a cofactor, there also exist amine dehydrogenases that do not have TTQ as a cofactor and contain a covalently bound 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) and a [4Fe-4S] cluster as a redox cofactor that catalyzes the oxidation of histamine to produce indole acetaldehyde and ammonia ions, hence the name histamine dehydrogenase [9]. Most of these enzymes contain two subunits, such as histamine dehydrogenase (MSMADH) in Nocardioides simplex, a homodimeric protein that oxidizes mainly histamine, but also oxidizes putrescine, but catalyzes putrescine with only 0.7% of the efficiency of histamine [9]. Histamine dehydrogenase (HDH-R) from Rhizobium sp. is also a homodimer, and its optimal substrate is histamine; it shows the highest enzyme activity at pH 9.0 and 70 °C and is considered to be the most histamine-specific of the amine oxidases and amine dehydrogenases [10]. There are also a small number of such enzymes that contain more than two subunits, such as histamine dehydrogenase (HADH) from the halophilic archaeon Natrinema gari, which is a heterotrimer, and the enzyme was found to be active at pH 6.5-8.5, 40-60 °C under high-salt conditions (3.5-5.0 mol/L NaCl) with high solubility and catalytic activity [11]. In addition, 3-phosphoglyceraldehyde dehydrogenase from L. plantarum SGJ-24 can also degrade histamine, and its optimal reaction pH and temperature were 7.5 and 40 °C, respectively, and it was able to stabilize at conditions lower than 55 °C and pH 6.5-8.5, and its degradation rate of histamine could reach 52.2% [12]. It has been confirmed that amine dehydrogenase is an alkaline oxidase with an optimal reaction pH in the range of 7.5-9.0, which cannot play an effective role in amine degradation in acidic fermented foods, and the enzyme activity is susceptible to strong inhibition by carbonyl reagents, such as amino ureas and aminoguanidine, and thus is not suitable for the degradation of biogenic amines in fermented foods [12-13].
Experimental Approach
To construct the plasmids, we commissioned the company to generate gene templates for FAD. These gene fragments were then inserted into the pAO815 vector, a specific type of plasmid. We successfully constructed the pAO815-AocI-FAD plasmid and introduced it into Escherichia coli bacteria. After agarose gel electrophoresis and gel extraction, we transferred the plasmid into the yeast cells. Then, we confirmed the expression of our target protein, the amine oxidase, in the yeast cells. Finally, we will lyse and obtain the DH5α Escherichia coli cells containing the target plasmids. After agarose gel electrophoresis, we will recover the gel and reintroduce these plasmids into the GS115 yeast strain selected for this experiment. After a few days of cultivation, we will use SDS-PAGE gel electrophoresis to determine whether the yeast has successfully synthesized our target protein - Saccharomyces cerevisiae.
In Figure 4C, the alignment results demonstrate a high similarity between the observed gene fragment and the expected sequence. Precisely the inserted gene fragment perfectly matches the desired sequence.
Figure 5C shows the yeast colonies that have grown after cultivation. Figures 5A and 5B display the experimental results of the pAO815-AocI-FAD insertion into the vector pAO815 linear, indicating a highly successful construction step.
Figure 6 illustrates the experimental results of SDS-PAGE gel electrophoresis under different methanol concentrations. The experimental samples were treated with various methanol concentrations to induce yeast protein expression. The red portion in the gel represents the target protein, and successful practical outcomes are indicated by the blue staining with Coomassie Brilliant Blue, as shown in the blue-colored regions. As shown in Fig 6, protein AocI has a size of 85 kDa. There was a clear difference between the protein band of yeast containing the AocI plasmid and the blank control group, indicating that the AocI gene was successfully expressed.
Characterization/Measurement
We tested the enzyme’s function of decomposing the biogenic amine, and it turns out it worked well because there is yellow around the yeast in Figure 7.
The specific instructions are as follows: bromocresol purple, ampicillin (that is, antibiotics), and histamine with different concentrations were added to these five Petri dishes, and the concentrations were 0 μ g/ml, 20 μ g/ml, 50 μ g/ml, 100μ g/ml and 200μ g/ml respectively. Then, the PDA was placed in four areas of five Petri dishes, and the Petri dishes were sealed. Two regions were GS115 blank, and the other was pAO815-AocI so that they could stand for one night. After that, a yellow circle appeared around the PDF in pAO815-AocI. Because bromocresol purple will change from purple to yellow when it meets histamine, it also means that we have successfully cracked biogenic amines into amines and aldehydes under the catalysis of amine oxidase.
In addition to this, we also performed HPLC tests. Histamine concentration was measured at five different temperatures (20,25,30,37,45) at different sampling times (12,24,36,48,72h). We first cultured the bacterial solution to OD0.6-0.8, so that the bacteria had the highest viability. Histamine was then added, and the initial concentration of histamine added was 200ug/ml. Use HPLC, that is, high-performance liquid chromatography to detect changes in histamine content. According to the results of the image, the content of histamine decreased gradually with the extension of time, and the degradation of histamine was the fastest at 30 degrees. At 72h, the histamine content was the lowest.
References
- Yamada H, Adachi O, Ogata K. Amine oxidases of microorganisms: part I. Formation of amine oxidase by fungi. Agricultural and biological chemistry, 1965, 29(2): 117-123.
- Song Y, Dong Q. Research progress in formation and control of the biogenic amine in Chinese rice wine. Science and Technology of Food Industry, 2016, 37(8): 387-391.
- Li BB, Lu SL. The importance of amine-degrading enzymes on the biogenic amine degradation in fermented foods: a review. Process Biochemistry, 2020, 99: 331-339.
- Mathias B, Urs M, Helmut K, et al. The potential of the yeast Debaryomyces hansenii H525 to degrade biogenic amines in food. Microorganisms, 2015, 3(4): 839-850.
- Yoshinori S, Hiroko M, Akira Y, et al. A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007. Journal of Bioscience and Bioengineering, 2004, 97(2): 104-110.
- Lee JI, Kim YW. Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines. World J Microbiol Biotechnol, 2013, 29(4): 673-682.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 286
Illegal NheI site found at 1233
Illegal NotI site found at 8485 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 4114
Illegal BamHI site found at 86
Illegal BamHI site found at 7832
Illegal BamHI site found at 8222
Illegal BamHI site found at 10833
Illegal XhoI site found at 7142
Illegal XhoI site found at 7531
Illegal XhoI site found at 7671
Illegal XhoI site found at 7717 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3111
Illegal NgoMIV site found at 6956
Illegal NgoMIV site found at 7702
Illegal NgoMIV site found at 8425
Illegal NgoMIV site found at 8535
Illegal NgoMIV site found at 8651
Illegal NgoMIV site found at 9740
Illegal AgeI site found at 52
Illegal AgeI site found at 6550
Illegal AgeI site found at 6565
Illegal AgeI site found at 7083
Illegal AgeI site found at 8507 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2573
Illegal BsaI site found at 3421
Illegal BsaI.rc site found at 5487
Illegal BsaI.rc site found at 6869
Illegal BsaI.rc site found at 9824
Illegal SapI site found at 4404
Illegal SapI site found at 8087
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