Cloning, Expression And Analysis Of GAD And GABA-T Gene From Monascus Ruber Mr-5

Monascus ruber is a filamentous fungus （family Monascaceae, order Eurotiales）. In industry, Monascus species are important sources of pigments or bioactive compounds, like y-aminobutyric acid （GABA）, which is a non-proteinaceous amino acid known to be a major inhibitory neurotransmitter in mammalian brain tissues. GABA has therapeutic properties. It improves headache, ringing in the ears, and amnesia caused by apoplexy and arteriosclerosis of the brain, increases the supply of oxidants, and accelerates metabolism of brain cells. In Monascus, the metabolism of GABA involves two enzymes called glutamate decarboxylase （GAD） and4-aminobutyrate aminotransferase （GABA-T）. GAD catalyzes the conversion of glutamate to GABA and then GABA-T catalyzes the conversion of GABA to succinic semialdehyde （SSA）. In recent years, numerous studies have reported that the GABA production of Monascus has been improved through strain selection and optimization of fermentation conditions. However, the regulation of GABA production at the molecular level was rarely reported. Furthermore, it was unable to query gene sequences of GAD and GABA-T, which were two major enzymes related to the metabolism of GABA.In this study, Monascus ruber strains Mr-5（CGMCC NO. M208043）, which was screened and stored by our lab previously, was as experimental material. The total RNA was extracted and cDNA was synthesized. By comparing GAD and GABA-T gene sequences of several Aspergillus species respectively, degenerate primers was designed based on conserved regions; RACE and Touchdown PCR were used to obtain the GAD and GABA-T gene sequences of Monascus ruber. GAD and GABA-T were analyzed and predicted by the tools of bioinformatics which are available online or corresponding software in the following aspects:the composition, physical and chemical properties, signalpeptide and higher-order structure, etc. Using real-time quantitative PCR, the relative expression of GAD and GABA-T gene were analyzed. The GAD gene was expressed in E. coli and the target protein was obtained after isolation and purification. The research contents are as follows:1. GAD gene sequence of Monascus ruber Mr-5The full-length cDNA of GAD gene of Monascus ruber Mr-5is1784bp, containing an open reading frame of1536bp. In addition, it has a5’-UTR sequence of152bp and a3’-UTR sequence of96bp. The nucleotide sequence of GAD gene has sequence identity of80%with that of Aspergillus niger and79%with that of Neosartorya fischeri and Aspergillus clavatus.2. The bioinformatics analysis of GADThe full-length GAD gene of Monascus ruber Mr-5is1536bp with a start codon of ATG and a stop codon of TAA, encoding511amino acids, which contains67negatively charged residues and59positively charged residues. The Molecular weight is about57.8kDa and theoretical pI is6.05. The formula is C2562H4004N704O768S26-Aliphatic index is82.41and grand average of hydropathicity （GRAVY） is-0.330, which indicates the GAD may be a hydrophilic protein. In the predicted protein secondary structure, the ratio of α-helical, extended strand,(3-turn and random coil is54.41%,30.72%,7.24%and7.63%, respectively. The model prediction of tertiary structure is conforming to secondary structure predictions. The results of searching for conserved domains show that the sequence has a specific hit of glutamate decarboxylase （TIGR01788）. The catalytic residue is Lys （K） and the chemical binding site is composed of8residues. Phylogenetic tree analysis reveals that GAD of Monascus ruber is more closely related to those of Aspergillus species than to other fungi. The blastp results indicate that the amino acid sequence of Mr-5GAD has84%sequence identity with those of Aspergillus furnigatus and Neosartorya fischeri.3. The GABA-T gene sequence of Monascus ruber Mr-5The full-length cDNA of GABA-T gene of Monascus ruber Mr-5is1563bp. The result of blastn show that the nucleotide sequence of GABA-T gene has sequence identity of82%with that of Aspergillus clavatus, Aspergillus niger and Aspergillus terreus and81%with that of Aspergillus fumigatus.4. The bioinformatics analysis of GABA-TThe full-length GABA-T gene of Monascus ruber Mr-5is1563bp with a start codon of ATG and a stop codon of TAA, encoding520amino acids, which contains49negatively charged residues and56positively charged residues. The Molecular weight is about57.5kDa and theoretical pI is9.12. The formula is C2595H4063N715O740S13. Aliphatic index is87.50and grand average of hydropathicity （GRAVY） is-0.216, which indicates the GABA-T may be a hydrophilic protein, In the predicted protein secondary structure, the ratio of a-helical, extended strand, P-turn and random coil is47.12%,31.15%,9.04%and12.69%, respectively. The model prediction of tertiary structure is conforming to secondary structure predictions. The results of searching for conserved domains show that the sequence has a specific hit of4-aminobutyrate aminotransferase （cd0061₀）. The catalytic residue is Lys （K） and the chemical binding site is composed of8residues. Phylogenetic tree analysis reveals that GABA-T of Monascus ruber is more closely related to those of Aspergillus species than to other fungi. The blastp results indicate that the amino acid sequence of Mr-5GABA-T has82%sequence identity with those of Aspergillus terreus （XP₀01217977.1） and Aspergillus oryzae （EIT80762.1）.5. The expression analysis of GAD and GABA-T geneThe relative expression of GAD and GABA-T gene of Monascus ruber Mr-5with different culture time was detected using qRT-PCR. With group one （culture time was10d） as control group, the mRNA level of GAD and GABA-T was standardize by the transcriptional level of reference gene actin. The results showed that mRNA level of GAD increased with the extension of incubation time, and groups whose culture time is16d,19d and22d respectively had significant difference （p<0.05） by comparing with control group; On the contrary, the relative transcript level of GABA-T reduced with the extension of incubation time, and it went to the minimum value when the culture time was19d. All groups had significant difference （p<0.05） by comparing with control group. 6. Prokaryotic expression of GADThe GAD gene of Monascus Mr-5connected with a pET-21a （+） vector was transformed into E.coli BL21（DE3） for induced expression. The analysis result of SDS-PAGE showed that when the IPTG was1mM and inducing temperature were37℃,28℃and16℃respectively, the recombinant proteins were inclusion body and at28℃,it had the most expression quantity. Moreover, its molecular weight was about58kDa, which was consistent with the predicted value. After purifying by nickel ion affinity chromatography, the recombinant protein had one band only and the elution effect was best with250mM imidazole. Measured by Bradford, the purified protein concentration was0.2956mg/mL.