ABSTRACT
1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.
Subject(s)
Escherichia coli/metabolism , Carboxy-Lyases/metabolism , Catalysis , Cadaverine/metabolismABSTRACT
Objective To establish a method of detecting the expression of Lysine decarboxylase (LDC) -a key enzyme for the synthesis of alkaloid in the host promoted by the endophytic fungal elicitor of Sophora alopecuroides by using real-time fluorescence quantitative PCR (qRT-PCR). Methods Target gene primers QLDC-F/QLDC-R and reference gene primers Lectin-F/Lectin-R were designed according to LDC and Lectin gene sequences of S. alopecuroids; Five-fold gradient dilution of cDNA was used as the standard sample for the construction of the standard curve of target gene and the reference gene. Reaction system and reaction conditions of qRT-PCR were optimized, and the sensitivity of semi-quantitative PCR and qRT-PCR were analyzed and compared. Under different eliciting time of endophytic fungal elicitors NDZKDF13 of S. alopecuroides, the content of oxymatrine in the host was determined by HPLC, the expression of LDC gene was detected by qRT-PCR, and the relationship between LDC gene expression and the accumulation of OMA was analyzed. Results The results of qRT-PCR were better when the cDNA content in the system was 200 ng/μL and the annealing temperature was 61 ℃. The standard curve of the target gene and the reference gene was constructed, in which the cycle threshold and template concentration showed a good linear relationship, the amplification efficiency was above 99%, and the sensitivity was 25 times that of semi-quantitative PCR. Under the induction effect of endophytic fungal elicitor NDZKDF13, expression of host LDC gene reached the peak on the 6th day, which was 25.58 times that of the control. The increase of OMA content lagged the change of the LDC gene expression and reached the highest amount on the 9th day after the induction. Conclusion The qRT-PCR technique was successfully applied to the functional gene research of S. alopecuroides. Through the optimization of various conditions, a platform for accurate and simple detection of functional gene expression in S. alopecuroides was established.
ABSTRACT
Establishing the genetic transformation system of medicinal plant is important to study their functional genes. Based on the established regeneration system of Sophra alopecuroides, 6 factors of genetic transformation were optimized, that was the concentration of Agrobacterium tumefaciens, the infection time, the co-cultivation time of agrobacterium tumefaciensand S.alopecuroides callus, the preculture time of S.alopecuroides callus, the adding method ofacetosyringone (AS) and the concentration of AS, respectively. The results showed that a maximum genetic transformation efficiency of 83.33% was achieved with 15d-precultured of S.alopecuroides callus, which was infected by A600=0.9 A. tumefaciens for 15 minutes and then co-cultivated for 48 hours with 200 μmol•L-1AS. The promoter sequence (1 260 bp) of upstream SaLDC was cloned from S.alopecuroides genomic DNA (gene bank accession number: KY038928). The deletion fragment of SaLDC promoter with different length (310,594,765,924,1 260 bp) were ligated with the GUS reporter gene to form five plant expression vectors named P310,P594,P765,P924,P1260, which were then transferred into S.alopecuroides callus. The GUS transient expression showed that all 5 different deletion fragment of SaLDC promoter can drive the GUS gene expression in S. alopecuroides callus. The SaLDC promoter we cloned has high promoter activity, and they may facilitate its function analysis in the future.
ABSTRACT
Cadaverine is a biogenic amine that has the potential to become an important platform chemical for the production of industrial polymers, such as polyamides and polyurethanes. We reported here a lysine decarboxylase from Klebsiella oxytoca. The lysine decarboxylase from Klebsiella oxytoca was cloned to Escherichia coli to get the strain LN18. The specific activity of the crude protein from LN18 reached 30 000 U. The molecular weight was about 80 kDa. The optimum temperature and pH of the crude protein were 55 ℃ and 5.5 respectively. The specific activity could keep over 30% at pH 8.0 compared the one at pH 5.5, much difference from Escherichia coli lysine decarboxylase CadA. Mg²⁺ was positive to the specific activity, whereas Fe²⁺, Zn²⁺ and Ca²⁺ were negative.
Subject(s)
Bacterial Proteins , Genetics , Metabolism , Cadaverine , Carboxy-Lyases , Genetics , Metabolism , Escherichia coli , Metabolism , Hydrogen-Ion Concentration , Klebsiella oxytoca , Genetics , TemperatureABSTRACT
OBJECTIVE: To study the effect of lysine decarboxylase (LDC) gene on the accumulation of matrine (MA) and oxymatrine (OMA) in cotyledon of Sophora alopecuroides L germinating seeds. METHODS: The S. alopecuroides germinating seeds were stressed by different mass fractions of PEG 6000, and the contents of MA and OMA were determined by high performance liquid chromatography (HPLC) and the expression level of LDC was analyzed by real-time fluorescence quantitative PCR (qPCR) after 72 h treatment. RESULTS: The contents of MA and OMA decreased in the cotyledon under light stress (PEG mass fraction 20%). The analysis of qPCR revealed that the LDC expression level was decreased first, and then increased with the stress rising. The changes of the contents of MA and OMA were parallel with the expression level of LDC especially under light and severe stress. CONCLUSION: There is certain association between the accumulation of MA and OMA and the gene expression quantity of LDC. The results is of significance for illustrating the role of LDC in the biosynthetic pathways of MA and OMA.
ABSTRACT
Objective: To obtain and analyze the full-length L-lysine decarboxylase (LDC) gene sequence of Huperzia serrata var. longipetiolata and predictively analyze its protein structure on the basis of cloning the coding region of LDC gene from four species of Huperziaceae. The species are Huperzia serrata var. longipetiolata, Phlegariurus minchegensis, Phlegariurus austrosinicus, and Phlegariurus petiolatus. Methods: The LDC coding region sequences were cloned by RT-PCR strategy with the template of total RNA extracted from the leaves. Then the sequences were analyzed by means of BLAST and MEGA 5.0. The full-length of LDC gene sequence of H. serrata var. longipetiolata was obtained by RACE technology. And then the secondary structure and three-dimensional structure of LDC protein were predictively analyzed. Results: The coding region sequences were highly similar to the lysine decarboxylase in the database. And the encoding protein of H. serrata var. longipetiolata was highly similar to the amino acid sequences of H. serrata in NCBI, and with high homology to Selaginella moellendorffii. The full-length LDC gene sequence of H. serrata var. longipetiolata contained 1 266 bp open reading frame and encodes a predicted protein of 403 amino acids. The GenBank accession number for this gene is KF040056. Conclusion: The LDC genes of the four species of Huperziaceae are cloned in this study. The full-length LDC gene sequence of H. serrata var. longipetiolata is obtained and analyzed, and its protein structure is predictively analyzed. The result will provide a foundation for exploring the mechanism of huperzine A biosynthesis in the plants of Huperziaceae.