Part:BBa_K4940003
Prα-POL-his
Prα-pol is a gene including a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. Its coding product Prα-POL is an alpha-pinene oxide lyase, which can convert α-pinene oxide to isonovalal.
Usage and Biology
Pseudomonas rhodesiae CIP107491 leads first to the epoxidation of the α-pinene substrate by a NADH-dependent monooxygenase before its decyclizing by a α-pinene oxide lyase without cofactors to generate cis-dimethyl-5-isopropylhexa-2,5dienal (isonovalal). Finally, isonovalal is probably degraded in isonovalic acid by a NAD+-dependent dehydrogenase. This NADH-dependent monooxygenase was named as alpha-pinene oxide lyase (Prα-POL), which was extracted successfully and the sequence of the gene was obtained (1). There was also a study showed that Prα-POL can be expressed in E. coli as an active recombinant protein, with enzymatic activities close to those obtained with native Prα-POL of P. Rhodesiae.
Plasmid design and construction
We inserted Prα-POL into the pET-28a vector to form pET-28a prα-pol, degrading α-pinene oxide efficiently.
Figure 1. The construction of plasmid pET-28a prα-pol.
Competent cell transformation
The recombinant plasmid was successfully transformed to the E. coli BL21 star (DE3) for gene expression, indicated by the colonies on the LB agar plate with antibiotics.
Figure 2. Colony growth of E. coli BL21 star (DE3) transformed with prα-pol plasmid.
Cultivation, Purification and SDS-PAGE
The recombinant E.coli was cultured and induced by IPTG to allow GlcDH-II protein expression. Since the protein was fused to a his tag, we purified the protein via affinity chromatography. The protein samples were tested by SDS-PAGE. We have already confirmed the expression of GlcDH-II based on the corresponding bands.
Figure 3. The general pattern of E. coli growth. The change of OD600 of the bacterial culture along with time was shown. OD600 was recorded at 0, 1, 2, 3 and 4 h, and IPTG was added at 4 h.
Figure 4. The results of the SDS-PAGE. The corresponding protein bands were indicated by the red box.
Determination of degradation effects
In order to test whether the engineered bacteria can degrade the target substances efficiently, an aqueous-organic two-phase system was constructed. The mass spectrum showed that both substrates, α-pinene oxides, and product, isonovaval were detected. In addition, an obvious difference can be seen between 6-8 min and 13-16 min in the Total ion chromatogram (TIC) of the IPTG group. This suggests that Prα-POL has expected function as alpha-pinene oxide lyase.
Figure 5. The structure of simplified aqueous-organic two-phase system. The components of the device are indicated in the figure.
Figure 6. The mass spectrum was generated for analysis. This revealed that both substrate, α-pinene oxides (A), and product, isonovaval (B) were detected in the GC-MS analysis.
Figure 7. TIC spectrum of four groups. During the ion abundance was the highest in the IPTG group (A) compared to that in the group without IPTG induction (D) during 6-8 min and 13-16 min. However, the ion abundance was similar in the PBS group (C) and pure α-pinene oxides group (B), both of which were lower than the other groups.
Reference
1. Dubessay P, Larroche C, Fontanille P. Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli. Appl Biochem Biotechnol. 2018 Jul;185(3):676–90.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 71
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 277
- 1000COMPATIBLE WITH RFC[1000]
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