Design
Overview
We combine a system that can degrade α-pinene stably and efficiently, focusing on introducing exogenous α-pinene monooxygenase and α-pinene oxide lyase into E. coli. Theoretically we divided the degradation pathway into two steps. We want to:
• Oxidize α-pinene efficiently by a P450 variant combined with NADH regenerative system, which is constructed by a membrane-bound protein GLF and intracellular enzyme GLCDH.
• Degrade α-pinene oxide through an enzyme, Prα-POL from Pseudomonas rhodesiensis CIP107491.
Here is described the rationale behind the design of the parts of our system as well as an overview of our experimental design.
Figure 1. The overall workflow of our project.
Theoretical basis: Biodegradation process of α-pinene
Figure 2. The whole process of α-pinene oxidation and degradation.
A) Potential oxidative pathways of α-pinene: summarized from the intracellular degradation pathway of Pseudomonas rhodesiae CIP 107491 and P450 oxidation experiments (1–3)
B) Degradation pathways of α-pinene oxide: summarized from the natural degradation pathway of Pseudomonas rhodesiae CIP 10749 (3,4).
C) Glucose-dependent NADPH regeneration system: constructed by experiments according to the characteristics of P450 (1,5).
Thick arrows: key reactions referred to in our project, thin arrows: side reactions, dashed arrows: possible reactions, orange substances: key substances involved in our project.
We planned to introduce the four genes into E. coli BL21 star (DE3) to form three systems (oxidation, degradation and regeneration) to make the engineered bacteria have the potential to degrade α-pinene efficiently. In the process of plasmid construction and gene selection, an in vivo degradation system of Pseudomonas rhodesiensis CIP107491, which can use α-pinene as carbon source, provided us with the inspiration (Figure 2A, Figure 2C). This bacterium can degrade α-pinene to two aldehydes, which may be oxidized to more non-toxic carbon dioxide by oxidants such as manganese dioxide, in line with our idea of green degradation. In addition, to enhance the oxidation efficiency of P450, we wanted to establish a synergistic system consistent with the properties of this enzyme. The glucose-dependent NADPH regeneration system based on GlcdH-II and GLF protein provided a solid theoretical basis for our synergistic system (Figure 2B).
Components of our system
Module 1：improved α-pinene oxidation system
pQE-80L-Kan glf plasmid
Elevated expression of glucose facilitator (GLF) from Zymomonas mobilis dramatically increase glucose uptake (6), providing foundation for Glucose-dependent NADPH regeneration and α- pinene oxidation (1). A glf gene was inserted into pQE-80L-Kan T7WT vector to form recombinant plasmid pQE-80L-Kan glf (Figure 3). We transformed this plasmid into E. coli BL21 star (DE3) to achieve high efficiency of glucose uptake.
Figure 3. Plasmid map of pQE-80L-Kan glf
Figure 4. Plasmid map of pCDFDUET-1 p450bm-3qm glcdh.
pCDFD UET-1 p450bm-3qm glcdh plasmid
P450BM-3is a wild type of monooxygenase from Bacillus megaterium that has been reported as the most potent oxidant for P450s (7). We adopted a quintuple mutant of this gene called P450BM-3 QM that can be expressed in E. coli BL21 star (DE3), combining with glucose dehydrogenase 2 (GlcDH-II) from B. megaterium (7). We inserted P450BM-3 QM and GlcDH-II into pCDFDUET-1 vector to form pCDFDUET-1 p450bm-3qm glcdh (Figure 4). Ultimately achieve efficient α-pinene oxidation.
Module 2: α-pinene oxide lyses system
Figure 5. Plasmid map of pET-28a prα-pol.
pET-28a prα-pol plasmid
α-pinene oxide lyase (Prα-POL) from P. Rhodesiense CIP107491 catalyzes the decyclization of α-pinene oxides to produce cis-dimethyl-5-isopropylhexa-2,5-dienal (also called Isonovalal). In our project, Prα-POL gene was inserted into empty vector pET-28a to form pET-28a prα-pol (Figure 5). Finally, Isonovalal may be degraded by NAD-dependent dehydrogenase into isovaleric acid (8).
Note: every designed plasmid contains a C-terminal his-tag serving for protein purification based on Ni column affinity chromatography.
Experimental Procedure
Our experiment can be roughly divided into the following steps, including a complete construction, expression and detection process of our two systems (Figure 6).
• Step1- Collection of genes, design and construction of our three recombinant plasmids.
• Step2- Transformation of recombinant plasmids into E. coli BL21 star (DE3).
√ Chemical transformation
√ Electro-transformation
• Step3- Expression and purification of GLF, GLcDH, P450BM-3 QM and Prα-POL proteins.
√ Nickel column affinity chromatography
√ Protein validation with SDS-PAGE
• Step4- Pre-experiment: Detecting the susceptibility of the constructed engineered bacteria to their organic substrates and defining their concentration limit.
• Step5- Simplification and construction of aqueous-organic two-phase systems for the whole cell catalysis.
• Step6- Using Gas Chromatograph-Mass Spectrometer (GC-MS) to detect the efficiency of oxidation and degradation.
For detailed experimental procedures, please refer to the protocol section.
Figure 6. Flow chart of our detailed experimental procedure.
Protocols
Transformation of Competent Cells
Prokaryotic Protein Expression and Purification
References
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