Part:BBa_K4395013
NADPH-dependent aldehyde reductase YahK catalyzes the reduction of a wide range of aldehydes into their corresponding alcohols. Has a strong preference for NADPH over NADH as the electron donor. Cannot use a ketone as substrate. Is a major source of NADPH-dependent aldehyde reductase activity in E.coli[1]. In addition, this enzyme has been actively expressed in E.coli[2].These characteristics of RsAs attract us and thus we choose it as aldehyde reductase of our experiment.
Reference: 1.Pick A, Rühmann B, Schmid J, Sieber V. Novel CAD-like enzymes from Escherichia coli K-12 as additional tools in chemical production. Appl Microbiol Biotechnol. 2013;97(13):5815-5824. doi:10.1007/s00253-012-4474-5 2.Kramer L, Le X, Rodriguez M, Wilson MA, Guo J, Niu W. Engineering Carboxylic Acid Reductase (CAR) through a Whole-Cell Growth-Coupled NADPH Recycling Strategy. ACS Synth Biol. 2020;9(7):1632-1637. doi:10.1021/acssynbio.0c00290
Contribution From NJTech-China-B 2023
Group:iGEM NJTech-China-B
Author: Yao Yao
Characterization from iGEM23-NJTech-China-B
YahK, an aldehyde reductase from Escherichia coli, catalyzes the reduction of various aldehydes to corresponding alcohols. YahK has been found to be important in NADP/NADPH homeostasis, lipid biosynthesis, amino acid metabolism, or the formation of fuel alcohols. The broad substrate range of YahK implies its more universal function. In our experiment, YahK was used to reduce Glutaraldehyde to produce pentanediol..
1.1 The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)
The gene of YahK was amplified from the genome of E. coli MG1655, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-YahK. By sequencing, the correct plasmid pRSFDuet-YahK was then transformed to E. coli BL21(DE3) (Figure 1).
Figure1The transformation of palsmid pRSFDuet-YahK into E. coli BL21(DE3)
1.2 The colony PCR of pRSFDuet-YahK in E. coli BL21(DE3)
The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 1500bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-YahK was successful obtained (Figure 2).
Figure2. Colony PCR results of recombinant strain that containig the plasmid pRSFDuet-YahK.
M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containg plasmid pRSFDuet-YahK
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-YahK was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. We transformed the plasmid pRSFDuet without YahK gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of YahK protein was correct, which was consistent with the expected molecular weight of 38.0 kDa (Figure 3).
Figure3. SDS-PAGE analysis of YahK expression in E. coli BL21(DE3)
Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containg pRSFDuet; lanes 3 and 4：supernatant
and precipitation of E. coli BL21(DE3) containg PRSFDuet-YahK
YahK is an NADPH dependent aldehyde reductase. The enzyme of Yahk was purified by with a Ni-nitrilotriacetic acid affinity chromatography (Ni-NTA) column. Subsequently, we tested its activity by detecting NADPH consumption with the purified YahK enzyme. The results showed that YahK can consume NADPH, indicating that YahK has active enzyme activity.
Figure4 The enzymatic activity of purified YahK
5-adminovalerate is produced from L-lysine through the 5-aminovalerate pathway. L-lysine monooxygenase (DavB) plays a key role in the 5-aminivalerate pathway of various microorganisms. In our experiment, DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide.
1.1 The transformation of palsmid pRSFDuet-DavB into E. coli BL21(DE3)
The gene of DavB was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavB. By sequencing, the correct plasmid pRSFDuet-DavB was then transformed to E. coli BL21(DE3) (Figure 1).
Figure1The transformation of palsmid pRSFDuet-DavB into E. coli BL21(DE3)
1.2 The colony PCR of pRSFDuet-DavB in E. coli BL21(DE3)
The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was between 1000 bp and 2000 bp. The results showed that the recombinant strain that containing plasmid pRSFDuet-DavB was successful obtained (Figure 2).
Figure2. Colony PCR results of recombinant strain that containig the plasmid pRSFDuet-DavB.
M represents the band of DNA marker; Lane 1,2 and 3 represent the band of different colonies containg plasmid pRSFDuet-DavB
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. We transformed the plasmid pRSFDuet without DavB gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavB protein was correct, which was consistent with the expected molecular weight of 62.4 kDa (Figure 3).
Figure3. SDS-PAGE analysis of DavB expression in E. coli BL21(DE3)
Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containg pRSFDuet; lanes 3 and 4：supernatant
and precipitation of E. coli BL21(DE3) containg PRSFDuet-DavB
3.1 Determination of DavB whole-cells catalytic activity
DavB catalyzes the oxidation of L-lysine to produce 5-aminovaleramide. The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB was inoculated and cultured in the LB medium at 37 ℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. Subsequently, we tested its activity by detecting L-lysine consumption with the whole-cells containing DavB enzyme. The results showed that whole-cells containing DavB enzyme can consume L-lysine, indicating that whole-cells containing DavB have active enzyme activity (Figure 4).
Figure4 The enzymatic activity of whole-cells
3.2 Detemination of crude enzyme of DavB
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavB was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. Subsequently, we tested its activity by detecting L-lysine consumption with the crude enzyme of DavB. The results showed that crude enzyme of DavB can consume L-lysine, indicating that crude enzyme of DavB has active enzyme activity (Figure 5).
Figure5 The enzymatic activity of crude enzyme
5-Aminovalerateis produced from L-lysine through the 5-aminovalerate pathway. 5-aminovaleramide amidohydrolase (DavA) plays a key role in the 5-aminovalerate pathway of various microorganisms. In our experiment, DavA catalyzes 5-aminovaleramide into 5-aminovalerate.
1.1 The transformation of palsmid pRSFDuet-DavA into E. coli BL21(DE3)
The gene of DavA was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavA. By sequencing, the correct plasmid pRSFDuet-DavA was then transformed to E. coli BL21(DE3) (Figure 1). The gene of DavB was amplified from the genome of Pseudomona, and integrated into pRSFDuet-1 vector to obtain the plasmid pRSFDuet-DavB. By sequencing, the correct plasmid pRSFDuet-DavB was then transformed to E. coli BL21(DE3) (Figure 1).
Figure1The transformation of palsmid pRSFDuet-DavA into E. coli BL21(DE3)
1.2 The colony PCR of pRSFDuet-DavA in E. coli BL21(DE3)
The colony PCR was then performed. The results of colony PCR showed the correct length of the gene fragment, which was 750 bp approximately. The results showed that the recombinant strain that containing plasmid pRSFDuet-DavA was successful obtained (Figure 2).
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. We transformed the plasmid pRSFDuet without DavA gene into E.coli BL21(DE3) strain and induced protein expression under the same conditions as a control experiment. SDS-PAGE results confirmed that the molecular weight of DavA protein was correct, which was consistent with the expected molecular weight of 29.2 kDa (Figure 3).
Figure3. SDS-PAGE analysis of DavA expression in E. coli BL21(DE3)
Lane M: protein molecular weight marker; lanes 1 and 2: supernatant and precipitation of E. coli BL21(DE3) containg pRSFDuet; lanes 3 and 4：supernatant
and precipitation of E. coli BL21(DE3) containg PRSFDuet-DavA
3.1 Determination of DavA whole-cells catalytic activity
DavA catalyzes 5-aminovaleramide into 5- aminovalerate.The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA was inoculated and cultured in the LB medium at 37 ℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. Subsequently, we tested its activity by detecting production of 5-aminovalerate with the whole-cells containing DavA enzyme. The results showed that whole-cells containing DavA enzyme can produce 5-aminovalerate, indicating that whole-cells containing DavA have active enzyme activity (Figure 4).
Figure4 The enzymatic activity of whole-cells
3.2 Detemination of crude enzyme of DavA
The correct colony of recombinant E. coli BL21(DE3) containing pRSFDuet-DavA was inoculated and cultured in the LB medium at 37℃ and 200 rpm. 0.5 mM of IPTG was added into the culture to induce protein expression when cells grew into an OD600 of 0.6-0.8. After overnight induction and cultivation, the cells were harvested by centrifugation and resuspended in 50 mM Phosphate Buffered Saline (pH 8.0) containing 300 mM Na2HPO4 and 100 mM NaH2PO4. The sespended cells were then lysed by ultrasonication to release the intracellular proteins. Subsequently, we tested its activity by detecting production of 5-aminovalerate with the crude enzyme of DavA. The results showed that crude enzyme of DavA can produce 5-aminovalerate, indicating that crude enzyme of DavA has active enzyme activity (Figure 5).
Figure5 The enzymatic activity of crude enzyme
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