Part:BBa_K4800008
Ptrc-sfp-MmCAR(Q302E)-SpyTag-EutM-SpyCatcher-YahK
Ptrc is the promoter, YciA-sfp-Q302E is a mutant gene derived from BBa_K1655000, EutM is the protein scaffold, Yahk is the aldo-keto reductase, and 'molecular glue' SpyTag/SpyCatcher can be used to construct multi-enzyme complexes.
Adaptation of the original part in cell factory
The existing part BBa_K1655000 contains carboxylate reductase (CAR) and its activation protein of sfp. By reading literature and consulting experts in related fields, we found that CARs could catalyse a broad spectrum of carboxylic acids into the corresponding aldehydes. Combined with our own project requirements, we tested its activity to catalyze a C5 carboxylic acid of 5-hydroxyvalerate with a final goal used for the 1,5-pentadiol production.
The existing part BBa_K1655000 contains the promoter T7, which only can be adapted to strains containing the lambda DE3 lysogen. As the results shown in Figure 1, when we synthesized the part BBa_K1655000, ligated it into the plasmid of pRSFDuet, and transformed it into Escherichia coli BL21 without the lambda DE3 lysogen, the ability of the part to catalyze 5-hydroxyvalerate was not detected.
The chassis of E. coli NT1003 we engineered for 1,5-PDO production is also a strain that does not carry the lambda DE3 lysogen. Therefore, to make the part expression efficiently in the chassis, the promoter in the part was required to be changed. As we just need to employ the components of CAR and sfp in BBa_K1655000 for 1,5-PDO production, a trc promoter was added between YciA and RBS of sfp. The new fragments were subcloned into the plasmid pRSFDuet and transformed into the strain of E. coli BL21 without the lambda DE3 lysogen to evaluate its activity. As the product of 5-hydroxypentanal generated by CAR is not stable. Aldehyde reductase (YahK) that converts 5-hydroxypentanal to 1,5-PDO was also ligated into the plasmid pRSFDuet for a co-expression to evaluate activity by detecting the bioconversion of 5-hydroxyvalerate to 1,5-PDO in a whole-cell process. As the results shown in Figure 1, when the CAR and sfp were expressed under the control of trc promoter in E. coli BL21, we successfully detected the 5-hydroxyvalerate consumption and 1,5-PDO production. After reaction of 6 h, 63.2 mM 5-hydroxyvalerate was consumed and 48.9 mM 1,5-PDO was synthesized. These results indicated that the sfp and CAR components in part BBa_K1655000 also exhibited the ability to catalyze C5 5-hydroxyvalerate. We successfully expanded the application scenarios of the part BBa_K1655000.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Unknown
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal NheI site found at 5786
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1313
Illegal EcoRI site found at 5098
Illegal XbaI site found at 5016
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604
Illegal NgoMIV site found at 3572
Illegal NgoMIV site found at 3634
Illegal NgoMIV site found at 3851
Illegal NgoMIV site found at 4298
Illegal AgeI site found at 1478
Illegal AgeI site found at 2489
Illegal AgeI site found at 2786
Illegal AgeI site found at 3671
Illegal AgeI site found at 3680
Illegal AgeI site found at 4355 - 1000COMPATIBLE WITH RFC[1000]
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