Our team aims to use kitchen waste, shrimp shells, as raw material to yield a biofuel, butyl butyrate, through the efficient biological production of engineered Clostridium tyrobutyricum (C. tyrobutyricum) and a catalyzed reaction by a lipase from engineered E. coli. In this project, our team has successfully made 9 parts to engineer C. tyrobutyricum and E. coli. BBa_K4408008 is constructed to express aldehyde dehydrogenase encoded by adhE2 in C. tyrobutyricum so that butanol can be produced by the bacteria. BBa_K4408009 is constructed to overexpress thl and hbd in order to enhance the synthesis pathways of butyrate and butanol. BBa_K4408005, BBa_K4408006 and BBa_K4408010 are constructed to conjugate thiolysis enzyme (thl) and β-hydroxybutyryl-coenzyme (hbd) through a SpyTag/SpyCatcher system to further improve the synthesis of butyrate and butanol. For the engineered E. coli, CALB gene was inserted to express CALB lipase from Candida antarcticum via a pet25b-T7-pelB-CALB-ChBD plasmid. By adding an affinity tag ChBD and pelB leader to CALB, we enhanced the reaction rate of CALB catalyzed esterification. BBa_K4408007 and BBa_K4408011 are constructed for this purpose.
| No. | Name | Type | Description | Length |
|---|---|---|---|---|
| 1 | BBa_K4408004 | Scar | 19aa CALB-ChBD linker | 57 bp |
| 2 | BBa_K4408005 | Composite | thl modified by oligopeptide SpyCatcher | 1569 bp |
| 3 | BBa_K4408006 | Composite | hbd modified by oligopeptide SpyTag | 921 bp |
| 4 | BBa_K4408007 | Composite | Lipase CALB connected with chitin binding domain | 1440 bp |
| 5 | BBa_K4408008 | Device | Pthl-adhE2, Expression of adhE2 with Pthl promoter | 3244 bp |
| 6 | BBa_K4408009 | Device | Pthl-adhE2-thl-hbd, Expression of adhE2, thl and hbd with Pthl promoter | 5309 bp |
| 7 | BBa_K4408010 | Device | Pthl-adhE2-Spythl-Spyhbd, Expression of adhE2, thl and hbd, modified by SpyCatcher/SpyTag oligopeptides |
5788 bp |
| 8 | BBa_K4408011 | Device | T7-CALB-ChBD, Expression of CALB connected with chitin binding domain | 1363 bp |