Abstract
Our team, the Worldshaper-NJBIOX, aims to use kitchen waste, shrimp and crab 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.
Figure 1 Synthesis pathway of butyrate and butanol by in our engineered C. tyrobutyricum
Table 1 Part list
| 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 |
1. Plasmid construction
1.1. Plasmids for C. tyrobutyricum
1.1.1 adhE2 (aldehyde dehydrogenase) expression: pMTL-Pthl-adhE2 recombinant plasmid
We took a recombinant plasmid pMTL-Bs2 constructed previously by our research group as template, and used PCR to obtain a linearized pMTL-Pthl vector. adhE2 gene fragment was amplified from the genome of Clostridium acetobutylicum by PCR. DNA electrophoresis confirmed the lengths of the two PCR products (5461bp, and 2577bp). adhE2 gene fragment was ligated with pMTL-Pthl linearized vector into a pMTL-Pthl-adhE2 recombinant plasmid by Gibson assembly method. pMTL-Pthl-adhE2 was transformed into E. coli JM109 strain. Colony PCR and DNA electrophoresis (750 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMTL-Pthl-adhE2 by gene sequencing.
Figure 2 Genetic circuit of pMTL-Pthl-adhE2
1.1.2 Expression of adhE2 (aldehyde dehydrogenase), thl (thiolase) and hbd (β-hydroxybutyryl-coenzyme) : pMTL-Pthl-adhE2-thl-hbd recombinant plasmid
The recombinant plasmid pMTL-Pthl-adhE2 is cleaved using XbaⅠ enzyme to obtain the linear vector. The thl fragment (1182bp) and hbd fragment (849bp) were amplified from the C. tyrobutyricum genome by PCR. The thl fragment was ligated to the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. Similar as for pMTL-Pthl-adhE2, through transformation, colony PCR and gene sequencing, we obtained the pMTL-Pthl-adhE2-thl recombinant plasmid and confirmed it. pMTL-Pthl-adhE2-thl was linearized by reverse PCR. The hbd fragment and the linear vector pMTL-Pthl-adhE2-thl were ligated by Gibson assembly method. Using the same method as in 1.1.1, we obtained the pMTL-Pthl-adhE2-thl recombinant plasmid and confirmed it. The recombinant plasmid pMTL-Pthl-adhE2-thl-hbd was also verified by enzymatic cleavage using Hind III enzyme.
Figure 3 Genetic circuit of pMTL-Pthl-adhE2-thl-hbd
1.1.3 Conjugation of thiolysis (thl) and β-hydroxybutyryl-coenzyme (hbd) by a SpyTag/SpyCatcher system: pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmids
pMTL-Pthl-adhE2 recombinant plasmid was digested with XbaI enzyme to obtain the linear vector. Using C. tyrobutyricum genome as template, PCR amplification was performed by introducing SpyCatcher and SpyTag sequences through primers, resulting in fragment Spythl (1182bp) (i.e., SpyCatcher-thl) and fragment Spyhbd (849bp) (i.e., SpyTag-hbd). The fragment Spythl was ligated with the linear vector pMTL-Pthl-adhE2 by Gibson assembly method. The correct pMTL-Pthl-adhE2-Spythl recombinant plasmid was obtained and verified by the same method as in 1.1.
The linear vector pMTL-Pthl-adhE2-Spythl was obtained by reverse PCR, and ligated with the fragment Spyhbd by Gibson assembly method. The correct pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmid was obtained and verified by the same method as in 1.1.1. The recombinant plasmid was also verified by enzymatic cleavage using Hind III enzyme.
Figure 4 pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd
Figure 5 Gel electrophoresis results of colony PCR verification of pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd plasmid (1062 bp)
Figure 6 Gel electrophoresis results of enzymatic cleavage verification of pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd plasmid (2512 bp and 8011 bp)
1.2. Plasmid for E. coli: pet25b-T7-pelB-CALB-ChBD plasmid for the expression of lipase CALB
The linear vector pet25b was obtained by double digestion of pet25b using NdeI and EcoRⅠ enzymes. CALB fragment (966 bp) was amplified from the Pseudomonas aeruginosa genome by PCR. ChBD fragment (417 bp) was obtained from a synthetic gene by PCR. The two fragments were fused together by fusion PCR, and then ligated to the linearized vector pet25b by T4 ligation. The pet25b-T7-pelB-CALB-ChBD recombinant plasmid was obtained and verified by the same method as in 1.1.1.
Figure 7 Genetic circuit of pet25b-T7-pelB-CALB-ChBD
2. Construction of engineered bacteria
2.1. Engineering C. tyrobutyricum
The three constructed plasmids for C. tyrobutyricum were conjugatively transferred to C. tyrobutyricum using E. coli CA434 as the donor strain, respectively, and the corresponding engineered strains were achieved as following.
2.1.1 Expression of adhE2 to establish a butanol synthesis pathway
The plasmid pMTL-Pthl-adhE2 was transferred to C. tyrobutyricum. Protein gel electrophoresis showed that aldehyde dehydrogenase (94 kDa) encoded by adhE2 was expressed in the engineered C. tyrobutyricum (Figure 8). Fermentation experiment showed that adhE2 overexpression did not affect the growth of the strain (Figure 7).
HPLC experiment showed that 3.0 g/L butanol and 1.0 g/L butyrate were obtained from this strain, thus realizing a de novo synthesis pathway of butanol while maintaining the native synthesis pathway of butyrate (Figure 10).
Figure 8 Protein gel electrophoresis of adhE2 expression in C. tyrobutyricum transformed with pMTL-Pthl-adhE2
Figure 9 Growth performance of recombinant plasmid pMTL-Pthl-adhE2 overexpression in C. tyrobutyricum
Figure 10 Butyrate and butanol yields of C. tyrobutyricum transformed with pMTL-Pthl-adhE2
2.1.2 Overexpression of thl and hbd to optimize the butanol and butyrate synthesis pathways
The plasmid pMTL-Pthl-adhE2-thl-hbd was transformed into C. tyrobutyricum. Protein gel electrophoresis showed that thiolysis (thl) and β-hydroxybutyryl-coenzyme (hbd) were expressed in the transformed strain (Figure 11). Fermentation experiment showed that the overexpression of these key enzymes effectively improved the growth performance of the strain, the maximum OD600 increased by 21% (Figure 12).
HPLC experiment showed that the yields of butyrate and butanol were increased by 30% and 33%, respectively, in this strain (Figure 11). Since they are the precursors for butyl butyrate synthesis catalyzed by lipase, the final butyl butyrate production was up to 590 mg/L in this strain, an increase by 1.1 fold compared to the strain without thl and hbd overexpression (Figure 13).
Figure 11 Enhanced expression of thl and hbd in C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd
Figure 12 Growth performance of C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd
Figure 13 Yields of butyrate and butanol of C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd
Figure 14 Butyl butyrate yield from C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd vs. that from C. tyrobutyricum transformed with pMTL-Pthl-adhE2
2.1.3 Conjugation of thl and hbd via a SpyCatcher-SpyTag system to optimize the butanol and butyrate synthesis pathways
The plasmid pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd was transformed into C. tyrobutyricum. And the experiments are still in process.
2.2. Engineering E.coli: Expression of CALB lipase
The plasmid pet25b-T7-pelB-CALB-ChBD was transformed into E. coli Rosetta(DE3). Protein gel electrophoresis showed that E. coli was successfully constructed to produce Candida antarctica Lipase B (CALB) (Figure 14).
CALB expressed by the plasmid has an affinity tag ChBD fused to it. ChBD-fusion CALB can facilitate the purification of CALB by chitin pellets, thus enhancing the reaction rate of CALB catalyzed esterification.
In the enzyme activity assay, the exogenously expressed lipase was found to have good activity, showing the highest enzyme activity of 110 U/mL at 120 min (Figure 13). 220 mg/L butyl butyrate was obtained by catalyzing C. tyrobutyricum transformed with pMTL-Pthl-adhE2. After immobilization by chitin pellets, the enzymatic activity of CALB increased by 24% to 136 U/mL, and the final catalytic butyl butyrate production was increased by 28% to 280 mg/L.
Figure 15 Lipase expression in E. coli
Figure 16 Enzyme activity assay of exogenously expressed lipase CALB
