Difference between revisions of "Part:BBa K4885002"

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This part is responsible for the expression of adhE2 gene with Pthl promotor and Dac gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Dac gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence  (BBa_K103015),  adhE2 sequence(BBa_K1462060), Dac gene sequence (BBa_K4885000) and terminator sequence  (BBa_K3585002). adhE2 gene codes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Dac gene codes a NAD-dependent deacetylase which is an enzyme removing acetyl groups from the N-terminal of protein substrates and weakening the N-terminal acetylation of proteins. The enzyme is responsible for the removal of N-terminal acetylation modification in the protein post-translational modification system.
 
This part is responsible for the expression of adhE2 gene with Pthl promotor and Dac gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Dac gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence  (BBa_K103015),  adhE2 sequence(BBa_K1462060), Dac gene sequence (BBa_K4885000) and terminator sequence  (BBa_K3585002). adhE2 gene codes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Dac gene codes a NAD-dependent deacetylase which is an enzyme removing acetyl groups from the N-terminal of protein substrates and weakening the N-terminal acetylation of proteins. The enzyme is responsible for the removal of N-terminal acetylation modification in the protein post-translational modification system.
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===Usage and biology===
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This is the part collection that used to engineer C. tyrobutyricum to overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885006 were used to overexpress rate-limiting
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enzymes (bcd and crt). BBa_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate. BBa_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.
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===Usage and Biology===
 
===Usage and Biology===
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We constructed pMTL-Pthl-adhE2-Dac plasmid responsible for the expression of adhE2 and Dac with Pthl promotor in C. tyrobutyricum L319.  
 
We constructed pMTL-Pthl-adhE2-Dac plasmid responsible for the expression of adhE2 and Dac with Pthl promotor in C. tyrobutyricum L319.  
 
Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template, VAD-F and VDAC-R as primers, Vdac vector (8004 bp) was amplified by PCR. Using C. tyrobutyricum genome as template and DAC-F and DAC-R as primers, Dac gene fragment (767 bp) was amplified. Gibson assembly method was used to link Dac gene fragment to Vdac vector linearized vector. Colony PCR was performed on the transformed colonies (1050 bp) with DAC-PF and Pb-PR as primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Pthl-adhE2-Dac was obtained.
 
Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template, VAD-F and VDAC-R as primers, Vdac vector (8004 bp) was amplified by PCR. Using C. tyrobutyricum genome as template and DAC-F and DAC-R as primers, Dac gene fragment (767 bp) was amplified. Gibson assembly method was used to link Dac gene fragment to Vdac vector linearized vector. Colony PCR was performed on the transformed colonies (1050 bp) with DAC-PF and Pb-PR as primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Pthl-adhE2-Dac was obtained.
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/002-fig1.png">
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  <div class="unterschrift"><b>Figure 1 Verification of pMTL-Pthl-adhE2-Dac plasmid by colony PCR</b>
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===(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Dac===
 
===(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Dac===
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HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing Dac in C. tyrobutyricum improved the yield of butyrate (1.73 g/L) by 50% while did not change the butanol yield (5 g/L) (Figure 4-5), increasing the butyrate-to-butanol molar ratio from 0.19 to 0.29. The increased product ratio was more favorable for the successive esterification of butyrate and butanol into butyl butyrate.  
 
HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing Dac in C. tyrobutyricum improved the yield of butyrate (1.73 g/L) by 50% while did not change the butanol yield (5 g/L) (Figure 4-5), increasing the butyrate-to-butanol molar ratio from 0.19 to 0.29. The increased product ratio was more favorable for the successive esterification of butyrate and butanol into butyl butyrate.  
 
Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to determine the yield. The yield of butyl butyrate in Ct(adhE2::Dac) increased 27% compared with that in Ct(adhE2) (Figure 6).
 
Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to determine the yield. The yield of butyl butyrate in Ct(adhE2::Dac) increased 27% compared with that in Ct(adhE2) (Figure 6).
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/002-fig2.png">
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  <div class="unterschrift"><b>Figure 2 Verification of Dac protein overexpression in Ct(adhE2::Dac) by SDS-PAGE</b>
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/002-fig3.png">
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  <div class="unterschrift"><b>Figure 3 Growth performance of Ct(adhE2::Dac)</b>
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/002-fig4.png">
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  <div class="unterschrift"><b>Figure 4 Butyrate fermentation performance of Ct(adhE2::Dac)</b>
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/002-fig5.png">
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  <div class="unterschrift"><b>Figure 5 Butanol fermentation performance of Ct(adhE2::Dac)</b>
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  <img class="bild" src="https://static.igem.wiki/teams/4885/wiki/parts/parts/002-fig6.png">
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  <div class="unterschrift"><b>Figure 6 Production of butyl butyrate by Ct(adhE2::Dac)</b>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4885002 parameters</partinfo>
 
<partinfo>BBa_K4885002 parameters</partinfo>
 
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Latest revision as of 13:28, 12 October 2023


Pthl-adhE2-Dac

This part is responsible for the expression of adhE2 gene with Pthl promotor and Dac gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Dac gene is derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319. It consists of Pthl sequence (BBa_K3443002), strong ribosomal binding site (RBS) sequence (BBa_K103015), adhE2 sequence(BBa_K1462060), Dac gene sequence (BBa_K4885000) and terminator sequence (BBa_K3585002). adhE2 gene codes for the alcohol/aldehyde bifunctional dehydrogenase. Its role is to convert acyl-CoA to aldehyde then to alcohol in two reductive steps using NADH as cofactor. Dac gene codes a NAD-dependent deacetylase which is an enzyme removing acetyl groups from the N-terminal of protein substrates and weakening the N-terminal acetylation of proteins. The enzyme is responsible for the removal of N-terminal acetylation modification in the protein post-translational modification system.

Usage and biology

This is the part collection that used to engineer C. tyrobutyricum to overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885006 were used to overexpress rate-limiting enzymes (bcd and crt). BBa_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate. BBa_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3300
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Results

(1)Plasmid construction

We constructed pMTL-Pthl-adhE2-Dac plasmid responsible for the expression of adhE2 and Dac with Pthl promotor in C. tyrobutyricum L319. Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template, VAD-F and VDAC-R as primers, Vdac vector (8004 bp) was amplified by PCR. Using C. tyrobutyricum genome as template and DAC-F and DAC-R as primers, Dac gene fragment (767 bp) was amplified. Gibson assembly method was used to link Dac gene fragment to Vdac vector linearized vector. Colony PCR was performed on the transformed colonies (1050 bp) with DAC-PF and Pb-PR as primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Pthl-adhE2-Dac was obtained.

Figure 1 Verification of pMTL-Pthl-adhE2-Dac plasmid by colony PCR

(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Dac

Ct(adhE2::Dac) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Dac using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. C. tyrobutyricum L319 transfected with pMTL-Pthl-adhE2 plasmid was notated as Ct(adhE2). Ct(adhE2) was used as the control. SDS-PAGE confirmed the overexpression of Dac protein (26 kDa) in Ct(adhE2::Dac) (Figure 2). Growth performance analysis showed that overexpressing Dac in C. tyrobutyricum improved the growth of the strain, with the maximum OD600 increased by 33% (Figure 3). HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing Dac in C. tyrobutyricum improved the yield of butyrate (1.73 g/L) by 50% while did not change the butanol yield (5 g/L) (Figure 4-5), increasing the butyrate-to-butanol molar ratio from 0.19 to 0.29. The increased product ratio was more favorable for the successive esterification of butyrate and butanol into butyl butyrate. Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to determine the yield. The yield of butyl butyrate in Ct(adhE2::Dac) increased 27% compared with that in Ct(adhE2) (Figure 6).

Figure 2 Verification of Dac protein overexpression in Ct(adhE2::Dac) by SDS-PAGE

Figure 3 Growth performance of Ct(adhE2::Dac)

Figure 4 Butyrate fermentation performance of Ct(adhE2::Dac)

Figure 5 Butanol fermentation performance of Ct(adhE2::Dac)

Figure 6 Production of butyl butyrate by Ct(adhE2::Dac)