Part:BBa_K4885006

Pthl-adhE2-bcd-crt

This part is responsible for the expression of bcd and crt enzymes derived from Clostridium tyrobutyricum (C. tyrobutyricum) L319 and adhE2 gene derived from Clostridium tyrobutyricum with Pthl promotor. It consists of Pthl sequence (BBa K3443002), strong ribosomal binding site (RBS) for adhE2 sequence (BBa_K4408001), adhE2 sequence(BBa_K1462060), bcd sequence(BBa_K4119027)， crt sequence (BBa_K4119028), and the Cpa fdx 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. To introduce adhE2 in C. tyrobutyricum, we can construct a butanol synthesis pathway based on the native butyrate synthesis pathway. Crt gene codes for crotonase which converts 3-hydroxybutyryl-CoA to crotonyl-CoA in the butyrate synthesis pathway in C. tyrobutyricum. bcd gene codes for butyryl-CoA dehydrogenase which converts crotonyl-CoA to butyryl-CoA in the butyrate synthesis pathway in C. tyrobutyricum. The crotonase and butyryl-CoA dehydrogenase are rate-limiting enzymes in the butyrate and butanol synthesis pathway.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3367
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4546
  Illegal SapI.rc site found at 3496
  

Results

(1)Plasmid construction and transformation

Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template and VEAD-F and veAD-r as primers, Vp-adhE2 vector (8001 bp) was amplified. Using C. tyrobutyricum genome as template, bcd gene fragment (1196 bp) was amplified with bcd-f and BCD-R as primers, and crt gene fragment (813 bp) was amplified with CRT-F and crt-R as primers. bcd gene fragment and crt gene fragment were fused into bcd-crt fragment by fusion PCR. Gibson assembly method was used to link bcd-crt gene fragment to Vp-adhE2 linearized vector. Colony PCR (1249 bp) was performed on the transformed colonies with primers bcd-PF and Pb-PR. The positive colonies were transferred and the plasmid was extracted. After gene sequencing verification, the recombinant plasmid was obtained: pMTL-Pthl-adhE2-bcd-crt.

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

Ct(adhE2::bcd::crt) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-bcd-crt, 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 bcd and crt proteins in Ct(adhE2::bcd::crt) (Figure1). The growth performance of Ct(adhE2::bcd::crt) was significantly better than Ct(adhE2), with the maximum OD600 reaching 10.2, an increase of 58% (Figure 2). HPLC experiment showed that after fermentation for 215 hours, compared to the yields in Ct(adhE2), the yield of butyrate increased by 20% and the yield of butanol increased by 40% in Ct(adhE2::bcd::crt) (Figure 3). 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. 620 mg/L butyl butyrate was produced by Ct(adhE2::bcd::crt), which was 59% higher than the 390 mg/L yield by Ct(adhE2)(Figure 4).