Part:BBa_K4885004

Pthl-adhE2-Pcat1-Cat1

This part is responsible for the expression of adhE2 gene with Pthl promotor and Cat1 gene with Pcatl promotor. adhE2 gene is derived from Clostridium acetobutylicum. Cat1 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), terminator sequence (BBa_K3585002), Pcat1 sequence (BBa_K4119011), strong ribosomal binding site (RBS) sequence (BBa_K103015), Cat1 gene sequence (BBa_K4119013) and terminator sequence (BBa_K3585002). adhE2 gene encodes 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. Cat1 gene codes the butyryl-CoA/acetate CoA transferase.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Results

(1)Plasmid construction and transformation

We used the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 as the template, Vad-f and Vad-r as the primers, and amplified to obtain the Vpthl-adhE2 vector (7985 bp). Using the genomic DNA of C. tyrobutyricum as a template, with primers Cat-f and Cat-r, we amplified a fragment of the butyryl-CoA/acetate CoA transferase (cat1) gene (1378 bp). The cat1 fragment and Vpthl-adhE2 linearized vector were ligated by Gibson assembly method. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transfected, plasmid was extracted, and the recombinant plasmid, pMTL-Pthl-adhE2-Pcat1-Cat1, was verified by gene sequencing.

(2)Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-cat1

Ct(adhE2::cat1) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-cat1 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 cat1 protein (47 kDa) in Ct(adhE2::cat1) (Figure1). HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing cat1 in C. tyrobutyricum improved the yield of butyrate by 130%, and decreased the yield of butanol by 26% (Figure 2), increasing the butyrate-to-butanol molar ratio from 0.3 to 1.1. The increased product ratio was more favorable for the esterification of butyrate and butanol into butyl butyrate. In addition, the yield of acetate as a byproduct decreased significantly from 4 g/L to 1.2 g/L by overexpressing cat1.