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Overview

This project is aimed to reduce the carbon emission during the fermentation of a valuable industrial strain, Clostridium tyrobutyricum (C. tyrobutyricum). F/Xpk gene sequences well fitted for C. tyrobutyricum L319 were obtained by strain screening and sequence optimization. Plasmids of the gene were constructed and transferred into C. tyrobutyricum L319 to construct a non-oxidative glycolysis (NOG) pathway. Unlike the native Embden-Meyerhof-Parnas (EMP) pathway which emits CO2 during glycolysis, this alternative NOG pathway constructed had efficient carbon conservation and reduced carbon emission while metabolizing glucose.

We have reached the following achievements.

1.Characterization of the part BBa_K4119002 (Bs2) from iGEM22_NJTech_China

We constructed pMTL-Pfba-Bs2 plasmid using Pfba promoter and Bs2 gene. We transformed the plasmid into Clostridium tyrobutyricum (C. tyrobutyricum) and detected the fluorescence intensity of Bs2 to characterize whether the Bs2 gene is regulated transcriptionally by the Pfba promoter.

Our results showed that Pfba achieved efficient expression of Bs2 gene with detectable fluorescence intensity. Under OD600=0.8 and OD600=1.2, the fluorescence intensity of the transfected strain was 33204.00 and 24397.00, much higher than that of the blank control (4720.67 and 1474.33).

Characterization：BBa_K4119002

2.Improvement of the part F/Xpk (BBa_K4119076)

Our team improved F/Xpk (BBa_K4119076, https://parts.igem.org/Part:BBa_K4119076) to a better version.

F/Xpk (BBa_K4119076) encoding phosphoketolase is derived from Clostridium acetobutylicum ATCC824. We employed ancestral sequence reconstruction (ASR) to reconstruct the ancestral phosphoketolase (F/Xpk), using FireProt-ASR with F/Xpk sequence (BBa_K4119076) as the input sequence. The ancestral F/Xpk predicted was named F/Xpk(ASR). The pMTL-Pthl-F/Xpk(ASR) recombinant plasmid was subsequently constructed and transferred into Clostridium tyrobutyricum (C. tyrobutyricum). The experiments showed that C. tyrobutyricum expressing F/Xpk(ASR) had slightly better growth, a 20% increase in butyrate yield and a decrease in acetate yield compared with C. tyrobutyricum expressing FXpk (BBa_K4119076). This indicated that using F/Xpk(ASR) instead of FXpk (BBa_K4119076) could greatly improve the butyrate fermentation performance and carbon conservation efficiency in C. tyrobutyricum.

Improved Part: BBa_K4119076

New Improved Part: BBa_K4886007

3. Plasmids constructed

We successfully constructed five plasmids pMTL-Pthl-F/Xpk(QS), pMTL-Pthl-F/Xpk(BD), pMTL-Pfba-F/Xpk(BD), pMTL-Ptkt-F/Xpk(BD) and pMTL-Pthl-F/Xpk(ASR). Future teams that need to express F/Xpk gene in Clostridium acetobutylicum strains can utilize these plasmids. F/Xpk originates from Bifidobacterium adolescentis and encodes phosphoketolase, an enzyme with has both the Fpk and Xpk activity. Phosphoketolase catalyzes the conversion of F6P to erythrose-4-phosphate (E4P) and acetyl-phosphate (AcP), as well that of xylulose-5-phosphate (X5P) to glyceraldehydes-3-phosphate (G3P) and AcP.