Butyrate production in engineered Escherichia coli with synthetic scaffolds

Abstract

Butyrate pathway was constructed in recombinant Escherichia coli using the genes from Clostridium acetobutylicum and Treponema denticola. However, the pathway constructed from exogenous enzymes did not efficiently convert carbon flux to butyrate. Three steps of the productivity enhancement were attempted in this study. First, pathway engineering to delete metabolic pathways to by-products successfully improved the butyrate production. Second, synthetic scaffold protein that spatially co-localizes enzymes was introduced to improve the efficiency of the heterologous pathway enzymes, resulting in threefold improvement in butyrate production. Finally, further optimizations of inducer concentrations and pH adjustment were tried. The final titer of butyrate was 4.3 and 7.2 g/L under batch and fed-batch cultivation, respectively. This study demonstrated the importance of synthetic scaffold protein as a useful tool for optimization of heterologous butyrate pathway in E. coli.

Photoautotrophic synthesis of butyrate by metabolically engineered cyanobacteria

“…Therefore, more well‐studied organisms, such as Escherichia coli are engineered into butyrate producers. Through expressing heterologous genes (Fischer, Tseng, Tai, Prather, & Stephanopoulos, ; Joung, Kurumbang, Sang, & Oh, ), optimizing 5′‐UTR for gene expression (Lim, Seo, Kim, & Jung, ), constructing protein fusions (Baek et al, ), cosubstrate fermentation (Saini, Wang, Chiang, & Chao, ), and engineering cofactor reference (Kataoka, Vangnai, Pongtharangkul, Yakushi, & Matsushita, ), the highest butyrate production titer has been demonstrated to around 10 g/L.…”

Spatial modulation and cofactor engineering of key pathway enzymes for fumarate production in Candida glabrata

“…Among these, scaffold engineering is used to improve pathway transportation efficiency by designing and synthesizing protein scaffolds, RNA scaffolds, and DNA scaffolds (Chen, Li, & Liu, ). For example, butyrate production was showed a three‐fold increase by attaching three pathway enzymes, 3‐hydroxybutyryl‐CoA dehydrogenase, 3‐hydroxybutyryl‐CoA dehydratase, and trans‐enoyl‐CoA reductase, to a protein scaffold via cognate ligand tags (Baek et al, ). Hydrogen production was increased by 24‐fold when hydrogenase was fused to a single copy of PP7 and ferredoxin to a dimer of MS2 in two‐dimensional RNA scaffolds (Delebecque, Lindner, Silver, & Aldaye, ).…”

Metabolic pathway balancing and its role in the production of biofuels and chemicals

“…This is accomplished through the use of protein fusions for enzyme cascades and synthetic scaffolding proteins to dock enzymes in close proximity to one another [45]. The development and implementation of synthetic protein scaffolds has achieved a 77-fold improvement in mevalonate production [46], a fivefold improvement in glucaric acid production [47], and a threefold improvement in butyrate production [48 ] over solely plasmid-based expression of pathway enzymes. Like other methods of pathway optimization, post-translational balancing does not benefit from reducing the production of surplus quantities of RNA or protein, but rather, by improving the efficiency of substrate transfer from enzyme to enzyme, minimizing diffusion, before the substrate reacts with the enzyme.…”