Synthetic protein scaffolds provide modular control over metabolic flux

Abstract

Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.

Gene Arrangements in Expression Vector Affect 3-Hydroxypropionic Acid Production in Klebsiella pneumoniae

“…Apart from enzymatic activity, the intrinsic compatibility of puuC to K. pneumoniae is also a key factor for 3-HP biosynthesis. Given the evolving nature of ALDH family [10,13], upcoming work may be the determination of efficient enzymes and engineering of their spatial organization [5,6]. Collectively, this study has provided keys for better understanding of metabolic engineering which involves multiple enzyme genes.…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…[72] Overproduction of mevalonate by recruiting the enzymes through synthetic protein scaffolds for substrate channeling was attempted as well ( Figure 7c). [54] The MEV upper pathway genes were assembled by the synthetic protein scaffold which increased the production up to 77-fold. These strategies can lead to the increased terpenoids production in E. coli.…”

“…c) Synthetic protein scaffold for the formation of enzyme assembly pipelines. [54] For making the synthetic protein scaffolds, protein-protein interactions are the basis for domain and ligand complex formation. Various enzyme assembly line configurations are possible through modulating the enzyme complex stoichiometry.…”

“…[21,44] Since these genes were brought from S. cerevisiae, codon-optimization was beneficial for its expression in E. coli. [54] Among the MEV pathway enzymes, mevalonate kinase (MK) was considered as a rate-limiting enzyme which was overexpressed by promoter exchange and plasmid copy number increment. [38] Hydroxymethylglutaryl-CoA (HMG-CoA), which is an intermediate in the MEV pathway, turned out to be accumulated in the cytoplasm and thus toxic to E. coli.…”

Advanced biofuel production in microbes

“…To address this imbalance, the authors overexpressed the "upper" part of the MEV pathway to overproduce MEV. However, this upregulation led to 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) accumulation, which was toxic and could be relieved by introducing extra copies of tHMGR.To balance the pathway flux gen-erated by the flux-limiting HMGR, Dueber et al [35] scaffolded the three enzymes in the "upper" MEV pathway and obtained a 77-fold increase in MEV production over the unscaffolded pathway.…”