A Synthetic Reaction Cascade Implemented by Colocalization of Two Proteins within Catalytically Active Inclusion Bodies

Abstract

In nature, enzymatic reaction cascades, i.e., realized in metabolic networks, operate with unprecedented efficacy, with the reactions often being spatially and temporally orchestrated. The principle of "learning from nature" has in recent years inspired the setup of synthetic reaction cascades combining biocatalytic reaction steps to artificial cascades. Hereby, the spatial organization of multiple enzymes, e.g., by coimmobilization, remains a challenging task, as currently no generic principles are available that work for every enzyme. We here present a tunable, genetically programmed coimmobilization strategy that relies on the fusion of a coiled-coil domain as aggregation inducing-tag, resulting in the formation of catalytically active inclusion body coimmobilizates (Co-CatIBs). Coexpression and coimmobilization was proven using two fluorescent proteins, and the strategy was subsequently extended to two enzymes, which enabled the realization of an integrated enzymatic two-step cascade for the production of (1 R,2 R)-1-phenylpropane-1,2-diol (PPD), a precursor of the calicum channel blocker diltiazem. In particular, the easy production and preparation of Co-CatIBs, readily yielding a biologically produced enzyme immobilizate renders the here presented strategy an interesting alternative to existing cascade immobilization techniques.

Enzyme assembly guided by SPFH‐induced functional inclusion bodies for enhanced cascade biocatalysis

“…However, presumably due to the lack of an inherent organized structure, only marginal improvements were reported when this approach was used for other metabolic pathways (Horn & Sticht, 2015; Lee, DeLoache, & Dueber, 2012; Young et al, 2017). Interestingly, functional inclusion bodies can retain the catalytic activity of enzymes and play a unique role in the process of biocatalysis, such as excellent stability and recoverability in aqueous or micro‐aqueous organic solvent systems (Diener et al, 2016; Jäger et al, 2018; Kloss et al, 2018a). Herein, whole‐cell catalysis shows that the fusion of SPFH and enzyme could significantly improve the conversion rate of products and effectively reduce the generation of intermediate products.…”

“…Therefore, developing different types of domains with aggregation tendency is essential to enhance cascade biocatalysis efficiency by functional inclusion bodies in metabolic engineering. In addition, the reported catalytic reactions of functional inclusion bodies are done in vitro (Jäger et al, 2018; Kloss et al, 2018a), but if functional inclusion bodies can directly play a cascade effect in living cells remains unknown.…”

“…Traditionally, inclusion bodies are regarded as waste repositories for unfolded or misfolded proteins without biological function. However, some studies indicate that inclusion bodies produced by fusion of the target enzymes and the coiled-coil domain or aggregation-prone protein in Escherichia coli can retain some enzyme functions (Arié, Miot, Sassoon, & Betton, 2006;Diener, Kopka, Pohl, Jaeger, & Krauss, 2016;Jäger et al, 2018). Now, functional inclusion bodies begin to appear in biocatalysis (Domach, 2015;Kloss et al, 2018aKloss et al, , 2018b.…”

“…Organizing interrelated cellular components on a temporal and spatial scale is crucial for improving the efficiency of the cellular processes such as metabolism and signal transduction (Agapakis, Boyle, & Silver, 2012;Young et al, 2017). With the advent and development of synthetic biology, the design of efficient microbial cell factories using spatial organization within cellular environment to enhance the metabolic flux has attracted extensive attentions (Kang et al, 2019;Lee et al, 2018;Young et al, 2017). To develop a scaffold that can be better applied to the living cells, the researchers paid attention to the proteins that can self-assemble into defined, nano to macromolecular architectures (Howorka, 2011).…”

Enzyme Assembly for Compartmentalized Metabolic Flux Control

“…However, as early as 1989, Worrall and Goss found that IBs of beta-galactosidase contain correctly folded proteins, and the enzyme activity in the IBs is one-third that of soluble galactosidase [48]. In recent years, catalytically active inclusion bodies (CatIBs) formed by fusion of the target enzyme and the coiled-coil domain or aggregation-prone protein have been proposed [49][50][51][52][53][54]. CatIBs represent a promising new form of carrier-free enzyme immobilization, and might have important implications for protein aggregation in vitro or in vivo.…”