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MEMO: A Method for Computing Metabolic Modules for Cell-Free Production Systems.

ABSTRACT: Cell-free bioproduction systems represent a promising alternative to classical microbial fermentation processes to synthesize value-added products from biological feedstocks. An essential step for establishing cell-free production systems is the identification of suitable metabolic modules with defined properties. Here we present MEMO, a novel computational approach to find smallest metabolic modules with specified stoichiometric and thermodynamic constraints supporting the design of cell-free systems in various regards. In particular, one key challenge for a sustained operation of cell-free systems is the regeneration of utilized cofactors (such as ATP and NAD(P)H). Given a production pathway with certain cofactor requirements, MEMO can be used to compute smallest regeneration modules that recover these cofactors with required stoichiometries. MEMO incorporates the stoichiometric and thermodynamic constraints in a single mixed-integer linear program, which can then be solved to find smallest suitable modules from a given reaction database. We illustrate the applicability of MEMO by calculating regeneration modules for the recently published synthetic CETCH cycle for in vitro carbon dioxide fixation. We demonstrate that MEMO is very flexible in taking into account the diverse constraints of the CETCH cycle (e.g., regeneration of 1 ATP, 4 NADPH and of 1 acetyl-group without net production of CO₂ and with permitted side production of malate) and is able to determine multiple solutions in reasonable time in two large reaction databases (MetaCyc and BiGG). The most promising regeneration modules found utilize glycerol as substrate and require only 8 enzymatic steps. It is also shown that some of these modules are robust against spontaneous loss of cofactors (e.g., oxidation of NAD(P)H or hydrolysis of ATP). Furthermore, we demonstrate that MEMO can also find cell-free production systems with integrated product synthesis and cofactor regeneration. Overall, MEMO provides a powerful method for finding metabolic modules and for designing cell-free production systems as one particular application.

SUBMITTER: Kamp AV

PROVIDER: S-EPMC7308072 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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MEMO: A Method for Computing Metabolic Modules for Cell-Free Production Systems.

Kamp Axel von AV Klamt Steffen S

ACS synthetic biology 20200302 3

Cell-free bioproduction systems represent a promising alternative to classical microbial fermentation processes to synthesize value-added products from biological feedstocks. An essential step for establishing cell-free production systems is the identification of suitable metabolic modules with defined properties. Here we present MEMO, a novel computational approach to find smallest metabolic modules with specified stoichiometric and thermodynamic constraints supporting the design of cell-free s ...[more]

PMID: 32069395

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Project description:Cell-free protein synthesis (CFPS) represents a versatile key technology for the production of toxic proteins. As a cell lysate, rather than viable cells, is used, the toxic effects on the host organism can be circumvented. The open nature of cell-free systems allows for the addition of supplements affecting protein concentration and folding. Here, we present the cell-free synthesis and functional characterization of two AB5 toxins, namely the cholera toxin (Ctx) and the heat-labile enterotoxin (LT), using two eukaryotic cell-free systems based on Chinese hamster ovary (CHO) and Spodoptera frugiperda (Sf21) cells. Through an iterative optimization procedure, the synthesis of the individual AB5 toxins was established, and the formation of multimeric structures could be shown by autoradiography. A functional analysis was performed using cell-based assays, thereby demonstrating that the LT complex induced the characteristic cell elongation of target cells after 24 h. The LT complex induced cell death at higher concentrations, starting at an initial concentration of 5 nM. The initial toxic effects of the Ctx multimer could already be detected at 4 nM. The detection and characterization of such AB5 toxins is of utmost importance, and the monitoring of intracellular trafficking facilitates the further identification of the mechanism of action of these toxins. We showed that the B-subunit of LT (LTB) could be fluorescently labeled using an LTB-Strep fusion protein, which is a proof-of-concept for future Trojan horse applications. Further, we performed a mutational analysis of the CtxA subunit as its template was modified, and an amber stop codon was inserted into CtxA's active site. Subsequently, a non-canonical amino acid was site-specifically incorporated using bio-orthogonal systems. Finally, a fluorescently labeled CtxA protein was produced using copper-catalyzed click reactions as well as a Staudinger ligation. As expected, the modified Ctx multimer no longer induced toxic effects. In our study, we showed that CFPS could be used to study the active centers of toxins by inserting mutations. Additionally, this methodology can be applied for the design of Trojan horses and targeted toxins, as well as enabling the intracellular trafficking of toxins as a prerequisite for the analysis of the toxin's mechanism of action.

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MEMO: A Method for Computing Metabolic Modules for Cell-Free Production Systems.

Publications

MEMO: A Method for Computing Metabolic Modules for Cell-Free Production Systems.

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