SynPharm and the guide to pharmacology database: A toolset for conferring drug control on engineered proteins

Abstract

Optimizing synthetic biological systems, for example novel metabolic pathways, becomes more complicated with more protein components. One method of taming the complexity and allowing more rapid optimization is engineering external control into components. Pharmacology is essentially the science of controlling proteins using (mainly) small molecules, and a great deal of information, spread between different databases, is known about structural interactions between these ligands and their target proteins. In principle, protein engineers can use an inverse pharmacological approach to include drug response in their design, by identifying ligand‐binding domains from natural proteins that are amenable to being included in a designed protein. In this context, “amenable” means that the ligand‐binding domain is in a relatively self‐contained subsequence of the parent protein, structurally independent of the rest of the molecule so that its function should be retained in another context. The SynPharm database is a tool, built on to the Guide to Pharmacology database and connected to various structural databases, to help protein engineers identify ligand‐binding domains suitable for transfer. This article describes the tool, and illustrates its use in seeking candidate domains for transfer. It also briefly describes already‐published proof‐of‐concept studies in which the CRISPR effectors Cas9 and Cpf1 were placed separately under the control of tamoxifen and mefipristone, by including ligand‐binding domains of the Estrogen Receptor and Progesterone Receptor in modified versions of Cas9 and Cpf1. The advantages of drug control or the rival protein‐control technology of optogenetics, for different purposes and in different situations, are also briefly discussed.

Artificial multi-enzyme cascades and whole-cell transformation for bioconversion of C1 compounds: Advances, challenge and perspectives

“…However, it also bears major drawbacks including the requirement for enzyme synthesis, the provision of cofactors, the existence of side reactions and the expensive process of scaling up for industrial production. In addition, as the number of reaction steps increases, the in vitro enzyme cascades also become more complex and more susceptible to inhibition of substrate, intermediates or products [ 40 , 41 ]. In generally, in vitro systems are especially suitable when: (1) ATP is unrequired; (2) none or only one kind of cofactor is involved; (3) all individual steps are thermodynamically favorable.…”

Workflows for optimization of enzyme cascades and whole cell catalysis based on enzyme kinetic characterization and pathway modelling

“…Additionally, also enzyme systems show a certain degree of complexity and are often subject to regulation by feedforward or feed-back loops and/or allosteric control by (co) substrates, intermediates, or products. And this becomes more and more pronounced the more complex the cascade gets [33,34].…”