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ANR funded project

Chimie durable, génie chimique et biotechnologie (DS0304) 2014
Projet SYNPATHIC

Robust engineering and directed evolution of synthetic pathways by integration of microfluidics and genomics

A primary goal of Synthetic Biology is to engineer or re-engineer or add additional new functionalities to a biological system by constructing new parts, or modifying an existing biological system. In White Biotechnology, a clear objective is to enrich the metabolic repertoire of a microbial cell with new metabolic enzymes or pathways that enable the production of chemicals from renewable carbon resource as an alternative cost-effective replacement of chemicals derived from fossil resources. In brief, the creation a bio-based chemical process for a given product follows a three step procedure, namely (i) pathway construction, (ii) engineering and optimization of the microbial cell factory and (iii) development of industrially-oriented process fermentation. Partner 1 of this consortium recently innovated in constructing a complex synthetic pathway composed of 8 reactions steps that leads to the production of two high added-value synthons, 2,4 dihydroxybutyrate (DHB) and 1,3 propanediol (PDO) from malate, the latter being produced from glucose using classical (natural) pathways. In this pathway, 5 reactions are catalyzed by non-natural enzymes, and three of them exhibit very poor catalytic activity and specificity towards their non-natural substrates, which renders the pathway poorly efficient. In addition, the synthetic pathway is expressed on plasmids as synthetic operons made of 3 to 8 genes. Although using plasmids, it is simple to manipulate gene expression through copy number or promoter strength, plasmids suffer from genetic instability and usually cause growth inhibition due to the ‘protein burden' resulting from their overexpression, which makes them poorly adapted for industrial applications, and raises fundamental questions about the way these synthetic operons can be to stably integrate into the host cell’s genome to eventually become a full constituent of natural cellular physiology and satisfy at the same time the ‘non-natural’ objectives.
The purpose of SYNPATHIC is to provide solutions to these two major bottlenecks, enabling major advances in the field of Synthetic Biology by (i) optimizing the catalytic efficiency of individual steps in pathways, (ii) optimizing the flux through whole pathways, and (iii) simultaneously adapting the microbial cell factory to a novel ‘non-natural’ objective. These solutions will be exemplified by optimizing the pathways for DHB and PDO production from glucose. The first scientific and technical hurdle will be tackled using innovative ultrahigh-throughput screening systems for directed evolution of ‘rate-limiting’ enzymes in pathways using droplet-based microfluidics. This technology, pioneered by Partner 2, uses monodisperse aqueous droplets in a continuous oil phase as independent bio-compatible microreactors for ultrahigh-throughput biological assays. Microorganisms/genes can be screened with 1,000-fold increase in speed and a 1,000,000-fold reduction in volume (and cost) compared to screening in microtitre plates. The second bottleneck, due to genetic instability of plasmid-borne synthetic operons, will be tackled through clever genomic integration of the synthetic module. This integration will be guided by a genome design software developed by partner 3 of this consortium, based on a threefold interdependence of genome layout, DNA 3-D conformation, and co-regulation of genes functioning in common metabolic pathways. Finally, process optimization will benefit from the combination of genomic integration and droplet-based microfluidic to monitor production variability within the genome-reengineered microbial population and select for optimized producers.



Partners

CBI (ESPCI UMR 8231) Chimie, Biologie, Innovation

iSSB institute of Systems and Synthetic Biology — Genopole, UEVE, CNRS

LISBP Laboratoire d'Ingenierie des Systèmes Biologiques et procédés

ANR grant: 449 948 euros
Beginning and duration: octobre 2014 - 36 mois

 

ANR Programme: Chimie durable, génie chimique et biotechnologie (DS0304) 2014

Project ID: ANR-14-CE06-0024

Project coordinator:
Monsieur Jean Marie François (Laboratoire d'Ingenierie des Systèmes Biologiques et procédés)

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.