Engineering metabolic pathways and bacterial chassis
Once a compound has been identified with a given antimicrobial or biological activity, it needs to be produced for purification, characterization and future use. To achieve production, the synthetic biology unit uses proprietary bacterial chassis that act as microbial factories and for which a series of genetic engineering tools are available or have been developed in-house. Genetic engineering aims to rewire the metabolism of the chassis strain in order to enhance the production of the compound if it is also produced endogenously, or to endow the strain with the ability to produce a new compound. Because each bacteria has evolved a robust and stable metabolic network to adapt to its very own environment, reprogramming chassis strains to become efficient microbial factories is challenging and requires fine manipulation of the strain’s metabolism.
Identification of the genetic region responsible for an antimicrobial or biological activity
During the compound screening process and in collaboration with the data science and activity testing units, the synthetic biology works towards the identification of the genomic locus or loci responsible for the activity observed using knock-out strategies (gene inactivation).
Selection of a chassis strain for compound production
The choice of a chassis strain mainly depends on its evolutionary proximity to the bacterial species in which the compound of interest was identified. It is indeed well known that expression units (gene clusters) are better expressed in evolutionarily close species due to the resemblance in their structure, their promoters and their ribosome binding sites. Together with the biodiversity farming and data science units, the synthetic biology hub therefore defines what the best chassis is for the production of a given compound, based on available phylogenetic information and deep-sequencing data.
Determination of the pathways to engineer
In addition to introducing or modifying the gene cluster(s) responsible for the production of a compound, several other genetic modifications are required in the chassis strain to optimize its biosynthesis. First, it is crucial to understand the metabolic routes used in both the original strain and the chassis strain to define which pathways need to be modified. The synthetic biology team collaborates with the advanced analytics team to interprete the data obtained from metabolomic analyses and identify these key bricks. Second, modifications may need to be introduced to bypass toxicities generated by the production of new molecules. Finally, genetic modifications will also be crucial at later steps in the development of the compound to improve its production at a large scale.
Fully automated high-throughput strain construction
To optimise the metabolic network of a bacterial strain, the activity the target genes must be modulated simultaneously. Hundreds if not thousands of strains with different expression levels of these genes or gene clusters often need to be constructed before landing the one strain with the optimal production of the compound of interest. The synthetic biology team developed a custom made computer-aided design software (CAD4Bio) based on an enriched database of biobricks (genes, gene clusters, promoters) genomes and molecular pathways. The software designs a battery of relevant genetic constructs in silico, and operates a robot that is capable of performing large-scale DNA cloning and high-throughput strain construction. This automated system also includes a machine-learning component: using the outcomes of previous experiments, artificial intelligence predicts the feasibility and probability of success of new constructs, therefore accelerating and derisking this key development step. Thanks to this approach, the unit now produces on average 1,000 original bacterial strains per month.
Design an development of new genetic tools and bacterial micro-factories
Although a myriad of genetic approaches have been developed by researchers and developers over the past fifty years, these tools are often designed for commonly used bacterial species and usualy don’t work in rare and difficult to tame microorganisms, in which an extensive work of optimization needs to be performed before attempting genetic engineering. Because common bacteria such as E. coli are rarely the most appropriate chassis to produce metabolites of interest, the synthetic biology unit has designed and developed a set of genetic tools to support genome engineering in rare and poorly described bacteria. To date, the team has implemented efficient genetic tools for several new species and phyla across the prokaryotic phylogenetic tree, an effort that it pursues to continuously enrich the battery of technologies and of chassis strains available.
The activity testing unit sometimes requires specific reporter strains to perform certain biological activity assays; the synthetic biology hub is in charge of engineering these reporter strains.
Once a compound has been successfully produced in a chassis strain and its interest has been confirmed, it will be necessary to scale its biosynthesis up to a pre-industrial and then an industrial scale. The synthetic biology team works hand in hand with the fermentation engineering team to introduce additional genetic modifications in the strain that will be important to ensure compound production remains stable in large fermentation volumes.
Mitousis, L., Thoma, Y., & Musiol-Kroll, E. M. (2020). An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds. Antibiotics, 9(8), 494.