Engineering genetic and metabolic pathways of rare bacteria and micro-organisms dedicated to bio-production
The synthetic biology actively contributes to the identification compounds of interest notably via the elucidation of genetic circuits involved in their production. Once a compound has been identified with a given antimicrobial or biological activity, it needs to be produced for purification, characterization and future use. Genetic engineering aims to rewire the bacterial metabolism in order to enhance the production of the compound. If the strain turns out to be non-genetically editable, compound biosynthesis pathways are transferred in organisms specialized in bio-production, called chassis. . During evolution, each bacteria has acquired a robust and stable metabolic network perfectly adapted to its own environment. Thus, 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) or genes transfers into chassis strain.
Selection of a chassis strain for compound production
The choice of a chassis strain is guided by its evolutionary proximity to the bacterial species in which the compound of interest was identified. It is indeed well known that expression units of gene clusters are better expressed in evolutionarily close species due to the resemblance. 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.
Engineering of the genetic and metabolic circuits
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 circuits 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. These genetic modifications are crucial at later steps in the development of the compound to improve its production at a large scale and the stability of the producer strain. In addition, additional modifications may need to be introduced to bypass toxicities generated by the production of new molecules.
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 co-developed a custom-made computer-aided design software (CAD4Bio®) based on an enriched genetic database (biobricks, genomes and molecular pathways). The software assists the design of relevant genetic constructions 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 is now able to produce on average 1,000 original bacterial strains per month.
Design and 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 Escherichia 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 requires specific reporter strains to highlight biological and antimicrobial activities; 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 is 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.