Molecular and Metabolic Engineering (EAD 11)

A multidisciplinar approach of molecular and metabolic engineering, from basic research to applications.

 

The I2M team covers multiple fields at the crossroad between biology, chemistry and physics focusing on molecular and metabolic engineering. The team is heavily involved in collaborative works both nationally and internationally with academic partners, but it also had a strong tradition of interacting with industrial partners. Research carried out at I2M mostly concerns synthetic biology. One aspect deals with to introduce or to even design in yeast cells metabolic pathways such as triterpenoid, steroid, flavonoid and carotenoid pathways, based both on natural enzymes but also on artificial steps. To do that, some innovating methods are set up upstream for efficient genome engineering, this engineering being both combinatorial and evolvable. Another aspect of I2M research deals with the functional engineering of enzymes aiming structure-activity relationships analysis. The constraints of protein structural dynamics are of most importance in interpreting these studies. The last aspect concerns the engineering of molecular nanocomplexes to control autoassembling of hybrid complexes composed of protein associated to DNA and to non-biological compounds (such as quantum dots).

 

 

Metabolic engineering

This research consists to transfer a metabolic pathway, taken in what natural biodiversity proposes, into a host microorganism with the constraint that the transferred pathway remains active, producing high-value molecules. The different ongoing projects are both applied (industrial production in yeast of steroid hormones, activated vitamin D, drug metabolites, etc.) but also quite fundamental in that I2M scrutinizes the plasticity of branched pathways and the unavoidable crosstalk occurring between the introduced heterologous pathway and the endogenous pathways of the host. The techniques to introduce heterologous metabolic pathways in yeast cells led us to develop upstream new innovative techniques of genome engineering.

More specifically, we tackle:

  • engineering cellular compartments in yeast for metabolic pathways that are spread in different cell compartment, this aspect being critical for the production of highly lipophilic chemicals;
  • controlling the branched points between the introduced metabolic pathway and all endogenous enzymatic activities together with controlling the alternative metabolic flux occurring;
  • controlling the parasite endogenous activities of the host and their effect on the efficiency of the flux within the introduced heterologous pathway;
  • controlling the putative toxicity and the possible genetic interferences of the introduced pathway on yeast viability.

Engineering an artificial metabolic pathway in the host triggers physiological constraints which are frequently balanced by spontaneous genetic and metabolic adaptive changes. A multiscale understanding of these phenomena requires using the different “omics” tools in an effort that associates resources of synthetic biology to those of system biology.

Prior to these analysis, a powerful metabolic engineering should take advantage of combinatorial approaches for choosing alternative enzymes in metabolic pathway that are only partially described, or for choosing the most efficient relative expression levels of the enzymes at each step in order to avoid bottleneck(s) or metabolic shortcuts. The team developed new engineering tools based on combinatorial and evolving approaches of metabolic engineering based on genome engineering techniques quite similar in their principles to the techniques of directed molecular evolution. The object evolved being not a protein, as in molecular evolution, but part of a metabolic pathway. A first approach uses libraries of host microorganisms expressing each a combinatorial random association of some metabolic steps reconstituting a part of a metabolism. The association of metabolic steps expressed is different from one host to the next in the library. The second approach consists to use a genome shuffling controlled both in nature and position, enabling evolution and functional optimization of the best candidates found in the first approach.

 

Engineering of proteins and of complexes

The I2M team is also well known for its know-how developed in the field of protein engineering, especially membrane oxidoreduction proteins. Historically, most work was devoted to understand the flexibility and the modes of discrimination for substrates in cytochromes P450.

We developed innovative approach of functional mapping of both libraries of combinatorial P450 mosaic variants obtained by in vivo recombination of their coding sequences, and combinatorial libraries of substrates. This combinatorial approach allows interpretation of classes of variation in substrate specificities in terms of variations of amino acid sequences combinations. The purpose of these studies is to determine any predictive rule suitable to semi-rational engineering of enzymatic functions. Another type of protein engineering developed at I2M is the analysis of how the domain dynamics of a multi-domain redox protein may control electron transfers. The presence in the same protein of both relatively rigid structural elements (catalytic core, structural blocks) and of rather mobile elements (loops, substrate channels, hinge domains) is crucial for functional diversity. By using the observed coevolution in gene families and structural modeling of the interface between domains, a rationale design of sequence elements to exchange can be proposed. The objective is to understand how domain mobility might control electron transfer rates. This analysis relies on several biophysical techniques such as FRET, RMN, SAXS and modeling.

The I2M team also engineers artificial molecular complexes for building functional molecular nanotools by using auto-assembled templates of DNA covalently bound to small reporter protein to spatially organize the proteins in regular motives. More recently, we investigated associative proteins that serves as a scaffold for non-covalent association of several enzymes based on highly affine protein-protein association. In an approach of synthetic biology, we used some of the cellulosome proteins to engineer the system of integrity maintenance in eukaryotic genomes.