Next steps in Synthetic Biology: towards the generalization of genome transplantation methods – SYNBIOMOLL

SB methods were initially developed with mycoplasmas (Mcap and Mmc) that have small genomes and use UGA as stop codon. Therefore, they now need to be adapted to other bacteria to become widely useful for both fundamental and applied research.
First, we will apply genome transplantation technology to the Spiroplasma group using Mcap as recipient cell and different members of Spiroplasma group as a source of donor genomes. We will test the importance of the phylogenetic distance between donor and recipient genomes to determine the limit of compatibility in this system. Once this limit determined, the most distant transplantable genome and the closest non-transplantable genome will be cloned in yeast in order to be modified using available yeast genetic tools. Genetic factors involved in the compatibility phenomena (as the origin of replication or genes involved in transcription) will be exchanged between these two genomes and the impact of these modifications on transplantation efficiency will be evaluated. Advanced knowledge on key genetic determinants involved in the transplantation process will help us to engineer a new Mcap recipient cell with a wider capacity to receive and boot-up foreign genomes.
Secondly, the transfer of genome transplantation technology within the Acholeplasma/Phytoplasma phylogenetic group will be developed and optimized using well-characterized A. laidlawii species. Unlike mycoplasmas, A. laidlawii presents features shared by most common bacteria of biotechnical interest (larger genomes and standard genetic code). Similarly to what will be done with Mcap recipient cell, the tolerance of A. laidlawii recipient cell to accept the genome of more and more distant Acholeplasma species will be studied. This will be extended up to test A. laidlawii recipient cell for its capacity to replicate and express in-yeast part of the genomes of different phytoplasmas species.

We primarily focused our attention on the degree of relatedness necessary between a donor cell and a recipient cell using Mcap as a recipient cell. Seven different donor genomes with increasing phylogenetic distance from the recipient cell, all belonging to the Spiroplasma phylogenetic group, were engineered and cloned into the yeast S. cerevisiae. Once these genomes were constructed, we tested their compatibility with Mcap during genome transplantation experiments using both isolated chromosomes (from-bacteria transplantation) and genomes cloned into yeast (from-yeast transplantation). We observed a direct correlation between the transplantation efficiency and the phylogenetic distance. The closest a donor genome was from the recipient cell, the highest was the transplantation efficiency. We succeeded to transplant all genomes tested up to Mesoplasma florum (M. florum). We thereby established that the transplantation limit in this system was located between M. florum and spiroplasmas genomes for both from-bacteria and from-yeast transplantations. This implies that we have now a fully operational platform for genetic engineering of all Mollicutes tested in this study except spiroplasmas. The only caveat encountered concerns Mycoplasma mycoides subsp. mycoides (Mmm). Unknown and probably genome-specific problems prevented the success of all from-yeast transplantations attempted so far.
Identification of M. florum as most-distant compatible organism is extremely interesting. Even if it is still belonging to the same phylogenetic group, it shares only ~90% of its core proteome with Mcap. This will facilitate our search for genetic factors responsible for this compatibility. Work toward this goal has already been started and promising results have been recorded using plasmids carrying optimized origin of replication (oriC) allowing a highly significant increase in their transformation efficiency in comparison to unmodified oriC.

First, this project will give to our team a leading position in the transplantation of entire bacterial genome, an highly promising aspect of SB. Our laboratory will then have an international visibility in the field of SB, which holds a great promise for the design and construction of biological systems with high potential on human and veterinary medicine, environmental protection and production of molecules with high added value. US DOE estimated that the global market of SB represented 0.5 billion euros in 2006 and should reach at least 3 billion euros in 2016.
The project is centered on the understanding of mechanisms involved into the compatibility between donor and recipient genomes. This key aspect will produce new compatible donor/recipient cell pairs, some of them being of direct interests for academic or biotechnological developments. In particular, we believe that the transfer of genome transplantation methods to the genus Acholeplasma will establish this organism as a new platform for the study of other bacteria of interest with applied and economically-relevant prospects. Acholeplasma laidlawii is of interest because it is not pathogenic and it is amenable to genetic transformation. Acholeplasmas are closely related to low G+C Gram-positive bacteria, some of them (lactic acid bacteria and related species) being used in agro-industry because they are «Generally Recognized As Safe» as defined by the FDA. Therefore the success of genome transplantation in A. laidlawii would be a key step towards sound biotechnology applications.
Furthermore, acholeplasmas by themselves could also be recognized as a suitable platform for the production of molecules with high value-added: these bacteria have a reduced metabolism without having the high requirements of mycoplasmas for their growth. Therefore, it is possible that the addition of new pathways in these simple organisms would not lead to perturbations observed in more complex organisms.

This project started early 2014 allow us to have one publication already accepted:
- C. Lartigue, A. Lebaudy, A. Blanchard, B. El Yacoubi, S. Rose, H. Grosjean, and S. Douthwaite. 2014. The flavoprotein Mcap0476 (RlmFO) catalyzes m5U1939 modification in Mycoplasma capricolum 23S rRNA. Nucleic Acids Res. 42 (12) :8073-8082.
In addition, recent developments on (i) the importance of some genetic factors (phylogenetic distance and the origin of replication of bacterial chromosomes) between donor and recipient genomes and (ii) our new capacity to extend genome transplantation to the model organism Mesoplasma florum using Mcap as a recipient cell will lead to the publication of two new scientific papers in highly-ranked journals in our field.

- F. Labroussaa, A. Lebaudy, G. Gourgues, V. Baby, D. Matteau, S. Rodrigue, A. Blanchard, S. Vashee, P. Sirand-Pugnet and C. Lartigue. Extending genome transplantation knowledge in bacterial cells. In preparation.
- F. Labroussaa, V. Baby, G. Gourgues, D. Matteau, C. Lartigue and S. Rodrigue. In-yeast cloning and genome transplantation of modified Mesoplasma organisms. In preparation.

This project and the results gathered so far have been presented in two international congresses, one in Toulouse (Biosynsys, July 2-4 2014) and the other one in Blumenau (Brazil) during the International Congress of Mycoplasmology (June 1-6, 2014).

Recently, new approaches referred to as Synthetic Biology (SB) have emerged. The work done at the J. C. Venter Institute (JCVI) has significantly contributed to the development of SB by showing: (1) the synthesis of complete bacterial genomes starting from chemically produced oligonucleotides, (2) the genome-scale engineering using yeast as a platform, (3) the transplantation of isolated bacterial genomes to other bacteria or from yeast to bacteria. Mycoplasma capricolum subsp. capricolum (Mcap) and Mycoplasma mycoides subsp. capri (Mmc), which are related species, were used as recipient cell and donor genome, respectively.
Mycoplasmas belong to the class Mollicutes, a group of wall-less bacteria phylogenetically related to low G+C% Firmicutes. Mycoplasmas are characterized by the use of UGA as a tryptophan codon and a small genome. Mollicutes include several pathogens affecting humans, animals and plants. One of the limiting factors to better understand the molecular basis of their pathogenicity is the lack of efficient genetic tools.
The goal of SYNBIOMOLL is to extend Synthetic Biology key technologies to other Mollicutes species of interest. Because several bacterial genomes have already been cloned in yeast and the synthetic assembly of large DNA fragments is in current use, the major technological limitation to a much wider use of these SB tools is our ability to extend the genome transplantation technology to other microbial species.
In task 2 (task 1 is the coordination of the project), we propose to study the mechanisms of genome transplantation by evaluating the degree of relatedness necessary between a donor cell and a recipient cell and also by identifying some of the genetic factors responsible for this compatibility. First, we will transplant in Mcap recipient cell the genomes from species with increasing phylogenetic distance from the recipient cell to identify the transplantation compatibility limit. Then, using a troubleshooting approach, we will determine whether non-compatibility is a problem of replication and/or expression of the donor genome in the recipient cell. Specific genome regions, including the chromosomal replication origin, will be exchanged between Mcap and a non-compatible genome to evaluate the impact on transplantation. As a control, the same exchanges will be performed with a compatible genome. For this sub-task, genomes will be cloned into yeast to be modified accordingly. The expected results should help identifying trans-acting proteins and/or cis-acting sequences required to boot-up heterologous genomes into recipient cells.
In task 3, we plan to develop genome transplantation in another phylogenetic group using Acholeplasma laidlawii (AL) as a recipient cell. AL can grow in much simpler culture media than mycoplasmas and is considered as an intermediate species between mycoplasmas and low G+C% firmicutes. Genome transplantations will first be performed with homologous donor genome and then with heterologous donor genomes from two other acholeplasmas. Once the conditions of genome transplantation in AL optimized, specific developments will be pursued to evaluate its capacities to replicate and express the genome of more distant non-cultivable, insect-transmitted plant-pathogenic phytoplasmas. The impossibility to cultivate these bacteria is a major barrier to their study and the development of plant protection strategies. In the SYNBIOMOLL project, we will evaluate AL ability to recognize and initiate replication of phytoplasma chromosomes using phytoplasma oriC plasmids and by replacing the oriC of AL with that of the phytoplasma. We will also use proteomic analyses to evaluate the expression of phytoplasma genes in AL.
Succeeding genome transplantation in AL would be important per se but also would serve as a launching pad for the culture and the study of non-cultivable phytoplasmas. At the end of SYNBIOMOLL, Carole Lartigue intends to propose this project for an ERC Consolidator Grants.