JCJC SVSE 5 - JCJC - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

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

SYNBIOMOLL

SYNBIOMOLL aims at extending Synthetic Biology key technologies to 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 is our ability to extend the genome transplantation technology to other microbial species. This project could therefore lead to a better comprehension of the biology of pathogenic bacteria and the development of new vaccines.

Extend genome transplantation methods to bacterial species with higher biotechnological, agronomic or medical interest than the pair (Mmc/Mcap) used at JCVI.

SYNBIOMOLL is a project within the scope of synthetic biology (SB) that aims at building new biological systems. Some of the recent SB achievements include the synthesis of whole bacterial genome from chemically synthesized oligonucleotides, the genome assembly and engineering in yeast and the whole genome transplantation. However, the impact of the initial studies performed with two related bacteria of the class Mollicutes (mycoplasmas) depends on our ability to extend these techniques to other bacteria that are significant for biotechnology, agronomy or medicine. Extending the techniques of genome transplantation to other bacteria is considered in the field as a technological bolt; until now, these techniques have been restricted to a small number of laboratories using the same pair of mycoplasmas as genome donor and recipient cell. <br />In this project, we propose to improve our understanding of genome transplantation mechanisms using Mycoplasma capricolum subsp. capricolum (Mcap) as a recipient cell. Optimization of transplantation methods using phylogenetically more distant genomes will facilitate the identification of key elements involved in the compatibility between the recipient cell and heterologous genomes. The comprehension of the genetic factors controlling this compatibility will be the first step toward the design of genetically optimized donor genomes for their successful transplantation into a more “universal” recipient cell. In addition, expansion of genome transplantation and genome engineering methods to Acholeplasma laidlawii, an organism using the universal genetic code unlike mycoplamas, will allow us to transfer this technology to other bacteria of interest. This objective will be greatly facilitated by the fundamental knowledge previously gathered with mycoplasmas.

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 modificati

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.

Project coordinator

Madame Carole Lartigue-Prat (UMR Biologie et Pathologie du Fruit)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

UMR BFP-INRA UMR Biologie et Pathologie du Fruit

Help of the ANR 299,965 euros
Beginning and duration of the scientific project: December 2013 - 42 Months

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