Despite the growing number of chemicals successfully engineered in host organisms, bioproduction R&D is slow and expensive, as the process is mostly based on trial-and-error. To overcome this critical hindrance, we propose to implement a generic automated design-build-test and learn cyclic pipeline for the production of targeted chemicals. As an illustration, we will apply the pipeline for the metabolic engineering of a library of new antimicrobials against Gram-positive bacteria. The pipeline comprises state-of-the-art bioproduction pathway design tools, robotized strain engineering, and high throughput product quantification via biosensors. The whole process is driven by an original computational machine learning component that determines the next set of constructions that needs to be processed by the pipeline with the goal of increasing product yield. In the specific approach we will be using, named active learning, a growing training set of experimental results is acquired on the fly in an iterative process between learning and measurements. The remarkable advantage of active learning is to yield performances comparable to classical machine learning with training sets sizes that can be several orders of magnitude smaller. Active learning can thus drastically reduce the cost of performing measurements, and in the present application significantly reduce the number of iterations for strain optimization. We propose to apply the pipeline for the production of nutritional and antimicrobial flavonoids. Precisely, the pipeline will be run for four research objectives that complement each other: (RO1) to learn enzyme sequences that maximize flavonoid titers, (RO2) to determine enzyme expression levels limiting intermediates accumulation and increasing final product yields, (RO3) to regulate the expression of the genes of the host strain to optimize both growth and flavonoid titers, and (RO4) to produce novel flavonoid structures with maximal toxicity against Gram-positive bacteria. While moving toward optimizing strains and producing novel flavonoids, our project will offer a technological rupture to industrial biotechnology where machine learning is driving experimental implementation and measurement. We anticipate this innovative solution will bring tremendous gains in throughput and speed. The project will be illustrated with the production of a library of flavonoids, but the design-build-test-learn pipeline is general enough to be applied to other molecules of interest to the health, food, chemistry and energy industrial sectors, including commodity chemicals, and fine and specialty chemicals. Our approach could for instance be extended to other pharmaceutical applications beyond the search for antimicrobial activity, as long as there exists a screening method relevant to the problem. Beyond small molecule bioproduction a similar pipeline could also be implemented to metabolize alternative but commercially attractive feedstock and to develop biosensors for environmental pollutants. The expertise gained in the project will drastically improve our SME partner strain development platform and in return the SME partner will bring the technology to the market seeking for industrial collaborations through a specific exploitation task. While we plan to release our computational methods to the academic community through web services, for specific applications, our know-how and software products will be packaged in an integrated pipeline and commercialized as a service. We foresee large industrial groups will want to customize development of the pipeline for their own application. The service we will provide to the industry will generate revenues and will also be a source for job creation.

The future challenge in animal production will be to provide food to a growing human population by respecting a balance between quality products, consumer acceptance and safety, as well as animal welfare. In a perspective of safe and sustainable food systems, reducing the use of antibiotics in livestock is a major concern. In fact, antibiotic resistance is one of the major medical challenges of the 21st century. The transfer of genes conferring resistance through the environment and the food chain, the potential for development of resistant bacteria and the appearance of therapeutic failures in human medicine, notably due to zoonotic bacteria, constitute major health issues for livestock farming sectors. In the pig breeding industry, the weaning period is often accompanied by a decreased growth rate caused by disparate food intake and diarrhoea due to digestive disorders that might be associated with bacterial population disequilibrium (i.e. dysbiosis) and/or opportunistic intestinal infections. Alarmingly, during this transition period the prophylactic use of antibiotics is still very frequent in order to limit piglet morbidity and mortality. Thus, reducing the prophylactic use of antibiotics in weaning pigs is a main issue and there is a strong need for alternatives. In this context, we have built a public-private partnership that gathers INRA scientists and industries from economic sectors of both animal feeding and pig breeding. PigletBiota is a precompetitive project that will study the physiological and genetic bases of the piglet sensitivity at weaning, as a prerequisite to identify innovative actions to adapt animals and pig production systems to a reduction of antibiotic use. The global aim of the PIGLETBIOTA project is to develop research that will contribute to adapt pig production systems to a reduction of antibiotics. The project proposes an integrative biology approach to determine the main factors influencing the variability of the individual’s robustness at weaning. We will monitor piglets for health, immune, stress and zootechnical traits and will characterize the intestinal microbiota diversity and composition as well as the contribution of host’s genotypes. The experimental design will combine various environments, including experimental and commercial farms, and ages at weaning and all animals will be fed without antibiotics. Animals (n~1000) will be clinically surveyed, measured for various traits related to production, immunity and stress, and genotyped with high-density SNP chips. The genetic parameters of the sensitivity at weaning will be estimated and genetic association studies performed. Faecal samples before and after the weaning date will be collected for characterizing the dynamics of the gut microbiota and studying its influence on the individual sensitivity at weaning. Animal and microbiota data will be vertically integrated in order to better understand the interplay between the these two levels of this biological system, and to develop robust indicators of weaning sensitivity. Finally, a functional screening using INRA platforms dedicated to human studies will be performed in order to detect active molecules to be tested in vivo and by using an axenic pigs model. The PigletBiota public-private consortium will favor translational research and innovation.

Anti-UV compounds represent a huge market in the cosmetics industry for their capacity to protect humans from sun damages. Unfortunately, current anti-UVs are being criticized for their toxicity towards the endocrine system of human, animals and fishes, and for their allergenicity; replacing them is thus a public health and environment preservation matter. Moreover, being fossil-based, these molecules are very difficult to degrade in the environment and their cost is extremely fluctuant. There is thus a high industrial/societal demand for cutting-edge technologies enabling the production of renewable and safer alternatives. Nevertheless, to achieve this, one must overcome the following major hurdles: (1) availability of natural raw materials at low cost and large volumes, (2) highly selective and efficient (no/limited wastes, high yields and purity), sustainable, safe and cost-effective production process, (3) anti-UVs must exhibit complimentary biological activities as the marketing approval for compounds exhibiting only anti-UV activity is a complex, lengthy and costly procedure, (4) molecules must not exhibit any toxicity for the consumers, and be environmentally friendly, (5) molecules must (i) possess the required physico-chemical properties to be efficiently incorporated in the cosmetic formulation (e.g., hydrophilic-lipophilic balance (HLB)), and (ii) must be photostable for a period of time compatible with the destination of the cosmetic. SINAPUV builds on the pioneer work of Chaire ABI and its partners from Purdue and Warwick universities that demonstrated that naturally occurring sinapoyl malate exhibits promising high anti-UV activity due to a very peculiar mechanism allowing the absorption of all wavelengths within the UV-B range. Unfortunately, sinapoyl malate extraction from plants is not feasible as it is present in very small quantities. SINAPUV is precisely designed to overcome not only this supply issue but also the hurdles described above by proposing a sustainable integrated approach that aims at producing biobased sinapoyl malate analogs directly from sugars and identifying the ones able to advantageously replace criticized commercial anti-UVs. To achieve this ambitious objective, the project relies on the simultaneous investigation of (1) a synthetic biology strategy for the engineering of microorganisms (bacteria and yeast) capable of producing two chemical intermediates - sinapic acid and sinapoyl malate - from carbohydrates, (2) the development of an integrated microbial production using the engineered strains and agro-industrial byproducts as fermentation medium, and purification processes allowing the production intensification of the intermediates, (3) the development of sustainable (chemo-)enzymatic pathways to sinapoyl malate analogs with tunable HLB from the previous intermediates, (3) the study of their spectral/biological properties both at the molecular and at the formulation level, (4) the determination of the toxicity of the most promising analogs, and (5) a life cycle assessment allowing the most durable analogs and integrated process(es) to be identified. Finally, the analogs exhibiting the highest anti-UV/biological activities and the lowest toxicity will be validated as proof of concept before their industrialization. To rise to this challenge, academic actors and industrials have decided to build a private public partnership gathering all the scientific and industrial expertise required to fully address this multidisciplinary project. Internationally recognized French academic laboratories in synthetic biology (MICALIS), fermentation/downstream processing/green chemistry/LCA (Chaire ABI), and endocrine disruption (HSC) will work hand in hand with a start-up specialized in the construction of industry compliant genetically engineered micro-organisms (Abolis) and a world-leading company specialized in the production of biobased cosmetic ingredients (Givaudan Active Beauty).

Lactic acid bacteria (LAB)-infecting bacteriophages (phages, or bacterial viruses) use diverse host-binding mechanisms, yet the overall picture of the interactions between LAB phages and their host remains incomplete. Unraveling the molecular details of phage-LAB interactions is essential not only for decoding phage biology, but also for industrial and public health purposes since LAB are important micro-organisms in food fermentations and in the human gut microbiota. Phages infecting the LAB species Lactococcus lactis and Streptococcus thermophilus have enjoyed extensive scientific scrutiny since they may disrupt LAB-dependent processes in dairy plants with serious concomitant economic losses. In contrast, there is a significant knowledge gap on the interactions between plant-associated LAB and their phage, even though they may also significantly impact fermentation processes. This is true for fermented beverages as exemplified by the emblematic field of winemaking that heavily relies on the LAB species Oenococcus oeni. Recently, we have shown that representative phages that infect O. oeni possess host-binding devices of distinct composition and morphology, and being different from those of lactococcal and streptococcal phages, that likely employ novel host-binding mechanisms. Moreover, we have observed that wine polyphenolic compounds (PCs), which are abundant in the O. oeni ecological niche, can interfere with the phage infection process. These organic compounds, being sterically similar to cell surface saccharides recognized by phages, may occupy phage receptor-binding sites, thereby preventing host binding. Moreover, PCs could also induce modifications in the cell wall saccharide composition, which would also prevent phages from binding to their host. In this context, our overall aim is to unravel molecular interactions between the wine LAB O. oeni, their viral predators, and PCs. We will work on three representative oenophages, all of which infect the same strain but use different host-binding devices differently affected by wine PCs. We will leverage complementary approaches covering the fields of structural biology, biochemistry, and microbiology, to meet our stated aim. We will 1) determine structure-function relationships of distinct host-binding devices combining cryo-electron microscopy, X-ray crystallography, biophysical characterization of protein-ligand interactions, and host cell-binding assays, 2) explore host-binding capabilities of these phages and the impact of PCs combining phenotypic analyses (generation of bacteriophage-insensitive mutants, phage plaque assays, adsorption tests) and comparative genomics, and 3) map phage-specific host cell saccharide receptors and examine the potential effects of PCs on the synthesis of these receptors through the analysis of gene expression, cell wall biochemical composition, and chemical structure of surface polysaccharides. Investigating molecular interactions between the wine LAB O. oeni and its phages, as a model system of the interactions between plant-related LABs and their phages, will significantly advance current knowledge of phage biology and structure, while simultaneously defining the role and potential inhibitory action of plant PCs on phage infection. We will produce important knowledge of LAB-phage interactions with expected high gains for the wine industry, as well as other plant-fermented products. Of note, plant-based fermented products are currently one of the most innovative and dynamic food categories, in response to the increasing popularity of vegetarian and vegan diets in western countries. Lastly, addressing the role of plant PCs on phage-host interactions may also lead to a better understanding of gut microbiota dynamics and to the rational development of ‘green’ phage-based biocontrol strategies, thereby opening perspectives in the socio-economically important fields of human health and agriculture.

Clostridioides difficile, an anaerobic Gram-positive spore-forming bacterium, is responsible for a wide spectrum of infections ranging from diarrhea to life-threatening pseudomembranous colitis. The use of antibiotic therapy raises concerns about the selection of antibiotic-resistant bacteria at hospitals. New therapeutic targets should be investigated as alternatives to antibiotic treatments. Polysaccharides (PS) biosynthesis enzymes are encoded in a PS locus where most genes are essential for bacterial viability. We propose therefore the enzymes involved in PS biosynthesis as new therapeutic targets. Moreover, vaccines currently under development target the toxins and may not prevent C. difficile colonization and dissemination. We also propose in this project to evaluate PSII and/or LTA as vaccine component(s) of a vaccine that may prevent C. difficile colonization and dissemination. To that aim, the project will (i) define if either one or both PSII and LTA are essential for bacterial viability, ii) identify specific enzymes involved in PSII or LTA biosynthesis, (iii) target them with inhibitors and (iv) test PSII and LTA as vaccine candidates using an innovative approach. We recently developed a new genetic conditional lethal mutant system and have already obtained antibodies directed against PSII. Using these tools, we showed that the PSII seems to be essential for bacterial viability. The project will be divided into three tasks. The first will determine whether PSII, its anchoring and/or LTA are essential and define at least two enzymes as new therapeutic targets, one of each involved in each PS biosynthesis. The second task will look for inhibitors able to target these specific enzymes, using in silico models and a chemistry approach. The last task will test PSII and LTA as vaccine candidates. This project aims to combat C. difficile infections and prevent them using vaccination.