MS-AFM-IR platform for structural analysis of lipid inclusions in oleaginous micro-organisms – BioSound-IR

Biodiesel is an interesting alternative renewable energy source with numerous environmental benefits compared to fossil fuels: it is carbon-neutral over its lifetime and is far less toxic than the petro diesel derivatives. Bio-fuel could be an alternative way to reduce the dependency on mineral oil whose prices and provision strongly depends on political situations and climatic environment etc… Nowadays bio-diesel is mostly produced by the trans-esterification of the TriAcylGlycerols (TAG) present in the seeds of oleaginous plants, yielding glycerol and fatty acid methyl esters (bio-diesel). However this producing route is being criticized since it mobilizes agricultural land that could be used to produce human or animal food. As an alternative, a world-wide interest turned toward microorganisms that could be engineered to make biofuels at an industrial scale (ethanol, methane, oil…). Several micro-organisms were shown to have the natural abilities to store TAG in specific organelles, called the lipid bodies (fungi/yeast, algae, actinobacteria…). TAGs are direct precursors of methyl esters of fatty acids (bio-diesel) that are chemically and structurally identical to those found in commercial fuels. The engineering of oil-producing microbes to enhance their natural capacities to store oil is a very active and competitive field word-wide. The ultimate goal of these efforts is to create sustainable microbial-based industrial production of bio-diesel.
Since the bacteria Streptomyces, a well-known antibiotic producer, has been grown for decades, it was chosen as a model oleaginous micro-organism in the ProBio3 project, a project “Investment for the Futur” Biotechnology/Bioresources, besides several model strains of yeast. Another interesting feature of Streptomyces, a soil-born bacteria, is its natural ability to metabolize various wastes of the agro-industry (lignocellulose, beat pulp...) for growth and TAG production, limiting the consumption of products used for human alimentation for bio-fuel production. Furthermore, considering the intimate link existing between storage lipid metabolism and antibiotic production demonstrated by the IGM/MES team, this project would certainly also benefit to the discovery of novel antibiotics. Indeed a low TAG content is a very good indicator of high antibiotics producing strains and can be used as an efficient screening procedure of the latter.
To meets its objectives, BioSound-IR proposes a multidisciplinary approach associating expertise in microbial physiology and genetics, imaging technics, spectroscopy techniques, analytical chemistry and chemioinformatic. A specific three-dimensional (3D) acoustic tomography BioSound-IR platform coupled with the AFM-IR technique (atomic force microscope coupled with a tunable infrared laser source, patented by LCP group) has already been developed. Spontaneous Raman and fluorescence imaging have been already implemented on 3D acoustic MS-AFM tomography platform. The most appropriate imaging and spectroscopy modes will be developed in fast imaging. The Project will manage the technical improvements of microwave sensor, BioWave-Pen, to achieve fast screening of the mutants with high TAG content. Furthermore, this technique will be used to follow the consumption of key nutrients of the growth medium and the release of intracellular metabolites (over-flow metabolism) and will constitute a great support for the optimization of the fermentation conditions. TAG content as well as size and localization of lipid bodies will be studied in natural and mutants Streptomyces strains. Volumetric information about lipid pockets will be obtained in acoustic mode while the high-resolution analysis will be obtained by cryo-SEM on the platform ICB-ARCEN of the ICB/OSNC team. BioSound-IR is thus a fundamental research project likely to support the efforts of entities (including partners ProBio3) engaged in the development of alternative and profitable modes of biodiesel production.