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International audience; 3-Hydroxypropanoic acid (3-HP) is an important biomass-derivable platform chemical that can be converted into a number of industrially relevant compounds. There have been several attempts to produce 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from K. pneumoniae. Genetic engineering, driven by in silico optimization, and optimization of cultivation conditions resulted in a 3-HP titer of 10 g/L, in a standard batch cultivation. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis. With this relatively high titer in batch, and the robustness of B. subtilis in high density fermentation conditions, we expect that our production strains may constitute a solid basis for commercial production of 3-HP.

International audience; The aim of this study was to determine the probiotic potential of autochthonous oral lactobacilli. For this, 66 strains were screened for antibacterial activity against two cariogenic strains (Streptococcus mutans and Actinomyces viscosus) and two periodontopathogenic strains (Fusobacterium nucleatum and Porphyromonas gingivalis). The inhibitory activity was investigated with the agar overlay technique. Positive results led us to explore some mechanisms of action. The ability to produce H(2)O(2) and the glycerol dehydratase gene were searched among all the strains. The gassericin A gene was checked among the Lactobacillus gasseri. All the tested strains inhibited S. mutans and A. viscosus; only one did not inhibited F. nucleatum and 52 strains inhibited slightly the growth of P. gingivalis. No inactivation of antibacterial activity was observed after treatment with proteinase K. The gene of the gassericin A was not found in any strain. Only one strain showed a 275-bp amplicon corresponding to the Glycerol Dehydratase gene. This strain has been identified by DNA 16S sequencing as a L. gasseri. Among the 66 tested strains, 7 produced hydrogen peroxide. Our findings suggest that in addition to the previous results, some of the autochthonous oral lactobacilli tested could be considered as suitable probiotics.

International audience; Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 °C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Detection of lactic acid bacteria producing 3-hydroxypropionaldehyde (acrolein precursor) from glycerol by molecular tests. Glycerol, one of the major product of yeasts metabolism during cider and wine alcoholic fermentation is important for sensorial quality of fermented bever- ages. Some lactic acid bacteria convert glycerol to 3-hydroxypropionaldehyde by glycerol dehy- dratase. This reaction originates acrolein which produces bitter compounds by combination with tannins. Thirty nine strains of lactic acid bacteria were isolated from spoiled ciders where glycerol was totally degraded. Lactobacillus collinoideswas the dominant isolated species, Lactobacillus hilgar- dii and Lactobacillus yamanashiensis (Lb . mali) were also identified. Glycerol dehydratase activity was shown. Two oligonucleotide primers (GD1 and GD2) were chosen in the most conserved encod- ing region of one of the glycerol dehydratase subunit of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella typhimuriumand Clostridium pasteurianum. The primers led to a 279 bp amplicon (GD) in PCR amplification with the genomic DNA of Lb. collinoides IOEB 9527 as template. The amino acid sequence deduced from the amplicon nucleotide sequence showed a very high similarity with the glycerol dehydratase genes of Gram negative and Cl. pasteurianum species. PCR using GD1 and GD2 primers, only revealed Lb. collinoides strains and Lb. hilgardii strains, which degrade glycerol. The amplified fragment was used as DNA probe in dot-blot hybridiza- tion with the genomic DNA of all the isolated strains from ciders. Only glycerol-degrading strains hybridized. The same probe allowed to isolate glycerol-degrading lactic acid bacteria strains from wine by colony hybridization. Moreover with GD1 and GD2 a 279 bp fragment was also amplified from genomic DNA of those wine strains. Some were Lb. hilgardii strains others could not be identified yet. lactic acid bacteria / glycerol / acrolein

Summary: Continuous cultures ofEnterobacter agglomeransCNCM 1210 were performed under regulated pH conditions (pH 7.0) with glycerol or glucose (20 gl−1) as carbon source. Cultures grown on glucose produced mainly acetate, ethanol and formate. In contrast, 1,3-propanediol (PPD) was the main product with glycerol. The carbon flow distribution at branching metabolic points was investigated. Higher PPD yields with increased dilution rate were correlated with an important increase in the relative ratio of glycerol dehydratase to glycerol dehydrogenase. Determination of intracellular triose-phosphate and fructose 1,6-biphosphate concentrations demonstrated that glyceraldehyde-3-phosphate dehydrogenase is the limiting step in glycerol dissimilation. At the pyruvate branching point, pyruvate dehydrogenase (PDH) activity was systematically detected. The pyruvate flow shifted to PDH is suspected to represent up to 22% of the acetyl-CoA formed. In addition, this enzyme pattern combined with the enhancedin vivolactate dehydrogenase activity at high growth rates, was correlated with a decrease in the pyruvate formate-lyase activity. A regulation of this latter enzyme by the accumulation of triose-phosphate is suspected.

Glycerol fermentation by Enterobacter agglomerans revealed that both growth and 1,3-propanediol production ceased after consumption of about 430 mM glycerol, irrespective of the initial glycerol content. This phenomenon was assigned to the production of 3-hydroxypropionaldehyde, which was identified by proton nuclear magnetic resonance and which showed a bacteriostatic effect. The accumulation during glycerol fermentation was also observed with two other enterobacterial species, i.e., Klebsiella pneumoniae and Citrobacter freundii.