Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated <i>Flavobacteriia</i> and <i>Gammaproteobacteria</i>

Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two “plurigenomic” (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the “great screen anomaly”. Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon “plurigenomic” library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes

Prevotella Buccae Bacteremia and Febrile Neutropenia: Report of one Case

An increasing number of anaerobic bacteremias of oral origin have been described during the recent years as a cause of fever in neutropenic cancer patients. In this paper, we describe a case of Prevotella buccae bacteremia, the first one to our knowledge, emphasizing thus the importance of considering the oral commensals when selecting antimicrobial regimens

Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions

Background: The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. Results: The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL-1) within 24 h as compared to the mono-copy strain. Conclusions: The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica. © 2019 The Author(s)

Strategies at Bioreactor Scale for the Production of Recombinant Proteins in Yarrowia lipolytica

Recombinant protein production represents a multibillion-dollar market. Therefore, it constitutes an important research field both in academia and industry. The use of yeast as cell factory presents several advantages such as ease of genetic manipulation, growth at high cell density, and possibility of posttranslational modifications. Yarrowia lipolytica is considered as one of the most attractive hosts due to its ability to metabolize raw substrate, to express genes at high level, and to secrete protein in large amounts. In the recent years, several reviews were dedicated to genetic tools developed for this purpose. Although the construction of efficient cell factory for recombinant protein synthesis is important, the development of an efficient process for protein production constitutes an equally vital aspect. Indeed, a sports car could not drive fast on a gravel road. The aim of this review is to provide a comprehensive snapshot of process tools to consider for recombinant protein production in bioreactor using Y. lipolytica as a cell factory, in order to facilitate the decision-making for future strain and process engineering