Screening Strategies for Biosurfactant Discovery.

The isolation and screening of bacteria and fungi for the production of surface-active compounds has been the basis for the majority of the biosurfactants discovered to date. Hence, a wide variety of well-established and relatively simple methods are available for screening, mostly focused on the detection of surface or interfacial activity of the culture supernatant. However, the success of any biodiscovery effort, specifically aiming to access novelty, relies directly on the characteristics being screened for and the uniqueness of the microorganisms being screened. Therefore, given that rather few novel biosurfactant structures have been discovered during the last decade, advanced strategies are now needed to widen access to novel chemistries and properties. In addition, more modern Omics technologies should be considered to the traditional culture-based approaches for biosurfactant discovery. This chapter summarizes the screening methods and strategies typically used for the discovery of biosurfactants and highlights some of the Omics-based approaches that have resulted in the discovery of unique biosurfactants. These studies illustrate the potentially enormous diversity that has yet to be unlocked and how we can begin to tap into these biological resources.

Correction to: Novel metagenome-derived ornithine lipids identified by functional screening for biosurfactants.

The published online version contains mistake in the author list.

Novel metagenome-derived ornithine lipids identified by functional screening for biosurfactants.

Biosurfactants are amphiphilic molecules that interact with the surfaces of liquids leading to many useful applications. Most biosurfactants have been identified from cultured microbial sources, leaving a largely untapped resource of uncultured bacteria with potentially novel biosurfactant structures. To access the uncultured bacteria, a metagenomic library was constructed in Escherichia coli from environmental DNA within an E. coli, Pseudomonas putida and Streptomyces lividans shuttle vector. Phenotypic screening of the library in E. coli and P. putida by the paraffin spray assay identified a P. putida clone with biosurfactant activity. Sequence analysis and transposon mutagenesis confirmed that an ornithine acyl-ACP N-acyltransferase was responsible for the activity. Although the fosmid was not active in E. coli, overexpression of the olsB gene could be achieved under the control of the inducible T7 promoter, resulting in lyso-ornithine lipid production and biosurfactant activity in the culture supernatants. Screening for activity in more than one host increases the range of sequences that can be identified through metagenomic, since olsB would not have been identified if only E. coli had been used as a host. The potential of lyso-ornithine lipids as a biosurfactant has not been fully explored. Here, we present several biosurfactant parameters of lyso-ornithine lipid to assess its suitability for industrial application.

Reconstruction of the carnitine biosynthesis pathway from Neurospora crassa in the yeast Saccharomyces cerevisiae.

Industrial synthesis of L-carnitine is currently performed by whole-cell biotransformation of industrial waste products, mostly D-carnitine and cronobetaine, through specific bacterial species. No comparable system has been established using eukaryotic microorganisms, even though there is a significant and growing international demand for either the pure compound or carnitine-enriched consumables. In eukaryotes, including the fungus Neurospora crassa, L-carnitine is biosynthesized through a four-step metabolic conversion of trimethyllysine to L-carnitine. In contrast, the industrial yeast, Saccharomyces cerevisiae lacks the enzymes of the eukaryotic biosynthesis pathway and is unable to synthesize carnitine. This study describes the cloning of all four of the N. crassa carnitine biosynthesis genes and the reconstruction of the entire pathway in S. cerevisiae. The engineered yeast strains were able to catalyze the synthesis of L-carnitine, which was quantified using hydrophilic interaction liquid chromatography electrospray ionization mass spectrometry (HILIC-ESI-MS) analyses, from trimethyllysine. Furthermore, the yeast threonine aldolase Gly1p was shown to effectively catalyze the second step of the pathway, fulfilling the role of a serine hydroxymethyltransferase. The analyses also identified yeast enzymes that interact with the introduced pathway, including Can1p, which was identified as the yeast transporter for trimethyllysine, and the two yeast serine hydroxymethyltransferases, Shm1p and Shm2p. Together, this study opens the possibility of using an engineered, carnitine-producing yeast in various industrial applications while providing insight into possible future strategies aimed at tailoring the production capacity of such strains.

Secular trends in pediatric bloodstream infections over a 20-year period at a tertiary care hospital in Germany.

OBJECTIVE: Over the last 20 years, a number of medical innovations with impact on the incidence of bacterial and fungal bloodstream infections (BSIs) in children have been developed and implemented. Although appropriate empirical antimicrobial therapy is a prerequisite to the successful treatment of BSIs, to date, epidemiological data on long-term microbiological trends in BSIs of hospitalized children have not been available. METHODS: Two cohorts of pediatric patients who were hospitalized in a single-center tertiary care hospital in Germany over a 20-year time span (period A from 1985 to 1995 vs. period B from 1997 to 2006) were retrospectively analyzed and compared with respect to the epidemiology and microbiology of BSIs. RESULTS: A total of 1,646 cases of monomicrobial BSIs were detected. The rate of positive blood culture results dropped from 4.5% in period A to 2.0% in period B. The proportion of gram-positive vs. gram-negative pathogens recovered from blood cultures remained stable. Among gram-positive pathogens, an increase in enterococci (3.3% vs. 8.2%) and in coagulase-negative staphylococci (CoNS) (22.9 vs. 28.2%) was observed. In contrast, BSIs caused by Staphylococcus aureus (16.4% vs. 11.7%), Streptococcus agalactiae (4.9% vs. 2.1%), Haemophilus influenzae (7.3% vs. 0.7%), and Neisseria meningitidis (1.9% vs. 0.5%) diminished. In analyzing subgroups, an increase of enterococcal and CoNS infections was noted among patients with immunosuppression and neonatal early-onset sepsis (EOS), while a decrease was found among late-onset sepsis (LOS) cases with S. viridans. Notably, aminopenicillin-resistant enterococci and aminopenicillin- and fluoroquinolone-resistant Enterobacteriaceae all increased over time, while the overall resistance pattern was still favorable. The overall mortality rate of BSIs decreased (5.2% vs. 2.6%). CONCLUSIONS: Over the 20-year study period, the spectrum of specific microorganisms among BSIs shifted, with opportunistic pathogens becoming predominant. Despite an increase in the proportion of antibiotic-resistant organisms, however, the mortality rate decreased.