Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

Production of a Bacteriocin Like Protein PEG 446 from Clostridium tyrobutyricum NRRL B-67062.

Clostridium tyrobutyricum strain NRRL B-67062 was previously isolated from an ethanol production facility and shown to produce high yields of butyric acid. In addition, the cell-free supernatant of the fermentation broth from NRRL B-67062 contained antibacterial activity against certain Gram-positive bacteria. To determine the source of this antibacterial activity, we report the genome and genome mining of this strain. The complete genome of NRRL B-67062 showed one circular chromosome of 3,242,608 nucleotides, 3114 predicted coding sequences, 79 RNA genes, and a G+C content of 31.0%. Analyses of the genome data for genes potentially associated with antimicrobial features were sought after by using BAGEL-4 and anti-SMASH databases. Among the leads, a polypeptide of 66 amino acids (PEG 446) contains the DUF4177 domain, which is an uncharacterized highly conserved domain (pfam13783). The cloning and expression of the peg446 gene in Escherichia coli and Bacillus subtilis confirmed the antibacterial property against Lactococcus lactis LM 0230, Limosilactobacillus fermentum 0315-25, and Listeria innocua NRRL B-33088 by gel overlay and well diffusion assays. Molecular modeling suggested that PEG 446 contains one alpha-helix and three anti-parallel short beta-sheets. These results will aid further functional studies and facilitate simultaneously fermentative production of both butyric acid and a putative bacteriocin from agricultural waste and lignocellulosic biomass materials.

Biophysical characterization of α-glucan nanoparticles encapsulating feruloylated soy glycerides (FSG).

Water insoluble α-glucans that were enzymatically synthesized using glucansucrase that was cloned from Leuconostoc mesenteroides NRRL B-1118 were previously shown to form nanoparticles via high pressure homogenization. These α-glucan nanoparticles were previously shown capable of encapsulating a small hydrophobic molecule. This work demonstrates that the same α-glucan can be formed into nanoparticles that encapsulate feruloylated soy glycerides from modified soybean oil, a product of interest to the cosmetic and skin care industries because of the UV absorbance and antioxidant properties of the feruloyl moiety. It is demonstrated that the feruloylated soy glyceride/α-glucan nanoparticles have distinct size, zeta potential and thermal profiles from that of nanoparticles made from α-glucan alone or feruloylated soy glyceride alone. Thermal analysis also demonstrates the release of feruloylated soy glycerides from the α-glucan nanoparticles.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation.

BACKGROUND: Traditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials. RESULTS: We demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v). CONCLUSION: LysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations.

Control of bacterial contamination in bioethanol fermentation facilities has traditionally relied on chemical-based products such as hop acids and use of antibiotics. Recent emphasis on antibiotic stewardship has prompted new research into the development of alternative approaches to microbial remediation strategies. We recently described a recombinant peptidoglycan hydrolase, endolysin LysKB317, which inhibited Limosilactobacillus fermentum strains in corn mash fermentation. Here, Saccharomyces cerevisiae EBY100 was used to anchor recombinant LysKB317 using cell surface display with the a-agglutinin proteins Aga1p-Aga2p. Immunostaining and confocal fluorescence were used for localization of the extracellular interface of the cells. Yeast surface-expressed endolysin demonstrated an 83.8% decrease in bacterial cell counts compared to a 9.5% decrease in control yeast. Recombinant S. cerevisiae expressing LysKB317 used for small-scale corn mash fermentation, when infected with L. fermentum, could proactively control bacterial infection for 72 h with at least 1-log fold reduction. Analysis of fermentation products showed improved ethanol concentrations from 3.4% to at least 5.9% compared to the infection-only control and reduced levels of lactic and acetic acid from 34.7 mM to 13.8 mM and 25.5 mM to 18.1 mM, respectively. In an optimized yeast surface display system, proactive treatment of bacterial contaminants by endolysin LysKB317 can improve fermentation efficiency in the presence of L. fermentum contamination.

Description of Cohnella zeiphila sp. nov., a bacterium isolated from maize callus cultures.

A Gram-stain positive, aerobic, motile, rod-shaped bacterium designated as strain CBP-2801T was isolated as a contaminant from a culture containing maize callus in Peoria, Illinois, United States. The strain is unique relative to other Cohnella species due to its slow growth and reduced number of sole carbon sources. Phylogenetic analysis using 16S rRNA indicated that strain CBP-2801T is a Cohnella bacterium and showed the highest similarity to Cohnella xylanilytica (96.8%). Genome-based phylogeny and genomic comparisons based on average nucleotide identity confirmed the strain to be a novel species of Cohnella. Growth occurs at 15-45 °C (optimum 40 °C), pH 5-7 (optimum pH 6) and with 0-1% NaCl. The predominant fatty acids are anteiso-15:0 and 18:1 ω6c. Genome mining for secondary metabolites identified a putative biosynthetic cluster that encodes for a novel lasso peptide. In addition, this study contributes five new genome assemblies of type strains of Cohnella species, a genus with less than 30% of the type strains sequenced. The DNA G + C content is 58.7 mol %. Based on the phenotypic, phylogenetic and biochemical data strain CBP-2801T represents a novel species, for which the name Cohnella zeiphila sp. nov. is proposed. The type strain is CBP-2801T (= DSM 111598 = ATCC TSD-230).

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Recombinant bacteriophage LysKB317 endolysin mitigates Lactobacillus infection of corn mash fermentations.

BACKGROUND: Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. RESULTS: In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing 1 species of Acetobacter, 8 species of Lactobacillus, 1 species of Pediococcus, 3 species of Streptococcus, and 1 species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5-7.5 up to at least 48 h. Maximum activity was observed at 50 °C up to at least 72 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 3-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 was similar to uncontaminated controls. CONCLUSION: Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.

Inhibition of Erwinia amylovora by Bacillus nakamurai.

A variety of potential inhibitors were tested for the first time for the suppression of Erwinia amylovora, the causal agent of fire blight in apples and pears. Strain variability was evident in susceptibility to inhibitors among five independently isolated virulent strains of E. amylovora. However, most strains were susceptible to culture supernatants from strains of Bacillus spp., and particularly to the recently described species B. nakamurai. Minimal inhibitory concentrations (MICs) were 5-20% (vol/vol) of culture supernatant from B. nakamurai against all five strains of E. amylovora. Although Bacillus species have been previously reported to produce lipopeptide inhibitors of E. amylovora, matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and column chromatography indicated that the inhibitor from B. nakamurai was not a lipopeptide, but rather a novel inhibitor.

Antimicrobial properties of amylose-fatty ammonium salt inclusion complexes.

Amylose-fatty (C12-C16) ammonium salt inclusion complexes are effective antimicrobial polymers causing growth inhibition of microbes at concentrations as low as 40 µg/mL of the complex (2 µg/mL active cationic ligand). The complex was more effective at controlling microbes than the uncomplexed ligand. The complexes were found to be particularly effective at inhibiting the growth of fungi, yeast, gram (+) bacteria, and algae; its performance was affected by pH. The complexes were not hemolytic at concentrations up to 2000 µg/mL. These agents were determined to be surface active polymers and their antimicrobial mode of action may involve cell membrane thinning or disruption, causing moderate leakage. Increased ligand concentration provided increased antimicrobial activity. Solutions of amylose complexes were found to be stable, retaining their antimicrobial efficacy after autoclaving, or after storage at room temperature for 6 months. Antimicrobial amylose complexes were produced using readily available inexpensive materials via an easily scalable process.

Inhibition of Streptococcus mutans and S. sobrinus biofilms by liamocins from Aureobasidium pullulans.

The aim of this study was to determine if the novel anti-streptococcal inhibitors, liamocins, also inhibit biofilm formation by S. mutans and S. sobrinus. S. mutans strain ATCC 25175 and S. sobrinus strain ATCC 33478 were tested for biofilm formation in a rapid microtiter plate (MTP) assay and the effects of added liamocins were determined. This assay measures relative biofilm growth on pin lids. Results were verified in a biofilm flow cell assay, using hydroxyapatite-coated coupons to simulate dental material. Planktonic cultures of S. mutans and S. sobrinus were inhibited by 0.1 mg liamocins/ml. When liamocins were added after the adhesion phase in a rapid microtiter plate assay, S. mutans was inhibited 53% by 5 mg liamocins/ml, while S. sobrinus was more sensitive, showing 100% inhibition at 0.5 mg liamocins/ml. When liamocins were added during the adhesion phase, biofilms of S. mutans showed 78% inhibition at 3.0 mg liamocins/ml. In a biofilm flow cell assay, liamocins added after the adhesion phase at 0.5 mg liamocins/ml inhibited biofilms of S. sobrinus, and appeared to remove biofilms over time. Liamocins were shown for the first time to inhibit biofilm formation by S. mutans and S. sobrinus. Since liamocins are specific for Streptococcus spp., they are potential new inhibitors of oral streptococcal biofilms that should not affect normal oral microflora.

Genome Sequences of Five Lactobacillus sp. Isolates from Traditional Turkish Sourdough.

A high level of variation in microflora can be observed in profiles of lactic acid bacteria (LAB) from sourdoughs. Here, we present draft genome sequences of Lactobacillus reuteri E81, L. reuteri LR5A, L. rhamnosus LR2, L. plantarum PFC-311, and the novel Lactobacillus sp. strain PFC-70, isolated from traditional Turkish backslopped wheat sourdoughs.

Resolving bacterial contamination of fuel ethanol fermentations with beneficial bacteria - An alternative to antibiotic treatment.

Fuel ethanol fermentations are not performed under aseptic conditions and microbial contamination reduces yields and can lead to costly "stuck fermentations". Antibiotics are commonly used to combat contaminants, but these may persist in the distillers grains co-product. Among contaminants, it is known that certain strains of lactic acid bacteria are capable of causing stuck fermentations, while other strains appear to be harmless. However, it was not previously known whether or how these strains interact one with another. In this study, more than 500 harmless strains of lactic acid bacteria were tested in a model system in combination with strains that cause stuck fermentations. Among these harmless strains, a group of beneficial strains was identified that restored ethanol production to near normal levels. Such beneficial strains may serve as an alternative approach to the use of antibiotics in fuel ethanol production.

Isomelezitose formation by glucansucrases.

Several glucansucrases were surveyed for their ability to produce isomelezitose, a trisaccharide with the structure α-D-glucopyranosyl (1 â†’ 6) ß-D-fructofuranosyl (2 â†” 1) α-D-glucopyranoside. Nearly all strains tested, with one exception, produced at least trace levels of isomelezitose. Yields were low but significant, ranging from less than 1% to approximately 5% based on sucrose. This trisaccharide may arise in either of two ways: glucopyranosyl transfer to the 6Fru-OH position of sucrose, or to the anomeric OH position of isomaltulose. This study indicates that isomelezitose formation may be a general phenomenon of many glucansucrase reactions.

An integrated genomic and transcriptomic survey of mucormycosis-causing fungi.

Mucormycosis is a life-threatening infection caused by Mucorales fungi. Here we sequence 30 fungal genomes, and perform transcriptomics with three representative Rhizopus and Mucor strains and with human airway epithelial cells during fungal invasion, to reveal key host and fungal determinants contributing to pathogenesis. Analysis of the host transcriptional response to Mucorales reveals platelet-derived growth factor receptor B (PDGFRB) signaling as part of a core response to divergent pathogenic fungi; inhibition of PDGFRB reduces Mucorales-induced damage to host cells. The unique presence of CotH invasins in all invasive Mucorales, and the correlation between CotH gene copy number and clinical prevalence, are consistent with an important role for these proteins in mucormycosis pathogenesis. Our work provides insight into the evolution of this medically and economically important group of fungi, and identifies several molecular pathways that might be exploited as potential therapeutic targets.

Effect of a single point mutation on the interaction of glucans with a glucansucrase from Leuconostoc mesenteroides NRRL B-1118.

Our previous work showed that substitution of an amino acid that is coupled with the +2 subsite adjacent to the transition stabilizer of a glucansucrase, which produces a water-insoluble glucan, resulted in significant changes in the structures and yields of the water-insoluble glucans produced. We now describe how these changes affect the ability of the glucansucrase to bind to exogenous glucans, and how these glucans can influence the yield, product structures, and kinetics of the mutant glucansucrases. The activity of the wild-type enzyme, with threonine at position 654, is not significantly activated by added dextran, and the yield of water-insoluble glucan from sucrose is only slightly increased by dextran. Mutant T654Y is not affected at all by the addition of dextran. However, several mutant enzymes exhibit markedly lower yields of glucan relative to the wild type; these lower yields can be partially or completely overcome by the addition of water-soluble dextran. Although evidence indicates that the soluble dextran is incorporated into water-insoluble glucan, the increased yields cannot be accounted for solely by incorporation of the dextran into insoluble product. Furthermore, these DsrI mutants are significantly activated by exogenous glucans. The addition of dextran does not markedly change the KM for sucrose in the mutant enzymes, but does increase the Vmax of the reaction. These effects apparently depend on the presence of unbranched sequences of α1→6-linked D-glucose units in the glucan.

The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores.

Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection.

Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Efficient and rapid production of value-added chemicals from lignocellulosic biomass is an important step toward a sustainable society. Lactic acid, used for synthesizing the bioplastic polylactide, has been produced by microbial fermentation using primarily glucose. Lignocellulosic hydrolysates contain high concentrations of cellobiose and xylose. Here, we constructed a recombinant Saccharomyces cerevisiae strain capable of fermenting cellobiose and xylose into lactic acid. Specifically, genes (cdt-1, gh1-1, XYL1, XYL2, XYL3, and ldhA) coding for cellobiose transporter, ß-glucosidase, xylose reductase, xylitol dehydrogenase, xylulokinase, and lactate dehydrogenase were integrated into the S. cerevisiae chromosomes. The resulting strain produced lactic acid from cellobiose or xylose with high yields. When fermenting a cellulosic sugar mixture containing 10 g/L glucose, 40 g/L xylose, and 80 g/L cellobiose, the engineered strain produced 83 g/L of lactic acid with a yield of 0.66 g lactic acid/g sugar (66% theoretical maximum). This study demonstrates initial steps toward the feasibility of sustainable production of lactic acid from lignocellulosic sugars by engineered yeast.

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media.

OBJECTIVES: To compare production of antibacterial liamocins (polyol lipids) by diverse strains of Aureobasidium pullulans grown on different culture media. RESULTS: Liamocins produced by strains of A. pullulans have potential agricultural and pharmaceutical applications as antibacterials with specificity against Streptococcus spp. Six strains of A. pullulans were characterized for liamocin production on four different culture media. The choice of strain and culture medium affected growth, liamocin yields, and production of contaminating pigments. Best growth and highest liamocin yields were obtained using A. pullulans strain NRRL 50384 grown on a sucrose basal medium. Unexpectedly, the choice of strain and culture medium also affected the structure of liamocins produced, providing novel types of liamocins. Liamocins varied not only in the ratios of trimer and tetramer polyester tail groups, but also in the nature of the polyol headgroup, which could include mannitol, arabitol, or glycerol. CONCLUSIONS: The ability to conveniently produce novel types of liamocins in good yields will provide novel antibacterials for applied uses, and facilitate structure-function studies on the mechanism of antibacterial activity.