During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by ¹H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear β-2,6-fructofuranosyl polysaccharide with occasional (5.7%) β-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC ₅₀ values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs.

Significance: Streptomyces genomes harbor a trove of biosynthetic gene clusters (BGCs) that encode for drug-like molecules. However, only a fraction of these readily yield expected products. To investigate why this is, we used polycyclic tetramate macrolactam (PTM) antibiotic production as a model system. By comparing the genomes and PTM production profiles of several closely related Streptomyces griseus clade members, we uncovered two mechanisms that differentiate more robust producers from weaker ones. The first involves small insertion–deletion lesions in PTM BGC promoters that significantly modulate production. The second mechanism involves biosynthetic pathway interactions, in which robust PTM producers unexpectedly benefit from griseorhodin coproduction, and weaker producers lack the pathway. We highlight comparative metabologenomics as a powerful approach to understand antibiotic crypticity.

Amplicon-based sequencing was utilized to address long-ignored gaps in microbiological knowledge about the diversity of microbes present in processing streams at Louisiana sugarcane raw sugar factories. These results support an emerging model where diverse organisms contribute to sugarcane juice degradation, help to contextualize microbial contamination problems faced by raw sugar factories, and will guide future studies on biocontrol measures to mitigate sucrose losses and operational challenges due to exopolysaccharide production.

Sugar beet is a major source of consumable sugar in domestic and industrial applications in the United States. Sugar beet roots are stored for up to 280 days under cold ambient temperature conditions in the Red River Valley (RRV) of Minnesota and North Dakota. During this period, storage rots often develop that can reduce the sucrose content and processing quality of the roots. Knowledge of the pathogens responsible for this sucrose loss is limited but crucial for devising management strategies. This study was conducted to re-examine and identify storage pathogens infecting sugar beet roots in outdoor nonventilated piles in the RRV of Minnesota and North Dakota and characterize their prevalence and geographic distribution. In the first year of the study, more than 50 roots exhibiting storage rot were collected from an outdoor nonventilated pile (ONVP) in Minnesota. In successive years, 150 storage rot and damaged symptomatic roots were collected from five geographically distinct ONVPs with three located in Minnesota and two located in North Dakota. Penicillium spp., Geotrichum candidum, and Mucor spp. were common filamentous fungal species identified, whereas Pichia membranifaciens was the dominant yeast identified in both years of the study. Leuconostoc mesenteroides and Gluconobacter cerinus were common bacterial isolates in both years of the study. Most of the bacterial isolates obtained from ONVP-4 (North Dakota) (63%) and ONVP-5 (North Dakota) (99%) during the second year of the study were identified as L. mesenteroides. This study identified fungal and bacterial species associated with storage diseases of sugar beet roots in the RRV of Minnesota and North Dakota, which is pivotal for implementing management strategies to minimize postharvest sucrose losses during sugar beet storage.