Characterization of Levan Fructan Produced by a Gluconobacter japonicus Strain Isolated from a Sugarcane Processing Facility.

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 ß-2,6-fructofuranosyl polysaccharide with occasional (5.7%) ß-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.

Microbiome Analysis of Sugarcane Juices and Biofilms from Louisiana Raw Sugar Factories.

During postharvest processing of sugarcane for raw sugar, microbial activity results in sucrose loss and undesirable exopolysaccharide (EPS) production. Historically, culture-based approaches have focused on the bacterium Leuconostoc mesenteroides as the main contributor to both processes. However, recent studies have shown that diverse microbes are present in sugarcane factories and may also contribute to sugarcane juice deterioration. In the present study, high-throughput amplicon-based sequence profiling was applied to gain a more comprehensive view of the microbial community in Louisiana raw sugar factories. Microbial profiling of the bacterial and fungal microbiomes by 16S V4 and ITS1 sequences, respectively, identified 417 bacterial amplicon sequence variants (ASVs) and 793 fungal ASVs. While Leuconostoc was indeed the most abundant bacterial genus overall (40.9% of 16S sequences), multiple samples were dominated by other taxa such as Weissella and Lactobacillus, underscoring the microbial diversity present in sugarcane factories. Furthermore, flask cultures inoculated with the same samples demonstrated differences in the rate of sucrose consumption, as well as the production of exopolysaccharides and other organic acids, which may result from the observed differences in microbial composition. IMPORTANCE 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.

Recovery of Aconitic Acid from Sweet Sorghum Plant Extract Using a Solvent Mixture, and Its Potential Use as a Nematicide.

Trans-aconitic acid (TAA) is naturally present in sweet sorghum juice and syrup, and it has been promoted as a potential biocontrol agent for nematodes. Therefore, we developed a process for the extraction of aconitic acid from sweet sorghum syrup. The process economics were evaluated, and the extract was tested for its capability to suppress the motility of the nematodes Caenorhabditis elegans and Meloidogyne incognita. Aconitic acid could be efficiently extracted from sweet sorghum syrup using acetone:butanol:ethanol mixtures, and it could be recovered from this solvent with a sodium carbonate solution, with an overall extraction and recovery efficiency of 86%. The estimated production cost was USD 16.64/kg of extract and this was highly dependent on the solvent cost, as the solvent was not recycled but was resold for recovery at a fraction of the cost. The extract was effective in reducing the motility of the parasitic M. incognita and causing over 78% mortality of the nematode when 2 mg/mL of TAA extract was added. However, this positive result could not conclusively be linked solely to TAA. An unidentified component (or components) in the acetone:butanol:ethanol sweet sorghum extract appears to be an effective nematode inhibitor, and it may merit further investigation. The impact of aconitic acid on C. elegans appeared to be entirely controlled by pH.

Aconitic Acid Recovery from Renewable Feedstock and Review of Chemical and Biological Applications.

Aconitic acid (propene-1,2,3-tricarboxylic acid) is the most prevalent 6-carbon organic acid that accumulates in sugarcane and sweet sorghum. As a top value-added chemical, aconitic acid may function as a chemical precursor or intermediate for high-value downstream industrial and biological applications. These downstream applications include use as a bio-based plasticizer, cross-linker, and the formation of valuable and multi-functional polyesters that have also been used in tissue engineering. Aconitic acid also plays various biological roles within cells as an intermediate in the tricarboxylic acid cycle and in conferring unique survival advantages to some plants as an antifeedant, antifungal, and means of storing fixed pools of carbon. Aconitic acid has also been reported as a fermentation inhibitor, anti-inflammatory, and a potential nematicide. Since aconitic acid can be sustainably sourced from renewable, inexpensive sources such as sugarcane, molasses, and sweet sorghum syrup, there is enormous potential to provide multiple streams of additional income to the sugar industry through downstream industrial and biological applications that we discuss in this review.

CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar.

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

Comparative genome-wide analysis of extracellular small RNAs from the mucormycosis pathogen Rhizopus delemar.

Rhizopus delemar is an emerging fungal pathogen causing devastating mucormycosis in immunocompromised individuals. The organism remains understudied and there are urgent needs for new methods of rapid disease diagnosis for timely therapy. Extracellular vesicles with encapsulated RNAs have recently been discovered to have great potential applications for disease diagnoses and treatments. To explore the utilization of ex-RNA in studies of mucormycosis, we have performed RNA-Seq of ex-sRNAs from two clinical strains of R. delemar. Approximately 3.3 and 3.2 million clean reads were obtained from FGSC-9543 and CDC-8219 strains, respectively. The median sequence length of the sRNAs was 22 nts, with a minimum of 18 and a maximum of 30 nts. Further annotation identified 560 and 526 miRNAs from FGSC-9543 and CDC-8219 strains, respectively. miRNA target prediction and analysis of GO and KEGG pathways have revealed that the regulation of metabolism, secondary metabolite biosynthesis, and two-component system signaling are important during growth. We have also validated RNA-Seq by qRT-PCR and Northern blotting analysis of randomly selected miRNAs. Our results show that R. delemar has a rich reservoir of secreted ex-sRNAs and our studies could facilitate the development of improved diagnostic methods as well as elucidating virulence mechanisms for R. delemar infection.

Comparative proteomic analysis of Gib2 validating its adaptor function in Cryptococcus neoformans.

Cryptococcus neoformans causes often-fatal fungal meningoencephalitis in immunocompromised individuals. While the exact disease mechanisms remain elusive, signal transduction pathways mediated by key elements such as G-protein α subunit Gpa1, small GTPase Ras1, and atypical Gß-like/RACK1 protein Gib2 are known to play important roles in C. neoformans virulence. Gib2 is important for normal growth, differentiation, and pathogenicity, and it also positively regulates cAMP levels in conjunction with Gpa1. Interestingly, Gib2 displays a scaffold protein property by interacting with a wide variety of cellular proteins. To explore Gib2 global regulatory functions, we performed two-dimensional differential gel electrophoresis (DIGE) analysis and found that GIB2 disruption results in an increased expression of 304 protein spots (43.4%) and a decreased expression of 396 protein spots (56.6%). Analysis of 96 proteins whose expression changes were deemed significant (≥ +/- 1.5- fold) revealed that 75 proteins belong to at least 12 functional protein groups. Among them, eight groups have the statistical stringency of p ≤ 0.05, and four groups, including Hsp70/71 heat shock protein homologs and ribosomal proteins, survived the Bonferroni correction. This finding is consistent with earlier established roles for the human Gß-like/RACK1 and the budding yeast Saccharomyces cerevisiae Asc1. It suggests that Gib2 could also be part of the complex affecting ribosomal biogenesis and protein translation in C. neoformans. Since eukaryotic Hsp70/71 proteins are involved in the facilitation of nascent protein folding, processing, and protection of cells against stress, we also propose that Gib2-regulated stress responses are linked to fungal virulence. Collectively, our study supports a conserved role of Gß-like/RACK/Gib2 proteins in the essential cellular process of ribosomal biogenesis and protein translation. Our study also highlights a multifaceted regulatory role of Gib2 in the growth and pathogenicity of C. neoformans.