The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Genome-wide DNase I-hypersensitive site assay reveals distinct genomic distributions and functional features of open chromatin in autopolyploid sugarcane.

The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.

First investigation into the genetic control of meiosis in sugarcane.

The sugarcane (Saccharum spp.) genome is one of the most complex of all. Modern varieties are highly polyploid and aneuploid as a result of hybridization between Saccharum officinarum and S. spontaneum. Little research has been done on meiotic control in polyploid species, with the exception of the wheat Ph1 locus harboring the ZIP4 gene (TaZIP4-B2) which promotes pairing between homologous chromosomes while suppressing crossover between homeologs. In sugarcane, despite its interspecific origin, bivalent association is favored, and multivalents, if any, are resolved at the end of prophase I. Thus, our aim herein was to investigate the purported genetic control of meiosis in the parental species and in sugarcane itself. We investigated the ZIP4 gene and immunolocalized meiotic proteins, namely synaptonemal complex proteins Zyp1 and Asy1. The sugarcane ZIP4 gene is located on chromosome 2 and expressed more abundantly in flowers, a similar profile to that found for TaZIP4-B2. ZIP4 expression is higher in S. spontaneum a neoautopolyploid, with lower expression in S. officinarum, a stable octoploid species. The sugarcane Zip4 protein contains a TPR domain, essential for scaffolding. Its 3D structure was also predicted, and it was found to be very similar to that of TaZIP4-B2, reflecting their functional relatedness. Immunolocalization of the Asy1 and Zyp1 proteins revealed that S. officinarum completes synapsis. However, in S. spontaneum and SP80-3280 (a modern variety), no nuclei with complete synapsis were observed. Importantly, our results have implications for sugarcane cytogenetics, genetic mapping, and genomics.

Limited allele-specific gene expression in highly polyploid sugarcane.

Polyploidy is widespread in plants, allowing the different copies of genes to be expressed differently in a tissue-specific or developmentally specific way. This allele-specific expression (ASE) has been widely reported, but the proportion and nature of genes showing this characteristic have not been well defined. We now report an analysis of the frequency and patterns of ASE at the whole-genome level in the highly polyploid sugarcane genome. Very high depth whole-genome sequencing and RNA sequencing revealed strong correlations between allelic proportions in the genome and in expressed sequences. This level of sequencing allowed discrimination of each of the possible allele doses in this 12-ploid genome. Most genes were expressed in direct proportion to the frequency of the allele in the genome with examples of polymorphisms being found with every possible discrete level of dose from 1:11 for single-copy alleles to 12:0 for monomorphic sites. The rarer cases of ASE were more frequent in the expression of defense-response genes, as well as in some processes related to the biosynthesis of cell walls. ASE was more common in genes with variants that resulted in significant disruption of function. The low level of ASE may reflect the recent origin of polyploid hybrid sugarcane. Much of the ASE present can be attributed to strong selection for resistance to diseases in both nature and domestication.

Sugarcane mosaic virus employs 6K2 protein to impair ScPIP2;4 transport of H2O2 to facilitate virus infection.

Sugarcane mosaic virus (SCMV), one of the main pathogens causing sugarcane mosaic disease, is widespread in sugarcane (Saccharum spp. hybrid) planting areas and causes heavy yield losses. RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) NADPH oxidases and plasma membrane intrinsic proteins (PIPs) have been associated with the response to SCMV infection. However, the underlying mechanism is barely known. In the present study, we demonstrated that SCMV infection upregulates the expression of ScRBOHs and the accumulation of hydrogen peroxide (H2O2), which inhibits SCMV replication. All eight sugarcane PIPs (ScPIPs) interacted with SCMV-encoded protein 6K2, whereby two PIP2s (ScPIP2;1 and ScPIP2;4) were verified as capable of H2O2 transport. Furthermore, we revealed that SCMV-6K2 interacts with ScPIP2;4 via transmembrane domain 5 to interfere with the oligomerization of ScPIP2;4, subsequently impairing ScPIP2;4 transport of H2O2. This study highlights a mechanism adopted by SCMV to employ 6K2 to counteract the host resistance mediated by H2O2 to facilitate virus infection and provides potential molecular targets for engineering sugarcane resistance against SCMV.

Lactobacillus casei-fermented Amomum xanthioides ameliorates metabolic dysfunction in high-fat diet-induced obese mice.

Amomum xanthioides (AX) has been used as an edible herbal medicine to treat digestive system disorders in Asia. Additionally, Lactobacillus casei is a well-known probiotic commonly used in fermentation processes as a starter. The current study aimed to investigate the potential of Lactobacillus casei-fermented Amomum xanthioides (LAX) in alleviating metabolic disorders induced by high-fat diet (HFD) in a mouse model. LAX significantly reduced the body and fat weight, outperforming AX, yet without suppressing appetite. LAX also markedly ameliorated excessive lipid accumulation and reduced inflammatory cytokine (IL-6) levels in serum superior to AX in association with UCP1 activation and adiponectin elevation. Furthermore, LAX noticeably improved the levels of fasting blood glucose, serum insulin, and HOMA-IR through positive regulation of glucose transporters (GLUT2, GLUT4), and insulin receptor gene expression. In conclusion, the fermentation of AX demonstrates a pronounced mitigation of overnutrition-induced metabolic dysfunction, including hyperlipidemia, hyperglycemia, hyperinsulinemia, and obesity, compared to non-fermented AX. Consequently, we proposed that the fermentation of AX holds promise as a potential candidate for effectively ameliorating metabolic disorders.

Amino acid metabolism and MAP kinase signaling pathway play opposite roles in the regulation of ethanol production during fermentation of sugarcane molasses in budding yeast.

Sugarcane molasses is one of the main raw materials for bioethanol production, and Saccharomyces cerevisiae is the major biofuel-producing organism. In this study, a batch fermentation model has been used to examine ethanol titers of deletion mutants for all yeast nonessential genes in this yeast genome. A total of 42 genes are identified to be involved in ethanol production during fermentation of sugarcane molasses. Deletion mutants of seventeen genes show increased ethanol titers, while deletion mutants for twenty-five genes exhibit reduced ethanol titers. Two MAP kinases Hog1 and Kss1 controlling the high osmolarity and glycerol (HOG) signaling and the filamentous growth, respectively, are negatively involved in the regulation of ethanol production. In addition, twelve genes involved in amino acid metabolism are crucial for ethanol production during fermentation. Our findings provide novel targets and strategies for genetically engineering industrial yeast strains to improve ethanol titer during fermentation of sugarcane molasses.

Transcriptome dynamics provides insights into divergences of the photosynthesis pathway between Saccharum officinarum and Saccharum spontaneum.

Saccharum spontaneum and Saccharum officinarum contributed to the genetic background of modern sugarcane cultivars. Saccharum spontaneum has shown a higher net photosynthetic rate and lower soluble sugar than S. officinarum. Here, we analyzed 198 RNA-sequencing samples to investigate the molecular mechanisms for the divergences of photosynthesis and sugar accumulation between the two Saccharum species. We constructed gene co-expression networks based on differentially expressed genes (DEGs) both for leaf developmental gradients and diurnal rhythm. Our results suggested that the divergence of sugar accumulation may be attributed to the enrichment of major carbohydrate metabolism and the oxidative pentose phosphate pathway. Compared with S. officinarum, S. spontaneum DEGs showed a high enrichment of photosynthesis and contained more complex regulation of photosynthesis-related genes. Noticeably, S. spontaneum lacked gene interactions with sulfur assimilation stimulated by photorespiration. In S. spontaneum, core genes related to clock and photorespiration displayed a sensitive regulation by the diurnal rhythm and phase-shift. Small subunit of Rubisco (RBCS) displayed higher expression in the source tissues of S. spontaneum. Additionally, it was more sensitive under a diurnal rhythm, and had more complex gene networks than that in S. officinarum. This indicates that the differential regulation of RBCS Rubisco contributed to photosynthesis capacity divergence in both Saccharum species.

Genomic investigation of duplication, functional conservation, and divergence in the LRR-RLK Family of Saccharum.

BACKGROUND: Sugarcane (Saccharum spp.) holds exceptional global significance as a vital crop, serving as a primary source of sucrose, bioenergy, and various by-products. The optimization of sugarcane breeding by fine-tuning essential traits has become crucial for enhancing crop productivity and stress resilience. Leucine-rich repeat receptor-like kinases (LRR-RLK) genes present promising targets for this purpose, as they are involved in various aspects of plant development and defense processes. RESULTS: Here, we present a detailed overview of phylogeny and expression of 288 (495 alleles) and 312 (1365 alleles) LRR-RLK genes from two founding Saccharum species, respectively. Phylogenetic analysis categorized these genes into 15 subfamilies, revealing considerable expansion or reduction in certain LRR-type subfamilies. Compared to other plant species, both Saccharum species had more significant LRR-RLK genes. Examination of cis-acting elements demonstrated that SsLRR-RLK and SoLRR-RLK genes exhibited no significant difference in the types of elements included, primarily involved in four physiological processes. This suggests a broad conservation of LRR-RLK gene function during Saccharum evolution. Synteny analysis indicated that all LRR-RLK genes in both Saccharum species underwent gene duplication, primarily through whole-genome duplication (WGD) or segmental duplication. We identified 28 LRR-RLK genes exhibiting novel expression patterns in response to different tissues, gradient development leaves, and circadian rhythm in the two Saccharum species. Additionally, SoLRR-RLK104, SoLRR-RLK7, SoLRR-RLK113, and SsLRR-RLK134 were identified as candidate genes for sugarcane disease defense response regulators through transcriptome data analysis of two disease stresses. This suggests LRR-RLK genes of sugarcane involvement in regulating various biological processes, including leaf development, plant morphology, photosynthesis, maintenance of circadian rhythm stability, and defense against sugarcane diseases. CONCLUSIONS: This investigation into gene duplication, functional conservation, and divergence of LRR-RLK genes in two founding Saccharum species lays the groundwork for a comprehensive genomic analysis of the entire LRR-RLK gene family in Saccharum. The results reveal LRR-RLK gene played a critical role in Saccharum adaptation to diverse conditions, offering valuable insights for targeted breeding and precise phenotypic adjustments.

Genetic association analysis in sugarcane (Saccharum spp.) for sucrose accumulation in humid environments in Colombia.

BACKGROUND: Sucrose accumulation in sugarcane is affected by several environmental and genetic factors, with plant moisture being of critical importance for its role in the synthesis and transport of sugars within the cane stalks, affecting the sucrose concentration. In general, rainfall and high soil humidity during the ripening stage promote plant growth, increasing the fresh weight and decreasing the sucrose yield in the humid region of Colombia. Therefore, this study aimed to identify markers associated with sucrose accumulation or production in the humid environment of Colombia through a genome-wide association study (GWAS). RESULTS: Sucrose concentration measurements were taken in 220 genotypes from the Cenicaña's diverse panel at 10 (early maturity) and 13 (normal maturity) months after planting. For early maturity data was collected during plant cane and first ratoon, while at normal maturity it was during plant cane, first, and second ratoon. A total of 137,890 SNPs were selected after sequencing the 220 genotypes through GBS, RADSeq, and whole-genome sequencing. After GWAS analysis, a total of 77 markers were significantly associated with sucrose concentration at both ages, but only 39 were close to candidate genes previously reported for sucrose accumulation and/or production. Among the candidate genes, 18 were highlighted because they were involved in sucrose hydrolysis (SUS6, CIN3, CINV1, CINV2), sugar transport (i.e., MST1, MST2, PLT5, SUT4, ERD6 like), phosphorylation processes (TPS genes), glycolysis (PFP-ALPHA, HXK3, PHI1), and transcription factors (ERF12, ERF112). Similarly, 64 genes were associated with glycosyltransferases, glycosidases, and hormones. CONCLUSIONS: These results provide new insights into the molecular mechanisms involved in sucrose accumulation in sugarcane and contribute with important genomic resources for future research in the humid environments of Colombia. Similarly, the markers identified will be validated for their potential application within Cenicaña's breeding program to assist the development of breeding populations.

Biomass and energy potential of Erianthus arundinaceus and Saccharum spontaneum-derived novel sugarcane hybrids in rainfed environments.

BACKGROUND: Energy canes are viable feedstocks for biomass industries due to their high biomass production potential, lower susceptibility to insects and diseases, better ability to adapt to extreme conditions and clean bioenergy. Interspecific hybrids (ISH) and intergeneric hybrids (IGH) have great potential to meet the growing demand of biomass, biomass-derived energy and feedstock. RESULTS: In this study, two types of energy canes, Type I and Type II, derived from S. spontaneum and E. arundinaceous background were evaluated for high biomass, fiber and bioenergy potential under subtropical climate along with the check varieties Co 0238 and CoS 767. Out of 18 energy canes studied, six energy canes, viz., SBIEC11008 (204.15 t/ha), SBIEC11005 (192.93 t/ha), SBIEC13008 (201.26 t/ha), SBIEC13009 (196.58 t/ha), SBIEC13002 (170.15 t/ha), and SBIEC13007 (173.76 t/ha), consistently outperformed the check varieties under Type-I, whereas in type-II, SBIEC11004 (225.78 t/ha), SBIEC11006 (184.89 t/ha), and SBIEC14006 (184.73 t/ha) energy canes produced significantly higher biomass than commercial checks, indicating their superior potential for cogeneration. Estimated energy output from the energy canes (700-1300 GJ/ha/year) exceeded the range of co-varieties (400-500 GJ/ha/year) and energy utilization efficiency in plants and ratoon crops for energy canes viz., SBIEC11008 (3%, 1.97%), SBIEC14006 (1.93%, 2.4%), SBIEC11005 (1.7%, 1.9%), and SBIEC11001 (1.01%, 1.03%), was higher than best checks Co 0238 (0.77, 0.9%). Additionally, energy canes SBIEC 13001 (22.35%), SBIEC 11008 (22.50%), SBIEC 14006 (28.54%), SBIEC 11004 (30.17%) and SBIEC 11001 (27.03%) had higher fiber contents than the co-varieties (12.45%). CONCLUSION: The study gives insight about the potential energy canes for higher biomass and energy value. These energy cane presents a vital option to meet the future demand of bioenergy, fiber and fodder for biomass due to their versatile capacity to grow easily under marginal lands without competing with cultivated land worldwide.

Transcriptome and small RNA analysis unveils novel insights into the C4 gene regulation in sugarcane.

MAIN CONCLUSION: This study reveals miRNA indirect regulation of C4 genes in sugarcane through transcription factors, highlighting potential key regulators like SsHAM3a. C4 photosynthesis is crucial for the high productivity and biomass of sugarcane, however, the miRNA regulation of C4 genes in sugarcane remains elusive. We have identified 384 miRNAs along the leaf gradients, including 293 known miRNAs and 91 novel miRNAs. Among these, 86 unique miRNAs exhibited differential expression patterns, and we identified 3511 potential expressed targets of these differentially expressed miRNAs (DEmiRNAs). Analyses using Pearson correlation coefficient (PCC) and Gene Ontology (GO) enrichment revealed that targets of miRNAs with positive correlations are integral to chlorophyll-related photosynthetic processes. In contrast, negatively correlated pairs are primarily associated with metabolic functions. It is worth noting that no C4 genes were predicted as targets of DEmiRNAs. Our application of weighted gene co-expression network analysis (WGCNA) led to a gene regulatory network (GRN) suggesting miRNAs might indirectly regulate C4 genes via transcription factors (TFs). The GRAS TF SsHAM3a emerged as a potential regulator of C4 genes, targeted by miR171y and miR171am, and exhibiting a negative correlation with miRNA expression along the leaf gradient. This study sheds light on the complex involvement of miRNAs in regulating C4 genes, offering a foundation for future research into enhancing sugarcane's photosynthetic efficiency.

Silencing ScGUX2 reduces xylan glucuronidation and improves biomass saccharification in sugarcane.

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases.

H3K27 demethylase SsJMJ4 negatively regulates drought-stress responses in sugarcane.

Sugarcane (Saccharum spp.), a leading sugar and energy crop, is seriously impacted by drought stress. However, the molecular mechanisms underlying sugarcane drought resistance, especially the functions of epigenetic regulators, remain elusive. Here, we show that a S. spontaneum KDM4/JHDM3 group JmjC protein, SsJMJ4, negatively regulates drought-stress responses through its H3K27me3 demethylase activity. Ectopic overexpression of SsJMJ4 in Arabidopsis reduced drought resistance possibly by promoting expression of AtWRKY54 and AtWRKY70, encoding two negative regulators of drought stress. SsJMJ4 directly bound to AtWRKY54 and AtWRKY70, and reduced H3K27me3 levels at these loci to ensure their proper transcription under normal conditions. Drought stress down-regulated both transcription and protein abundance of SsJMJ4, which was correlated with the reduced occupancy of SsJMJ4 at AtWRKY54 and AtWRKY70 chromatin, increased H3K27me3 levels at these loci, as well as reduced transcription levels of these genes. In S. spontaneum, drought stress-repressed transcription of SsWRKY122, an ortholog of AtWRKY54 and AtWRKY70, was associated with increased H3K27me3 levels at these loci. Transient overexpression of SsJMJ4 in S. spontaneum protoplasts raised transcription of SsWRKY122, paralleled with reduced H3K27me3 levels at its loci. These results suggest that the SsJMJ4-mediated dynamic deposition of H3K27me3 is required for an appropriate response to drought stress.

Unveiling the predominance of Saccharum spontaneum alleles for resistance to orange rust in sugarcane using genome-wide association.

KEY MESSAGE: Six QTLs of resistance to sugarcane orange rust were identified in modern interspecific hybrids by GWAS. For five of them, the resistance alleles originated from S. spontaneum. Altogether, they efficiently predict disease resistance. Sugarcane orange rust (SOR) is a threatening emerging disease in many sugarcane industries worldwide. Improving the genetic resistance of commercial cultivars remains the most promising solution to control this disease. In this study, an association panel of 568 modern interspecific sugarcane hybrids (Saccharum officinarum x S. spontaneum) from Réunion's breeding program was evaluated for its resistance to SOR under natural conditions of infection. Two genome-wide association studies (GWAS) were conducted between disease reactions and 183,842 single nucleotide polymorphism (SNP) markers obtained by targeted genotyping-by-sequencing. Five resistance quantitative trait loci (QTLs), named Oru1, Oru2, Oru3, Oru4 and Oru5, were identified using a single-locus GWAS (SL-GWAS). These five QTLs all originated from the species S. spontaneum. A multi-locus GWAS (ML-GWAS) uncovered an additional but less significant resistance QTL named Oru6, which originated from S. officinarum. All six QTLs had a moderate to major phenotypic effect on disease resistance. Prediction accuracy estimated with linear regression models based on each of the five QTLs identified by SL-GWAS was between 0.16-0.41. Altogether, these five QTLs provided a relatively high prediction accuracy of 0.60. In comparison, accuracies obtained with six genome-wide prediction models (i.e., GBLUP, Bayes-A, Bayes-B, Bayes-C, Bayesian Lasso and RKHS) reached only 0.65. The good prediction accuracy of disease resistance provided by the QTLs and the predominant S. spontaneum origin of their resistance alleles pave the way for effective marker-assisted breeding strategies.

First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Taxonomic identification, phenol biodegradation and soil remediation of the strain Rhodococcus sacchari sp. nov. Z13T.

Phenols are highly toxic chemicals that are extensively used in industry and produce large amounts of emissions. Notably, phenols released into the soil are highly persistent, causing long-term harm to human health and the environment. In this study, a gram-positive, aerobic, and rod-shaped bacterial strain, Z13T, with efficient phenol degradation ability, was isolated from the soil of sugarcane fields. Based on the physiological properties and genomic features, strain Z13T is considered as a novel species of the genus Rhodococcus, for which the name Rhodococcus sacchari sp. nov. is proposed. The type strain is Z13T (= CCTCC AB 2022327T = JCM 35797T). This strain can use phenol as its sole carbon source. Z13T was able to completely degrade 1200 mg/L phenol within 20 h; the maximum specific growth rate was µmax = 0.93174 h-1, and the maximum specific degradation rate was qmax = 0.47405 h-1. Based on whole-genome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, strain Z13T contains a series of phenol degradation genes, including dmpP, CatA, dmpB, pcaG, and pcaH, and can metabolize aromatic compounds. Moreover, the potential of strain Z13T for soil remediation was investigated by introducing Z13T into simulated phenol-contaminated soil, and the soil microbial diversity was analyzed. The results showed that 100% of the phenol in the soil was removed within 7.5 d. Furthermore, microbial diversity analysis revealed an increase in the relative species richness of Oceanobacillus, Chungangia, and Bacillus.

ß-Carotene production from sugarcane molasses by a newly isolated Rhodotorula toruloides L/24-26-1.

Production of carotenoids by yeast fermentation is an advantaged technology due to its easy scaling and safety. Nevertheless, carotenoid production needs an economic culture medium and other efficient yeast stains. The study aims to isolate and identify a yeast strain capable of producing carotenoids using a cost-effective substrate. A new strain was identified as Rhodotorula toruloides L/24-26-1, which can produce carotenoids at different pretreated and unpretreated sugarcane molasses concentrations (40 and 80 g/L). The highest biomass concentration (18.6 ± 0.6 g/L) was reached in the culture using 80 g/L of hydrolyzed molasses. On the other hand, the carotenoid accumulation reached the maximum value using pretreated molasses at 40 g/L (715.4 ± 15.1 µg/g d.w). In this case, the ß-carotene was 1.5 times higher than that on the control medium. The yeast growth in molasses was not correlated with carotenoid production. The most outstanding production of The DPPH, ABTS, and FRAP tests demonstrated the antioxidant activity of the obtained carotenogenic extracts. This research demonstrated the R. toruloides L/24-26-1 strain biotechnological potential for carotenoid compounds. The yeast produces carotenoids with antioxidant activity in an inexpensive medium, such as sulfuric acid pretreated and unpretreated molasses.

Pioglitazone reduces serum ketone bodies in sodium-glucose cotransporter-2 inhibitor-treated non-obese type 2 diabetes: A single-centre, randomized, crossover trial.

AIM: To examine the effects of the thiazolidinedione (TZD) pioglitazone on reducing ketone bodies in non-obese patients with T2DM treated with the sodium-glucose cotransporter-2 (SGLT2) inhibitor canagliflozin. METHODS: Crossover trials with two periods, each treatment period lasting 4 weeks, with a 4-week washout period, were conducted. Participants were randomly assigned in a 1:1 ratio to receive pioglitazone combined with canagliflozin (PIOG + CANA group) versus canagliflozin monotherapy (CANA group). The primary outcome was change (Δ) in ß-hydroxybutyric acid (ß-HBA) before and after the CANA or PIOG + CANA treatments. The secondary outcomes were Δchanges in serum acetoacetate and acetone, the rate of conversion into urinary ketones, and Δchanges in factors related to SGLT2 inhibitor-induced ketone body production including non-esterified fatty acids (NEFAs), glucagon, glucagon to insulin ratio, and noradrenaline (NA). Analyses were performed in accordance with the intention-to-treat principle. RESULTS: Twenty-five patients with a mean age of 49 ± 7.97 years and a body mass index of 25.35 ± 2.22 kg/m2 were included. One patient discontinued the study during the washout period. Analyses revealed a significant increase in the levels of serum ketone bodies and an elevation in the rate of conversion into urinary ketones after both interventions. However, differernces in levels of ketone bodies (except for acetoacetate) in the PIOG + CANA group were significantly smaller than in the CANA group (219.84 ± 80.21 µmol/L vs. 317.69 ± 83.07 µmol/L, p < 0.001 in ß-HBA; 8.98 ± 4.17 µmol/L vs. 12.29 ± 5.27 µmol/L, p = 0.018 in acetone). NEFA, glucagon, glucagon to insulin ratio, and NA were also significantly increased after both CANA and PIOG + CANA treatments; while only NEFAs demonstrated a significant difference between the two groups. Correlation analyses revealed a significant association between the difference in Δchanges in serum NEFA levels with the differences in Δchanges in ketones of ß-HBA and acetoacetate. CONCLUSION: Supplementation of pioglitazone could alleviate canagliflozin-induced ketone bodies. This benefit may be closely associated with decreased substrate NEFAs rather than other factors including glucagon, fasting insulin and NA.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.