Pathogen Profile of Klebsiella variicola, the Causative Agent of Banana Sheath Rot.

Banana (Musa spp.) is an important fruit and food crop worldwide. In recent years, banana sheath rot has become a major problem in banana cultivation, causing plant death and substantial economic losses. Nevertheless, the pathogen profile of this disease has not been fully characterized. Klebsiella variicola is a versatile bacterium capable of colonizing different hosts, such as plants, humans, insects, and animals, and is recognized as an emerging pathogen in various hosts. In this study, we obtained 12 bacterial isolates from 12 different banana samples showing banana sheath rot in Guangdong and Guangxi Provinces, China. Phylogenetic analysis based on 16S rRNA sequences confirmed that all 12 isolates were K. variicola strains. We sequenced the genomes of these strains, performed comparative genomic analysis with other sequenced K. variicola strains, and found a lack of consistency in accessory gene content among these K. variicola strains. However, prediction based on the pan-genome of K. variicola revealed 22 unique virulence factors carried by the 12 pathogenic K. variicola isolates. Microbiome and microbial interaction network analysis of endophytes between the healthy tissues of diseased plants and healthy plants of two cultivars showed that Methanobacterium negatively interacts with Klebsiella in banana plants and that Herbaspirillum might indirectly inhibit Methanobacterium to promote Klebsiella growth. These results suggest that banana sheath rot is caused by the imbalance of plant endophytes and opportunistic pathogenic bacteria, providing an important basis for research and control of this disease.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Endophytic Bacillus subtilis TR21 Improves Banana Plant Resistance to Fusarium oxysporum f. sp. cubense and Promotes Root Growth by Upregulating the Jasmonate and Brassinosteroid Biosynthesis Pathways.

The banana (Musa spp.) industry experiences dramatic annual losses from Fusarium wilt of banana disease, which is caused by the fungus Fusarium oxysporum f. sp. cubense (FOC). Pisang Awak banana 'Fenza No. 1' (Musa spp. cultivar Fenza No. 1), a major banana cultivar with high resistance to F. oxysporum f. sp. cubense race 4, is considered to be ideal for growth in problematic areas. However, 'Fenza No. 1' is still affected by F. oxysporum f. sp. cubense race 1 in the field. TR21 is an endophytic Bacillus subtilis strain isolated from orchids (Dendrobium sp.). Axillary spraying of banana plants with TR21 controls Fusarium wilt of banana, decreasing the growth period and increasing yields in the field. In this study, we established that TR21 increases root growth in different monocotyledonous plant species. By axillary inoculation, TR21 induced a similar transcriptomic change as that induced by F. oxysporum f. sp. cubense race 1 but also upregulated the biosynthetic pathways for the phytohormones brassinosteroid and jasmonic acid in 'Fenza No. 1' root tissues, indicating that TR21 increases Fusarium wilt of banana resistance, shortens growth period, and increases yield of banana by inducing specific transcriptional reprogramming and modulating phytohormone levels. These findings will contribute to the identification of candidate genes related to plant resistance against fungi in a nonmodel system and facilitate further study and exploitation of endophytic biocontrol agents.

Cross resistance, inheritance and fitness advantage of cyetpyrafen resistance in two-spotted spider mite, Tetranychus urticae.

Cyetpyrafen belonging to mitochondrial electron transport inhibitors of complex II (METI II) has been widely applied to manage pest mites in China. To investigate the adaption of Tetranychus urticae in the evolution of cyetpyrafen resistance, a study of cross resistance, mode of inheritance and fitness comparison of resistance using indoor cyetpyrafen resistant strain (resistance ratio, RR > 2, 000-fold) was executed. Cyet-R showed serious cross resistance to cyenopyrafen (>2500-fold) and cyflumetofen (~190-fold). The number of resistant genes was evaluated via chi-square (χ2) test and the concentration-response curve regarding goodness-of-fit between observed and the expected mortality. The LC50s of F1RS (Cyet-R♀ × Tu-YN♂) and F1SR (Tu-YN♀ × Cyet-R♂) were 3126.30 mg/L and 2743.97 mg/L, respectively, without significance, suggesting autosomal inheritance. The degree of dominance (D) values of F1RS and F1SR ranged between 0 and 1, revealing an incompletely dominant inheritance in the tested population of Tetranychus urticae. Plots of concentration-response data for the orthogonal backcross and reverse backcross progenies showed a significant deviation from the expected lines, pointing out a polygenic inheritance. Besides, lifetable analysis showed a fitness advantage of Cyet-R with a significantly decreased adult preadult period and significantly increased total fecundity. This study suggested that cyetpyrafen resistance against T. urticae was inherited as autosomal, incompletely dominant and multigenetic and characterized with serious cross resistance and fitness advantage. Therefore, rational application and preventive strategy should be considered to sustain the efficacy of cyetpyrafen against T. urticae.

Comparative genomics analysis of two banana Fusarium wilt biocontrol endophytes Bacillus subtilis R31 and TR21 provides insights into their differences on phytobeneficial trait.

Fusarium wilt of banana is considered one of the most destructive plant diseases. Bacillus subtilis R31 and TR21, isolated from Dendrobium sp. leaves, exhibit different phytobeneficial effects on banana Fusarium wilt bio-controlling. Here, we performed genome sequencing and comparative genomics analysis of R31 and TR21 to enhance our understanding of the different phytobeneficial traits. These results revealed that the strain-specific genes of R31 involved in sporulation, quorum sensing, and antibiotic synthesis allow R31 to present a better capacity of sporulation, rhizosphere adaptation, and quorum sensing than TR21. Selective pressure analysis indicated that the glycosylase and endo-alpha-(1- > 5)-L-arabinanase genes were strong positive selected, which may contribute to the TR21 to colonize well in banana's vascular bundles. Altogether, our findings presented here should advance further agricultural application of R31 and TR21 as two promising resources of plant growth promotion and biological control via genetic engineering.

Metabolomics integrated with transcriptomics: assessing the central metabolism of marine red yeast Sporobolomyces pararoseus under salinity stress.

Salinity stress is one of the most serious environmental issues in agricultural regions worldwide. Excess salinity inhibits root growth of various crops, and results in reductions of yield. It is of crucial to understand the molecular mechanisms mediating salinity stress responses for enhancing crops' salt tolerance. Marine red yeast Sporobolomyces pararoseus should have evolved some unique salt-tolerant mechanism, because they long-term live in high-salt ecosystems. However, little research has conducted so far by considering S. pararoseus as model microorganisms to study salt-tolerant mechanisms. Here, we successfully integrated metabolomics with transcriptomic profiles of S. pararoseus in response to salinity stress. Screening of metabolite features with untargeted metabolic profiling, we characterized 4862 compounds from the LC-MS/MS-based datasets. The integrated results showed that amino acid metabolism, carbohydrate metabolism, and lipid metabolism is significantly enriched in response to salt stress. Co-expression network analysis showed that 28 genes and 8 metabolites play an important role in the response of S. pararoseus, which provides valuable clues for subsequent validation. Together, the results provide valuable information for assessing the central metabolism of mediating salt responses in S. pararoseus and offer inventories of target genes for salt tolerance improvement via genetic engineering.

Comparing the efficiency of RNAi after feeding and injection of dsRNA in spider mites.

Pesticide resistance in spider mites drives the development of acaricides with novel mode of action, which could benefit from RNAi as a screening tool in search of new molecular targets. RNAi via oral delivery of dsRNA has been frequently reported in spider mites, but injection of dsRNA is rarely reported. We compare here the efficiency of oral delivery versus injection of dsRNA in female adult mites. When comparing silencing efficiency, oral delivery of dsRNAs silenced 40.6 ± 8.9% of CPR, 63.8 ± 6.9% of CHMP2A, and 37.7 ± 5.7% of CHMP3 genes. Similar silencing efficiencies were found for injection (48.6 ± 3.7% of CPR, 70.2 ± 4.1% of CHMP2A, 59.8 ± 2.2% of CHMP3), but with much lower quantities of dsRNAs. Oral delivery of dsRNA failed to silence the expression of the CHMP4B gene, but this could be accomplished by injection of dsRNA (23.1 ± 1.0%). When scoring the phenotypic effects of silencing, both oral delivery and injection of CHMP2A- and CHMP3-dsRNA influenced the locomotion speed of mites significantly. For CPR, silencing could only be accomplished by dsRNA injection, not by feeding. CPR silencing significantly impacted the toxicity of a typical acaricide, pyridaben, as the susceptibility of mites raised 2.75-fold. Last, injection of Eya-dsRNA in adults produced transgenerational phenotypic effects on 3.59% of offspring, as quantified by an observed deviation in eye development, while oral delivery of Eya-dsRNA did not. In conclusion, injection of dsRNA is superior to oral delivery in silencing the expression of the selected genes in this study and could be considered the method of choice to study gene function in reverse genetic approaches.

Genomics and lipidomics analysis of the biotechnologically important oleaginous red yeast Rhodotorula glutinis ZHK provides new insights into its lipid and carotenoid metabolism.

BACKGROUND: Rhodotorula glutinis is recognized as a biotechnologically important oleaginous red yeast, which synthesizes numerous meritorious compounds with wide industrial usages. One of the most notable properties of R. glutinis is the formation of intracellular lipid droplets full of carotenoids. However, the basic genomic features that underlie the biosynthesis of these valuable compounds in R. glutinis have not been fully documented. To reveal the biotechnological potential of R. glutinis, the genomics and lipidomics analysis was performed through the Next-Generation Sequencing and HPLC-MS-based metabolomics technologies. RESULTS: Here, we firstly assemble the genome of R. glutinis ZHK into 21.8 Mb, containing 30 scaffolds and 6774 predicted genes with a N50 length of 14, 66,672 bp and GC content of 67.8%. Genome completeness assessment (BUSCO alignment: 95.3%) indicated the genome assembly with a high-quality features. According to the functional annotation of the genome, we predicted several key genes involved in lipids and carotenoids metabolism as well as certain industrial enzymes biosynthesis. Comparative genomics results suggested that most of orthologous genes have underwent the strong purifying selection within the five Rhodotorula species, especially genes responsible for carotenoids biosynthesis. Furthermore, a total of 982 lipids were identified using the lipidomics approaches, mainly including triacylglycerols, diacylglyceryltrimethylhomo-ser and phosphatidylethanolamine. CONCLUSION: Using whole genome shotgun sequencing, we comprehensively analyzed the genome of R. glutinis and predicted several key genes involved in lipids and carotenoids metabolism. By performing comparative genomic analysis, we show that most of the ortholog genes have undergone strong purifying selection within the five Rhodotorula species. Furthermore, we identified 982 lipid species using lipidomic approaches. These results provided valuable resources to further advance biotechnological applications of R .glutinis.

Whole genome sequencing and comparative genomic analysis of oleaginous red yeast Sporobolomyces pararoseus NGR identifies candidate genes for biotechnological potential and ballistospores-shooting.

BACKGROUND: Sporobolomyces pararoseus is regarded as an oleaginous red yeast, which synthesizes numerous valuable compounds with wide industrial usages. This species hold biotechnological interests in biodiesel, food and cosmetics industries. Moreover, the ballistospores-shooting promotes the colonizing of S. pararoseus in most terrestrial and marine ecosystems. However, very little is known about the basic genomic features of S. pararoseus. To assess the biotechnological potential and ballistospores-shooting mechanism of S. pararoseus on genome-scale, the whole genome sequencing was performed by next-generation sequencing technology. RESULTS: Here, we used Illumina Hiseq platform to firstly assemble S. pararoseus genome into 20.9 Mb containing 54 scaffolds and 5963 predicted genes with a N50 length of 2,038,020 bp and GC content of 47.59%. Genome completeness (BUSCO alignment: 95.4%) and RNA-seq analysis (expressed genes: 98.68%) indicated the high-quality features of the current genome. Through the annotation information of the genome, we screened many key genes involved in carotenoids, lipids, carbohydrate metabolism and signal transduction pathways. A phylogenetic assessment suggested that the evolutionary trajectory of the order Sporidiobolales species was evolved from genus Sporobolomyces to Rhodotorula through the mediator Rhodosporidiobolus. Compared to the lacking ballistospores Rhodotorula toruloides and Saccharomyces cerevisiae, we found genes enriched for spore germination and sugar metabolism. These genes might be responsible for the ballistospores-shooting in S. pararoseus NGR. CONCLUSION: These results greatly advance our understanding of S. pararoseus NGR in biotechnological potential and ballistospores-shooting, which help further research of genetic manipulation, metabolic engineering as well as its evolutionary direction.

Multimetal Borides Nanochains as Efficient Electrocatalysts for Overall Water Splitting.

The development of cost-efficient, active, and stable electrode materials as bifunctional catalysts for electrochemical water splitting is crucial to high-performance renewable energy storage and conversion devices. In this work, the synthesis of Co-based multi-metal borides nanochains with amorphous structure is reported for boosting the oxygen evolution (OER) and hydrogen evolution reactions (HER) by one-pot NaBH4 reduction of Co2+ , Ni2+ , and Fe2+ under ambient temperature. In all the investigated Co-based metal borides, NiCoFeB nanochains show the excellent OER performance with a low overpotential of 284 mV at 10 mA cm-2 and Tafel slope of 46 mV dec-1 , respectively, together with excellent catalytic stability, and robust HER performance with an overpotential of 345 mV at 10 mA cm-2 . The density functional theory (DFT) calculations reveal that the excellent electrocatalytic performance is mainly attributed to optimal electronic structure by tuning the Co-3d band activities by the incorporation of Ni and Fe for enhanced water splitting via the potentially existed Co0 state. Moreover, the electrolyzer using NiCoFeB nanochains as anode and cathode offers 10 mA cm-2 at a cell voltage of 1.81 V, comparable to commercial Pt/C // Ir/C, providing a simple method to design and explore highly efficient and cheap bifunctional electrocatalysts for overall water splitting.

[Advances in phytoene dehydrogenase - A review].

Carotenoids, as a group of over 700 valuable unsaturated terpene compounds classified as carotene and xanthophyll family, are endowed with powerful nutritional value. Phytoene dehydrogenase is the key rate-limiting enzyme in carotenoids biosynthesis pathway, involved in catalyzing the conversion from colorless hydrocarbon phytoene to other pigmented carotenoids, and plays an essential central regulation role. The function of phytoene dehydrogenases from different organisms exist diversity. CrtP, CrtQ and isomerase CrtH are essential for the formation of lycopene in most Cyanobacteria, whereas PDS, ZDS and isomerase Z-ISO, CrtISO are in charge of producing lycopene in most algae and plants. Nevertheless, there is only one CrtI-type for the formation of neurosporene, lycopene or dehydrolycopene in most bacteria and fungi. In this review, isolation, characterization, functional diversity, transcription regulatory mechanisms and phylogenetic analysis of phytoene dehydrogenase from different organisms are illustrated. This paper will provide insights into phytoene dehydrogenase and may facilitate the optimization of carotenoids production in genetic engineering strategy.

Fungal biofilm formation and its regulatory mechanism.

Fungal biofilm is a microbial community composed of fungal cells and extracellular polymeric substances (EPS). In recent years, fungal biofilms have played an increasingly important role in many fields. However, there are few studies on fungal biofilms and their related applications and development are still far from enough. Therefore, this review summarizes the composition and function of EPS in fungal biofilms, and improves and refines the formation process of fungal biofilms according to the latest viewpoints. Moreover, based on the study of Saccharomyces cerevisiae and Candida albicans, this review summarizes the gene regulation network of fungal biofilm synthesis, which is crucial for systematically understanding the molecular mechanism of fungal biofilm formation. It is of great significance to further develop effective methods at the molecular level to control harmful biofilms or enhance and regulate the formation of beneficial biofilms. Finally, the quorum sensing factors and mixed biofilms formed by fungi in the current research of fungal biofilms are summarized. These results will help to deepen the understanding of the formation process and internal regulation mechanism of fungal biofilm, provide reference for the study of EPS composition and structure, formation, regulation, group behavior and mixed biofilm formation of other fungal biofilms, and provide strategies and theoretical basis for the control, development and utilization of fungal biofilms.

Effect and Safety of Herbal Medicine Foot Baths in Patients with Diabetic Peripheral Neuropathy: A Multicenter Double-Blind Randomized Controlled Trial.

OBJECTIVE: To evaluate the effect and safety of foot baths with Tangbi Waixi Decoction (TW) in treating patients with diabetic peripheral neuropathy (DPN). METHODS: It is a multicenter double-blinded randomized controlled trial. Participants with DPN were recruited between November 18, 2016 and May 30, 2018 from 8 hospitals in China. All patients received basic treatments for glycemic management. Patients received foot baths with TW herbal granules either 66.9 g (intervention group) or 6.69 g (control group) for 30 min once a day for 2 weeks and followed by a 2-week rest, as a therapeutic course. If the Toronto Clinical Scoring System total score (TCSS-TS) ⩾6 points, the patients received a total of 3 therapeutic courses (for 12 weeks) and were followed up for 12 weeks. The primary outcome was change in TCSS-TS score at 12 and 24 weeks. Secondary outcomes included changes in bilateral motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) of the median and common peroneal nerve. Safety was also assessed. RESULTS: Totally 632 patients were enrolled, and 317 and 315 were randomized to the intervention and control groups, respectively. After the 12-week intervention, patients in both groups showed significant declines in TCSSTS scores, and significant increases in MNCV and SNCV of the median and common peroneal nerves compared with pre-treatment (P<0.05). The reduction of TCSS-TS score at 12 weeks and the increase of SNCV of median nerve at 24 weeks in the control group were greater than those in the intervention group (P<0.05). The number of adverse events did not differ significantly between groups (P>0.05), and no serious adverse event was related with treatment. CONCLUSION: Treatment of TW foot baths was safe and significantly benefitted patients with DPN. A low dose of TW appeared to be more effective than a high dose. (Registry No. ChiCTR-IOR-16009331).

Integrative analysis of genomic and metabolomic data reveals key metabolic pathways involved in lipid and carotenoid biosynthesis in oleaginous red yeast Rhodosporidiobolus odoratus XQR.

Rhodosporidiobolus odoratus, one of the oleaginous red yeasts, is gaining biotechnological importance for their ability to produce microbial lipids and carotenoids. However, to date, the genomic resource underling lipid and carotenoid biosynthesis in R. odoratus has not been reported. Here, we reported the first genome assembly of R. odoratus using a combination of PacBio and Illumina techniques. The final genome assembly is 23.74 Mb in size, containing 52 scaffolds with a N50 length of 1200,460 bp and a GC content of 56.90%. Benchmarking Universal Single-Copy Orthologs (BUSCO) assessment showed that our assembly contains 94.23% of Basidiomycota universal single-copy orthologs. The genome was predicted to contain 4986 protein-coding genes, 4967 of which were functionally annotated. Metabolomic profiling identified 574 lipids, 3 carotenoids, and 208 volatile organic compounds synthesized by R. odoratus. Integrative analysis of genomics and metabolomics provides insights into the biosynthesis of lipid, carotenoid, and other bioactive compounds in R. odoratus. Collectively, the results presented herein greatly enhance our understanding of R. odoratus in lipids and carotenoids biosynthesis, and thus further accelerate its fundamental molecular investigations and biotechnological applications.

Electrodeposited copper enhanced removal of 2,4-dichlorophenol in batch and flow reaction in Cu@CC-PS-MFC system.

Combination of microbial fuel cell (MFC) and advanced oxidation process (AOP) is promising for pollutant removal. In this paper, Cu0-loaded carbon cloth cathode by electrodeposition (Cu@CC-PS-MFC) was applied to enhance 2,4-dichlorophenol (2,4-DCP) degradation based on persulfate (PS) activation in microbial fuel cell. Cu0 exhibited a typical structure of face-centered cubic metal polyhedron on carbon cloth. The removal of 2,4-DCP by Cu@CC-PS-MFC (75.6%) was enhanced by more than 50% compared to CC-PS-MFC (49.2%) after 1 h of reaction. 30 mg/L 2,4-DCP in Cu@CC-PS-MFC was completely removed and achieved a high mineralization (80.6%) after 9 h of reaction under optimized condition with low dissolved copper ion concentration (0.615 mg/L). Meanwhile, more than 90% removal of 2,4-DCP was stably achieved with flow operation condition (hydraulic residence time of 7.2 h). The change of copper valent state Cu0/Cu2O/CuO was the main mechanism of PS activation with main reactive species of O•H and O21. The bioanode of MFC enhanced the in-situ regeneration of ≡Cu+ and ≡Cu0 on the catalyst surface by transporting electrons, which was believed to contribute to good catalyst lifetime and excellent 2,4-DCP removal. Electrodeposited copper contributes to the enhanced degradation of 2,4-DCP with energy recovery at the same time which can further broaden the application MFC.

Complete genome sequence of the Pogostemon cablin bacterial wilt pathogen Ralstonia solanacearum strain SY1.

BACKGROUND: Ralstonia solanacearum causes bacterial wilt of Pogostemon cablin which is an important aromatic herb and also the main materials of COVID-19 therapeutic traditional drugs. However, we are lacking the information on the genomic sequences of R. solanacearum isolated from P. cablin. OBJECTIVE: The acquisition and analysis of this whole-genome sequence of the P. cablin bacterial wilt pathogen. METHODS: An R. solanacearum strain, named SY1, was isolated from infected P. cablin plants, and the complete genome sequence was sequenced and analyzed. RESULTS: The SY1 strain contains a 3.70-Mb chromosome and a 2.18-Mb megaplasmid, with GC contents of 67.57% and 67.41%, respectively. A total of 3308 predicted genes were located on the chromosome and 1657 genes were located in the megaplasmid. SY1 strain has 273 unique genes compared with five representative R. solanacearum strains, and these genes were enriched in the plant-pathogen interaction pathway. SY1 possessed a higher syntenic relationship with phylotype I strains, and the arsenal of type III effectors predicted in SY1 were also more closely related to those of phylotype I strains. SY1 contained 14 and 5 genomic islands in its chromosome and megaplasmid, respectively, and two prophage sequences in its chromosome. In addition, 215 and 130 genes were annotated as carbohydrate-active enzymes and antibiotic resistance genes, respectively. CONCLUSION: This is the first genome-scale assembly and annotation for R. solanacearum which isolated from infected P. cablin plants. The arsenal of virulence and antibiotic resistance may as the determinants in SY1 for infection of P. cablin plants.

Biocontrol endophytes Bacillus subtilis R31 influence the quality, transcriptome and metabolome of sweet corn.

During colonization of soil and plants, biocontrol bacteria can effectively regulate the physiological metabolism of plants and induce disease resistance. To illustrate the influence of Bacillus subtilis R31 on the quality, transcriptome and metabolome of sweet corn, field studies were conducted at a corn experimental base in Zhuhai City. The results show that, after application of B. subtilis R31, sweet corn was more fruitful, with a 18.3 cm ear length, 5.0 cm ear diameter, 0.4 bald head, 403.9 g fresh weight of single bud, 272.0 g net weight of single ear, and 16.5 kernels sweetness. Combined transcriptomic and metabolomic analyses indicate that differentially expressed genes related to plant-pathogen interactions, MAPK signaling pathway-plant, phenylpropanoid biosynthesis, and flavonoid biosynthesis were significantly enriched. Moreover, the 110 upregulated DAMs were mainly involved in the flavonoid biosynthesis and flavone and flavonol biosynthesis pathways. Our study provides a foundation for investigating the molecular mechanisms by which biocontrol bacteria enhance crop nutrition and taste through biological means or genetic engineering at the molecular level.

Rhodotorula mucilaginosa-alternative sources of natural carotenoids, lipids, and enzymes for industrial use.

Biotechnologically useful yeast strains have been receiving important attention worldwide for the demand of a wide range of industries. Rhodotorula mucilaginosa is recognized as a biotechnologically important yeast that has gained great interest as a promising platform strain, owing to the diverse substrate appetites, robust stress resistance, and other gratifying features. Due to its attractive properties, R. mucilaginosa has been regarded as an excellent candidate for the biorefinery of carotenoids, lipids, enzymes, and other functional bioproducts by utilizing low-cost agricultural waste materials as substrates. These compounds have aroused great interest as the potential alternative sources of health-promoting food products, substrates for so-called third-generation biodiesel, and dyes or functional ingredients for cosmetics. Furthermore, the use of R. mucilaginosa has rapidly increased as a result of advancements in fermentation for enhanced production of these valuable bioactive compounds. This review focuses on R. mucilaginosa in these advancements and summarizes its potential prospects as alternative sources of natural bioproducts.

Transcriptomic and metabolomic analysis reveals the potential mechanisms underlying the improvement of ß-carotene and torulene production in Rhodosporidiobolus colostri under low temperature treatment.

Carotenoids are a group of versatile isoprenoid pigments widely utilized in the food, pharmaceutical and cosmetic industries. Rhodosporidiobolus colostri is a cold-adapted yeast that has piqued interest as a natural source of microbial carotenoids, including ß-carotene, torulene and torularhodin. Here, the effect of low temperature on carotenoid production in R. colostri was investigated. The results indicated that the total carotenoid production was significantly increased at the low temperature (16 ℃) treatment (29.016 mg/L) as compared to control (25 ℃) (17.147 mg/L) after 5 days of cultivation. Among them, the increase in ß-carotene and torulene serve as the main contributors to the improvement in total carotenoid production. Integrative analyses of the transcriptome and metabolome suggested that the up-regulation of the terpenoid backbone biosynthesis pathway and the down-regulation of the TCA cycle flux allow more acetyl-CoA to be diverted to carotenogenesis, which might be the reason for the increased production of ß-carotene and torulene in R. colostri under low temperature treatment. Our results presented herein should not only provide an effective strategy for increasing total carotenoids production in R. colostri, but lay the molecular groundwork to further facilitate genetic engineering to enhance the yield of certain carotenoids.

Functional verification and characterization of a type-III geranylgeranyl diphosphate synthase gene from Sporobolomyces pararoseus NGR.

Carotenoids, a group of natural pigments, have strong antioxidant properties and act as precursors to vitamin A, which have garnered attention from industry and researchers. Sporobolomyces pararoseus represents a hyper-producer of carotenoids, mainly including ß-carotene, torulene, and torularhodin. Geranylgeranyl diphosphate synthase (GGPPS) is regarded as a key enzyme in the carotenoid biosynthesis pathway. However, the precise nature of the gene encoding GGPPS in S. pararoseus has not been reported yet. Here, we cloned a cDNA copy of the GGPPS protein-encoding gene crtE from S. pararoseus NGR. The crtE full-length genomic DNA and cDNA are 1,722 and 1,134 bp, respectively, which consist of 9 exons and 8 introns. This gene encodes 377 amino acids protein with a predicted molecular mass of 42.59 kDa and a PI of 5.66. Identification of the crtE gene encoding a functional GGPPS was performed using heterologous complementation detection in Escherichia coli. In vitro enzymatic activity experiments showed that CrtE utilized farnesyl diphosphate (FPP) as an allylic substrate for the condensation reaction with isopentenyl diphosphate (IPP), generating more of the unique product GGPP compared to other allylic substrates. The predicted CrtE 3D-model was analyzed in comparison with yeast GGPPS. The condensation reaction occurs in the cavity of the subunit, and three bulky amino acids (Tyr110, Phe111, and His141) below the cavity prevent further extension of the product. Our findings provide a new source of genes for carotenoid genetic engineering.

High-Quality Genome Assembly of Oleaginous Red Yeast Sporobolomyces roseus CGMCC 2.4355.

Sporobolomyces roseus is an important oleaginous red yeast with critical biotechnological applications and has received significant recognition as a valuable source of industrial enzymes, carotenoids, and lipids. To reveal the genetic basis and functional components underlying its biotechnological applications, a high-quality genome assembly is required. Here, we present a novel genome assembly of S. roseus CGMCC 2.4355 using a combination of Illunima and Oxford Nanopore technologies. The genome has an assembly size of 21.4 Mb and consists of 15 scaffolds with an N50 length of 2,126,566 bp and GC content of 49.52%. The assembly is of high integrity, comprising 95.2% complete Benchmarking Universal Single-Copy Orthologs (BUSCOs) as evaluated by a genome completeness assessment. The genome was predicted to contain 8,124 protein-coding genes, 6,890 of which were functionally annotated. We believe that the combination of our analyses and high-quality genome assembly will promote the basic development of S. roseus as an agent for biotechnological applications and make a significant contribution to assess the evolutionary relationship of Sporobolomyces species.