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Hygromycin B (HYG-B) is a 5-glycosylated 2-dexoystreptamine- aminoglycoside antibiotic-(2DOS-AGA) produced by Streptomyces hygroscopicus subspecies hygroscopicus NRRL ISP-5578 with broad-spectrum activity against many pathogenic bacteria, fungi and helminths. In the literature, limited studies are concerned with the biosynthetic pathway and different cultural conditions affecting the production of HYG-B. This study aimed to optimize key environmental conditions influencing its production as one-factor-at-a-time (OFAT) and as a statistical model of response surface D-optimal design (DOD). Moreover, the biosynthetic pathway of HYG-B in light of the identified genes/proteins of the HYG-B gene cluster was proposed and elucidated. The effect of culture media composition and incubation time were studied OFAT, and the results showed that both culture media 6 (CM6) and CM4 gave the highest specific productivity, 5.88 and 3.99 µg/mg, respectively, and 7 days as incubation time. So, using CM6 and 7 days incubation resulted in a sevenfold increase (190 µg/mL) compared to the original unoptimized condition (CM1 and 6 days incubation; 26.9 µg/mL). Three important factors—initial pH, incubation temperature, and agitation—were tested using a DOD quadratic model generating 20 experimental runs. An initial pH of 6.4, an incubation temperature, of 28 ℃, and agitation. of 295 rpm were predicted and experimentally verified, resulting in a 13-fold increase (371.5 µg/mL) compared to the unoptimized condition and a sevenfold increase compared to that obtained as OFAT. In conclusion, DOD design is an efficient tool for optimizing HYG-B. However, the optimized conditions should be scaled up in a bioreactor for industrial production of HYG-B by S. hygroscopicus NRRL ISP-5578.

In this study, we have demonstrated the applicability of electroporation for the stable nuclear transformation of Coccomyxa solorinae-saccatae. An antibiogram revealed that Hygromycin B and G418 are the most effective selective agents among eight different antibiotics tested. We have shown that a plasmid vector containing the hptII gene, coding for hygromycin B phosphotransferase, with expression driven by the strong cauliflower mosaic virus CaMV35S promoter ensures sufficient protection of transformed algal cells against high concentrations of Hygromycin B. The ability to drive transgene expression in the alga C. solorinae-saccatae offers unique opportunities to study the physiology of lichenic algae, as it is one of the symbiotic strains of the Coccomyxa simplex group. Furthermore, our findings demonstrate that electroporation is a convenient and effective technique for the transformation of algae in the Coccomyxa genus.

Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene ( aph7” ). Integration of aph7” in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7” . The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7” cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase ( UpAPT ). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.

Callus browning, a common trait derived from the indica rice cultivar ( Oryza sativa L.), is a challenge to transformation regeneration. Here, we report the map-based cloning of BROWNING OF CALLUS1 ( BOC1 ) using a population derived from crossing Teqing, an elite indica subspecies exhibiting callus browning, and Yuanjiang, a common wild rice accession ( Oryza rufipogon Griff.) that is less susceptible to callus browning. We show that BOC1 encodes a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE (SRO) protein. Callus browning can be reduced by appropriate upregulation of BOC1 , which consequently improves the genetic transformation efficiency. The presence of a Tourist -like miniature inverted-repeat transposable element ( Tourist MITE) specific to wild rice in the promoter of BOC1 increases the expression of BOC1 in callus. BOC1 may decrease cell senescence and death caused by oxidative stress. Our study provides a gene target for improving tissue culturability and genetic transformation. Callus browning heavily affects indica rice transformation regeneration. Here, the authors show transposon insertion in the promoter of BOC1 gene, encoding a SIMILAR TO RADICAL-INDUCED CELL DEATH ONE protein, can upregulate its expression and decrease callus browning in cultivated rice by releasing oxidative stress.

The ever-growing rise of antibiotic resistance among bacterial pathogens is one of the top healthcare threats today. Although combination antibiotic therapies represent a potential approach to more efficiently combat infections caused by susceptible and drug-resistant bacteria, only a few known drug pairs exhibit synergy/cooperativity in killing bacteria. Here, we discover that well-known ribosomal antibiotics, hygromycin A (HygA) and macrolides, which target peptidyl transferase center and peptide exit tunnel, respectively, can act cooperatively against susceptible and drug-resistant bacteria. Remarkably, HygA slows down macrolide dissociation from the ribosome by 60-fold and enhances the otherwise weak antimicrobial activity of the newest-generation macrolide drugs known as ketolides against macrolide-resistant bacteria. By determining a set of high-resolution X-ray crystal structures of drug-sensitive wild-type and macrolide-resistant Erm-methylated 70S ribosomes in complex with three HygA-macrolide pairs, we provide a structural rationale for the binding cooperativity of these drugs and also uncover the molecular mechanism of overcoming Erm-type resistance by macrolides acting together with hygromycin A. Altogether our structural, biochemical, and microbiological findings lay the foundation for the subsequent development of synergistic antibiotic tandems with improved bactericidal properties against drug-resistant pathogens, including those expressing erm genes. The authors discovered that hygromycin A not only enhances the cell-killing properties of macrolides but also renders them active against resistant bacteria. The provided structures of antibiotic pairs in complex with WT and macrolide-resistant ribosomes rationalize binding cooperativity of these drugs.

We previously reported Agrobacterium-mediated transformation methods for the liverwort Marchantia polymorpha using the hygromycin phosphotransferase gene as a marker for selection with hygromycin. In this study, we developed three additional markers for M. polymorpha transformation: the gentamicin 3'-acetyltransferase gene for selection with gentamicin; a mutated acetolactate synthase gene for selection with chlorsulfuron; and the neomycin phosphotransferase II gene for selection with G418. Based on these four marker genes, we have constructed a series of Gateway binary vectors designed for transgenic experiments on M. polymorpha. The 35S promoter from cauliflower mosaic virus and endogenous promoters for constitutive and heat-inducible expression were used to create these vectors. The reporters and tags used were Citrine, 3×Citrine, Citrine-NLS, TagRFP, tdTomato, tdTomato-NLS, GR, SRDX, SRDX-GR, GUS, ELuc(PEST), and 3×FLAG. These vectors, designated as the pMpGWB series, will facilitate molecular genetic analyses of the emerging model plant M. polymorpha.

Madurella mycetomatis is the main cause of mycetoma, a chronic granulomatous infection for which currently no adequate therapy is available. To improve therapy, more knowledge on a molecular level is required to understand how M . mycetomatis is able to cause this disease. However, the genetic toolbox for M . mycetomatis is limited. To date, no method is available to genetically modify M . mycetomatis . In this paper, a protoplast-mediated transformation protocol was successfully developed for this fungal species, using hygromycin as a selection marker. Furthermore, using this method, a cytoplasmic-GFP-expressing M . mycetomatis strain was created. The reported methodology will be invaluable to explore the pathogenicity of M . mycetomatis and to develop reporter strains which can be useful in drug discovery as well as in genetic studies.

On the basis of the one strain-many compounds (OSMAC) strategy, two new hygromycin A derivatives (3, 4), together with six known compounds were isolated from a medicinal plant inter rhizospheric Streptomyces in Pulsatilla chinensis. The structures of 3 and 4 were elucidated using NMR and HRESIMS analyses. A plausible biosynthetic pathway for these compounds was discussed. All the compounds were evaluated for their antimicrobial and cytotoxic activities. Compound 5 exhibited potent inhibitory activity against S. aureus and B. subtilis with the MICs of 16 and 8 μg ml−1, while 4 showed weak inhibitory activity against S. aureus.

Background Cutaneotrichosporon oleaginosus is an oleaginous yeast that can produce up to 80% lipid per dry weight. Its high capacity for the biosynthesis of single cell oil makes it highly interesting for the production of engineered lipids or oleochemicals for industrial applications. However, the genetic toolbox for metabolic engineering of this non-conventional yeast has not yet been systematically expanded. Only three long endogenous promoter sequences have been used for heterologous gene expression, further three dominant and one auxotrophic marker have been established. Results In this study, the structure of putative endogenous promoter sequences was analyzed based on more than 280 highly expressed genes. The identified motifs of regulatory elements and translational initiation sites were used to annotate the four endogenous putative promoter sequences D9FADp, UBIp, PPIp, and 60Sp. The promoter sequences were tested in a construct regulating the known dominant marker hygromycin B phosphotransferase. The four newly described promoters and the previously established GAPDHp successfully initiated expression of the resistance gene and PPIp was selected for further marker development. The geneticin G418 resistance (aminoglycoside 3’-phosphotransferase, APH) and the nourseothricin resistance gene N-acetyl transferase (NAT) were tested for applicability in C. oleaginosus . Both markers showed high transformation efficiency, positive rate, and were compatible for combined use in a successive and simultaneous manner. Conclusions The implementation of four endogenous promoters and one novel dominant resistance markers for C. oleaginosus opens up new opportunities for genetic engineering and strain development. In combination with recently developed methods for targeted genomic integration, the established toolbox allows a wide spectrum of new strategies for genetic and metabolic engineering of the industrially highly relevant yeast.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG 4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium -mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD 600 of the infection solution(suspension of A . tumefaciens ) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.