High Transformation Efficiency Research Articles

Organophotocatalyst Enabled Deoxycyclopropanation of Alcohols.

Cyclopropane fragments, which widely exist in marketed drugs and natural products, can confer special pharmacological properties to small-molecule drugs. Therefore, developing methods to construct cyclopropanes is of great significance. Nevertheless, the introduction of cyclopropane primarily relies on already-formed cyclopropyl groups, which significantly restricts the diversity of cyclopropane skeletons. Late-stage direct cyclopropanation is still a challenging task. Herein, a photo-induced intermolecular deoxycyclopropanation reaction that employs alcohols as substrates, and 1 mol.% of 2,3,5,6-tetrakis(carbazol-9-yl)-1,4-dicyanobenzene (4CzTPN)as organophotocatalyst is reported. This method proceeds with high transformation efficiency (up to 98% yield) and exhibits broad functional group tolerance, such as primary, secondary, and tertiary alcohols as well as various activated β-halogenated alkenes. This process is mild, easy to operate, and has low equipment requirements. The power of this technology is demonstrated by the late-stage functionalization of five marketed drugs and five natural products.

Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.

Novel Approach of Tackling Wax Deposition Problems in Pipeline Using Enzymatic Degradation Process: Challenges and Potential Solutions

Anthropogenic activities have led to hydrocarbon spills, and while traditional bioremediation methods are costly and time-consuming, recent research has focused on engineered enzymes for managing pollutant. The potential of enzymes for resolving wax flow problems in the petroleum industry remains unexplored. This paper offers a comprehensive review of the current state of research activities related to the bioremediation of petroleum-polluted sites and the biodegradation of specific petroleum hydrocarbons. The assayed enzymes that took part in the degradation were discussed in detail. Lipase, laccase, alkane hydroxylase, alcohol dehydrogenase, esterase, AlkB homologs and cytochrome P450 monooxygenase are among the enzymes responsible for the degradation of more than 50% of the hydrocarbons in contaminated soil and wastewater and found to be active on carbon C8 to C40. The possible biodegradation mechanism of petroleum hydrocarbons was also elucidated. The enzymes’ primary metabolic pathways include terminal, subterminal, and ω-oxidation. Next, given the successful evidence of the hydrocarbon treatment efficiency, the authors analyzed the opportunity for the enzymatic degradation approach if it were to be applied to a different scenario: managing wax deposition in petroleum-production lines. With properties such as high transformation efficiency and high specificity, enzymes can be utilized for the treatment of viscous heavy oil for transportability, evidenced by the 20 to 99% removal of hydrocarbons. The challenges associated with the new approach are also discussed. The production cost of enzymes, the characteristics of hydrocarbons and the operating conditions of the production line may affect the biocatalysis reaction to some extent. However, the challenges can be overcome by the usage of extremophilic enzymes. The combination of technological advancement and deployment strategies such as the immobilization of a consortium of highly thermophilic and halotolerant enzymes is suggested. Recovering and reusing enzymes offers an excellent strategy to improve the economics of the technology. This paper provides insights into the opportunity for the enzymatic degradation approach to be expanded for wax deposition problems in pipelines.

Optimized genome-wide CRISPR screening enables rapid engineering of growth-based phenotypes in Yarrowia lipolytica

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, we designed a compact, high-activity genome-wide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm and a large dataset of ∼50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering.

Next-generation AMA1-based plasmids for enhanced heterologous expression in filamentous fungi.

Episomal AMA1-based plasmids are increasingly used for expressing biosynthetic pathways and CRISPR/Cas systems in filamentous fungi cell factories due to their high transformation efficiency and multicopy nature. However, the gene expression from AMA1 plasmids has been observed to be highly heterogeneous in growing mycelia. To overcome this limitation, here we developed next-generation AMA1-based plasmids that ensure homogeneous and strong expression. We achieved this by evaluating various degradation tags fused to the auxotrophic marker gene on the AMA1 plasmid, which introduces a more stringent selection pressure throughout multicellular fungal growth. With these improved plasmids, we observed in Aspergillus nidulans a 5-fold increase in the expression of a fluorescent reporter, a doubling in the efficiency of a CRISPRa system for genome mining, and a up to a 10-fold increase in the production of heterologous natural product metabolites. This strategy has the potential to be applied to diverse filamentous fungi.

BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus

BackgroundTo effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process.ResultsTranscriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R−M− without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained.ConclusionsThe enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.Graphical

Electrocatalytic upcycling of plastic waste: Progress, challenges, and future

AbstractThe escalating accumulation of plastic waste has been developed into a formidable global environmental challenge. Traditional disposal methods such as landfilling and incineration not only exacerbate environmental degradation by releasing harmful chemicals and greenhouse gases, but also squander finite resources that could otherwise be recycled or repurposed. Upcycling is a kind of plastic recycling technology that converts plastic waste into high‐value chemicals and helps to avoid resource waste and environmental pollution. Electrocatalytic upcycling emerges as a novel technology distinguished by its mild operational conditions, high transformation efficiency and product selectivity. This review commences with an overview of the recycling and upcycling technology employed in plastic waste management and the respective advantages and inherent limitations are also delineated. The different types of plastic waste upcycled by electrocatalytic strategy are then discussed and the plastic waste transformation process is examined together with the mechanisms underlying the electrocatalytic upcycling. Furthermore, the structure‐activity relationships between electrocatalysts and plastic waste upcycling performance are also elucidated. The review aims to furnish readers with a comprehensive understanding of the electrocatalytic techniques for plastic waste upcycling and to provide a guidance for the design of electrocatalysts towards efficient plastic waste transformation.

An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 β-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.

Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production.

Genetic engineering at the genomic scale provides a rapid means to evolve microbes for desirable traits. However, in many filamentous fungi, such trials are daunted by low transformation efficiency. Differentially expressed genes under certain conditions may contain important regulatory factors. Accordingly, although manipulating these subsets of genes only can largely reduce the time and labor, engineering at such a sub-genomic level may also be able to improve the microbial performance. Herein, first using the industrially important cellulase-producing filamentous fungus Trichoderma reesei as a model organism, we constructed suppression subtractive hybridization (SSH) libraries enriched with differentially expressed genes under cellulase induction (MM-Avicel) and cellulase repression conditions (MM-Glucose). The libraries, in combination with RNA interference, enabled sub-genomic engineering of T. reesei for enhanced cellulase production. The ability of T. reesei to produce endoglucanase was improved by 2.8~3.3-fold. In addition, novel regulatory genes (tre49304, tre120391, and tre123541) were identified to affect cellulase expression in T. reesei. Iterative manipulation using the same strategy further increased the yield of endoglucanase activity to 75.6 U/mL, which was seven times as high as that of the wild type (10.8 U/mL). Moreover, using Humicola insolens as an example, such a sub-genomic RNAi-assisted strain evolution proved to be also useful in other industrially important filamentous fungi. H. insolens is a filamentous fungus commonly used to produce catalase, albeit with similarly low transformation efficiency and scarce knowledge underlying the regulation of catalase expression. By combining SSH and RNAi, a strain of H. insolens producing 28,500 ± 288 U/mL of catalase was obtained, which was 1.9 times as high as that of the parent strain.IMPORTANCEGenetic engineering at the genomic scale provides an unparalleled advantage in microbial strain improvement, which has previously been limited only to the organisms with high transformation efficiency such as Saccharomyces cerevisiae and Escherichia coli. Herein, using the filamentous fungus Trichoderma reesei as a model organism, we demonstrated that the advantage of suppression subtractive hybridization (SSH) to enrich differentially expressed genes and the convenience of RNA interference to manipulate a multitude of genes could be combined to overcome the inadequate transformation efficiency. With this sub-genomic evolution strategy, T. reesei could be iteratively engineered for higher cellulase production. Intriguingly, Humicola insolens, a fungus with even little knowledge in gene expression regulation, was also improved for catalase production. The same strategy may also be expanded to engineering other microorganisms for enhanced production of proteins, organic acids, and secondary metabolites.

A Highly Efficient Agrobacterium rhizogenes-Mediated Hairy Root Transformation Method of Idesia polycarpa and the Generation of Transgenic Plants.

Idesia polycarpa is a promising woody oilseed species because of its high oil yield. However, its use is greatly limited due to the lack of varieties with good qualities; additionally, gene function has been less studied in this plant because an efficient transformation method has not been established yet. In this study, we established a rapid and efficient hairy root transformation method by infecting the whole seedling, the rootless seedling, and the leaf petiole with Agrobacterium rhizogenes using different infection methods. Among these transformation methods, a higher transformation efficiency was obtained using the whole seedling, which could reach up to 71.91%. Furthermore, we found that the seedling age significantly affected the transformation efficiency, either using whole or rootless seedlings. Additionally, we found that the transgenic roots could regenerate transgenic shoots. Taken together, our study lays the foundation for future study and for genetically modifying wood traits in the future.

Development and characterization of tobacco suspension cell chassis NBS-1

Plant synthetic biology has significant theoretical advantages in exploration and production of plant natural products. However, its contribution to the field of biosynthesis is currently limited due to the lack of efficient chassis systems and related enabling technologies. Synthetic biologists often avoid tobacco as a chassis system because of its long operation cycle, difficulties in genetic and metabolic modification, complex metabolism and purification background, nicotine toxicity, and challenges in accurately controlling for agricultural production. Nevertheless, the tobacco suspension cell chassis system offers a viable solution to these challenges. The objective of this research was to develop a tobacco suspension cell chassis with high scientific and industrial potential. This chassis should exhibit rapid growth, high biomass, excellent dispersion, high transformation efficiency, and minimal nicotine content. Nicotiana benthamiana, which has high applicability in molecular technology, was used to induce suspension cells. The induced suspension cells, named NBS-1, exhibited rapid growth, excellent dispersion, and high biomass, reaching a maximum biomass of 476.39 g/L (fresh weight), which was significantly higher than that of BY-2. The transformation efficiency of the widely utilized pEAQ-HT transient expression system in NBS-1 reached 81%, which was substantially elevated compared to BY-2. The metabolic characteristics and bias of BY-2 and NBS-1 were analyzed using transcriptome data. It was found that the gene expression of pathways related to biosynthesis of flavonoids and their derivatives in NBS-1 was significantly higher, while the pathways related to alkaloid biosynthesis were significantly lower compared to BY-2. These findings were further validated by the total content of flavonoid and alkaloid. In summary, our research demonstrates NBS-1 possesses minimal nicotine content and provides valuable guidance for selecting appropriate chassis for specific products. In conclusion, this study developed NBS-1, a tobacco suspension cell chassis with excellent growth and transformation, high flavonoid content and minimal nicotine content, which has important guiding significance for the development of tobacco suspension cell chassis.

Tissue culture and high-efficiency transformation in an apomictic initial variety of Paspalum notatum Flüggé

Tissue culture and high-efficiency transformation in an apomictic initial variety of Paspalum notatum Flüggé

A versatile, rapid Agrobacterium-mediated transient expression system for functional genomics studies in cannabis seedling.

We have developed and optimized a rapid, versatile Agrobacterium-mediated transient expression system for cannabis seedlings that can be used in functional genomics studies of both hemp-type and drug-type cannabis. Cannabis (Cannabis sativa L.) holds great promise in the medical and food industries due to its diverse chemical composition, including specialized cannabinoids. However, the study of key genes involved in various biological processes, including secondary metabolite biosynthesis, has been hampered by the lack of efficient in vivo functional analysis methods. Here, we present a novel, short-cycle, high-efficiency transformation method for cannabis seedlings using Agrobacterium tumefaciens. We used the RUBY reporter system to monitor transformation results without the need for chemical treatments or specialized equipment. Four strains of A. tumefaciens (GV3101, EHA105, LBA4404, and AGL1) were evaluated for transformation efficiency, with LBA4404 and AGL1 showing superior performance. The versatility of the system was further demonstrated by successful transformation with GFP and GUS reporter genes. In addition, syringe infiltration was explored as an alternative to vacuum infiltration, offering simplicity and efficiency for high-throughput applications. Our method allows rapid and efficient in vivo transformation of cannabis seedlings, facilitating large-scale protein expression and high-throughput characterization studies.

Development of a chloroplast expression system for Dunaliella salina

Dunaliella salina is an innovative expression system due to its distinct advantages such as high salt tolerance, low susceptibility to contamination, and the absence of the cell wall. While nuclear transformation has been extensively studied, research on D. salina chloroplast transformation remains in the preliminary stages. In this study, we established an efficient chloroplast expression system for D. salina using Golden Gate assembly. We developed a D. salina toolkit comprising essential components such as chloroplast-specific promoters, terminators, homologous fragments, and various vectors. We confirmed its functionality by expressing the EGFP protein. Moreover, we detailed the methodology of the entire construction process. This expression system enables the specific targeting of foreign genes through simple homologous recombination, resulting in stable expression in chloroplasts. The toolkit achieved a relatively high transformation efficiency within a shorter experimental cycle. Consequently, the construction and utilization of this toolkit have the potential to enhance the efficiency of transgenic engineering in D. salina and advance the development of microalgal biofactories.

The coupling transformation process of biodegradation and pyrolysis for Dananhu low-rank coal

The study presented a novel approach for coupling biodegradation and pyrolysis to transform low-rank coal. In the first stage, four kinds of bacteria, Planomicrobium huatugouensis (P. huatugouensis), Sphingomonas polyaromaticivorans (S. polyaromaticivorans), Pseudomonas putida (P. putida), and Bacillus subtilis subsp. subtilis (B. subtilis subsp. subtilis), were used to transform the Dananhu low-rank coal. After the first stage, the coal that has not been biodegraded (residual coal) undergoes the second stage of pyrolytic transformation, which is carried out by a thermogravimetric analyzer combined with Fourier-transform infrared spectrometry (TG-FTIR). The results showed that the maximum coupling transformation rate of biodegradation and pyrolysis could reach 73.95%, which was 21.88% higher than the thermal transformation rate of Dananhu low-rank coal and 27.37% higher than the optimal microbial transformation rate. This indicated that coupling transformation could significantly improve the utilization rate of Dananhu low-rank coal. In the stage of biodegradation transformation, bacteria destroyed the carboxyl groups, ether bonds, and other oxygen-containing functional groups in Dananhu low-rank coal, converting the large molecules of coal into liquid molecules, meanwhile, the compact coal structure became loose, and the residual coal was also pyrolyzed easily than Dananhu low-rank coal. During the pyrolysis process, a large number of fatty chains, carboxyl groups, oxy groups, and hydroxy groups were decomposed to produce lots of gas-phase products. The pyrolysis products showed an increase in the release of CO and CH4, and an increase in the proportion of aliphatic hydrocarbons in the volatiles. The high efficiency of coupling transformation is due to the combination of the advantages of the two technologies, which is achieving the green and energy-efficient utilization of low-rank coal.

Optimization of factors affecting Agrobacterium-mediated hairy root induction in Vitex negundo L. (Lamiaceae)

Vitex negundo L. is an aromatic, woody, blooming shrub in the Verbenaceae family which can grow into a small tree. Traditionally, V. negundo root has been used to cure diabetes, colic, boils, leprosy, and rheumatism. Keeping the importance of its roots in mind, an attempt has been taken for development of a protocol for efficient hairy root proliferation system. The Agrobacterium rhizogenes strains (MTCC 532 and MTCC 2364) were used for hairy root induction. For A. rhizogenes infection, both in vitro and in vivo leaves as well as internodes were used as explant. In vitro leaves and internodal explants were obtained by the inoculation of matured nodal segments on the optimum medium [MS + 2.0 mg/L N6-Benzylaminopurine (BAP)] with c.a. 91.6% shoot regeneration and an average of 8.1 shoots per explants. In vitro leaf showed best hairy root induction followed by in vitro internode on ½ MS medium augmented with acetosyringone. Highest transformation efficiency was achieved using MTCC 2364 strain, while no transformation was observed in MTCC 532 strain. Different factors affecting transformation including co-cultivation period, infection time and optical density (O.D.) value were standardized. The highest efficacy, 88.8% hairy root induction was observed in in vitro leaves infected by MTCC 2364 for 60 minutes infection time with an O.D. value of 0.29 maintained over a 44-48 hours of co-cultivation period. The prescribed protocol may be used as a reference for development of industrial scale hairy root production for bioactive compound located in root of V. negundo.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

BackgroundMost recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors.ResultsIn micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct.ConclusionsFor the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

Establishment of an Efficient Sugarcane Transformation System via Herbicide-Resistant CP4-EPSPS Gene Selection

Sugarcane (Saccharum spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The intricate polyploid and aneuploid genome of sugarcane has shown various limits for conventional breeding strategies. Nonetheless, biotechnological engineering currently offers the best chance of introducing commercially significant agronomic features. In this study, an efficient Agrobacterium-mediated transformation system that uses the herbicide-resistant CP4-EPSPS gene as a selection marker was developed. Notably, all of the plants that were identified by PCR as transformants showed significant herbicide resistance. Additionally, this transformation protocol also highlighted: (i) the high yield of transgenic lines from calli (each gram of calli generated six transgenic lines); (ii) improved selection; and (iii) a higher transformation efficiency. This protocol provides a reliable tool for a routine procedure for the generation of resilient sugarcane plants.

Optimization of induction and hairy root culture establishment in two mullein species, Verbascum erianthum and Verbascum stachydiforme

The genus Verbascum, belonging to the family Scrophulariaceae, has a significant center of diversity in Iran. Two of its species, V. erianthum and V. stachydiforme, originate in the Iranian-Turanian region, but no studies have been conducted on the induction of their hairy roots. This genus is a valuable source of biologically active compounds such as iridoid glycosides and flavonoids. Hairy root culture is a suitable technique for the production and accumulation of secondary metabolites. Three different studies were conducted to optimize the induction and establishment of hairy roots. In the first experiment, the influence of explant type (leaf and hypocotyl), six infection methods, and co-culture time (48 and 72 h) on the efficiency of hairy root induction was investigated. The results showed that the highest hairy root induction (68.18%) was observed in the leaf explants inoculated by direct infection with three wounds within 72 h co-culture time. In the second experiment, the effect of four Agrobacterium rhizogenes strains (ATCC 15834, A4, A7, and A13) and leaf age (14, 21, and 28 days) on transformation efficiency and some morphological traits examined in both species were studied. The high transformation efficiency of hairy root (80.55%) was detected in the 21-day-old leaf explant of V. erianthum species that was inoculated with the A13 strain. The transformed hairy root colons were confirmed by PCR using rolB gene-specific primers. To optimize hairy root growth and avoid tissue browning, hairy roots were cultured in various media containing different antioxidants and improver agents (including ascorbic acid, citric acid, and NAA). The results showed that the highest fresh growth index (20.42) and the lowest tissue browning (9%) as well as total phenol (8.51 mg GA/g DW), and total flavonoid content (4.42 mg QUE/g DW) were obtained in medium B5 with 1.5 mg/l NAA.

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.