CRISPR-Cas13: Pioneering RNA Editing for Nucleic Acid Therapeutics.

The CRISPR-Cas13 system has emerged as a revolutionary tool for RNA editing, offering new opportunities for the development of nucleic acid therapeutics. Unlike DNA-targeting CRISPR-Cas9, Cas13 targets and cleaves RNA, enabling gene silencing and preventing genomic instability. Its applications include suppressing disease-causing genes, correcting splicing errors, and modulating immune responses. Despite these advances, challenges persist, such as the need to refine specificity, mitigate off-target impacts, and ensure effective delivery. This review provides an overview of the CRISPR-Cas13 mechanism, elucidating its role in RNA-targeted therapies and its transformative potential for disease treatment. Furthermore, it addresses the ongoing challenges that the scientific community is striving to overcome.

Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering.

Kauralexin A1 (KA1) is a key intermediate of the kauralexin A series metabolites of maize phytoalexins. However, their application is severely limited by their low abundance in maize. In this study, an efficient biosynthetic pathway was constructed to produce KA1 in Saccharomyces cerevisiae. Also, metabolic and enzyme engineering strategies were applied to construct the high-titer strains, such as chassis modification, screening synthases, the colocalization of enzymes, and multiple genomic integrations. First, the KA1 precursor ent-kaurene was synthesized using the efficient diterpene synthase GfCPS/KS from Fusarium fujikuroi, and optimized to reach 244.36 mg/L in shake flasks, which displayed a 200-fold increase compared to the initial strain. Then, the KA1 was produced under the catalysis of ZmCYP71Z18 from Zea mays and SmCPR1 from Salvia miltiorrhiza, and the titer was further improved by integrating the fusion protein into the genome. Finally, an ent-kaurene titer of 763.23 mg/L and a KA1 titer of 42.22 mg/L were achieved through a single-stage fed-batch fermentation in a 5 L bioreactor. This is the first report of the heterologous biosynthesis of maize diterpene phytoalexins in S. cerevisiae, which lays a foundation for further pathway reconstruction and biosynthesis of the kauralexin A series maize phytoalexins.

Enzyme and Metabolic Engineering Strategies for Biosynthesis of α-Farnesene in Saccharomyces cerevisiae.

α-Farnesene, a type of acyclic sesquiterpene, is an important raw material in agriculture, aircraft fuel, and the chemical industry. In this study, we constructed an efficient α-farnesene-producing yeast cell factory by combining enzyme and metabolic engineering strategies. First, we screened different plants for α-farnesene synthase (AFS) with the best activity and found that AFS from Camellia sinensis (CsAFS) exhibited the most efficient α-farnesene production in Saccharomyces cerevisiae 4741. Second, the metabolic flux of the mevalonate pathway was increased to improve the supply of the precursor farnesyl pyrophosphate. Third, inducing site-directed mutagenesis in CsAFS, the CsAFSW281C variant was obtained, which considerably increased α-farnesene production. Fourth, the N-terminal serine-lysine-isoleucine-lysine (SKIK) tag was introduced to construct the SKIK∼CsAFSW281C variant, which further increased α-farnesene production to 2.8 g/L in shake-flask cultures. Finally, the α-farnesene titer of 28.3 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Transcriptome Analysis and Identification of Sesquiterpene Synthases in Liverwort Jungermannia exsertifolia.

The liverwort Jungermannia exsertifolia is one of the oldest terrestrial plants and rich in structurally specific sesquiterpenes. There are several sesquiterpene synthases (STSs) with non-classical conserved motifs that have been discovered in recent studies on liverworts; these motifs are rich in aspartate and bind with cofactors. However, more detailed sequence information is needed to clarify the biochemical diversity of these atypical STSs. This study mined J. exsertifolia sesquiterpene synthases (JeSTSs) through transcriptome analysis using BGISEQ-500 sequencing technology. A total of 257,133 unigenes was obtained, and the average length was 933 bp. Among them, a total of 36 unigenes participated in the biosynthesis of sesquiterpenes. In addition, the in vitro enzymatic characterization and heterologous expression in Saccharomyces cerevisiae showed that JeSTS1 and JeSTS2 produced nerolidol as the major product, while JeSTS4 could produce bicyclogermacrene and viridiflorol, suggesting a specificity of J. exsertifolia sesquiterpene profiles. Furthermore, the identified JeSTSs had a phylogenetic relationship with a new branch of plant terpene synthases, the microbial terpene synthase-like (MTPSL) STSs. This work contributes to the understanding of the metabolic mechanism for MTPSL-STSs in J. exsertifolia and could provide an efficient alternative to microbial synthesis of these bioactive sesquiterpenes.

High auxin stimulates callus through SDG8-mediated histone H3K36 methylation in Arabidopsis.

Callus induction, which results in fate transition in plant cells, is considered as the first and key step for plant regeneration. This process can be stimulated in different tissues by a callus-inducing medium (CIM), which contains a high concentration of phytohormone auxin. Although a few key regulators for callus induction have been identified, the multiple aspects of the regulatory mechanism driven by high levels of auxin still need further investigation. Here, we find that high auxin induces callus through a H3K36 histone methylation-dependent mechanism, which requires the methyltransferase SET DOMAIN GROUP 8 (SDG8). During callus induction, the increased auxin accumulates SDG8 expression through a TIR1/AFBs-based transcriptional regulation. SDG8 then deposits H3K36me3 modifications on the loci of callus-related genes, including a master regulator WOX5 and the cell proliferation-related genes, such as CYCB1.1. This epigenetic regulation in turn is required for the transcriptional activation of these genes during callus formation. These findings suggest that the massive transcriptional reprogramming for cell fate transition by auxin during callus formation requires epigenetic modifications including SDG8-mediated histone H3K36 methylation. Our results provide insight into the coordination between auxin signaling and epigenetic regulation during fundamental processes in plant development.

Achievements and perspectives of synthetic biology in botanical insecticides.

Botanical insecticides are the origin of all insecticidal compounds. They have been widely used to control pests in crops for a long time. Currently, the commercial production of botanical insecticides extracted from plants is limited because of insufficient raw material supply. Synthetic biology is a promising and effective approach for addressing the current problems of the production of botanical insecticides. It is an emerging biological research hotspot in the field of botanical insecticides. However, the biosynthetic pathways of many botanical insecticides are not completely elucidated. On the other hand, the cytotoxicity of botanical pesticides and low efficiency of these biosynthetic enzymes in new hosts make it still challenging for their heterologous production. In the present review, we summarized the recent developments in the heterologous production of botanical insecticides, analyzed the current challenges, and discussed the feasible production strategies, focusing on elucidating biosynthetic pathways, enzyme engineering, host engineering, and cytotoxicity engineering. Looking to the future, synthetic biology promises to further advance heterologous production of more botanical pesticides.

Valorization of Food Processing Waste to Produce Valuable Polyphenolics.

Traditional incineration and landfill of food processing waste (FPW) have polluted the environment and underutilized valuable bioactive compounds, including polyphenols in food waste. As one of the most widely occurring compounds in the FPW, polyphenols possess high utilization value in many fields such as human health, energy, and environmental protection. Extracting polyphenols directly from FPW can maximize the value of polyphenols and avoid waste of resources. However, traditional polyphenol extraction methods mostly use the Soxhlet extraction, infiltration, and impregnation method, consuming a large amount of organic solvent and suffering from long extraction time and low extraction efficiency. Emerging green extraction methods such as supercritical fluid extraction, ultrasonic-assisted extraction, microwave-assisted extraction, and other methods can shorten the extraction time and improve the solvent extraction efficacy, resulting in the green and safe recovery of polyphenols from FPW. In this paper, the traditional treatment methods of FPW waste and the application of polyphenols in FPW are briefly reviewed, and the traditional extraction methods and emerging green extraction methods of polyphenols in FPW are compared to obtain insight into the start-of-the-art extraction approaches.

Exogenous artificial DNA forms chromatin structure with active transcription in yeast.

Yeast artificial chromosomes (YACs) are important tools for sequencing, gene cloning, and transferring large quantities of genetic information. However, the structure and activity of YAC chromatin, as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events, have not been widely explored. Here, we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported data-carrying chromosome (dChr). In addition, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional, and proteomic characteristics of the exogenous dChr. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. The dChr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs. The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids. A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.

TMK1-based auxin signaling regulates abscisic acid responses via phosphorylating ABI1/2 in Arabidopsis.

Differential concentrations of phytohormone trigger distinct outputs, which provides a mechanism for the plasticity of plant development and an adaptation strategy among plants to changing environments. However, the underlying mechanisms of the differential responses remain unclear. Here we report that a high concentration of auxin, distinct from the effect of low auxin concentration, enhances abscisic acid (ABA) responses in Arabidopsis thaliana, which partially relies on TRANS-MEMBERANE KINASE 1 (TMK1), a key regulator in auxin signaling. We show that high auxin and TMK1 play essential and positive roles in ABA signaling through regulating ABA INSENSITIVE 1 and 2 (ABI1/2), two negative regulators of the ABA pathway. TMK1 inhibits the phosphatase activity of ABI2 by direct phosphorylation of threonine 321 (T321), a conserved phosphorylation site in ABI2 proteins, whose phosphorylation status is important for both auxin and ABA responses. This TMK1-dependent auxin signaling in the regulation of ABA responses provides a possible mechanism underlying the high auxin responses in plants and an alternative mechanism involved in the coordination between auxin and ABA signaling.

[Analysis of proposals received and funded in discipline of microbiology of the National Natural Science Foundation of China in 2016].

We summarized proposals submitted and funded in the discipline of microbiology of the Department of Life Sciences of National Natural Science Foundation of China in 2016. The traits and concerns in different sub-disciplines as well as distinctive funding programs were addressed, and the prior funding fields were prospected. The information may provide references for researchers who apply funding at the discipline of microbiology.

[Analysis of proposals received and funded in discipline of microbiology of the National Natural Science Foundation of China from 2011 to 2015].

Based on a wrap-up of the research proposals received and awards made during 2011 through 2015 in the discipline of microbiology of the Department of Life Sciences, National Natural Science Foundation of China, this article presents a statistic analysis of award recipient institutions and main research trends, and attempts a prospective prioritization of the funding areas from the points of encouraging interdisciplinary research, optimizing funding instruments and strengthening talent training, with a view to providing reference for scientists and researchers in the field of microbiology.

[Funding for Division of Microbiology in 2014 by National Natural Science Foundation of China].

In this paper, we provided an overview of proposals submitted and projects funded in 2014 at the Division of Microbiology, Department of Life Sciences, National Natural Science Foundation of China. The traits and problems in different sub-disciplines were analyzed, the background, results and analysis of internet voting before panel meetings in Microbiology discipline were also introduced. The information will provide references for Chinese researchers to apply funding in microbiology discipline in the future.

[Funding for division of microbiology by National Natural Science Foundation of China in 2013].

We provide an overview of proposals applied and projects funded by the division of microbiology, department of life sciences, National Natural Science Foundation of China in 2013,. The traits and problems in different sub-disciplines were also analyzed, which provides reference for Chinese researchers to apply funding in microbiology next year.

[Funding in 2012 for Division of Microbiology by National Natural Science Foundation of China].

We provide here an overview of proposals applied and projects funded by the division of microbiology, department of life sciences, National Natural Science Foundation of China in 2012. We analyzed the traits and problems in different sub-disciplines, and illustrated the stimulating policy for future funding. This overview provides reference for Chinese researchers to apply relevant funding for projects in microbiology.

Cytokinin signaling regulates pavement cell morphogenesis in Arabidopsis.

The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue. Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways. To identify additional components or mechanisms, we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern. Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines, the ahk3cre1 cytokinin receptor mutant, and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation, whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon. Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern. Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

[Analysis and prospect of projects funded in discipline of microbiology (NSFC) from 2006 to 2010].

The overview of projects funded by general programs,key programs and national science fund for distinguished young scholars in discipline of microbiology, National Natural Science Foundation of China (NSFC) from 2006 to 2010 was recommended. Some important characters such as the distribution of projects in different subjects, organizations, regions and research fields were analyzed. Some important research fields which should be supported in "The Twelfth Five-Year Plan" was also put forward. The goal of the paper is to provide information of funding in NSFC for researchers in the field of microbiology.

Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.

Auxin is a multifunctional hormone essential for plant development and pattern formation. A nuclear auxin-signaling system controlling auxin-induced gene expression is well established, but cytoplasmic auxin signaling, as in its coordination of cell polarization, is unexplored. We found a cytoplasmic auxin-signaling mechanism that modulates the interdigitated growth of Arabidopsis leaf epidermal pavement cells (PCs), which develop interdigitated lobes and indentations to form a puzzle-piece shape in a two-dimensional plane. PC interdigitation is compromised in leaves deficient in either auxin biosynthesis or its export mediated by PINFORMED 1 localized at the lobe tip. Auxin coordinately activates two Rho GTPases, ROP2 and ROP6, which promote the formation of complementary lobes and indentations, respectively. Activation of these ROPs by auxin occurs within 30 s and depends on AUXIN-BINDING PROTEIN 1. These findings reveal Rho GTPase-based auxin-signaling mechanisms, which modulate the spatial coordination of cell expansion across a field of cells.

A ROP GTPase signaling pathway controls cortical microtubule ordering and cell expansion in Arabidopsis.

Cortical microtubules (MTs) participate in the spatial control of cell expansion and division that is required for plant growth and morphogenesis. Well-ordered transverse cortical MTs promote cell elongation and restrict radial cell expansion. The molecular mechanism controlling their ordering is poorly understood. We report the first known signaling pathway that promotes the organization of cortical MTs into parallel arrays oriented perpendicular to the axis of cell elongation in plants. Well-ordered MTs locally restrict cell expansion to promote indentation formation in the jigsaw-puzzle-shaped pavement cells of Arabidopsis leaves. Deleting ROP6, a Rho-family GTPase, randomized cortical MTs and released the localized restriction of cell expansion, whereas ROP6 overexpression enhanced MT ordering, turning the jigsaw-puzzle appearance of cells into a cylindrical shape. ROP6 directly binds and activates MT-associated RIC1 to achieve the MT ordering. The ROP6-RIC1 pathway also affects MT ordering of hypocotyl cells, showing a broad role for this pathway in the spatial regulation of cell expansion.