Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L-cysteine.

L-Cysteine production through fermentation stands as a promising technology. However, excessive accumulation of L-cysteine poses a challenge due to the potential to inflict damage on cellular DNA. In this study, we employed a synergistic approach encompassing atmospheric and room temperature plasma mutagenesis (ARTP) and adaptive laboratory evolution (ALE) to improve L-cysteine tolerance in Escherichia coli. ARTP-treated populations obtained substantial enhancement in L-cysteine tolerance by ALE. Whole-genome sequencing, transcription analysis, and reverse engineering, revealed the pivotal role of an effective export mechanism mediated by gene eamB in augmenting L-cysteine resistance. The isolated tolerant strain, 60AP03/pTrc-cysEf , achieved a 2.2-fold increase in L-cysteine titer by overexpressing the critical gene cysEf during batch fermentation, underscoring its enormous potential for L-cysteine production. The production evaluations, supplemented with L-serine, further demonstrated the stability and superiority of tolerant strains in L-cysteine production. Overall, our work highlighted the substantial impact of the combined ARTP and ALE strategy in increasing the tolerance of E. coli to L-cysteine, providing valuable insights into improving L-cysteine overproduction, and further emphasized the potential of biotechnology in industrial production.

Targeted mutagenesis of BnTTG1 homologues generated yellow-seeded rapeseed with increased oil content and seed germination under abiotic stress.

Yellow seed is one desirable trait with great potential to improve seed oil quality and yield. The present study surveys the redundant role of BnTTG1 genes in the proanthocyanidins (PA) biosynthesis, oil content and abiotic stress resistance. Stable yellow seed mutants were generated after mutating BnTTG1 by CRISPR/Cas9 genome editing system. Yellow seed phenotype could be obtained only when both functional homologues of BnTTG1 were simultaneously knocked out. Homozygous mutants of BnTTG1 homologues showed decreased thickness and PA accumulation in seed coat. Transcriptome and qRT-PCR analysis indicated that BnTTG1 mutation inhibited the expression of genes involved in phenylpropanoid and flavonoid biosynthetic pathways. Increased seed oil content and alteration of fatty acid (FA) composition were observed in homozygous mutants of BnTTG1 with enriched expression of genes involved in FA biosynthesis pathway. In addition, target mutation of BnTTG1 accelerated seed germination rate under salt and cold stresses. Enhanced seed germination capacity in BnTTG1 mutants was correlated with the change of expression level of ABA responsive genes. Overall, this study elucidated the redundant role of BnTTG1 in regulating seed coat color and established an efficient approach for generating yellow-seeded oilseed rape genetic resources with increase oil content, modified FA composition and resistance to multiple abiotic stresses.

Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum.

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3Pf , is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3Pf inner membrane immunity protein.

CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato.

CRISPR/Cas9 technology has become the most efficient method for genome editing in many plant species, including important industrial crops such as potatoes. This study used three target regions (T1, T2, and T3) in gbss exon I, whose sequences were first inserted into the BbsI sites in the appropriate guide RNA (gRNA) vector (pEn-Chimera, pMR203, pMR204, and pMR205), and then localized between the AtU6 promoter and the gRNA scaffold sequence. Expression vectors were constructed by introducing gRNA genes into the pMR287 (pYUCas9Plus) plasmids using the MultiSite Gateway system by attR and attL sites. The three target regions of mutant potato lines were analyzed. The use of CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis allowed tri- or tetra-allelic mutant potato lines to be generated. Multiple nucleotide substitutions and indels within and around the three target sites caused a frameshift mutation that led to a premature stop codon, resulting in the production of gbss-knockout plants. Mutation frequencies and analysis of mutation patterns suggested that the stably transformed Cas9/multiple guide RNA expression constructs used in this study can induce targeted mutations efficiently in the potato genome. Full knockout of the gbss gene was analyzed by CAPS, Sanger sequencing and iodine staining. The present study demonstrated successful CRISPR/Cas9-mediated multiple guide RNA-targeted mutagenesis in the potato gbss gene by Agrobacterium-mediated transformation, resulting in an amylose-free phenotype.

Nonsteroidal anti-inflammatory drug diclofenac accelerates the emergence of antibiotic resistance via mutagenesis.

Overuse of antimicrobial agents are generally considered to be a key factor in the occurrence of antibiotic resistance bacteria (ARB). Nevertheless, it is unclear whether ARB can be induced by non-antibiotic chemicals such as nonsteroidal anti-inflammatory drug (NSAID). Thus, the objective of this study is to investigate whether NSAID diclofenac (DCF) promote the emergence of antibiotic resistance in Escherichia coli K12 MG1655. Our results suggested that DCF induced the occurrence of ARB which showed hereditary stability of resistance. Meanwhile, gene variation was identified on chromosome of the ARB, and DCF can cause bacterial oxidative stress and SOS response. Subsequently, transcriptional levels of antioxidant (soxS, sodA, sodC, gor, katG, ahpF) and SOS (recA, lexA, uvrA, uvrB, ruvA, ruvB, dinB, umuC, polB) system-related genes were enhanced. However, the expression of related genes cannot be increased in high-dosage treatment compared with low-dosage samples because of cytotoxicity and cellular damage. Simultaneously, high-dosage DCF decreased the mutation frequency but enhanced the resistance of mutants. Our findings expand our knowledge of the promoting effect on the emergence of ARB caused by DCF. More attention and regulations should be given to these potential ecological and health risks for widespread DCF.

Pulsed Light Mutagenesis of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), for Enhanced Production of Flavonoids, Laccase, and Fermentation Biomass.

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

Aristolochic acid-associated cancers: a public health risk in need of global action.

Aristolochic acids (AAs) are a group of naturally occurring compounds present in many plant species of the Aristolochiaceae family. Exposure to AA is a significant risk factor for severe nephropathy, and urological and hepatobiliary cancers (among others) that are often recurrent and characterized by the prominent mutational fingerprint of AA. However, herbal medicinal products that contain AA continue to be manufactured and marketed worldwide with inadequate regulation, and possible environmental exposure routes receive little attention. As the trade of food and dietary supplements becomes increasingly globalized, we propose that further inaction on curtailing AA exposure will have far-reaching negative effects on the disease trends of AA-associated cancers. Our Review aims to systematically present the historical and current evidence for the mutagenicity and carcinogenicity of AA, and the effect of removing sources of AA exposure on cancer incidence trends. We discuss the persisting challenges of assessing the scale of AA-related carcinogenicity, and the obstacles that must be overcome in curbing AA exposure and preventing associated cancers. Overall, this Review aims to strengthen the case for the implementation of prevention measures against AA's multifaceted, detrimental and potentially fully preventable effects on human cancer development.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans.

Rhodanobacter has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The in situ stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the Rhodanobacter species, Rhodanobacter denitrificans strains have been reported to be capable of denitrification and contain abundant metal resistance genes. However, due to the lack of a mutagenesis system in these strains, our understanding of the mechanisms underlying low-pH resistance and the ability to dominate in the contaminated environment remains limited. Here, we developed an in-frame markerless deletion system in two R. denitrificans strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 Rhodanobacter strains. We then deleted the upp gene, which encodes uracil phosphoribosyltransferase, in R. denitrificans strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δupp and R5_Δupp and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. To test the developed protocol, the narG gene encoding nitrate reductase was knocked out in the R3_Δupp and R5_Δupp host strains. As expected, the narG mutants could not grow in anoxic medium with nitrate as the electron acceptor. Overall, these results show that the in-frame markerless deletion system is effective in two R. denitrificans strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in Rhodanobacter species. IMPORTANCE Rhodanobacter denitrificans is capable of denitrification and is also resistant to toxic heavy metals and low pH. Accordingly, the presence of Rhodanobacter species at a particular environmental site is considered an indicator of nitrate and uranium contamination. These characteristics suggest its future potential application in bioremediation of nitrate or concurrent nitrate and uranium contamination in groundwater ecosystems. Due to the lack of genetic tools in this organism, the mechanisms of low-pH and heavy metal resistance in R. denitrificans strains remain elusive, which impedes its use in bioremediation strategies. Here, we developed a genome editing method in two R. denitrificans strains. This work marks a crucial step in developing Rhodanobacter as a model for studying the diverse mechanisms of low-pH and heavy metal resistance associated with denitrification.

CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering.

Nanobodies provide important advantages over traditional antibodies, including their smaller size and robust biochemical properties such as high thermal stability, high solubility, and the ability to be bioengineered into novel multivalent, multi-specific, and high-affinity molecules, making them a class of emerging powerful therapies against SARS-CoV-2. Recent research efforts on the design, protein engineering, and structure-functional characterization of nanobodies and their binding with SARS-CoV-2 S proteins reflected a growing realization that nanobody combinations can exploit distinct binding epitopes and leverage the intrinsic plasticity of the conformational landscape for the SARS-CoV-2 S protein to produce efficient neutralizing and mutation resistant characteristics. Structural and computational studies have also been instrumental in quantifying the structure, dynamics, and energetics of the SARS-CoV-2 spike protein binding with nanobodies. In this review, a comprehensive analysis of the current structural, biophysical, and computational biology investigations of SARS-CoV-2 S proteins and their complexes with distinct classes of nanobodies targeting different binding sites is presented. The analysis of computational studies is supplemented by an in-depth examination of mutational scanning simulations and identification of binding energy hotspots for distinct nanobody classes. The review is focused on the analysis of mechanisms underlying synergistic binding of multivalent nanobodies that can be superior to single nanobodies and conventional nanobody cocktails in combating escape mutations by effectively leveraging binding avidity and allosteric cooperativity. We discuss how structural insights and protein engineering approaches together with computational biology tools can aid in the rational design of synergistic combinations that exhibit superior binding and neutralization characteristics owing to avidity-mediated mechanisms.

Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae.

Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains with improved malonyl-CoA synthesis. Here, using the biosensor we constructed previously, a growth-based screening system that links the availability of malonyl-CoA with cell growth is developed. Coupling this system with in vivo continuous mutagenesis enabled rapid generation of genome-scale mutation library and screening strains with improved malonyl-CoA availability. The mutant strains are analyzed by whole-genome sequencing and transcriptome analysis. The omics analysis revealed that the carbon flux rearrangement to storage carbohydrate and amino acids synthesis affected malonyl-CoA metabolism. Through reverse engineering, new processes especially reduced lysine and arginine synthesis were found to improve malonyl-CoA synthesis. Our study provides a valuable complementary tool to other high-throughput screening method for mutant strains with improved metabolite synthesis and improves our understanding of the metabolic regulation of malonyl-CoA synthesis. IMPORTANCE Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae, due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux. Here, we developed a growth-based screening system that linked the availability of malonyl-CoA with cell growth and manipulated DNA replication for rapid in vivo mutagenesis. The combination of growth-based screening with in vivo mutagenesis enabled quick evolution of strains with improved malonyl-CoA availability. The whole-genome sequencing, transcriptome analysis of the mutated strains, together with reverse engineering, demonstrated weakening carbon flux to lysine and arginine synthesis and storage carbohydrate can contribute to malonyl-CoA synthesis. Our work provides a guideline in simultaneous strain screening and continuous evolution for improved metabolic intermediates and identified new targets for improving malonyl-CoA downstream product synthesis.

The effect of alkyl substitution on the oxidative metabolism and mutagenicity of phenanthrene.

Alkyl-substituted PAHs may be present in certain petroleum-derived products and in the environment and may eventually end up in consumer products, such as foodstuffs, cosmetics and pharmaceuticals. Safety concerns over possible exposure to alkylated PAHs have emerged. Bioactivation is a prerequisite for the mutagenicity and carcinogenicity of PAHs and has been extensively studied for non-substituted PAHs, while data on the bioactivation of alkyl-substituted PAHs are scarce. The present study investigated the effect of alkyl substitution on the CYP 450-mediated metabolism of phenanthrene and eight of its alkylated congeners by quantifying metabolite formation in rat and human liver microsomal incubations. Furthermore, the mutagenicity of four selected methylated phenanthrenes was compared to that of phenanthrene using the Ames test. The obtained results support the hypothesis that alkyl substitution shifts the oxidative metabolism from the aromatic ring to the alkyl side chain. Increasing the length of the alkyl chain reduced overall metabolism with metabolic conversion for 1-n-dodecyl-phenanthrene (C12) being negligible. 1- and 9-methyl-phenanthrene, in which the methyl group generates an additional bay region-like structural motif, showed mutagenicity toward Salmonella typhimurium TA98 and TA 100, whereas phenanthrene and also 2- and 3-methyl-phenanthrene, without such an additional bay region-like structural motif, tested negative. It is concluded that the position of the alkylation affects the metabolism and resulting mutagenicity of phenanthrene with the mutagenicity increasing in cases where the alkyl substituent creates an additional bay region-like structural motif, in spite of the extra possibilities for side chain oxidation.

Revealing the effect of seed phosphorus concentration on seedling vigour and growth of rice using mutagenesis approach.

The harvested plant products, specifically, the grains of cereals are major drivers of soil phosphorus (P) depletion. However, the breeding or biotechnology efforts to develop low P seeds have not been attempted because of possible adverse effects on seedling vigour and crop establishment. Several studies have contradictory observations on influence of seed P on seedling vigour. Lack of appropriate genetic material has been the major bottleneck in reaching the consensus. In this study, we used 30 EMS induced mutants of rice cultivar Nagina22 to understand the role of seed P on seedling vigour and associated physiological processes. Seedling vigour, morpho-physiological characteristics, acid phosphatases, alpha-amylase, and expression of P transporter genes were analyzed in seedlings obtained from seeds of high and low grain P mutants. The study suggests that seed P has a significant role on seedling vigour, chlorophyll content and photosynthesis process of young seedlings, and P transport from roots. Notably, we identified few mutants such as NH4791, NH4785, NH4714, NH4663, NH4614, and NH4618 which showed least influence of low seed P on seedling vigour and other metabolic processes. Therefore, these mutants can be used in breeding programs aiming for development of low P grains. Also, these and other identified mutants can be used to decipher the genetic and molecular mechanisms regulating the differential response of seed P on germination, seedling vigour and several other physiological processes influencing the crop growth and establishment.

Iterative mutagenesis induced by atmospheric and room temperature plasma treatment under multiple selection pressures for the improvement of coenzyme Q10 production by Rhodobacter sphaeroides.

CoQ10, which has been widely applied in medicine by dietary supplement, possesses important functions in antioxidant process and bioenergy generation. Iterative mutagenesis introduced by atmospheric and room temperature plasma (ARTP) treatment was studied to improve the coenzyme Q10 (CoQ10) production of Rhodobacter sphaeroides (R. sphaeroides), and multiple selection pressures including vitamin K3 (VK3), Na2S and benzoic acid (BA) were adopted for the first time. After two rounds of mutation and screening, a mutant strain R.S 17 was obtained, and the product titer was increased by 80.37%. The CoQ10 titer and cell density reached 236.7 mg L-1 and 57.09 g L-1, respectively, in the fed-batch fermentation, and the CoQ10 content was 22.1% higher than that of the parent strain. In addition, the spectral scanning results indicated the metabolic flux improvement contributing to the CoQ10 production in R.S 17, and the genetic stability was validated. Based on the iterative mutagenesis introduced by ARTP under multiple selection pressures, the promotion of CoQ10 production by R. sphaeroides was achieved. The significant improvement in fermentation performances and the good genetic stability of R.S 17 indicate a potential way for the efficient biosynthesis of CoQ10.

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation.

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5 and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

Targeted mutagenesis of CYP76AK2 and CYP76AK3 in Salvia miltiorrhiza reveals their roles in tanshinones biosynthetic pathway.

Salvia miltiorrhiza Bunge, belonging to Lamiaceae family, is one of the most important Chinese medicinal herbs. The dried roots, also called Danshen in Chinese, are usually used in the formula of Chinese traditional medicine due to the bioactive constituents known as phenolic acids and tanshinones, which are a group of abietane nor-diterpenoid quinone natural products. Cytochrome P450 enzymes (CYPs) usually play crucial roles in terpenoids synthesis, especially in hydroxylation processes. Up to now, several important P450 enzymes, such as CYP76AH1, CYP76AH3, CYP76AK1, CYP71D373, and CYP71D375, have been functionally characterized in the tanshinones biosynthetic pathway. Nevertheless, the tanshinones biosynthesis is a so complex network that more P450 enzymes should be identified and characterized. Here, we report two novel P450 enzymes CYP76AK2 and CYP76AK3 that are involved in tanshinones biosynthetic pathway. These two P450 enzymes were highly homologous to previously reported CYP76AK1 and showed the same expression profile as CYP76AK1. Also, CYP76AK2 and CYP76AK3 could be stimulated by MeJA and SA, resulting in increased expression. We used a triple-target CRISPR/Cas9 system to generate targeted mutagenesis of CYP76AK2 and CYP76AK3 in S. miltiorrhiza. The content of five major tanshinones was significantly reduced in both cyp76ak2 and cyp76ak3 mutants, indicating that the two enzymes might be involved in the biosynthesis of tanshinones. This study would provide a foundation for the catalytic function identification of CYP76AK2 and CYP76AK3, and further enrich the understanding of the network of tanshinones secondary metabolism synthesis as well.

Structural basis for Ca2+ activation of the heteromeric PKD1L3/PKD2L1 channel.

The heteromeric complex between PKD1L3, a member of the polycystic kidney disease (PKD) protein family, and PKD2L1, also known as TRPP2 or TRPP3, has been a prototype for mechanistic characterization of heterotetrametric TRP-like channels. Here we show that a truncated PKD1L3/PKD2L1 complex with the C-terminal TRP-fold fragment of PKD1L3 retains both Ca2+ and acid-induced channel activities. Cryo-EM structures of this core heterocomplex with or without supplemented Ca2+ were determined at resolutions of 3.1 Å and 3.4 Å, respectively. The heterotetramer, with a pseudo-symmetric TRP architecture of 1:3 stoichiometry, has an asymmetric selectivity filter (SF) guarded by Lys2069 from PKD1L3 and Asp523 from the three PKD2L1 subunits. Ca2+-entrance to the SF vestibule is accompanied by a swing motion of Lys2069 on PKD1L3. The S6 of PKD1L3 is pushed inward by the S4-S5 linker of the nearby PKD2L1 (PKD2L1-III), resulting in an elongated intracellular gate which seals the pore domain. Comparison of the apo and Ca2+-loaded complexes unveils an unprecedented Ca2+ binding site in the extracellular cleft of the voltage-sensing domain (VSD) of PKD2L1-III, but not the other three VSDs. Structure-guided mutagenic studies support this unconventional site to be responsible for Ca2+-induced channel activation through an allosteric mechanism.

Mechanistic studies of non-canonical amino acid mutagenesis.

Over the past decade, harnessing the cellular protein synthesis machinery to incorporate non-canonical amino acids (ncAAs) into tailor-made peptides has significantly advanced many aspects of molecular science. More recently, groundbreaking progress in our ability to engineer this machinery for improved ncAA incorporation has led to significant enhancements of this powerful tool for biology and chemistry. By revealing the molecular basis for the poor or improved incorporation of ncAAs, mechanistic studies of ncAA incorporation by the protein synthesis machinery have tremendous potential for informing and directing such engineering efforts. In this chapter, we describe a set of complementary biochemical and single-molecule fluorescence assays that we have adapted for mechanistic studies of ncAA incorporation. Collectively, these assays provide data that can guide engineering of the protein synthesis machinery to expand the range of ncAAs that can be incorporated into peptides and increase the efficiency with which they can be incorporated, thereby enabling the full potential of ncAA mutagenesis technology to be realized.

Antimutagenic, Cytoprotective and Antioxidant Properties of Ficus deltoidea Aqueous Extract In Vitro.

Ficus deltoidea var. deltoidea is used as traditional medicine for diabetes, inflammation, and nociception. However, the antimutagenic potential and cytoprotective effects of this plant remain unknown. In this study, the mutagenic and antimutagenic activities of F. deltoidea aqueous extract (FDD) on both Salmonella typhimurium TA 98 and TA 100 strains were assessed using Salmonella mutagenicity assay (Ames test). Then, the cytoprotective potential of FDD on menadione-induced oxidative stress was determined in a V79 mouse lung fibroblast cell line. The ferric-reducing antioxidant power (FRAP) assay was conducted to evaluate FDD antioxidant capacity. Results showed that FDD (up to 50 mg/mL) did not exhibit a mutagenic effect on either TA 98 or TA 100 strains. Notably, FDD decreased the revertant colony count induced by 2-aminoanthracene in both strains in the presence of metabolic activation (p < 0.05). Additionally, pretreatment of FDD (50 and 100 µg/mL) demonstrated remarkable protection against menadione-induced oxidative stress in V79 cells significantly by decreasing superoxide anion level (p < 0.05). FDD at all concentrations tested (12.5-100 µg/mL) exhibited antioxidant power, suggesting the cytoprotective effect of FDD could be partly attributed to its antioxidant properties. This report highlights that F. deltoidea may provide a chemopreventive effect on mutagenic and oxidative stress inducers.