Tripterifordin and neotripterifordin are important ent-kaurane diterpenoids in the Chinese medicinal herb Tripterygium wilfordii, possessing significant anti-HIV(human immunodeficiency virus) activity. On the basis of elucidating the natural biosynthetic pathways of these compounds, heterologous production with microbial cell factories can help to alleviate the reliance on plant resources and provide abundant raw materials for sustainable production. TwKO is the first CYP450 enzyme involved in the biosynthesis of tripterifordin and neotripterifordin. This study aimed to enhance the catalytic activity of TwKO by site-directed mutagenesis to benefit the production of tripterifordin and neotripterifordin in yeast. The AlphaFold DB established based on the AlphaFold 2 was employed to obtain the protein model of TwKO. According to multiple sequence alignments and principles of natural evolution, the key residues influencing the binding of TwKO to the substrate were identified. Subsequently, functional characterization of the mutants were conducted in Saccharomyces cerevisiae. A total of 71 mutants were obtained, among which 11 and 11 mutants had the abilities of enhancing the production of 16α-hydroxy-ent-kaurenol and 16α-hydroxy-ent-kaurenoic acid, respectively. In addition, 10 mutants could increase the proportion of the oxidation product of 16α-hydroxy-ent-kaurenol. In particular, R304 was identified as a key residue affecting the catalytic specificity of TwKO, the mutation of which led to the specific prodiction of 16α-hydroxy-ent-kaurenol. This study was the first to reveal the key residue affecting the catalytic activity of TwKO and obtained the mutants with increased TwKO activity, lay a foundation for the biosynthesis of tripterifordin and neotripterifordin.
Tripterygium/chemistry* ; Mutagenesis, Site-Directed ; Diterpenes, Kaurane/chemistry* ; Plant Proteins/chemistry* ; Cytochrome P-450 Enzyme System/chemistry* ; Saccharomyces cerevisiae/metabolism*

In this study, a single base editing system was used to edit the FecB and GDF9 gene to achieve a targeted site mutation from A to G and from C to T in Ouler Tibetan sheep fibroblasts, and to test its editing efficiency. Firstly, we designed and synthesized sgRNA sequences targeting FecB and GDF9 genes of Ouler Tibetan sheep, followed by connection to epi-ABEmax and epi-BE4max plasmids to construct vectors and electrotransfer into Ouler Tibetan sheep fibroblasts. Finally, Sanger sequencing was performed to identify the target point mutation of FecB and GDF9 genes positive cells. T-A cloning was used to estimate the editing efficiency of the single base editing system. We obtained gRNA targeting FecB and GDF9 genes and constructed the vector aiming at mutating single base of FecB and GDF9 genes in Ouler Tibetan sheep. The editing efficiency for the target site of FecB gene was 39.13%, whereas the editing efficiency for the target sites (G260, G721 and G1184) of GDF9 gene were 10.52%, 26.67% and 8.00%, respectively. Achieving single base mutation in FecB and GDF9 genes may facilitate improving the reproduction traits of Ouler Tibetan sheep with multifetal lambs.
Animals ; Sheep/genetics* ; Gene Editing ; Tibet ; Mutation ; Phenotype ; Mutagenesis, Site-Directed

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.
Amino Acid Sequence ; Fallopia japonica/metabolism* ; Polyketide Synthases/chemistry* ; Acetone ; Mutagenesis, Site-Directed ; Flavonoids/metabolism* ; Acyltransferases/metabolism*

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.
Creatinine ; Mutagenesis, Site-Directed ; Ureohydrolases/genetics* ; Catalysis

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.
Clone Cells ; Mutagenesis, Site-Directed ; Mutation ; Plasmids/genetics* ; Polymerase Chain Reaction/methods*

A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (M
Antiviral Agents/therapeutic use* ; Binding Sites ; COVID-19/virology* ; Coronavirus Papain-Like Proteases/metabolism* ; Crystallography, X-Ray ; Drug Evaluation, Preclinical ; Drug Repositioning ; High-Throughput Screening Assays/methods* ; Humans ; Imidazoles/therapeutic use* ; Inhibitory Concentration 50 ; Molecular Dynamics Simulation ; Mutagenesis, Site-Directed ; Naphthoquinones/therapeutic use* ; Protease Inhibitors/therapeutic use* ; Protein Structure, Tertiary ; Recombinant Proteins/isolation & purification* ; SARS-CoV-2/isolation & purification*

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.
Base Sequence ; Cloning, Molecular ; Genetic Vectors ; genetics ; Mutagenesis, Site-Directed ; methods ; Nucleic Acid Amplification Techniques ; Plasmids ; Polymerase Chain Reaction

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.
DNA Primers ; genetics ; Escherichia coli ; Mutagenesis, Site-Directed ; methods ; Plasmids ; genetics ; Polymerase Chain Reaction

Genome editing is a genetic engineering technique that uses site-directed cleavage activity of specific artificial nucleases and endogenous DNA damage repair activity to generate insertions, deletions or substitutions in the targeted genomic loci. As the accuracy and efficiency of genome editing is improving and the operation is simple, the application of genome editing is expanding. This article provides an overview of the three major genome editing technologies and genome editing types, and the regulatory frameworks for genome-edited products were summarized in the United States, the European Union, and other countries. At the same time, based on the Chinese safety management principles and systems for genetically modified organisms (GMOs), the authors proposed a regulatory framework for genome-edited products. Genome-edited products should first be classified according to whether containing exogenous genetic components such as Cas9 editing enzymes or not. They should be regulated as traditional genetically modified organisms if they do. Otherwise, the regulation of genome-edited products depends on targeted modifications.
CRISPR-Cas Systems ; Endonucleases ; Gene Editing ; Genome ; Mutagenesis, Site-Directed

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.
Calcium ; Carbachol ; Humans ; Hydrogen-Ion Concentration ; Ion Channels ; Kidney ; Mass Screening ; Mutagenesis, Site-Directed ; Mutant Proteins ; Protein Kinase C ; Receptors, Muscarinic