The difference of transcriptome of HPV-infected patients contributes more to the occurrence of cervical cancer than the mutations of E6 and E7 genes in HPV16.

Human papillomavirus (HPV) E6 and E7 genes are biomarkers and drivers of the progression of cervical cancer (CxCa). The aim of this study was to investigate the relationship between HPV16 E6, E7 gene mutations and the occurrence and development of CxCa. Cervical exfoliated cells and clinical data of patients with cervical diseases were collected. Sample DNA was extracted, the E6 and E7 gene fragments were amplified by PCR, and the mutations were detected by Sanger sequencing and compared with standard sequences. Microarray was used to sequence the transcriptome of cells. Data of transcriptome analyzed and visualized using R software and its packages. Analysis of clinical characteristics demonstrated the association of HPV16 infection with CxCa (P < .05). Sanger sequencing results showed that the mutation sites of E6 gene included T178G/A, T350G, A131C, and T241G; among these, A131C and T241G were synonymous mutations. The mutation sites of E7 gene included A647G, T846C, G666A, T843C, and T760C, and all of them were synonymous mutations except A647G. There was no significant difference in the distribution of HPV16 E6, E7 mutations among CxCa, cervical intraepithelial neoplasia, and infection groups (P > .05). Compared with the non- CxCa group, gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes (DEGs) showed more significant enrichment of DEGs in the biological processes, pathways, and diseases closely related to cancer. Compared with the non-mutation group, the DEGs in the E6, E7 gene mutation group were significantly enriched in the events related to infection and immunity. To summarize, HPV16 may be associated with the occurrence and development of CxCa, but HPV16 E6 and E7 gene mutations have little effect on the occurrence and development of CxCa. Individual differences may have a greater effect on the progression of CxCa.

Synthesis of Trifluoromethylated Monoterpenes by an Engineered Cytochrome P450.

Protein engineering of cytochrome P450s has enabled these biocatalysts to promote a variety of abiotic reactions beyond nature's repertoire. Integrating such non-natural transformations with microbial biosynthetic pathways could allow sustainable enzymatic production of modified natural product derivatives. In particular, trifluoromethylation is a highly desirable modification in pharmaceutical research due to the positive effects of the trifluoromethyl group on drug potency, bioavailability, and metabolic stability. This study demonstrates the biosynthesis of non-natural trifluoromethyl-substituted cyclopropane derivatives of natural monoterpene scaffolds using an engineered cytochrome P450 variant, P411-PFA. P411-PFA successfully catalyzed the transfer of a trifluoromethyl carbene from 2-diazo-1,1,1-trifluoroethane to the terminal alkenes of several monoterpenes, including L-carveol, carvone, perilla alcohol, and perillartine, to generate the corresponding trifluoromethylated cyclopropane products. Furthermore, integration of this abiotic cyclopropanation reaction with a reconstructed metabolic pathway for L-carveol production in Escherichia coli enabled one-step biosynthesis of a trifluoromethylated L-carveol derivative from limonene precursor. Overall, amalgamating synthetic enzymatic chemistry with established metabolic pathways represents a promising approach to sustainably produce bioactive natural product analogs.

Study and design of amino acid-based radical enzymes using unnatural amino acids.

Radical enzymes harness the power of reactive radical species by placing them in a protein scaffold, and they are capable of catalysing many important reactions. New native radical enzymes, especially those with amino acid-based radicals, in the category of non-heme iron enzymes (including ribonucleotide reductases), heme enzymes, copper enzymes, and FAD-radical enzymes have been discovered and characterized. We discussed recent research efforts to discover new native amino acid-based radical enzymes, and to study the roles of radicals in processes such as enzyme catalysis and electron transfer. Furthermore, design of radical enzymes in a small and simple scaffold not only allows us to study the radical in a well-controlled system and test our understanding of the native enzymes, but also allows us to create powerful enzymes. In the study and design of amino acid-based radical enzymes, the use of unnatural amino acids allows precise control of pKa values and reduction potentials of the residue, as well as probing the location of the radical through spectroscopic methods, making it a powerful research tool. Our understanding of amino acid-based radical enzymes will allow us to tailor them to create powerful catalysts and better therapeutics.

Identification of two novel highly inducible promoters from Bacillus licheniformis by screening transcriptomic data.

Bacillus licheniformis TCCC11148 is an important industrial strain used to produce alkaline protease. In this study, the transcriptome of B. licheniformis TCCC11148 was analyzed by high throughput RNA sequencing (RNA-Seq) to identify genes that are expressed differentially in the different phases were detected using RNA-Seq. In total, 440 differentially expressed genes between the 12 h and 48 h groups were identified, including 267 up- and 173 downregulated genes. Additionally, 198 differentially expressed genes were identified in the 48 h vs. the 60 h group, including 182 up- and 16 downregulated genes. To screen for novel inducible promoters, an alkaline protease reporter gene was used to test 24 promoters from 66 candidate genes with obviously higher expression levels (RPKM values) than the control group based on the transcriptome data of B. licheniformis in different phases. Gene 707, related to coenzyme transport and metabolism, and gene 1004, related to posttranslational modification were identified as likely having inducible promoters. The expression level of recombinant strains with reporter genes under the control of promoters p707 and p1004 were 8 times higher than that of the control group. This study contributes a method for finding useful inducible promoters for industrial use based on transcriptomic data.

[Characterization of a novel bidirectional promoter in Bacillus subtilis].

Based on the transcriptome analysis data of a Bacillus licheniformis, a novel bidirectional promoter was identified from the strain and its transcriptional strength was analyzed. The expression level of a Bacillus clausii derived alkaline protease gene driven by the bidirectional promoter was studied by using the known strong constitutive promoter pShuttle-09 as a control. Three recombinant expression vectors and the corresponding recombinant bacteria were constructed. Under the control of the new promoter pLA and its reverse promoter pLB, the alkaline protease expression level respectively reached 164 U/mL and 111 U/mL. The results indicated that the transcription strength of pLA was significantly higher than that of pShuttle-09 and pLB, and both the pLA and pLB promoters could initiate the expression of the alkaline protease. Thus, it provides a new expression element for the heterogenous genes in Bacillus sp. and a new idea for the co-expression of two genes in one prokaryotic strain.

Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing.

A highly efficient genome editing system for Bacillus licheniformis was developed based on single-plasmid CRISPR/Cas9. For highly efficient genome editing the shuttle vector pWH1520 was selected to construct the knockout plasmids. A construct harboring a pS promoter driving cas9 endonuclease expression, a strong pLY-2 promoter driving the transcription of a single guide RNA was demonstrated as being the most effective. To verify the feasibility of the method the uprT gene coding uracil phosphoribosyltransferase was selected as the reporter gene. The efficiency of introducing nucleotide point mutations and single gene deletion reached an editing efficiency of up to 99.2% and 97.3%, respectively. After a upp-deficient strain was engineered, the system and strain were applied to introduce genomic deletions of another two genes, amyL and chiA (encoding amylase and chitinase, respectively) with about 90% deletion efficiency. As two native extracellular proteins with relatively high secretion in the host, amylase and chitinase can hamper the secretion and expression of alkaline protease. It was demonstrated that the mutant with deletions of the two genes effectively improved the alkaline protease yield by 24.8%. The results illustrated that the establishment of a CRISPR/Cas9 system for Bacillus licheniformis is of significance, and confirmed the system's high efficiency. The system provides support for effective molecular modification and metabolic regulation of Bacillus licheniformis, and offers promise for applications in genetic modification of other industrially relevant Bacillus species.

Identification and characterization of a novel cold-tolerant extracellular protease from Planococcus sp. CGMCC 8088.

A psychrophilic extracellular protease was isolated from the marine bacterium Planococcus sp. M7 found in the deep-sea mud of the Southern Indian Ocean. The mature protease is about 43 kDa and contains 389 amino acids. Sequence alignment revealed that the protease whose catalytic triad was comprised of Ser224, Lys249, and Gln253 contains a catalytic module belonging to the serralysin-type protease family 41, and displays 46.55% identity with the experimentally verified serine protease from Bacillus subtilis str. 168. The enzyme displayed an alkaline mesophilic preference with an optimum pH of 10.0 and an optimum temperature of 35 °C. The enzyme retained its activity from 5 to 35 °C and was resistant to repeated freezing and thawing, but was completely inactivated at 55 °C. Calcium ions had a protective effect against thermal denaturation. More than 60% of the maximum activity was retained at pH values in the range of 5.0-11.0. Almost no activity loss was detected after 1 h of incubation at pH 8.0-10.0 and 20 °C, or with 1.0% SDS. Most important, this protease also showed good stability and compatibility with the standard enzyme-free detergent, which indicates its special interest for applications in detergent industry.