Xiuping Qian

Vancomycin is a clinically important glycopeptide antibiotic against Gram-positive pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus. In the mutant strain of Amycolatopsis keratiniphila HCCB10007 Δeco-cds4-27, the production of ECO-0501 was disrupted, but enhanced vancomycin yield by 55% was observed compared with the original strain of A. keratiniphila HCCB10007. To gain insights into the mechanism of the enhanced production of vancomycin in the mutant strain, comparative metabolomics analyses were performed between the mutant strain and the original strain, A. keratiniphila HCCB10007 via GC–TOF–MS and UPLC-HRMS. The results of PCA and OPLS-DA revealed a significant distinction of the intracellular metabolites between the two strains during the fermentation process. 64 intracellular metabolites, which involved in amino acids, fatty acids and central carbon metabolism, were identified as differential metabolites. The high-yield mutant strain maintained high levels of glucose-1-phosphate and glucose-6-phosphate and they declined with the increases of vancomycin production. Particularly, a strong association of fatty acids accumulation as well as 3,5-dihydroxyphenylacetic acid and non-proteinogenic amino acid 3,5-dihydroxyphenylglycine (Dpg) with enhancement of vancomycin production was observed in the high-yield mutant strain, indicating that the consumption of fatty acid pools might be beneficial for giving rise to 3,5-dihydroxyphenylacetic acid and Dpg which further lead to improve vancomycin production. In addition, the lower levels of glyoxylic acid and lactic acid and the higher levels of sulfur amino acids might be beneficial for improving vancomycin production. These findings proposed more advanced elucidation of metabolomic characteristics in the high-yield strain for vancomycin production and could provide potential strategies to enhance the vancomycin production.

Vancomycin is a clinically important glycopeptide antibiotic against Gram-positive pathogenic bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA). In the mutant strain of A.keratiniphila HCCB10007 Δeco-cds4-27, the production of ECO-0501 was disrupted, but enhanced vancomycin yield by 55% was observed compared with the original strain of A.keratiniphila HCCB10007. To gain insights into the mechanism about enhancement production of vancomycin in the mutant strain, comparative metabolomics analyses were performed between the mutant strain and the original strain A.keratiniphila HCCB1007 via GC-TOF-MS and UPLC-HRMS. The results of PCA and OPLS-DA revealed the significant distinction of the intracellular metabolites between the two strains during the fermentation process. 64 intracellular metabolites, which involved in amino acids, fatty acids and central carbon metabolism, were identified as differential metabolites. The high-yield mutant strain maintained high levels of glucose-1-phosphate and glucose-6-phosphate and they declined with the increases of vancomycin productions. Particularly, a strong association of fatty acids accumulation as well as 3,5-dihydroxyphenylacetic acid and non-proteinogenic amino acid 3,5-dihydroxyphenylglycine (Dpg) with enhancement of vancomycin production was observed in the high-yield mutant strain, indicating that the consumption of fatty acid pools might be benefit for giving rise to 3,5-dihydroxyphenylacetic acid and Dpg which further lead to improve vancomycin production. In addition, the lower levels of glyoxylic acid and lactic acid and higher levels of sulfur amino acids might be benefit for improving vancomycin production. These findings proposed more advanced elucidation of metabolomic characteristics in the high-yield strain for vancomycin production and could provide potential strategies to enhance the vancomycin production.

The glucagon-like peptide-1 (GLP-1) is a peptide with incretin activity and plays an important role in glycemic control as well as the improvement of insulin resistance in type 2 diabetes mellitus (T2DM). However, the short half-life of the native GLP-1 in circulation poses difficulties for clinical practice. To improve the proteolytic stability and delivery properties of GLP-1, a protease-resistant modified GLP-1 (mGLP-1) was constructed with added arginine to ensure the structural integrity of the released mGLP-1 in vivo. The model probiotic Lactobacillus plantarum WCFS1 was chosen as the oral delivery vehicle with controllable endogenous genetic tools driven for mGLP-1 secretory constitutive expression. The feasibility of our design was explored in db/db mice which showed an improvement in diabetic symptoms related to decreased pancreatic glucagon, elevated pancreatic β-cell proportion, and increased insulin sensitivity. In conclusion, this study provides a novel strategy for the oral delivery of mGLP-1 and further probiotic transformation.

Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%–97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%–40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.

Porphyrin derivatives are popular photodynamic therapy (PDT) agents; however, their typical insolubility in water has made it challenging to separate cells of organisms in a liquid water environment. Herein, a novel water-soluble 5,10,15,20-tetrakis(4-methoxyphenyl-3-sulfonatophenyl) porphyrin (TMPPS) was synthesized with 95% yield by modifying the traditional sulfonation route. The reaction of TMPPS with AgNO₃ afforded AgTMPPS an unusual Ag(II) oxidation state (97% yield). The free base and Ag(II) complex were characterized by matrix-assisted laser desorption ionization-mass spectroscopy, and ¹H nuclear magnetic resonance, Fourier-transform infrared, UV-vis, fluorescence, and X-ray photolectron spectroscopies. Upon 460 nm laser irradiation, AgTMPPS generated a large amount of ¹O₂, whereas no ⦁OH was detected. Antibacterial experiments on methicillin-resistant Staphylococcus aureus (MRSA) revealed that the combined action of Ag^Ⅱ ions and PDT could endow AgTMPPS with a 100% bactericidal ratio for highly concentrated MRSA (10⁸ CFU/mL) at a very low dosage (4 μM) under laser irradiation at 360 J/cm². Another PDT response was demonstrated by photocatalytically oxidizing 1,4-dihydronicotinamide adenine dinucleotide to NAD⁺ with AgTMPPS. The structural features of the TMPPS and AgTMPPS molecules were investigated by density functional theory quantum chemical calculations to demonstrate the efficient chemical and photodynamical effects of AgTMPPS for non-invasive antibacterial therapy.

Micro/nanorobot, as a research field, has attracted interest in recent years. It has great potential in medical treatment, as it can be applied in targeted drug delivery, surgical operation, disease diagnosis, etc. Differently from traditional drug delivery, which relies on blood circulation to reach the target, the designed micro/nanorobots can move autonomously, which makes it possible to deliver drugs to the hard-to-reach areas. Micro/nanorobots were driven by exogenous power (magnetic fields, light energy, acoustic fields, electric fields, etc.) or endogenous power (chemical reaction energy). Cell-based micro/nanorobots and DNA origami without autonomous movement ability were also introduced in this article. Although micro/nanorobots have excellent prospects, the current research is mainly based on in vitro experiments; in vivo research is still in its infancy. Further biological experiments are required to verify in vivo drug delivery effects of micro/nanorobots. This paper mainly discusses the research status, challenges, and future development of micro/nanorobots.