Huanzhang Xia

Levonorgestrel is currently the most widely used contraceptive, with advantages that include rapid oral absorption and high bioavailability. Among the various synthetic methods employed in its production, Saccharomyces cerevisiae AS2.346 was first introduced by the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, in 1976, successfully catalyzing the conversion of ethyl secodione to (13R,17S)-ethyl secol. Presently, Saccharomyces cerevisiae AS2.346 whole-cell catalysis is still used in levonorgestrel industrial production. To identify the enzyme responsible for catalyzing the conversion of ethyl secodione to (13R,17S)-ethyl secol, we isolated two genes, Assdr1 and Assdr2, both encoding carbonyl reductases. The biochemical characterization of AsSDR1 and AsSDR2 showed that they had high activity for ethyl secodione. Functional analysis demonstrated that the deletion of Assdr1 almost completely abolished the production of (13R,17S)-ethyl secol, while the deletion of Assdr2 had little to no effect on the production, indicating that Assdr1 plays a pivotal role in the biosynthesis of (13R,17S)-ethyl secol. The reason for the difference in the function of AsSDR1 and AsSDR2 in vivo may be attributed to their different cellular localizations, with AsSDR1 located outside the mitochondria and AsSDR2 in the mitochondria. A metabolomics analysis of Assdr1 knockout strains revealed that AsSDR1 may act as a pterin reductase. Overexpression of the newly isolated gene Assdr1 led to an average 47.88 % higher (13R,17S)-ethyl secol yield compared to that of the wild-type strain. Furthermore, engineering the metabolism of the NADPH cofactor by overexpressing the truncated gene pos5Δ17 encoding mitochondrial NADH kinase produced a 111.25 % higher (13R,17S)-ethyl secol yield compared to that of the wild-type strain. These findings not only elucidate the key enzymatic players involved in the synthesis of (13R,17S)-ethyl secol but also provide a framework for optimizing the industrial biotransformation process for levonorgestrel production.

Background: Aminoglycosides (AGs) are one of the initial classes of antibiotics that have been used clinically and possess broad spectrum of activity. Nevertheless, their clinical utilization is restricted by safety issues associated with nephrotoxicity and ototoxicity.Methods: Glycosyltransferase (GT) KanM2 was introduced into M. echinospora to produce the gentamicin derivatives, in which a kanosamine sugar ring was introduced to replace the garosamine. The premature termination codon (PTC) readthrough activity of genkamicins (GKs) was compared using dual luciferase reporter assay. The toxicity of GK was assessed in vitro in HEK-293 and NCI-H1299 cells and determined based on cell viability calculated after 48 h of treatment with different concentrations of the compounds. The NCI-H1299 cells harbouring the R213X nonsense mutation were treated with different concentrations of the derivatives to compare their expression of p53 proteins. The expression of p53 and its downstream targets p21 and BAX was detected using Western blotting and qRT-PCR in NCI-H1299 cells containing the R213X nonsense mutation treated with different concentrations of GK-Ae and G418. Finally, immunofluorescence and flow cytometry were used to determine the subcellular localization of full-length p53 protein induced by GK-Ae treatment and its effect on apoptosis in cancer cells.Results: Eight gentamicin derivatives were obtained in this study. GK-Ae displayed similar PTC readthrough activity and reduced toxicity compared to natural aminoglycoside G418. Moreover, GK-Ae increased the levels of both p53 and its downstream targets p21 and BAX, and promoted apoptosis of cancer cells.Conclusion: These results demonstrate the potential of combinatorial biosynthesis to increase the diversity of structures of AGs and provide directions for the development of new AGs with low toxicity and high PTC readthrough activity.

Background: Currently, cancer is still a significant disease that seriously endangers human health. Therefore, advanced diagnostic technology and treatment protocols are urgently needed. The rapid development of nanotechnology is expected to provide new ideas for cancer diagnosis and treatment. Objective: The research aims to comprehensively demonstrate the hotspots of nanotechnology applications in cancer. Methods: In this study, an International Patent Classification codes co-occurrence network is constructed to visualize the technology landscape by simultaneously locating and ranking technologies that play an integral role in nanotechnology diffusion and bridging in the field of cancer. In addition, community identification and topic modeling highlight the latent topics in patent documents. Results: The visualization results of the patent network yield five main clusters: Cluster 0 is a nanoparticle composition delivery system with liposomes as the primary carrier. Cluster 1 is mainly represented by nano-immunotherapy with immune checkpoint inhibitors. Cluster 2 is nano phototherapy based on photodynamic therapy and photothermal therapy. Cluster 3 is diagnostic imaging involving nanotechnology. Cluster 4 is a drug delivery system with nanovesicles and albumin nanoparticles as carriers. Conclusion: It was found that carriers represented by liposomes, vesicles, and albumin nanoparticles are essential nanomaterials in the current anticancer drug delivery systems. Integrating next-generation immunosuppressants and nanotechnology will become an important development direction for future immunotherapy. Organic/inorganic nanomaterials are pivotal in cancer imaging diagnosis and phototherapy.

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7’-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%.

Tumor necrosis factor alpha (TNFα) is now regarding as a key role in the pathogenesis of immune-mediated disease such as Rheumatoid arthritis (RA), Crohn's Disease, Psoriatic arthritis and Plaque Psoriasis. HERE we have successfully developed an anti-hTNFα monoclonal rabbit antibody(HZ3M) with high binding and neutralizing activity based on RabMAbs platform. Rabbit hybridomas, immunized subcutanrously with 0.4 mg human TNFα, were generated from the rabbit splenocytes and a total of 142 hybridoma clones with specific binding to human TNFa were obtained. The anti-TNFa RabMAbs showed better neutralizing activity and higher antigen binding affinity compared to Humira and Remicade, the elimination phase half-life 58.2h respectively. In vivo efficay studies, normal mice or human TNF-alpha transgenic mice were injected with 1.0 mg/kg Humira (positive control), HZ3M at 0.33?1.0 or 3.0 mg/kg, or solvent (negative control), showed that HZ3M is able to bind and neutralize hTNFα in transgenic and normal mice as well as normal rabbits.Clearly dose-dependent response can be determined. Compared to marketed anti-TNFa drug Humira, the efficacy of HZ3M is seems to show significant longer holding time.Our observations indicate that the HZ3M derived from RabMAb preclinical safety study, and might have a therapeutic role in RA treatment.

Both kanamycin A and kanamycin B, antibiotic components produced by Streptomyces kanamyceticus, have medical value. Two different pathways for kanamycin biosynthesis have been reported by two research groups. In this study, to obtain an optimal kanamycin A-producing strain and a kanamycin B-high-yield strain, we first examined the native kanamycin biosynthetic pathway in vivo. Based on the proposed parallel biosynthetic pathway, kanN disruption should lead to kanamycin A accumulation; however, the kanN-disruption strain produced neither kanamycin A nor kanamycin B. We then tested the function of kanJ and kanK. The main metabolite of the kanJ-disruption strain was identified as kanamycin B. These results clarified that kanamycin biosynthesis does not proceed through the parallel pathway and that synthesis of kanamycin A from kanamycin B is catalyzed by KanJ and KanK in S. kanamyceticus. As expected, the kanamycin B yield of the kanJ-disruption strain was 3268±255 μg/mL, 12-fold higher than that of the original strain. To improve the purity of kanamycin A and reduce the yield of kanamycin B in the fermentation broth, four different kanJ- and kanK-overexpressing strains were constructed through either homologous recombination or site-specific integration. The overexpressing strain containing three copies of kanJ and kanK in its genome exhibited the lowest kanamycin B yield (128±20 μg/mL), which was 54% lower than that of the original strain. Our experimental results demonstrate that kanamycin A is derived from KanJ-and-KanK-catalyzed conversion of kanamycin B in S. kanamyceticus. Moreover, based on the clarified biosynthetic pathway, we obtained a kanamycin B-high-yield strain and an optimized kanamycin A-producing strain with minimal byproduct.

Three putative regulatory genes, namely ttmRI, ttmRII and ttmRIII, which are present in the tetramycin (ttm) biosynthetic gene cluster, were found in Streptomyces ahygroscopicus. Disruption of ttmRI, ttmRII or ttmRIII reduced tetramycin production, and their complementation restored production to varying degrees. Gene expression analysis of the wild-type (WT) and mutant strains through reverse transcriptase–polymerase chain reaction (RT-PCR) of the ttm gene cluster showed that the expression levels of most of the biosynthetic genes were reduced in ΔttmRI, ΔttmRII and ΔttmRIII. Electrophoretic mobility shift assays demonstrated that TtmRI, TtmRII and TtmRIII bound the promoters of several genes in the ttm gene cluster. This study found that these three proteins are a group of positive regulators that activate the transcription of the ttm gene cluster in S. ahygroscopicus. In addition, ΔttmRII had a reduced degree of grey pigmentation. Thus, TtmRII has a pleiotropic regulatory function in the tetramycin biosynthetic pathway and in development.

Isepamicin is a weakly toxic but highly active aminoglycoside antibiotic derivative of gentamicin B. Gentamicin B is a naturally occurring minor component isolated from Micromonospora echinospora. 2ʹ-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2ʹ-OH-containing gentamicin B is produced as a minor component. However, the biosynthetic pathway of gentamicin B remains unclear. Considering that gentamicin B shares a unique C2ʹ hydroxyl group with kanamycin A, we aimed to design a new biosynthetic pathway of gentamicin B by combining twelve steps of gentamicin biosynthesis and two steps of kanamycin biosynthesis. We blocked the biosynthetic pathway of byproducts and generated a strain overproducing JI-20A, which was used as a precursor of gentamicin B biosynthesis, by disrupting genK and genP. The amount of JI-20A produced in M. echinospora ∆K∆P reached 911 μg/ml, which was 14-fold higher than that of M. echinospora ∆P. The enzymes KanJ and KanK necessary to convert 2ʹ-NH2 into 2ʹ-OH from the kanamycin biosynthetic pathway were heterologously expressed in M. echinospora ΔKΔP to transform JI-20A into gentamicin B. The strain with kanJK under PermE* could produce 80 μg/ml of gentamicin B, which was tenfold higher than that of the wild-type strain. To enhance gentamicin B production, we employed different promoters and gene integration combinations. When a PhrdB promoter was used and kanJ and kanK were integrated in the genome through gene replacement, gentamicin B was generated as the major product with a maximum yield of 880 μg/ml. We constructed a new biosynthetic pathway of high-level gentamicin B production; in this pathway, most byproducts were removed. This method also provided novel insights into the biosynthesis of secondary metabolites via the combinatorial biosynthesis.