An adaptive drug-releasing contact lens for personalized treatment of ocular infections and injuries.

This research introduces an innovative solution to address the challenges of bacterial keratitis and alkali burns. Current treatments for bacterial keratitis and alkali burns rely on the frequent use of antibiotics and anti-inflammatory eye drops. However, these approaches suffer from poor bioavailability and fluctuating concentrations, leading to limited efficacy and potential drug resistance. Our approach presents an adaptive drug-releasing contact lens responsive to reactive oxygen species (ROS) at ocular inflammation sites, synchronously releasing Levofloxacin and Diclofenac. During storage, minimal drug release occurred, but over 7 days of wear, the lens maintained a continuous, customizable drug release rate based on disease severity. This contact lens had strong antibacterial activity and biofilm prevention, effectively treating bacterial keratitis. When combined with autologous serum, this hydrophilic, flexible lens aids corneal epithelial regeneration, reducing irritation and promoting healing. In summary, this ROS-responsive drug-releasing contact lens combines antibacterial and anti-inflammatory effects, offering a promising solution for bacterial keratitis and alkali burns.

Characterization and utilization of methyltransferase for apramycin production in Streptoalloteichus tenebrarius.

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%.

Development and validation of a novel reporter gene assay for determination of recombinant human thrombopoietin.

Recombinant human thrombopoietin (rhTPO) was approved by the National Medical Products Administration in 2010 for the treatment of thrombocytopenia in patients with immune thrombocytopenic purpura and chemotherapy-induced thrombocytopenia. Nevertheless, no method for determining rhTPO bioactivity has been recorded in different national/regional pharmacopoeia. Novel methods for lot release and stability testing are needed that are simpler, quicker, and more accurate. Here, we developed a novel reporter gene assay (RGA) for rhTPO bioassay with Ba/F3 cell lines that stably expressed human TPO receptor and luciferase reporter driven by sis-inducible element, gamma response region, and gamma-interferon activated sequence. During careful optimization, the RGA method demonstrated high performance characteristics. According to the International Council for Harmonization Q2 (R1) guidelines and the Chinese Pharmacopoeia 2020 edition, the validation results demonstrated that this method is highly time-saving, sensitive, and robust for research, development, manufacture, and quality control of rhTPO.

Optimization of tetramycin production in Streptomyces ahygroscopicus S91.

BACKGROUND: Tetramycin is a 26-member tetraene antibiotic used in agriculture. It has two components, tetramycin A and tetramycin B. Tetramycin B is obtained by the hydroxylation of tetramycin A on C4. This reaction is catalyzed by the cytochrome P450 monooxygenase TtmD. The two components of tetramycin have different antifungal activities against different pathogenic fungi. Therefore, the respective construction of high-yield strains of tetramycin A and tetramycin B is conducive to more targeted action on pathomycete and has a certain practical value. RESULTS: Streptomyces ahygroscopicus S91 was used as the original strain to construct tetramycin A high-yield strains by blocking the precursor competitive biosynthetic gene cluster, disrupting tetramycin B biosynthesis, and overexpressing the tetramycin pathway regulator. Eventually, the yield of tetramycin A in the final strain was up to 1090.49 ± 136.65 mg·L- 1. Subsequently, TtmD, which catalyzes the conversion from tetramycin A to tetramycin B, was overexpressed. Strains with 2, 3, and 4 copies of ttmD were constructed. The three strains had different drops in tetramycin A yield, with increases in tetramycin B. The strain with three copies of ttmD showed the most significant change in the ratio of the two components. CONCLUSIONS: A tetramycin A single-component producing strain was obtained, and the production of tetramycin A increased 236.84% ± 38.96% compared with the original strain. In addition, the content of tetramycin B in a high-yield strain with three copies of ttmD increased from 26.64% ± 1.97 to 51.63% ± 2.06%.

Pyridoxal-5'-phosphate-dependent enzyme GenB3 Catalyzes C-3',4'-dideoxygenation in gentamicin biosynthesis.

BACKGROUND: The C-3',4'-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3'-phosphotransferase (APH(3')) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. RESULTS: Here, we report that GenP catalyzes 3' phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5'-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3',4'-dideoxy-4',5'-ene-6'-oxo products. The following C-6'-transamination and the GenB4-catalyzed reduction of 4',5'-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. CONCLUSIONS: This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3') deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3') to activate hydroxyls as leaving groups for the 3',4'-dideoxygenation in gentamicin biosynthesis.

The bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3',4'-di-deoxygenation via reduction and transamination activities.

BACKGROUND: New semi-synthetic aminoglycoside antibiotics generally use chemical modifications to avoid inactivity from pathogens. One of the most used modifications is 3',4'-di-deoxygenation, which imitates the structure of gentamicin. However, the mechanism of di-deoxygenation has not been clearly elucidated. RESULTS: Here, we report that the bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3',4'-di-deoxygenation via reduction and transamination activities. Following disruption of genB4 in wild-type M. echinospora, its products accumulated in 6'-deamino-6'-oxoverdamicin (1), verdamicin C2a (2), and its epimer, verdamicin C2 (3). Following disruption of genB4 in M. echinospora ΔgenK, its products accumulated in sisomicin (4) and 6'-N-methylsisomicin (5, G-52). Following in vitro catalytic reactions, GenB4 transformed sisomicin (4) to gentamicin C1a (9) and transformed verdamicin C2a (2) and its epimer, verdamicin C2 (3), to gentamicin C2a (11) and gentamicin C2 (12), respectively. CONCLUSION: This finding indicated that in addition to its transamination activity, GenB4 exhibits specific 4',5' double-bond reducing activity and is responsible for the last step of gentamicin 3',4'-di-deoxygenation. Taken together, we propose three new intermediates that may refine and supplement the specific biosynthetic pathway of gentamicin C components and lay the foundation for the complete elucidation of di-deoxygenation mechanisms.

Improving gentamicin B and gentamicin C1a production by engineering the glycosyltransferases that transfer primary metabolites into secondary metabolites biosynthesis.

Gentamicin B and gentamicin C1a are the direct precursor for Isepamicin and Etimicin synthesis, respectively. Although producing strains have been improved for many years, both gentamicin B titer and gentamicin C1a titer in the fermentation are still low. Because all gentamicin components are biosynthesized using UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and UDP-xylose as precursors, we tried to explore strategies for development of strains capable of directing greater fluxes of these precursors into production of gentamicins. The glycosyltransferases KanM1 and GenM2, which are responsible for UDP-GlcNAc and UDP-xylose transfer, respectively, were overexpressed in gentamicin B producing strain Micromonospora echinospora JK4. It was found that gentamicin B could be improved by up to 54% with improvement of KanM1 and GenM2 expression during appropriately glucose feeding. To prove this strategy is widely usable, the KanM1 and GenM2 were also overexpressed in gentamicin C1a producing strain, titers of gentamicin C1a improved by 45% when compared with titers of the starting strain. These results demonstrated overexpression the glycosyltransferases that transfer primary metabolites into secondary metabolites is workable for improvement of gentamicins production.

Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering.

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.

Characterization of three positive regulators for tetramycin biosynthesis in Streptomyces ahygroscopicus.

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.

Biosynthesis of 3″-demethyl-gentamicin C components by genN disruption strain of Micromonospora echinospora and test their antimicrobial activities in vitro.

Gentamicin consists primarily of four components, which have different patterns of methylation at C-6' position. The methyl groups have a significant impact on gentamicin antimicrobial activity. Sequence analysis predicted that GenN was a methyltransferase in the gentamicin biosynthetic pathway. To study the function of genN, it was disrupted in Micromonospora echinospora. The genN disruption strains produced 3″-N-demethyl-gentamicin C complex instead of the gentamicin C complex. In this study, 3″-N-demethyl gentamicin C1a was purified from the broth of disruption strain, and its structure was elucidated using MS and NMR. Besides 3″-N-demethyl products corresponding to gentamicin C1a, C2, and C2a, two 3″-N-demethyl products corresponding to gentamicin C1 were detected, which were concluded as C-6' epimers originating from decreased substrate specificity of 6'-N methyltransferase. To explore the effects of 3″-N-methyl on gentamicin antimicrobial activity, antimicrobial activity of these demethyl gentamicin analogues were tested in vitro. 3″-N-Demethyl gentamicin components have identical activity with corresponding components of gentamicin. The results of bioassays showed that the 3″-N-methyl group has little impact on gentamicin activity. However, these highly bioactive compounds afforded a unique opportunity for creating new and high potent aminoglycoside antibiotics.

Assembly of a novel biosynthetic pathway for gentamicin B production in Micromonospora echinospora.

BACKGROUND: 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. 2'-NH2-containing gentamicin C complex is a dominant compound produced by wild-type M. echinospora; by contrast, 2'-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 C2' 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. RESULTS: 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 2'-NH2 into 2'-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. CONCLUSION: 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.

Functional manipulations of the tetramycin positive regulatory gene ttmRIV to enhance the production of tetramycin A and nystatin A1 in Streptomyces ahygroscopicus.

A putative regulatory gene ttmRIV located in the tetramycin biosynthetic gene cluster was found in Streptomyces ahygroscopicus. In-frame deletion of ttmRIV led to abolishment of tetramycin and significant enhancement of nystatin A1, whose production reached 2.1-fold of the H42 parental strain. Gene complementation by an integrative plasmid carrying ttmRIV restored tetramycin biosynthesis revealed that ttmRIV was indispensable to tetramycin biosynthesis. Gene expression analysis of the H42 strain and its mutant strain ΔttmRIV via reverse transcriptase-PCR of the tetramycin gene cluster demonstrated that the expression levels of most biosynthetic genes were reduced in ΔttmRIV. Results of electrophoretic mobility shift assays showed that TtmRIV bound the putative promoters of several genes in the tetramycin pathway. Thus, TtmRIV is a pathway-specific positive regulator activating the transcription of the tetramycin gene cluster in S. ahygroscopicus. Providing an additional copy of ttmRIV under the control of the ermEp* promoter in the H42 strain boosted tetramycin A production to 3.3-fold.

Biosynthesis of Epimers C2 and C2a in the Gentamicin C Complex.

Gentamicin is a broad-spectrum aminoglycoside antibiotic widely used to treat life-threatening bacterial infections. The gentamicin C complex consists of gentamicin C1, gentamicin C1a, and epimers gentamicin C2 and gentamicin C2a. At present there is a generally accepted pathway of gentamicin biosynthesis, except for detailed understanding of the epimerization process involving gentamicins C2 and C2a. Here we have investigated the biosynthesis of these epimers. JI-20B-an intermediate in the gentamicin biosynthetic pathway-and its epimer JI-20Ba were generated by in-frame deletion within genP, which encodes a phosphotransferase that catalyzes the first step of 3',4'-bisdehydroxylation in gentamicin biosynthesis. GenB1 and GenB2 are aminotransferases with different substrate specificities and enantioselectivities. JI-20Ba, containing a 6'S chiral amine, a precursor of gentamicin C2a, was synthesized from G418 by GenQ/GenB1 through sequential oxidation/transamination at C-6'. GenQ/GenB2 catalyzed the synthesis of JI-20B, containing a 6'R chiral amine, a precursor of gentamicin C2, from G418. GenB2 catalyzed the epimerization of JI-20Ba/JI-20B and of gentamicins C2a/C2.

Genetic engineering combined with random mutagenesis to enhance G418 production in Micromonospora echinospora.

G418, produced by fermentation of Micromonospora echinospora, is an aminoglycoside antibiotic commonly used in genetic selection and maintenance of eukaryotic cells. Besides G418, M. echinospora produces many G418 analogs. As a result, the G418 product always contains impurities such as gentamicin C1, C1a, C2, C2a, gentamicin A and gentamicin X2. These impurities are less potent but more toxic than G418, but the purification of G418 is difficult because it has similar properties to its impurities. G418 is an intermediate in the gentamicin biosynthesis pathway. From G418 the pathway proceeds via successive dehydrogenation and aminotransferation at the C-6' position to generate the gentamicin C complex, but genes responsible for these steps are still obscure. Through disruption of gacJ, which is deduced to encode a C-6' dehydrogenase, the biosynthetic impurities gentamicin C1, C1a, C2 and C2a were all removed, and G418 became the main product of the gacJ disruption strain. These results demonstrated that gacJ is in charge of conversion of the 6'-OH of G418 into 6'-NH2. Disruption of gacJ not only eliminates the impurities seen in the original strain but also improves G418 titers by 15-fold. G418 production was further improved by 26.6 % through traditional random mutagenesis. Through the use of combined traditional and recombinant genetic techniques, we produced a strain from which most impurities were removed and G418 production was improved by 19 fold.

Enhancement of nystatin production by redirecting precursor fluxes after disruption of the tetramycin gene from Streptomyces ahygroscopicus.

Complete and independent tetramycin and nystatin gene clusters containing varying lengths of type I polyketide synthase (PKS) genes were isolated from Streptomyces ahygroscopicus, a producer of tetramycin (a tetraene) in large amounts and nystatin A1 (a heptaene) in small amounts. Tetramycin was similar to pimaricin, and nystatin A1 was similar to amphotericin. All these polyene macrolide antibiotics possessed the same macrolactone ring biosynthesized from coenzyme A precursors by PKSs but had different number of atoms in the macrolactone ring and side groups. Because tetramycin and nystatin shared limited coenzyme A precursors in the same producer organism, blocking the consumption of precursors in tetramycin pathway may increase the coenzyme A pool. Thus, we genetically manipulated the tetramycin PKS to enhance nystatin production. The type I PKS ttmS1 gene mutant abolished production of tetramycin and had a beneficial effect on the production of nystatin A1. For the mutant, the yield of nystatin A1 was increased by 10-fold compared to that of the wild-type. Thus, deletion of the tetramycin pathway redirected precursor metabolic fluxes and provided an easy genetic approach to manipulate organisms and to increase production levels of a precise target.

Construction of a gentamicin C1a-overproducing strain of Micromonospora purpurea by inactivation of the gacD gene.

Gentamicin C1a is the precursor of the semi-synthetic antibiotic etimicin and has the highest antibacterial activity in the clinically important gentamicin C mixture. To obtain a gentamicin C1a-overproducing strain, we inactivated gacD gene in Micromonospora purpurea. The gacD was presumed to encode a C6' methyltransferase by sequence analysis, and plays a role in the conversion of the gentamicin intermediate X2 to G418. So the inactivation of gacD blocks the metabolic pathways from X2 to G418 and leads to the accumulation of gentamicin C1a.The resulting recombination strain produced gentamicin C1a more than 10-fold compared to the wild type strain. Moreover, the wild-type strain produced 4 main production components, C1a, C2, C2a and C1, while the recombination strain produced only 2 components, C1a and C2b, making the purification of gentamicin C1a easier. The recombination strain was genetically stable and should be useful for the industrial production of gentamicin C1a.

Construction of kanamycin B overproducing strain by genetic engineering of Streptomyces tenebrarius.

Genetic engineering as an important approach to strain optimization has received wide recognition. Recent advances in the studies on the biosynthetic pathways and gene clusters of Streptomyces make stain optimization by genetic alteration possible. Kanamycin B is a key intermediate in the manufacture of the important medicines dibekacin and arbekacin, which belong to a class of antibiotics known as the aminoglycosides. Kanamycin could be prepared by carbamoylkanamycin B hydrolysis. However, carbamoylkanamycin B production in Streptomyces tenebrarius H6 is very low. Therefore, we tried to obtain high kanamycin B-producing strains that produced kanamycin B as a main component. In our work, aprD3 and aprD4 were clarified to be responsible for deoxygenation in apramycin and tobramycin biosynthesis. Based on this information, genes aprD3, aprQ (deduced apramycin biosynthetic gene), and aprD4 were disrupted to optimize the production of carbamoylkanamycin B. Compared with wild-type strain, mutant strain SPU313 (ΔaprD3, ΔaprQ, and ΔaprD4) produced carbamoylkanamycin B as a single antibiotic, whose production increased approximately fivefold. To construct a strain producing kanamycin B instead of carbamoylkanamycin B, the carbamoyl-transfer gene tacA was inactivated in strain SPU313. Mutant strain SPU314 (ΔaprD3, ΔaprQ, ΔaprD4, and ΔtacA) specifically produced kanamycin B, which was proven by LC-MS. This work demonstrated careful genetic engineering could significantly improve production and eliminate undesired products.

Biosynthesis of 3'-deoxy-carbamoylkanamycin C in a Streptomyces tenebrarius mutant strain by tacB gene disruption.

Streptomyces tenebrarius H6 mainly produces three kinds of antibiotics: apramycin, carbamoyltobramycin and some carbamoylkanamycin B. In our present study, a dehydrogenase gene tacB in the tobramycin biosynthetic gene cluster was disrupted by in-frame deletion. The result of TLC bio-autograph analysis demonstrated the disruption mutant strain produced apramycin and a new antibiotic. The new antibiotic was identified as 3'-deoxy-carbamoylkanamycin C by MS and NMR analysis after isolation and purification. The disruption mutant was restored to produce carbamoyltobramycin in a complementation experiment by the intact tacB gene. Our studies suggested that the tacB gene encodes a 6'-dehydrogenase, which reduces the 6'-hydroxyl group of paromamine to a keto group, thus facilitating the transfer of an aminogroup to form neamine. This study is the first report on the generation of a tobramycin derivative by gene engineering, and will contribute to clarify the complete biosynthetic pathway of tobramycin.