Enzyme Screening Research Articles

Isolation and Screening Microorganisms from Black Soldier Fly Larvae (Hermetia illucens) that Producing Amylase, Protease and Cellulase

The Black Soldier Fly (BSF) has gained significant attention for its ability to decompose various types of organic waste and produce valuable enzymes related to its digestive system. These enzymes hold immense potential for applications across diverse industrial sectors, with promise in the livestock industry. This study aimed to extract and assess amylase, protease, and cellulase enzymes from microorganisms present in BSF larvae fermentation. The isolation technique employed was the spread plate method, followed by rigorous screening for the presence of these enzymes. The results showed 46 isolates of 26 bacterial isolates and 20 yeast isolates. In the amylase enzyme screening, a substantial 25 bacterial and 19 yeast isolates exhibited positive amylase activity. For cellulase, 20 bacterial and 14 yeast isolates displayed positive results. In the case of protease, 16 bacterial and 12 yeast isolates demonstrated protease enzyme activity. Notably, nine isolates exhibited the remarkable capability to produce multiple enzymes, including eight bacterial and one yeast isolate. These results showcase the rich enzymatic potential of BSF-associated microorganisms, offering exciting prospects for their application in various industrial sectors, especially in enhancing the efficiency and sustainability of livestock production

Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

Diadenosine Tetraphosphate (Ap4A) Serves as a 5′ RNA Cap in Mammalian Cells

AbstractThe recent expansion of the field of RNA chemical modifications has changed our understanding of post‐transcriptional gene regulation. Apart from internal nucleobase modifications, 7‐methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non‐canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non‐canonical RNA cap, diadenosine tetraphosphate (Ap4A), in human and rat cell lines. Ap4A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non‐canonical initiating nucleotide (NCIN). Using liquid chromatography‐mass spectrometry (LC–MS), we show that the amount of capped Ap4A‐RNA is independent of the cellular concentration of Ap4A. A decapping enzyme screen identifies two enzymes cleaving Ap4A‐RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4A‐RNA and show that although it is not translated, Ap4A‐RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

Whole-cell biosensor engineering based on the transcription factor XylS/Pm for sensitive detection of PCB intermediate chlorobenzoic acid

Chlorobenzoic acids (Cba) are toxic chloride pollutants commonly released by chemical industries or formed during the degradation pathway of organic chlorides, such as polychlorinated biphenyls (PCBs). Here, we successfully developed a highly sensitive Escherichia coli whole-cell Cba biosensor, named XyF10 (MG1655(DE3)-Ps-XylS (Step13)-Pm1-EGFP), by comprehensive engineering the elements of Cba sensing genetic circuit from Pseudomonas putida. Compared to the native sensing system, XyF10 exhibited a significantly reduced detection limit (<1 μM vs. 50 μM) and an increased signal-to-noise ratio (11.7-fold vs. 4.4-fold), making it a highly efficient whole-cell sensor with the lowest detection limit for Cba reported to date. Structural analysis and molecular dynamics simulations of XylS and its variants revealed that the increased flexibility of a ring structure in XylS (Step13) variant promoted the sensing efficiency of XyF10 system by fascinating the formation of active dimers and enabled more stable binding with inducer. Furthermore, the XyF10 system displayed an obvious preference for meta-substituted Cba (3-Cba) as substrates, which was attributed by the stronger interaction between 3-Cba and the active site of XylS. Overall, our research not only advanced the Cba detection techniques for large-scale detection of Cba in the environment, but also provided potential tools for high-throughput screening of PCB degradation pathways and enzymes.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

Biotransamination of Furan-Based Aldehydes with Isopropylamine: Enzyme Screening and pH Influence.

Furan-based amines are highly valuable compounds which can be directly obtained via reductive amination from easily accessible furfural, 5-(hydroxymethyl)furfural (HMF) and 2,5-diformylfuran (DFF). Herein the biocatalytic amination of these carbonyl derivatives is disclosed using amine transaminases (ATAs) and isopropylamine (IPA) as amine donors. Among the different biocatalysts tested, the ones from Chromobacterium violaceum (Cv-TA), Arthrobacter citreus (ArS-TA), and variants from Arthrobacter sp. (ArRmut11-TA) and Vibrio fluvialis (Vf-mut-TA), afforded high levels of product formation (>80 %) at 100-200 mM aldehyde concentration. The transformations were studied in terms of enzyme and IPA loading. The pH influence was found as a key factor and attributed to the imine/aldehyde equilibrium that can arise from the high reactivity of the carbonyl substrates with a nucleophilic amine such as IPA.

Identification of HDAC inhibitors as neutrophil recruitment modulators in zebrafish using a chemical library screen.

Tissue injury-induced neutrophil recruitment is a prerequisite for the initiation and amplification of inflammatory responses. Although multiple proteases and post-translational modification (PTM) enzymes regulate leukocyte recruitment, an unbiased functional screen of those enzymes regulating inflammatory leukocyte recruitment has yet to be undertaken. Here, using a zebrafish tail fin amputation (TFA) model to screen a chemical library consisting of 295 compounds that target proteases and PTM enzymes, we identified multiple histone deacetylase (HDAC) inhibitors that modulate inflammatory neutrophil recruitment. AR-42, a pan-HDAC inhibitor, was shown to inhibit neutrophil recruitment in three different zebrafish sterile tissue injury models: TFA, copper-induced neuromasts damage (CIND), and mechanical otic vesicle injury (MOVI), and a sterile murine peritonitis model. RNA sequencing analysis of AR-42-treated fish embryos revealed downregulation of neutrophil-associated cytokines/chemokines, and exogenous supplementation with recombinant human IL-1β and CXCL8 partially restored the defective neutrophil recruitment in AR-42-treated MOVI model fish embryos. We thus demonstrate that AR-42 non-cell autonomously modulates neutrophil recruitment by suppressing transcriptional expression of cytokines/chemokines, thereby identifying AR-42 as a promising anti-inflammatory drug for treating sterile tissue injury-associated diseases.

Optimized Single-Step Recovery of Lipophilic and Hydrophilic Compounds from Raspberry, Strawberry and Blackberry Pomaces Using a Simultaneous Ultrasound-Enzyme-Assisted Extraction (UEAE).

An ultrasound-enzyme-assisted extraction (UEAE) was optimized to extract, simultaneously, the hydrophilic and lipophilic compounds from three berry pomaces (raspberry, strawberry and blackberry). First, an enzyme screening designated a thermostable alkaline protease as the most suitable enzyme to recover, in an aqueous medium, the highest yields of polyphenols and oil in the most efficient way. Secondly, the selected enzyme was coupled to ultrasounds (US) in sequential and simultaneous combinations. The simultaneous US-alkaline enzyme combination was selected as a one-single-step process and was then optimized by definitive screening design (DSD). The optimized parameters were: US amplitude, 20% (raspberry pomace) or 70% (strawberry and blackberry pomaces); pH, 8; E/S ratio, 1% (w/w); S/L ratio, 6% (w/v); extraction time, 30 min; temperature, 60 °C. Compared to conventional extractions using organic solvents, the UEAE extracted all the polyphenols, with around 75% of the active polyphenols (measured by the DPPH● method) and up to 75% of the initial oil from the berry pomaces. Characterized lipophilic compounds were rich in polyunsaturated fatty acids (PUFAs), tocols and phytosterols. The polyphenolics were analyzed by UPLC-MS/MS; characteristic ellagitannins of the Rosaceae family (sanguiin H-6 or agrimoniin, sanguiin H-10, …) and ellagic acid conjugates were found as the major components.

Enzymatic biodegradation and detoxification of azo and anthraquinone dyes by indigenously isolated bacterial monocultures and their synergistic behaviour in developed consortia

AbstractWastewater generated by textile industry needs to be treated to reduce its toxicity before final disposal and/or for recycling purposes. In the current study, several bacterial strains were screened for dye decolorization potential. UV–visible spectroscopy was used to determine maximum absorption wavelength of disperse dyes. HPLC and MTS assay were used to confirm the degradation and detoxification of disperse dyes, respectively. Results revealed that indigenously isolated Bacillus licheniformis, Glutamicibacter uratoxydans and Pseudomonas aeruginosa showed strong decolorization of red, blue and violet, respectively in 6–9 h. MTS assay revealed 100% viability of NIH/3T3 cell lines in presence of treated dyes. Enzyme screening assay confirmed the production of intracellular and membrane bound oxidoreductases in presence of specific dye as substrate. To resolve this issue, bacterial consortia were prepared, and better decolorization of all dyes was achieved in synergistic behaviour of Consortia 1 and 4 with 85% and 88% decolorization potential, respectively.

Phosphorus-Containing Catalyst Impact on Furfural and Glucose Production during Consecutive Hydrothermal Pretreatment and Enzymatic Hydrolysis

Lignocellulosic biomasses have a very important role as raw materials to produce biobased chemicals. However, a sustainable, efficient, and economically competitive way to convert lignocellulosic biomass into these chemicals has still not been achieved. This study is related to the selective separation and conversion of birch wood C5 carbohydrates into furfural during the H3PO4–NaH2PO4-catalyzed hydrothermal pretreatment simultaneously preserving cellulose in the lignocellulosic leftover for glucose production by the enzymatic hydrolysis. The ratio of H3PO4–NaH2PO4 in the catalyst solution was changed (3:0, 2:1, 1:1, and 1:2). Results show that around 64.1 to 75.9% of available C5 carbohydrates were converted into furfural. The results of birch wood lignocellulosic leftover chemical composition analysis show that cellulose losses during the pretreatment stage did not reach more than 10% of the initial amount. Based on the enzymatic hydrolysis screening experiments, a suitable catalyst for pretreatment was selected and an in-depth study was carried out. Enzymatic hydrolysis experiments were organized based on the three-factor central composite face-centered design. The variable parameters were treatment time (24–72 h), enzyme load (10–20 U/g cellulose), and substrate amount in reaction media (10–20%). At optimal conditions, 49.9 ± 0.5% of available cellulose in lignocellulosic leftover was converted into glucose.

(Invited) Commercializing Single Walled Carbon Nanotube-Based Sensors - Enzyme Activity and Beyond

The ECS carbon nanostructures community is well informed on the potential of single walled carbon nanotube (SWCNT)-based sensors and likewise the challenges associated with realizing practical applications outside of an academic laboratory. This talk will discuss our latest efforts in facile separation and sensor preparation methods. It will discuss potential use cases in improving high throughput screening of custom enzymes, with a current focus on recalcitrant substrates such as conventional polymers. This talk will also focus on latest efforts of reducing to practice optical fluorimeters (single, multi-channel, spatial, and temperature controlled) for field use applications. Finally, this talk will detail the on-going adventure of commercialization at Zymosense Inc. which we hope will serve as a practical guide to other groups considering transferring their technology from the lab.

Recent Advances on Key Enzymes of Microbial Origin in the Lycopene Biosynthesis Pathway.

Lycopene is a common carotenoid found mainly in ripe red fruits and vegetables that is widely used in the food industry due to its characteristic color and health benefits. Microbial synthesis of lycopene is gradually replacing the traditional methods of plant extraction and chemical synthesis as a more economical and productive manufacturing strategy. The biosynthesis of lycopene is a typical multienzyme cascade reaction, and it is important to understand the characteristics of each key enzyme involved and how they are regulated. In this paper, the catalytic characteristics of the key enzymes involved in the lycopene biosynthesis pathway and related studies are first discussed in detail. Then, the strategies applied to the key enzymes of lycopene synthesis, including fusion proteins, enzyme screening, combinatorial engineering, CRISPR/Cas9-based gene editing, DNA assembly, and scaffolding technologies are purposefully illustrated and compared in terms of both traditional and emerging multienzyme regulatory strategies. Finally, future developments and regulatory options for multienzyme synthesis of lycopene and similar secondary metabolites are also discussed.

Development of a growth-coupled selection platform for directed evolution of heme biosynthetic enzymes in Corynebacterium glutamicum.

Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in k cat/K M was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.

Optimization of multi-enzyme cascade process for the biosynthesis of benzylamine.

Benzylamine is a valuable intermediate in the synthesis of organic compounds such as curing agents and antifungal drugs. To improve the efficiency of benzylamine biosynthesis, we identified the enzymes involved in the multi-enzyme cascade, regulated the expression strength by using RBS engineering in Escherichia coli, and established a regeneration-recycling system for alanine. This is a cosubstrate, coupled to cascade reactions, which resulted in E. coli RARE-TP and can synthesize benzylamine using phenylalanine as a precursor. By optimizing the supply of cosubstrates alanine and ammonia, the yield of benzylamine produced by whole-cell catalysis was increased by 1.5-fold and 2.7-fold, respectively, and the final concentration reached 6.21mM. In conclusion, we achieved conversion from l-phenylalanine to benzylamine and increased the yield through enzyme screening, expression regulation, and whole-cell catalytic system optimization. This demonstrated a green and sustainable benzylamine synthesis method, which provides a reference and additional information for benzylamine biosynthesis research.

Perbandingan Kadar SGPT Pada Sampel Serum Darah Yang Segera Diperiksa Dengan Ditunda Selama 24 Jam dan 48 Jam Pada Suhu Ruang

Serum Glutamic Pyruvic Transaminase (SGPT) examination is an examination to determine the presence of disturbances in liver function. The SGPT enzyme is often used as an enzyme screening of basic parameter for a diagnosis and follow-up of disorders liver function. The types of specimens were used for SGPT examinations were serum, plasma heparin ora EDTA, and this study used serum samples. This study aimsto determine whether there are differences between the examination of SGPT levels which are immediately examined of postponed for 24 hours and 48 hours. This research is a cross sectional research with Posttest Only Control Group Design. This research was conducted at the Palembang Health Laboratory Center (BBLK) of South Sumatra Province on 28-30 December 2021 with the respondents of Diploma Medical Laboratory Technology Students level 2 and 3 with 30 samples. The results showed that the average SGPT level which immediately checked was 0.14, the average SGPT level delayed 24 hours was 0.13 and the SGPT level delayed 48 hours was 0.11. the results of the Two Way ANOVA test showed p value &lt; 0,05, so it can be concluded that there was a change in the level of SGPT which immediately examined by being delayed for 24 hours and 48 hours at roon temperature

Isolation, identification, and enzyme screening of fungi infested various vegetables in the local markets of Visakhapatnam, Andhra Pradesh

This study aimed to examine the fungi present in fruits and vegetables obtained from the local vegetable markets, Visakhapatnam and assess their enzymatic activity. seven fungal pathogens responsible for vegetable spoilage were isolated using sterilization and spread plate techniques on potato dextrose agar (PDA) medium. The isolated fungi were identified based on macroscopic and microscopic characteristics. The identified fungal strains included Aspergillus, Curvularia, Alternaria, Cladosporium, Fusarium, sclerotinia, Mucor, Penicillium, and Rhizopus. Only three selected strains were tested for extracellular amylase, pectinase and cellulase activity, which were showing 15mm,20mm,30mm clear zone capability. These findings provide essential information about the degradative properties of filamentous fungi and their secretion of extracellular enzymes. The study also highlights the presence of toxigenic fungi in local vegetables, emphasizing the importance of removing spoiled fruits to prevent the accumulation of toxins associated with fungal growth.

Engineering Escherichia coli for selective 1-decanol production using the reverse β-oxidation (rBOX) pathway

1-Decanol has great value in the pharmaceutical and fragrance industries and plays an important role in the chemical industry. In this study, we engineered Escherichia coli to selectively synthesize 1-decanol by using enzymes of the core reverse β-oxidation (rBOX) pathway and termination module with overlapping chain-length specificity. Through screening for acyl-CoA reductase termination enzymes and proper regulation of rBOX pathway expression, a 1-decanol titer of 1.4 g/L was achieved. Further improvements were realized by engineering pyruvate dissimilation to ensure the generation of NADH through pyruvate dehydrogenase (PDH) and reducing byproduct synthesis via a tailored YigI thioesterase knockout, increasing 1-decanol titer to 1.9 g/L. The engineered strain produced about 4.4 g/L 1-decanol with a yield of 0.21 g/g in 36 h in a bi-phasic fermentation that used a dodecane overlay to increase 1-decanol transport and reduce its toxicity. Adjustment of pathway expression (varying inducer concentration) and cell growth (oxygen availability) enabled 1-decanol production at 6.1 g/L (0.26 g/g yield) and 10.05 g/L (0.2 g/g yield) using rich medium in shake flasks and bioreactor, respectively. Remarkably, the use of minimal medium resulted in 1-decanol production with 100% specificity at 2.8 g/L (0.14 g/g yield) and a per cell mass yield higher than rich medium. These 1-decanol titers, yields and purity are at least 10-fold higher than others reported to date and the engineered strain shows great potential for industrial production. Taken together, our findings suggest that using rBOX pathway and termination enzymes of proper chain-length specificity in combination with optimal chassis engineering should be an effective approach for the selective production of alcohols.

Versatile Product Detection via Coupled Assays for Ultrahigh-Throughput Screening of Carbohydrate-Active Enzymes in Microfluidic Droplets.

Enzyme discovery and directed evolution are the two major contemporary approaches for the improvement of industrial processes by biocatalysis in various fields. Customization of catalysts for improvement of single enzyme reactions or de novo reaction development is often complex and tedious. The success of screening campaigns relies on the fraction of sequence space that can be sampled, whether for evolving a particular enzyme or screening metagenomes. Ultrahigh-throughput screening (uHTS) based on in vitro compartmentalization in water-in-oil emulsion of picoliter droplets generated in microfluidic systems allows screening rates >1 kHz (or >107 per day). Screening for carbohydrate-active enzymes (CAZymes) catalyzing biotechnologically valuable reactions in this format presents an additional challenge because the released carbohydrates are difficult to monitor in high throughput. Activated substrates with large optically active hydrophobic leaving groups provide a generic optical readout, but the molecular recognition properties of sugars will be altered by the incorporation of such fluoro- or chromophores and their typically higher reactivity, as leaving groups with lowered pKa values compared to native substrates make the observation of promiscuous reactions more likely. To overcome these issues, we designed microdroplet assays in which optically inactive carbohydrate products are made visible by specific cascades: the primary reaction of an unlabeled substrate leads to an optical signal downstream. Successfully implementing such assays at the picoliter droplet scale allowed us to detect glucose, xylose, glucuronic acid, and arabinose as final products of complex oligosaccharide degradation by glycoside hydrolases by absorbance measurements. Enabling the use of uHTS for screening CAZyme reactions that have been thus far elusive will chart a route toward faster and easier development of specific and efficient biocatalysts for biovalorization, directing enzyme discovery by challenging catalysts for reaction with natural rather than model substrates.

Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases.

Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.

Delayed onset Cutibacterium (formerly Propionibecterium) acnes endophthalmitis triggered by intravitreal steroid injection.

We report a case of delayed onset Cutibacterium acnes (C. acnes, formerly Propionibacterium acnes or P. acnes) endophthalmitis with onset triggered by intravitreal steroid (triamcinolone acetonide; TA) injection in a pseudophakic patient, 7 years after cataract surgery.Methods/Patients: A 67 years old, male presented with gradual worsening of vision, eye redness and photosensitivity that started a month after intravitreal TA injection (Triescience, 4mg/0.1mL, Alcon Labs, Fort Worth, TX) for cystoid macular edema secondary to branch retinal vein occlusion in the right eye. Patient had undergone cataract surgery with intraocular lens (IOL) in that eye 7 years prior. Exam showed visual acuity of counting fingers at 3 feet, conjunctival injection, with 2+ anterior chamber cell and 2+ vitreous haze. Uveitis work up including Angiotensin converting enzyme (ACE), Quantiferon Gold, and syphilis IgG screen was negative. Diagnostic pars plana vitrectomy (PPV) with intravitreal injection of antibiotics was performed and vitreous sample was sent for detailed laboratory analysis. Vitreous fluid analysis was positive for C. acnes and negative for viral, fungal and malignant cells. Although the patient received intravitreal antibiotics injection twice, the intraocular infection and inflammation persisted which eventually required an IOL and capsular bag removal, followed by insertion of a secondary IOL later. Delayed onset C. acnes endophthalmitis may be triggered by an intravitreal steroid injection in pseudophakic patients.