Correction to: Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae.
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SUBMITTER: Wehrs M
PROVIDER: S-EPMC6936116 | biostudies-literature | 2019 Dec
REPOSITORIES: biostudies-literature
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Correction to: Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae.
Wehrs Maren M Prahl Jan-Philip JP Moon Jadie J Li Yuchen Y Tanjore Deepti D Keasling Jay D JD Pray Todd T Mukhopadhyay Aindrila A
Microbial cell factories 20191229 1
Following publication of the original article [1], the authors have noted that the standard curve in Additional file 1: Figure S7 is incorrect. ...[more]
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Project description:BackgroundBeyond pathway engineering, the metabolic state of the production host is critical in maintaining the efficiency of cellular production. The biotechnologically important yeast Saccharomyces cerevisiae adjusts its energy metabolism based on the availability of oxygen and carbon sources. This transition between respiratory and non-respiratory metabolic state is accompanied by substantial modifications of central carbon metabolism, which impact the efficiency of metabolic pathways and the corresponding final product titers. Non-ribosomal peptide synthetases (NRPS) are an important class of biocatalysts that provide access to a wide array of secondary metabolites. Indigoidine, a blue pigment, is a representative NRP that is valuable by itself as a renewably produced pigment.ResultsSaccharomyces cerevisiae was engineered to express a bacterial NRPS that converts glutamine to indigoidine. We characterize carbon source use and production dynamics, and demonstrate that indigoidine is solely produced during respiratory cell growth. Production of indigoidine is abolished during non-respiratory growth even under aerobic conditions. By promoting respiratory conditions via controlled feeding, we scaled the production to a 2 L bioreactor scale, reaching a maximum titer of 980 mg/L.ConclusionsThis study represents the first use of the Streptomyces lavendulae NRPS (BpsA) in a fungal host and its scale-up. The final product indigoidine is linked to the activity of the TCA cycle and serves as a reporter for the respiratory state of S. cerevisiae. Our approach can be broadly applied to investigate diversion of flux from central carbon metabolism for NRPS and other heterologous pathway engineering, or to follow a population switch between respiratory and non-respiratory modes.
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Project description:The growing demand for natural pigments in the industrial sector is a significant driving force in the development of production processes. The production of natural blue pigments, which have wide industrial applications, using microbial systems has been gaining significant attention. In this study, we used Aspergillus oryzae as a platform cell factory to produce the blue pigment indigoidine (InK), by genetic manipulation of its non-ribosomal peptide synthetase system to overexpress the indigoidine synthetase gene (AoinK). Phenotypic analysis showed that InK production from the engineered strain was growth associated, owing to the constitutive control of gene expression. Furthermore, the initial pH, temperature, and glutamine and MgSO4 concentrations were key factors affecting InK production by the engineered strain. The pigment secretion was enhanced by addition of 1% Tween 80 solution to the culture medium. The maximum titer of total InK was 1409.22 ± 95.33 mg/L, and the maximum productivity was 265.09 ± 14.74 mg/L·d. Moreover, the recombinant InK produced by the engineered strain exhibited antioxidant activity. These results indicate that A. oryzae has the potential to be used as a fungal platform for overproduction of extracellular non-ribosomal peptide pigments.
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Project description:[This corrects the article DOI: 10.1371/journal.pone.0099266.].
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Project description:BackgroundRapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL.MethodsIn silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate <1%) were identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously.ResultsFive unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS.ConclusionsThis proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.
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Project description:Despite the advances in modern medicine, antibiotic resistance is a persistent and growing threat to the world. Thus, the discovery and development of novel antibiotics have become crucial to combat multi-drug resistant pathogens. The goal of our research is to discover a small molecular peptide that can disrupt the synthesis of new ribosomes. Using the phage display technique, we have discovered a 7-mer peptide that binds to the second strand of 16S h34 RNA with a dissociation constant in the low micromolar range. Binding of the peptide alters RNA structure and inhibits the binding of the ribosomal RNA small subunit methyltransferase C (RsmC) enzyme that methylates the exocyclic amine of G1207. The addition of this peptide also increases the lag phase of bacterial growth. Introduction of chemical modifications to increase the binding affinity of the peptide to RNA, its uptake and stability can further improve the efficacy of the peptide as an antibiotic agent against pathogenic bacteria.
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Project description:PurposeThe purpose of this study was to evaluate a new inline motion compensation approach called image-based navigation with Constant Respiratory efficiency UsIng Single End-expiratory threshold (iNAV-CRUISE) for coronary MR angiography (CMRA).MethodsThe CRUISE gating technique was combined with iNAV motion correction and implemented inline for motion-compensated CMRA on a 1.5 Tesla scanner. The approach was compared to conventional diaphragmatic navigator gating (dNAVG) in 10 healthy subjects. The CMRA images were compared for vessel sharpness and visual score of the right coronary artery (RCA), left anterior descending artery (LAD), left circumflex, and scan time.ResultsThe scan time was similar between the methods (dNAVG : 6:32 ± 1:09 vs. iNAV-CRUISE: 6:58 ± 0:17, P = not significant). However, the vessel sharpness of the RCA (dNAVG : 60.2 ± 10.1 vs. iNAV-CRUISE: 71.8 ± 8.9, P = 0.001) and LAD (dNAVG : 58.0 ± 8.0 vs. iNAV-CRUISE: 67.4 ± 7.1, P = 0.008) were significantly improved using iNAV-CRUISE. The visual score of the RCA was higher using iNAV-CRUISE compared to dNAVG (dNAVG : 3,4,3 vs. iNAV-CRUISE: 4,4,3, P < 0.01).ConclusionThe iNAV-CRUISE approach out-performs the conventional respiratory motion compensation technique in healthy subjects. Although scan time was comparable, the image quality was improved using iNAV-CRUISE. Magn Reson Med 79:416-422, 2018. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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