Biology subjects

Nybo, S. E.

Publications and source records attributed to Nybo, S. E..

3 recordsLinked to original sources

bioRxiv2020

Next-generation sequencing of pIJ101-based Streptomyces-E. coli shuttle vectors

The sequences of two commonly used pIJ101-based Streptomyces-E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing and mapped. pEM4 was found to be 8.3 kbp long, containing genes encoding {beta}-lactamase, thiostrepton resistance, the lacZ{square} fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing genes encoding {beta}-lactamase, thiostrepton resistance, the lacZ{square} fragment, an E. coli pUC19 origin of replication, and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 kbp and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these two popular vectors, ensuring their compatibility with modern synthetic biology cloning methodologies.

genetics↗

bioRxiv2018

Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells

Purine metabolites have been implicated as clinically relevant biomarkers of worsening or improving Parkinsons disease (PD) progression. However, the identification of purine molecules as biomarkers in PD has largely been determined using non-targeted metabolomics analysis. The primary goal of this study was to develop an economical targeted metabolomics approach for the routine detection of purine molecules in biological samples. Specifically, this project utilized LC/MS/MS and LC/QTOF/MS to accurately quantify levels of six purine molecules in samples from cultured N2a murine neuroblastoma cells. The targeted metabolomics workflow was integrated with automated label-free digital microscopy, which enabled normalization of purine concentration per unit cell in the absence of fluorescent dyes. The established method offered significantly enhanced selectivity compared to previously published procedures. In addition, this study demonstrates that a simple, quantitative targeted metabolomics approach can be developed to identify and quantify purine metabolites in biological samples. We envision that this method could be broadly applicable to quantification of purine metabolites from other complex biological samples, such as cerebrospinal fluid or blood.

neuroscience↗

bioRxiv2017

Metabolic engineering of Escherichia coli for production of valerenadiene

Valeriana officinalis is a medicinal herb which produces a suite of compounds in its root tissue useful for treatment of anxiety and insomnia. The sesquiterpene components of the root extract, valerenic acid and valerena-1,10-diene, are thought to contribute to most of the observed anxiolytic of Valerian root preparations. However, valerenic acid and its biosynthetic intermediates are only produced in low quantities in the roots of V. officinalis. Thus, in this report, Escherichia coli was metabolically engineered to produce substantial quantities of valerena-1,10-diene in shake flask fermentations with decane overlay. Expression of the wildtype valerenadiene synthase gene (pZE-wvds) resulted in production of 12 g/mL in LB cultures using endogenous FPP metabolism. Expression of a codon-optimized version of the valerenadiene synthase gene (pZE-cvds) resulted in 3-fold higher titers of valerenadiene (32 g/mL). Co-expression of pZE-cvds with an engineered methyl erythritol phosphate (MEP) pathway improved valerenadiene titers 65-fold to 2.09 mg/L valerenadiene. Optimization of the fermentation medium to include glycerol supplementation enhanced yields by another 5.5-fold (11.0 mg/L valerenadiene). The highest production of valerenadiene resulted from engineering the codon-optimized valerenadiene synthase gene under strong Ptrc and PT7 promoters and via co-expression of an exogenous mevalonate (MVA) pathway. These efforts resulted in an E. coli production strain that produced 62.0 mg/L valerenadiene (19.4 mg/L/OD600 specific productivity). This E. coli production platform will serve as the foundation for the synthesis of novel valerenic acid analogues potentially useful for the treatment of anxiety disorders.

synthetic biology↗