The Wangikar Lab
New research from the Wangikar lab
Jaiswal, D., Prasannan, C. B., Hendry, J. I., & Wangikar, P. P. (2018). SWATH Tandem Mass Spectrometry Workflow for Quantification of Mass Isotopologue Distribution of Intracellular Metabolites and Fragments Labeled with Isotopic 13C Carbon. Analytical chemistry, 90(11), 6486-6493
Accurate quantification of mass isotopologue distribution (MID) of metabolites is a prerequisite for 13C-metabolic flux analysis. Currently used mass spectrometric (MS) techniques based on multiple reaction monitoring (MRM) place limitations on the number of MIDs that can be analyzed in a single run. Moreover, the deconvolution step results in amplification of error. Here, we demonstrate that SWATH MS/MS, a data independent acquisition (DIA) technique allows quantification of a large number of precursor and product MIDs in a single run. SWATH sequentially fragments all precursor ions in stacked mass isolation windows. Co-fragmentation of all precursor isotopologues in a single SWATH window yields higher sensitivity enabling quantification of MIDs of fragments with low abundance and lower systematic and random errors. We quantify the MIDs of 53 precursor and product ions corresponding to 19 intracellular metabolites from a dynamic 13C-labeling of a model cyanobacterium, Synechococcus sp. PCC 7002. The use of product MIDs resulted in an improved precision of many measured fluxes compared to when only precursor MIDs were used for flux analysis. The approach is truly untargeted and allows additional metabolites to be quantified from the same data.
Shah, S., Agera, R., Sharma, P., Sunder, A. V., Bajwa, H., James, H. M., ... & Wangikar, P. P. (2018). Development of biotransformation process for asymmetric reduction with novel anti-Prelog NADH-dependent alcohol dehydrogenases. Process Biochemistry.
Alcohol dehydrogenases or carbonyl reductases have been extensively developed for the asymmetric reduction of ketones to chiral alcohols, which are important pharmaceutical precursors. Ideal biotransformation using alcohol dehydrogenases requires (i) the identification of novel enzymes with broad substrate range, high substrate tolerance, enantioselectivity and preference for cheaper cofactor (NADH) and (ii) the development of an optimized biocatalytic process with a mechanism for efficient cofactor recycling. This report details the mining and identification of a subfamily of novel NADH-dependent alcohol dehydrogenases with anti-Prelog stereo selectivity, that exhibit high specific activity on β-ketoesters. Further, an efficient biocatalytic process has been developed using ADH from Acetobacter aceti mined in this study for the enantioselective reduction of up to 10 M ethyl 4-chloro-3-oxobutanoate to (S)-Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The process employed lyophilized cell-free extract and reduction was achieved with > 99% yield and high enantiomeric excess (> 99% ee) in 24 h using a biphasic reaction system. A space-time yield of approximately 650 g/L. d and cofactor TTN of 106 has been achieved using the process, with potential application in industrial biocatalysis.
Cyanobacteria are attractive hosts that can be engineered for the photosynthetic production of fuels, fine chemicals, and proteins from CO2. Moreover, the responsiveness of these photoautotrophs towards different environmental signals, such as light, CO2, diurnal cycle, and metals make them potential hosts for the development of biosensors. However, engineering these hosts proves to be a challenging and lengthy process. Synthetic biology can make the process of biological engineering more predictable through the use of standardized biological parts that are well characterized and tools to assemble them. While significant progress has been made with model heterotrophic organisms, many of the parts and tools are not portable in cyanobacteria. Therefore, efforts are underway to develop and characterize parts derived from cyanobacteria. In this review, we discuss the reported parts and tools with the objective to develop cyanobacteria as cell factories or biosensors. We also discuss the issues related to characterization, tunability, portability, and the need to develop enabling technologies to engineer this “green” chassis.
Recent Conference Attended:
Researchers from the Wangikar lab recently presented their work at the International conference ( https://www.aiche.org/sbe/conferences/metabolic-engineering-conference/2018) held in Munich, Germany from June 24-28, 2018
Prachi Varshney graduated with a PhD recently for her thesis on ‘CO2 CAPTURE WITH EXTREMOPHILIC MICROALGAE-ISOLATION AND CHARACTERIZATION OF NOVEL STRAINS’
areas of research expertise
ABOUT PRAMOD WANGIKAR
Pramod P Wangikar
Prof. Pramod P. Wangikar is a faculty of the Department of Chemical Engineering in the Institute of Technology- Bombay (IIT-B). Prof. Wangikar obtained his Bachelor of Chemical Engineering from the Institute of Chemical Technology (ICT), Mumbai and then earned his Ph.D. from the University of Iowa,