BLT Stages

Abstract Bachelor project 2019-2020: INVESTIGATION OF PUTATIVE FLAVIN-DEPENDENT PHENOLIC HALOGENASES FROM PSEUDOALTEROMONAS STRAINS

Traditionally nonenzymatic halogenation is used for pharmaceutical and agrochemical applications. This leads to deleterious reagents and lacks regiocontrol. Therefore developing new economical and environmentally friendly industrial processes are necessary. These processes would be more reliable, easy and cleaner methods for the regioselective halogenation of organic compounds. In nature halogenases are often used to regio-selectively halogenate a diverse range of biosynthetic precursors. This introduction of halogens often has a great effect on the biological activity of the resulting natural products. Flavin-dependent halogenases are such halogens but there is not much known about them yet. Therefor a study was started by Jan Gebauer the investigate two proteins that may be flavin-dependent halogenases. Jan Gebauer is doing his PhD on this topic. To investigate this, the two genes had to be expressed in Escherichia coli (E.coli). So, the goal of this study was to identify and characterize these two proteins.

To be able to express the genes in E.coli firstly cultures forming flavin reductase SSuE in E.coli TunerTM (DE3)pGEB-SSuE-His6 were made. Then the cells were harvest and overnight cultures were made. A NanoDrop UV-VIS spectrophotometer was used to measure the optical density of the cultures and the concentration of the PCR products.

Next PCR was used to multiply the uncharacterized deoxyribonucleic acid (DNA) sequences and PCR purification was done. Thereafter Gibson assembly and plasmid prep is done. Then Phaenic expression, cloning of the plasmids and plasmid isolation is done. The previous steps are done to combine the DNA sequences with the chosen expression vector for E. coli., to express them and isolate the plasmids in the cells. Thereafter sequencing could be done to compare the new constructs with the desired genes of flavin-dependent enzymes. Next the cells were grown on plates and transformation E.coli DH5α was done. Lastly gels and a SDS-page were ran to see if DNA was included in the vector and if unknown DNA sequences were successfully activated in BL21.

The results that were accomplished in this study are that NH153 and NC201 were successfully expressed in E.coli and NH153 and RadH were active in BL21. The experiment is not finished. MALDI-TOF analysis, conversion with expressed reductase and halogenase, optimizing reaction conditions, upscaling and rational substrate scope investigation should be done.