RESUMO
Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structural diversity prompted the development of microbial platforms to produce esters with novel chemical structures and properties. One successful strategy was the heterologous expression of the mycocerosic polyketide synthase-based biosynthetic pathway (MAS-PKS, PapA5 and FadD28 enzymes) from Mycobacterium tuberculosis in Escherichia coli. This recombinant strain has the ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures and high oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived of short-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5 acyltransferase-enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substrate recognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized both in vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that when substituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5 mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBE compared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into the MBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtained by the external supply of this alcohol. KEY POINTS: ⢠Mutation in channel 2 changes PapA5 acyltransferase bioconversion efficiency. ⢠Improved production of short-chain alcohol derived multimethyl-branched esters. ⢠Establishing ethanologenic pathways for de novo production of ethanol derived MBE. ⢠Characterization of a novel phenylethanol-derived MBE.
Assuntos
Aciltransferases , Mycobacterium tuberculosis , Aciltransferases/genética , Escherichia coli/genética , Ésteres , EtanolRESUMO
Se realizó un estudio inmunohistoquímico de la enzima tirosina hidroxilasa (marcador de neuronas dopaminérgicas) en el hipotálamo de ratas Wistar machos adultas en cortes flotantes de muestras fijadas por perfusión. Debido a que el número de cerebros que se procesaron fue superior al número que pueden ser cortados inmediatamente, el material debió almacenarse congelando los cerebrosenteros a -80ºC. Pero por un desperfecto técnico del equipo de refrigeración, las muestras debieron trasladarse a -20ºC resultando en el deterioro de las mismas. Ante este inconveniente, los sucesivos cerebros fueron almacenados en sacarosa al 30%p/v en buffer fosfato salino (PBS) con 0,01% de azida sódica y mantenidos a 4ºC durante tiempos variables (de semanas a meses) hasta ser congelados con gas clorofluorado y cortados. Estos cerebros no mostraron alteración en la estructura morfológica del tejido. Esta metodología de preservación aquí descrita sería una alternativa de elección válida para aquellos laboratorios que no cuenten con un equipo de refrigeración de -80ºC.
In central nervous system histological studies, free-floating sections of perfusion-fixed samples are frequently used. Samples storage may be performed freezing either the entire brain at -80ºC or sections at -20ºC. When studying hypothalamic tyrosine hydroxylase enzyme(dopaminergic neurons marker) by immunohistochemistry in adult male Wistar rats, entire brains were stored at -80ºC. Due to an abrupt freezer technical failure, samples should be thawed to -20ºC with the resulting samples damage. To avoid this situation, subsequent brains were stored in 30% sucrose in saline phosphate buffer (PBS) with 0.01% sodium azide and kept at 4ºC for different periods (weeks to months) until they were frozen with chlorofluorade gas and cut. These brains showed no morphological alterations of tissue structure. This preservation method appeared to be an alternative valid option to laboratories with no -80ºC freezing equipment.
