Banca de QUALIFICAÇÃO: PRISCILA AMARAL DINIZ

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
STUDENT : PRISCILA AMARAL DINIZ
DATE: 23/02/2022
TIME: 09:00
LOCAL: meet.google.com/kbo-ismh-fnc
TITLE:

DEVELOPMENT OF A HETEROLOGY EXPRESSION SYSTEM IN Penicillium camemberti

KEY WORDS:

biotechnology, Penicillium camemberti, genetic modifications, expression system, filamentous fungi

PAGES: 31
BIG AREA: Ciências Biológicas
AREA: Bioquímica
SUBÁREA: Bioquímica dos Microorganismos
SUMMARY:

Genetic manipulation of organisms has been shown to be one of the most
promising aspects of biotechnology, and it is possible to highlight the development of obtaining
products of economic and industrial interest. For the purpose of producing proteins from
interest, heterologous expression systems can be developed, which are
production of proteins generated through the genetic modification of an organism. Although the
Filamentous fungi are microorganisms that have a robust secretion system of
proteins, there is no commercially available heterologous expression system in this
organism, and there is a scarcity of scientific works using such systems. In this
work began to develop a heterologous expression system in Penicillium
camemberti. This is a filamentous fungus that has bioprocesses with GRAS status to
lipase production, performs post-translational modifications such as glycosylation, phosphorylation and
disulfide bonds, secretes large amounts of enzymes into the extracellular environment, in addition to
to be cultivated in low-cost media and to have potential for genetic improvement.
To study the expression system, a galactosidase, alpha-amylase (α-
amy), derived from Penicillium camemberti itself, as well as the components of an ORF,
open reading frame, such as promoter and terminator sequences. For this, we used the
fusion polymerase chain reaction (fusion PCR) technique, which makes it possible to obtain several
ORFs with different promoters and terminators, making it possible to optimize the constructs for
production of proteins of industrial interest. In order to test the level of
ORF expression with different constructs, constitutive promoters were chosen and
inducible drugs already used in expression vectors in other microorganisms such as
proteins citrate synthase, glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate
kinase. The terminator used was from the tryptophan biosynthesis protein. With
amplifications of promoter, target gene and terminator sequences, PCR was optimized
melting at 59°C due to the annealing of the megaprimers used in the technique, which also
allows the fusion of the fragments in an ORF with the target gene. At the same time, there was
in silico analysis of the alpha-amylase enzyme to obtain data such as physical characteristics -
chemical, structural, cellular localization and similarity. Some data obtained were
number of amino acids, molecular weight, theoretical isoelectric point, number of residues
negatively charged residues, number of positively charged residues and coefficient of
hydropathicity. Preliminary studies have indicated that alpha-amylase from P. camemberti is
an extracellular enzyme that is easily secreted from the intracellular environment, which already indicates that
this is an enzyme whose characteristics tend to be promising for large-scale production
for various purposes such as in the application of the textile and food industry.

BANKING MEMBERS:
Presidente - 1757978 - DANIEL BONOTO GONCALVES
Interno - 1367304 - ALEXSANDRO SOBREIRA GALDINO
Externo à Instituição - LEONARDO AUGUSTO DE ALMEIDA - UNIFAL-MG