Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris
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2016-08-31
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Universidade Federal de Goiás
Resumo
In the last decades the search for new renewable energy sources have increased and one of
the emerged technologies was the production of ethanol from lignocellulosic biomass (second
generation ethanol). For obtaining fermentable sugars from biomass, a complex of enzymes
and proteins that acts on polysaccharides of the plant cell wall is required. Expansins are
proteins that can help enzymes in the degradation process, promoting the loosening of the
cell wall of plants by breaking hydrogen bonds between cellulose fibers and hemicellulose. The
superfamily of expansins has been described mainly in plants, although they are also found in
fungi and bacteria. Four families are described to this superfamily: α-expansins, β-expansins,
expansins-like A and expansins-like B. The α-expansins are present in most eudicots, whereas
the β-expansins are involved in cellular expansion process of the monocots family Poaceae. To
analyze the sequence and to verify the functional activity of a β-expansin on filter paper, in
this work a gene of β-expansin (expb11) of sugarcane was isolated, cloned and expressed in
the yeast Pichia pastoris. Nucleotide sequences of expansin genes from sugarcane were
initially obtained from the public SUCEST database using reference sequences of expansins
from sorghum, maize and rice as query. A total of 227 ESTs were identified. These ESTs were
submitted to clustering and 30 contigs and 92 singletons were obtained. Specific oligos were
designed and used to capture the genomic fragment corresponding to the expb11 gene by
PCR. The coding DNA fragment of expb11 was obtained by RT-PCR from the total RNA
extracted from stalks of the RB867515 sugarcane cultivar. This fragment was cloned into the
pGEM-T Easy vector and subcloned into the pPIC9 vector. Genomic and cDNA fragments were
sequenced. The expression cassette generated by subcloning into the pPIC9 expression vector
was integrated into the P. pastoris strain GS115 genome, but it has not been possible to
detect a functional activity of expansin. The expb11 gene comprises 1367 bp, containing 3
introns, and its cDNA is 801 bp long, with an ORF of 776 bp. The alignment of sugarcane and
sorghum sequences of expb11 showed that the two species share a 95% sequence identity
along these genes and that the gene structure is highly conserved in these species. The in
silico analysis of the putative EXPB11 amino acid sequence allowed the detection of two
conserved domains, one similar to the GH45 family, and the other to the carbohydrate binding
module (CBM). The putative EXPB11 protein has a predicted molecular weight of
approximately 26.4 kDa, an isoelectric point (pI) of 4.88, and contains two glycosylation sites.
Our results confirmed that the isolated and cloned expb11 gene corresponds to a β-expansin
gene from sugarcane, paving the way to the expression, isolation and use of recombinant
EXPB11 in the mix of enzymes and proteins for lignocellulosic biomass degradation.
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COSTA, I. G. O. Clonagem e expressão de uma β-expansina de cana-de-açúcar na levedura Pichia pastoris. 2016. 95 f. Dissertação (Mestrado em Genética e Melhoramento de Plantas) - Universidade Federal de Goiás, Goiânia, 2016.