Bariloche protein symposium argentine society for biochemistry and molecular biology

58
BIOCELL, 27 (Suppl. I), 2003
BT-C2.
ERYTHROMYCIN A BIOSYNTHESIS IN
HETEROLOGOUS HOST
Peirú, Salvador; Menzella, Hugo G.; Kurth, Daniel G. and
Gramajo, Hugo C.
Facultad de Ciencias Bioquímicas y Farmacéuticas- IBR. UNR-
CONICET, Suipacha 531, Rosario, 2000. Argentina. E-mail:
[e-mail]
The increasing knowledge about the chemistry performed by
polyketide synthetases (PKSs) has lead to the construction of
polyketide libraries, generated by genetic modification of macrolide
PKSs. Since these libraries were constructed in heterologous hosts
lacking glycosylation pathways, only the corresponding aglycones
were produced. We wished to expand the capabilities of the
combinatorial biosynthesis strategies to incorporate post-PKS
“tailoring” steps, in particular the adition of deoxysugar
components. We decided to construct a strain capable to perform
the tailoring steps that lead to the formation of Erythromycin A
from the aglycon 6-dEB, wich is synthesized by the modular PKS
6-dEB synthetase (DEBS). These reactions include the attachment
of two deoxysugar moieties, L-mycarose and D-desosamine, two
hydroxilations and a methylation of the mycarose residue. One of
the most widely used heterologous host for genetic manipulation
of actinomycetes PKSs is Streptomyces coelicolor. We decided to
genetically engineer the S coelicolor A3(2) strain transferring the
metabolic pathways involved in the conversion of 6-dEB to
Erythromycin A from Micromonospora megalomicea. This implied
the construction of two operons including the genes involved in
the biosynthetic pathways of the deoxysugars TDP-L-mycarose
and TDP-D-desosamine and their corresponding
glycosyltransferases, two hydroxylases and a methyltransferase.
This strain could perform the bioconversion of the macrolide 6-
dEB to the different erythromycin intermediates in batch cultures
fed with this polyketide.
BT-C3.
EVALUATION OF THE EFFECT OF N-ACETYL-
GLUCOSAMINE ON THE PRODUCTIVITY AND THE
GLYCOSYLATION PATTERN OF RHEPO
Didier, Caroline; Etcheverrigaray, Marina and Kratje, Ricardo.
Laboratorio de Cultivos Celulares, FBCB-UNL, Santa Fe,
Argentina. E-mail: [e-mail]
Recombinant human erythropoietin (rhEPO) is a therapeutic
protein that promotes red-blood cells maturation. Its biological
activity strongly depends on its antennarity and sialylation, being
N-acetyl-glucosamine (GlcNAc) an aminosugar contained in
carbohydrate chains of rhEPO. In order to optimise the production
process, a rhEPO producing clone of CHO cells was adapted to
grow in protein free medium (PFM), achieving a 50% increment
in cell concentration compared to the standard medium
(supplemented with 0.2% FCS), and a similar rhEPO specific
production rate. Different culture conditions were then assayed to
evaluate their effect on productivity and glycosylation pattern. In
particular, four cultures of this clone were initiated in PFM
supplemented either without or with 0.2 , 2 and 20 mM GlcNAc.
All cultures showed the same specific growth rate, but a decrease
in the maximum cell concentration was achieved while increasing
the aminosugar concentration (1.9 10
6
 and 4.4 10
5 
cell/ml for 0 and
20 mM, respectively). Increasing GlcNAc caused increments in
specific glucose-consumption rate, and specific lactate-,
ammonium- and rhEPO-production rates. Analysis of the molecule
performed by isoelectric focusing - Western blot and band
densitometry indicated that the addition of GlcNAc caused the
disappearance of the most acidic isoform and a decrease in the
proportion of the next most sialylated isoform. This effect was
probably caused by ammonium accumulation.
BT-C4.
UNCULTURED 
γ- PROTEOBACTERIA DOMINATE 16S
RDNA CLONE LIBRARIES FROM NONYLPHENOL
ETHOXYLATE-ENRICHED ACTIVATED SLUDGE
Figuerola, Eva
 1
; Itria, Raúl
 2
; Lozada, Mariana
1
; de Tullio, Luis
2
;
Erijman, Leonardo
1
.
1
INGEBI-CONICET; 
2
CIIA-INTI, Buenos Aires, Argentina. E-mail:
[e-mail]
Nonylphenol ethoxylates (NPEO) are widely used surfactants that
enter the environment primarily through sewage and wastewater
treatment plants, causing adverse effects on aquatic organisms.
We investigated the link between bacterial population and NPEO
biodegradation in engineered environments, using cultured-
independent methods. Four clone libraries were constructed with
amplified full-length insert 16S rDNA from reactors fed with
synthetic effluent, two of which received additionally NPEO. A
total of 187 clones were digested with restriction enzymes RsaI
and HhaI. Changes of relative diversities under different
 
treatment
regimens, i.e. presence or absence of NPEO in the feeding solution,
within otherwise identical environments, were taken as
representative of qualitative changes in the microbial community.
NPEO had a significant effect in the increase in relative abundance
of a few RFLP patterns compared to control libraries. The
sequences of several clones corresponding to the dominant
phylotype in libraries from reactors treated with NPEO (ca. 35%
of total clones in each replicate reactor) were assigned to uncultured
gamma-proteobacteria subclass, suggesting the involvement of this
group in NPEO degradation. Two new specific rRNA-targeted
oligonucleotide probes were designed from our five 16S rDNA
sequences and four other sequences retrieved from public databases
of closely related environmental clones. Real time PCR using the
specific probes demonstrated their applicability to monitoring the
abundance of these so far uncultured species in natural and
engineered environments.
BT-C5.
PLASMID DESIGN TO PRODUCE MEMBRANE-
PERMEANT RECOMBINANT PROTEINS
Lopez, Cecilia; Magadán, Javier; Mesa, Rosana and Mayorga Luis
Lab. Biol. Celular y Molecular. IHEM (CONICET) Facultad de
Ciencias Médicas. UNCuyo. E-mail: [e-mail]
Arginine-rich peptides can permeate inside cells carrying
covalently bond proteins. Therefore, these peptides are useful tools
to study protein function in cells that cannot be transfected. In
order to produce membrane-permeant recombinant proteins the
plasmid pGEX-2T-R was designed. The plasmid pGEX-2T was
cut on BamHI and EcoRI sites and an oligonucleotide encoding
the amino-acid sequence RRRQRRRKRRRQ was ligated. Then
E. coli BL21(DE3) pLysS cells were transformed and were induced
to produce GST-R (arginine-rich peptide covalently bond to GST).
Then permeability assays were performed in CHO cells and human
spermatozoa. The cells were incubated either with GST-R or GST
for three hours and washed. The presence of the protein was
determined by indirect immuno-fluorescence and visualised by
fluorescence microscopy. Further experiments were carried out in
human spermatozoa using either rhodamine-labeled GST-R or GST
and visualised by fluorescence microscopy and confocal
microscopy. GST-R permeated into both CHO cells and human
spermatozoa while GST was excluded. These results suggest that
pGEX-2T-R can be used to produce membrane-permeant
recombinant proteins
.

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