Download Luiziana Ferreira da Silva Lab of Bioproducts Department of Microbiology

Luiziana Ferreira da Silva
Lab of Bioproducts
Department of Microbiology
Institute of Biomedical Sciences
University of São Paulo - Brazil
Microbial Genetics/Biotechnology
– Microbial anticancer compounds
Nitrogen Biological Fixation in
sugarcane
Bacterial Metabolism/Bacterial products
– biopolymers, biosurfactants
Bacterial Metabolism/Biotechnology –
bacterial biopolymers
Microbial ecology, Plant-microorganism
interactions/Biotechnology
Harmfull effects
Human
Animals
Agriculture
Environment
Benefits
Medicine
Pharmaceutical industry
Food Industry
Agriculture
Environment
Roles of microbiomes
Studies in humans
Skin protection
Obesity-associated gut microbiome
Brain development & behavior
Faecal bacteriotherapy in the
treatment of recurrent C. dificile
diarrhea.
Roles to plant and animals
A number of new surveys are helping scientists understand the
many ecosystems our bodies offer to microbes and their
interactions.
A relevant part of our society is still unaware
about the benefits and the contribution to our
lives that were achieved because of the action
of microorganisms
Country extension
Various climates
Different environments
Different crops cultivated
Variable and heterogeneous natural microbiota
New species discovered
New genes
Generation of bioproducts
Platforms for alternative products or alternative
routes
Nitrogen fixing bacteria
Antitumoral agents
Endophytes
Biopolymers
1986 isolated from Brazilian Savannah (cerrado) a
soil showing high acidity, low levels of N and organic matter –
Barbosa et al., 1986, Barbosa & Carvalhal, 1988
Physiologic characteristics related to the environment were studied in
this bacterium:
• Nitrogen fixing ability under adverse conditions: low pH and under
high concentrations of toxic compounds
• Role of exopolysaccharide in protecting the nitrogenase from oxygen
deleterious effects
• Stimulation of other bacteria in N-free medium
• Liberation of aminoacids in N-free culture medium
• Cyanophicin like intracelular reserves
Dr.Heloiza R Barbosa
E. coli pure &
associate cultures
B. derxii pure &
associate cultures
Stimulating both hetero
and autothrophs, usually
present in soil
Biotechnological
applications
Purified granules can be chemically
converted to a derivative with
reduced arginine content or to
completely biodegradable
poly(aspartic acid) , which can be
used as a substitute for
nonbiodegradable polyacrylates
with many technical and medical
applications
Nitrogenated reserve
granules
Cyanophycin-like?
Under study
Gonçalves de Lima et al. 1969
Retamycin
Production of antitumoral agents
Polyketydes (PKS)
Anthracyclines are
active against
• breast cancer,
• lymphomas,
• acute leukemias,
• neuroblastomas,
• bone and soft-tissue
sarcomas
Dr.Gabriel Padilla
Studies on the mechanism of action
Chemical structures of cosmomycins
produced by Streptomyces olindensis
Garrido LM1, Lombó F, Baig I, Nur-E-Alam M, Furlan RL, Borda
CC, Braña A, Méndez C, Salas JA, Rohr J, Padilla G.
Genetic organization of the sequenced DNA region and constructs used for the
generation of the different mutants. The black triangle indicates the apramycin
resistance cassette. Restriction sites indicated with an asterisk are not unique
sites. SaSau3AI; NcNcoI; BaBamHI; NrNruI
Chemical structures of
compounds isolated from
Streptomyces olindensis
ΔcosK (a) and S. olindensis
ΔcosG (b)
Proposed
pathway for
glycosylation
events in
cosmomycin D
biosynthesis
Dr.Welington L. Araujo
Characterization of mutants
a-1
b-1
c-1
f
a-2
b-2
c-2
g
a-3
d
e
h
Random mutagenesis to identify genes related to biosynthetic pathway
PKSi 1
KS
AT
PKSi 2
KS
AT
PKSi 3
KS
AT
PKSi 4
KS
AT
PKSi 5
KS
D
H
D
H
M
M
DH
K
R
(ER)
D
H
M
D
H
P
P
KR
2515 a.a.
P
P
2585 a.a.
P
P
KR
2576 a.a.
P
P
KR
1777 a.a.
AT
DH
ER
P
P
KR
2262 a.a.
PKSi 7
KS
P
P
AT
P
P
TE
2145 a.a.
PKSi 8
KS
AT
DH
M
ER
P
P
KR
K
S
A
T
K
R
D
H
E
R
T
E
Ketosynthase domain
Acyl transferase domain
Ketoreductase domain
Dehidrathese domain
Enoil reductase domain
Thioesterase domain
2488 a.a.
PKSi 9
KS
P
P
AT
M
R
2576 a.a.
PKSi 10
KS
P
P
AT
TE
M
Methyltransferase domain
P
P
Acyl Carrier protrein domain
H
Hydrolase domain
P
Acyl CoA reductase domain
R
Ketosynthase domain
2069 a.a.
PKSi 11
KS
P
P
AT
TE
2037 a.a.
PKSi 12
KS
P
P
AT
P
P
M
H
2637 a.a.
K
PKSIII
S
P
406 a.a.
PKS-NRPS (PKSi6)
KS
AT
D
H
M
KR
P
P
C
A
P
P
R
3981 a.a.
13 hypothetical PKS clusters identified in the E. nigrum P16 genome. Aminoacid numbers and domain positions are in
scale
Epicolactone
New metabolite from Epicoccum nigrum
Associated to microbial control
Two PKS regulators were identified
Identification of the PKS gene cluster
Dr. J. Gregório C Gomez
Dr. Luiziana F. Silva
PHA are accumulated as intracelular
granules by bacteria
C4 & C5
Short-chain length monomers
PHA scl
Naturally PHA-accumulating bacteria produce either
PHAscl or PHAmcl in large amounts
C6 & C12
Medium-chain length
monomers PHA mcl
Monomer composition is responsible
for PHA properties and applications
730
Features influencing PHA monomer
composition
Isolation & evaluation of soil bacteria on the ability to produce PHB
from Sucrose, Glucose and Fructose
Table 2. Production of P(3HB-co-3HV) from glucose plus propionic acid.
PHA
Residual
CDWc
3HB
mol%
3HV
mol%
Y3HV/Prpb
0,0
71,4
96,1
3,9
0,13
0,95
101,8
14,6
55,0
45,0
0,07
P.cepacia DSM 50181
3,35
1,9
38,4
97,3
2,7
0,04
IPT-040
3,77
1,7
32,3
97,1
2,9
0,05
IPT-044
3,92
1,7
51,1
97,1
2,9
0,07
IPT-045
3,73
0,0
49,4
96,2
3,8
0,08
IPT-048
2,97
0,0
44,3
96,2
3,8
0,06
IPT-055
4,27
72,2
1,5
100,0
0,0
0,00
IPT-056
3,60
31,3
30,9
98,5
1,5
0,02
IPT-076
5,06
1,9
56,8
97,1
2,9
0,10
IPT-083
4,89
5,2
56,8
96.9
3,1
0,10
IPT-086a
2,06
75,
39,0
89,9
10,1
0,09
IPT-098
5,90
0,0
17,7
94,7
5,3
0,07
IPT-101
2,98
41,8
32,3
95,4
4,6
0,05
CDWc
(g/L)
Carbohydrates
(%)
A. eutrophus DSM 54
3,92
A. latus DSM 1123
Strains
%
Gomez et al., 1996
P3HB-co-3HV from sucrose and propionic acid
Phenotype/
strategy
Strain
3HB 3HV Y3HV/prp*
g/g
mol% mol%
B. sacchari wild type
93.8
6.2
0.10
189
prp UV mutant
43.6
56.4
0.90
189
Sucrose:propionate
feeding rates
92.0
18.0
1.27
Improvement to
incorporate 3HV units
*Y3HV/prp = 3HV yield from propionic acid
Maximum theoretical yield = 1.35 g/g
3HB
C4-monomer PHA
3HV – C5
4kbp
Inactivation on the propionate
A
EcoRI
2.9 kbp
EcoRI
2 kbp
9 kbp
EcoRI
EcoRI
SalI SalI
EcoRI
catabolic pathway
2MCC = 2 methyl citrate cycle
B
ORF1
ORF2 ORF3
ORF4
ORF6
ORF5
ORF7 ORF8
52bp
ORF9
3HV content is dependent on propionic acid concentration
2MCC is more operative
at low prp concentrations.
A second prp catabolic
pathway do exist?
acnM/prpC deletion
Control on 3HV content
Sucrose:propionic ratio in the feeding media
Sucrose
Glucose
Xylose
Glycerol
Fatty acids
Plant oils
Soybean molasses
Others...
Pseudomonas sp.
B. sacchari
E. coli
Platforms for...
PHA
Rhamonolipids
1,3-Propanediol
Others...
Improving
product
yields
biotechnological processes:
in
•Knockout or gene overexpression is no
guarantee of success
•A better knowledge of the metabolism
is needed to engineer metabolism and
improve biotechnological processes.
Vallino & Stephanopoulos, 1992
Metabolic engineering
advanced analytical tools to identify appropriate targets for genetic modifications
mathematical models to perform in silico design of optimized cell factories.
Nielsen & Jewett, 2008 FEMS Yeast Res.
Fluxes analysis is
representative of
the phenotype of
biotechnological
interest
.C (negligible)
dC/dt = 0 (steady state)
Metabolic pathway
analysis
PHAmcl
Fluxes distribution for PHA production
by Pseudomonas sp. from glucose.
Optimal fluxes distribution for PHA production
by Pseudomonas sp. from glucose.
PHA production by wild type B. sacchari
Fluxes
under
the
theoretical
maximum global PHA yield
0,49
0,49
0,48
0,01
0,01
1,24
1.16
1,65
1,73
Glucose
Xylose
Glycerol
Fatty acids
Plant oils
Soybean
molasses
Pseudomonas sp.
B. sacchari
E. coli
Platforms for...
PHA
Rhamonolipids
1,3-Propanediol
Others...
Brazilian biodiversity as new
platforms
J.Gregório C. Gomez
Luiziana F. Silva
Marilda Keico Taciro
Karel Olavarria Gamez
Rogério S. Gomes
Johana K. Bocanegra-Rodriguez
Thatiane T. Mendonça
Gabriela C. Lozano
Linda P. Guaman Bautista
Liege A. Kawai
Lucas Garbini Cespedes
Diana Carolina Tusso Pizon
Bernardo Ferreira Camilo
Kelli Lopes Rodrigues
Cesar W. Guzman Moreno
Rafael Nahat
Carlos
Thandara Garcia Ravelli
Juliano Cherix
Edmar Ramos de Oliveira Filho
Ruben Sanchez (UENF)
Aline Carolina C. Lemos
Alexandre A. Alves
Aelson L. Santos
Amanda B. Flora
Karen L. Almeida
Odalys Rodriguez Gamez
Arelis Abalos Rodriguez
Jhoanne Hansen
Galo A. C. Le Roux - EPUSP
Carlos A. M. Riascos
Paulo Alexandrino
André Fujita IME USP
Juliana Cardinali Rezende
Andreas K. Gombert (UNICAMP)
Walter M. van Gulik
Aljoscha Wahl
Reza Maleki Seifar
J. J. (Sef) Heijnen
Kirsten Steinbusch – Waste2Chemical
Niels van Stralen – Waste2Chemical
NWO
MES-Cuba
TUDelft