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Biodeep October 12-15, 2003. “Nature has more imagination than our dreams”. Contents. Strains and clones transferred Microbial screening Proteases Esterases Xylanases Molecular screening Analysis of the cloned genomic fragments Construction/expression - PowerPoint PPT Presentation
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Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 1/30
Biodeep
October 12-15, 2003
“Nature has more imagination
than our dreams”
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 2/30
Contents Strains and clones transferred Microbial screening
Proteases Esterases Xylanases
Molecular screening Analysis of the cloned genomic fragments Construction/expression Biochemical characterization
Biodeep Database Communications Conclusion
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 3/30
Strains transferred
Milan University (Dr. T. Brusa)
Culturesreceived
Strainsstudied(1)
Strainscultures(2)
1st Batch 94 94 892nd Batch(anaerobes) 18 18 18
Total 112 112 107
1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefore stored in collection) and can be used for future work
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 4/30
Strains transferred Essex University (Dr. A. Sass)
Culturesreceived
Strainsstudied(1)
Strainscultures(2)
1st Batch 117 117(3) 117
2nd Batch 90 90 90
3rd Batch 40 39 35
Total 247 246 242
1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefore stored in collection) and can be used for future work3. The 19 strains of the 1st batch which could not be cultured in our lab have been sent a second time. The 40 strains of batch 3 are anaerobes (stricts or facultatives).4 enrichment cultures currently used
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 5/30
Strains transferred
LLM (Dr. F. Morel)Culturesreceived
Strainsstudied(1)
Strainscultures(2)
1st Batch 20 20 20
2nd Batch 21 16 16
Total 41 36 36
1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 6/30
Strains transferred Messina University (Dr. G. D’auria)
Culturesreceived
Strainsstudied(1)
Strainscultures(2)
1st Batch 59(3) 37 18
2nd Batch 42(3) 34(4) 4
Total 101(3) 71 22
1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab (and therefor stored in collection) and can be used for future work3. See next slide4. All the strains excepted the ones growing in P10 medium (petrol based medium)
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 7/30
Strains transferred Cultures from Univ. Messina
Enrichment cultures and pure strains No synthetic media defined (from the water point of view) We tried to grow the stains in a defined medium The growth conditions defined for the 22 strains cultured in our lab are availableabout 70 pure strains growing in ONR medium will be transfer soon
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 8/30
Strains transferred
Biodeep Partners Culturesreceived
Strainsstudied (1)
Strainscultured (2)
Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)
11224710141
1122467136
1072422236
Total 501 465 407
1. Strains studied means that we tried at least once to culture them2. Strains cultured means strains that have been cultured in our lab and can be used for future work
Review
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 9/30
Clones transferred clones from TUB (Dr. P. Golyshin)
11 genomic fragments encoding 11 different esterases Corresponding (partial or FL) sequences have been provided with some genomic fragments Subcloning of oil8 have been done in fusion with pelB leader for periplasmic expression. Expression successful
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 10/30
Biodeep Partners Culturesscreened
Positivecultures
Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)
2184012
183903
Total 117 60
Extracellular proteases
using skim milk (0.2%)
screening assay Review (aerobes)
Microbial screening : ProteaseP 1600
After 3 days at 25°C
1. Planned with the pure strains to be transferred
(1)
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 11/30
Taxonomic position Culturesscreened
positivecultures
Actinobacterium sp.Alcalinivorax sp.Alpha proteobacteria sp.Alteromonas sp (including P 1972)Bacillus sp. (including P 1600)Exiguobacterium sp.Halobacillus sp.Halomonas sp.Idiomarina sp.LactobacillalesMarinobacter sp.Pseudoalteromonas sp.Pseudomonas sp.Thiobacillus sp.Salmonella sp.Sporosarcina sp.Thalassospira sp.Vibrio sp.Virgibacillus sp.
21215242711311935111133
100121924130021001133
Unidentified strains 12 7Total 117 60
Taxonomic position according Biodeep partnersMicrobial screening : Protease
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 12/30
Microbial screening : Esterase Esterases Principle of the
CLIPS-O™ substratesR = CH3 C2H5 C9H19 C15H31
(1)(2)(3)(4)
O
OH
O
NO2
R
O
O
OH
HO
NO2
BSA
O
ONO2
H
H
O
H
HO
NO2
beta-elimination(BSA)
chemical oxidation(NaIO4)
Measurable at 414 nm
esterase / lipase
R = C2H5
C9 H19
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 13/30
Biodeep Partners Culturesscreened
Positivecultures
Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)
0600
0200
C3>C10
Total 6 2
Preliminary results : Non induced (aerobes)
Preliminary results : Induced (aerobes).
Microbial screening : Esterase
Biodeep Partners Culturesscreened
Positivecultures
Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)
3(1)
5(1)
00
1100
C10C10
Total 8 21. Measured only using C10 CLIPS-O™ substrates
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 14/30
Biodeep Partners Culturesscreened
Positivecultures
Univ. Milan (Dr. Brusa)Univ. Essex (Dr. Sass)Univ. Messina (Dr. D’auria)LMM Marseille (Dr. Morel)
41100
0200
Total 15 2
Xylanase using “xylan blue” (0.05%) screening assay
(xylan was used as carbon and energy sources) Preliminary results (aerobes)
Microbial screening : Xylanase
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 15/30
Taxonomic position according Biodeep partners
Microbial screening : Xylanase
Taxonomic position Culturesscreened
positivecultures
Alteromonas spAlcalinovorax sp.Bacillus sp.Halomonas sp.Pseudoalteromonas. sp.Sporosarcina sp.Vibrio sp.
1151211
0000010
Unidentified strains 3 1Total 15 2
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 16/30
Molecular screening Proteases from strain P 1972 (Alteromonas sp.)
and strain P 1600 (Bacillus sp.)
Genomic library construction : partial restriction
using Sau 3A I
Inserts size ranging from 2 to 5 kb
Quality control : less than 10% of empty clones
Screening performed
Positive hits identified and sequenced
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 17/30
Analysis of the genomic fragment (PP 1972)
JSC-PP1972-7d1 rev2984 bp
ORF Protéase P 1972
Cterm region
Pst I (19) Bgl II (2971)
Xba I (2974)Nde I (1541)Sac I (360)
Xho I (2807)
Msc I (1339)
Hin dIII (975)
Hin dIII (2356)Eco RV (5) Eco RV (869)
2989 bp
Identity/Similarity with Subtilisin (P00780) in amino acids :- FL gene : 29%-48%- Nterm region of the gene : 34%-54%
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 18/30
Constructions
promoter P1972 FL geneConstruction Activity
OK +
OK +pelB
1.
2.
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 19/30
Subcellular localization
0
20
40
60
80
100
Rel
ativ
e ac
tivi
ty
(%)
Growth medium Cytoplasmiqueand
Periplasmique
0
20
40
60
80
100
Rel
ativ
e ac
tivi
ty
(%)
Growth medium Cytoplasmiqueand
Periplasmique
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 20/30
Kinetic of expressionInduction at 28°C
0
0,5
1
1,5
2
2,5
3
0 10 20 30 40 50 60 70 80
Time (h)
Gro
wth
(O
D6
00
nm
)
0
20
40
60
80
100
120
Relative A
ctivity in th
e cultu
re m
ediu
m(%
)
Growth Relative activity (%)
Induction
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 21/30
Biochemical characterization
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Temperature (°C)
Re
lati
ve
ac
itiv
ity
(%
)
0
20
40
60
80
100
120
0 0,5 1 1,5 2 2,5
Incubation (h)
Rel
ativ
e ac
tivity
(%)
Thermostability at 40°C Thermostability at 30°C
0
20
40
60
80
100
120
7 8 9 10 11
pH
Rel
ativ
e A
ctiv
ity
(%)
0
20
40
60
80
100
120
0 4 8 12 16 20 24
Incubation time
Rel
ativ
e ac
tivi
ty (
%)
Thermostability at 30°C
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 22/30
JSC-PP16004212 bp
ORF protease P 1600
Potentiel Signal Peptide
Bam HI (452)
Bgl II (1161)
Eco RV (4172)
Sac II (10) Xho I (4197)
Nde I (1941) Nde I (4071)
Pst I (3506)
Pst I (4162)
Xba I (555) Xba I (1526)
Hin dIII (1491)
Hin dIII (1863)
Hin dIII (3549)Eco RI (2061)
Eco RI (3228)
Eco RI (3762)
Eco RI (4164)
Analysis of the genomic fragment (PP 1600)
4212 bp
Identity/Similarity with Intracellular Alkaline Protease (P29140)in amino acids :
- FL gene : 56%-75%
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 23/30
Potential SP
Construction Activity
OK +
In progress
In progress
Constructions
promoter P1600 FL gene
pelB
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 24/30
Biochemical characterization Optimum pH : 8-9
Other characteristics : in progress
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 25/30
Clones collection
Biodeeppartners
Genomic fragments Subcloned ORF Related activity Characterization ofenzymatic activities
TubTubTubTubTubTubTubTubTubTubTubProtéusProtéus
Oil2Oil7Oil8O02O04O08O09O12O14O16O21
PP 1600PP 1972
NoNo
Yes (pelB)NoNoNoNoNoNoNoNo
Yes (several)Yes (several)
EsteraseEsteraseEsteraseEsteraseEsteraseEsteraseEsteraseEsteraseEsteraseEsteraseEsteraseProteaseProtease
Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)Yes (PG)
Yes (in progress)Yes
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 26/30
Biodeep database
Protéus
Design of the data sheet model Data related to 347 strains transferred in July to Univ. of Milano
University of Milano
Design of the database
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 27/30
Communications
Our industrial partners
Congress :MINATEC 2003 (Grenoble, France)BIO2003 (Washington, USA)INDUSTRIAL APPLICATIONS OF BIOCATALYSIS (Boston, USA)
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 28/30
Conclusions and Outlook
Current status of the Biodeep microbial collection :
407 strains
Current status of the Biodeep clones collection :
13 cloned genomic fragments containing
enzymatic activities (3 ORF subcloned)
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 29/30
Timetable of future activities
Activities AvailableMicrobial database
for partnersData
availableCulture collection
(1st cruise and 2nd cruise)407 strains.
Complete end year 2003First screening
for productsResults available
In progressCloned collection(and expression)
13 dif. clonesIn progress
Confidential NDA 01/393/191 - 01/393/222 Meeting of October 12-15, 2003 - slide 30/30
…Thanks for your attention.
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