biodeep october 12-15, 2003

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”


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


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


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


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


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)


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


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


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


Biodeep Partners Culturesscreened

Positivecultures


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)


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


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



Positivecultures


0600

0200

C3>C10

Total 6 2

Preliminary results : Non induced (aerobes)

Preliminary results : Induced (aerobes).

Microbial screening : Esterase


Positivecultures


3(1)

5(1)

00

1100

C10C10

Total 8 21. Measured only using C10 CLIPS-O™ substrates



Positivecultures


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


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


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


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%


Constructions

promoter P1972 FL geneConstruction Activity

OK +

OK +pelB

1.

2.


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


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


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


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%


Potential SP

Construction Activity

OK +

In progress

In progress

Constructions

promoter P1600 FL gene

pelB


Biochemical characterization Optimum pH : 8-9

Other characteristics : in progress


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


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


Communications

Our industrial partners

Congress :MINATEC 2003 (Grenoble, France)BIO2003 (Washington, USA)INDUSTRIAL APPLICATIONS OF BIOCATALYSIS (Boston, USA)


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)


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


…Thanks for your attention.

www.proteus.fr

biodeep october 12-15, 2003

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