Transcript Screening of a metagenome library from an oil enrichment

WP6, WP7
Technical University
Braunschweig
German Research Centre for
Biotechnology, Braunschweig
Screening isolates for enzymatic activities
(WP6 and WP7)
Focus on enzymes from metagenomic
expression libraries (WP7)
Work objectives:
to explore the diversity of DHABs:
to isolate, hyperexpress and characterize novel enzymes
and other products
(DL27 – M34; DL29 - M34, DL30 – M26, DL 31 – M34)
Two approaches: analysis of expression libraries and
microbial isolates
Metagenomic expression library in lambda phage
DNA size-fractionated,
partially digested with Sau3A
Cosmid arms treated with
phosphatese
Ligation
Package in vitro
Library
of dozensselect
of thousands
phageresistans
particles
Transduce,
for antibiotics
with
kbp
inserts
and score
for0-12
white
phages
n X-Gal
Phage l expression system
The ZAP Express vector allows bouth eukaryotic
and prokaryotic expression and accomodates DNA
insert from 0 to 12 kb in length.
„Oil“ library = 1,8 x 106 phage particles. Average
insert size - 7.5 kbp
Clones in the ZAP Express vector
can be screend with either DNA
probes or antibody probes
Esterases
Cellulases and amylases
From phage library to enzyme
Excision
Screening of ca. 10000 phage clones
yields ca. 20 positives
Purification
100
Insert cloned into the ZAP Express
vector excised out of the phage in the form
of the Km-resistant pBK-CMV phagemid
vector
Expression
518.3
MALDI-TOF
80
60
569.2 637.2
598.1
40
826.0
20
673.4
775.3
711.3
500
Product,
enzymology
600
700
800
867.0
925.5
900
994.7
1035.4
1000
1100
Sequencing of
selected clones
Selected clones clustered
BIODIVERSITY FEATURES
Thirteen unique clones (pBK Oil2, pBK Oil7, pBK
Oil8, pBK O02, pBK O03, pBKO04, pBK O08,
pBKO09, pBK O10, pBK O11, pBKO12, pBKO14,
pBKO16, pBK O21, pBK O23) encoding esterases
have been cloned and sequenced, and their protein
products have been purified and characterized.
One gene (pBKOA3) encoding a amylase has been
detected and cloned and the sequence and
characterization is in progress
One gene (pBKOC1) encoding a endoglucanase
has been retrieved from phage library
Subcloned DNA fragments from positives
Plac
oil2
Plac
oil7
oil8
Plac
yafH (29 %)
1 kb
<45% similarity,
<30% identity
<40% similarity,
<30% identity
<45% similarity,
<30% identity
44 520 kDa
pI 10,88
32516 kDa
pI 10,25
32627 kDa
pI 9,26
pp-kinase (65 %)
molGC 58 %
Plac
esterase/deacetylase
Oil2
esterase
Conserved hypothetical
protein
October 03
Plac
Rhodanese
domain
bolA
putative para-nitrobenzyl/carboxyl esterase,
Ca. 500 aa, < 35 % seq. similarity,
AraC regulatory protein
Abo 80% similarity
O02
molGC 68 %
Rhodanese
domain protein
AraC
regulatory protein
Plac
COG0247, GlpC,
Fe-S oxidoreductase
Part of 980 aa,
50% simil.
bolA
Conserved
Hypothetical
Protein ca. 70 %
Cholesterol
oxidase pecursor
O04
molGC 59 %
COG0657, Aes,
Conserved hypoth.
Esterase/lipase Put. esterase,protein, proteobact.
ca. 130 aa ca 70 % a.a. sim
180 aa
Plac Ca.
29% similarity <30% smlr.
Consvd. membr
prot, 65 %
pfam02517, Abi, CAAX
amino terminal protease family
O08= Oil2
molGC 57 %
Plac
O09
molGC 56 %
2731-3550
GtPase
2029-1067
487-960
Cons‘d hyp. Protein (68%to PAO) Esterase (44% similar,
32 % ident)
Plac
COG0657, Aes, Esterase/lipase
[Lipid metabolism], <50 % similarity
Enoyl CoA
hydratase
O12
molGC 57 %
New:
Plac
O10
1072 to 104, 323 aa
Metallo-beta-lactamase
family protein, 65% sim
KT2440
1117-ca.280 aa (one read left)
enoyl-CoA hydratase/isomerase
family protein 89 % sim. Abo_141
70 % Caulobacter
molGC 56 %
October 03
204 to 1251-ca 350 aa (full length sequenced)
Putative acetyl esterase (lipase/hydrolase)
40 % sim. Bacillus
O11
New:
Plac
108 to 1526, 483 aa
Sulfate transporter/permease
family protein, Abo_1815, Abo_1817 80%sim
Arabidopsis-chloroplast- next (56%)
molGC 56 %
409-486 aa
Sulfate transporter/permease
family protein, Abo_1815, Abo_1817 80%sim
Arabidopsis-chloroplast- next (56%)
2414 to 296 aa (putative lipase/hydrolase
40 % sim. Bacillus
New:
O16
Low homology ORF ca 960 aa
October 03
molGC 44 %
Plac
Plac
Cons. hypothetical
Cons. hypothetical,
low homology
Putative membrane proteinHydroxyacyl
Low homology <30 %
dehydrogenase
O21
molGC 33 %
O23
New:
molGC 54 %
Plac
Glucosylhydrolase
family protein (junk sequence!)
-30 bp to the end of the fragment
Carboxylesterase
Ca 500 aa (one read left)
54% sim. Bacillus
Enzyme reaction products
TG
1,3-DG
1(3), 2-DG
MG
oil2 oil7 oil8
O.4
O.2 O.21 O.8 O.12 O.14
O.16
O.9
O
O
O
O
Tributyrin
O
Esterase in H2O:Acetonitrile
O
Enzyme purification:
i.e. Oil2, Oil7 and Oil8
Native gel
electrophoresis,
development with
a-naphtylbutyrate
Purification:
Cationic exchange on MonoS
Hydrophobic interaction (Phenylsuperose)
Gel filtration (Superose 12)
Specific activities with p-nitrophenol derivatives
6000
OIL 2
3000
2000
1000
µmol/min/µg protein
4000
2000
1000
5000
OIL 8
4000
3000
2000
1000
0
300
O.5
250
200
150
100
50
3000
2000
1000
1200
O.8
1000
800
600
400
200
0
0
300
250
O.9
200
150
100
50
0
µmol/min/µg protein
250
O.4
4000
0
1400
µmol/min/µg protein
µmol/min/µg protein
3000
6000
5000
OIL 7
4000
0
350
µmol/min/µg protein
5000
0
µmol/min/µg protein
µmol/min/µg protein
µmol/min/µg protein
5000
200
O.10
150
100
50
0
C2 C3 C4 C6 C8 C12C14C16
Carbon atoms
C2 C3 C4 C6 C8 C12C14C16
Carbon atoms
Specific activities with p-nitrophenol derivatives
160
3000
O.11
120
80
40
µmol/min/µg protein
µmol/min/µg protein
200
0
O.13
600
400
200
O.16
500
400
300
200
100
µmol/min/µg protein
µmol/min/µg protein
1500
1000
500
1200
O.14
1000
800
600
400
200
0
3500
0
3000
O.21
2500
2000
1500
1000
500
0
3000
O.23
4000
3000
2000
1000
0
µmol/min/µg protein
5000
µmol/min/µg protein
2000
1400
0
700
600
O.12
0
µmol/min/µg protein
µmol/min/µg protein
800
2500
2500
O.2
2000
1500
1000
500
0
C2 C3 C4 C6 C8 C12C14C16
Carbon atoms
C2 C3 C4 C6 C8 C12C14C16
Carbon atoms
Screening hydrolases using a pH indicator method:
Scheme:
CONDITIONS:
96 microtiter plate containing 5µ enzyme solution (20 mg/ml), and 100 µl of the following mixture:
420 µL of a 30 mM solution of the ester in acetonitrile,
470 µL acetonitrile
4-nitrophenol (6oooµL of a 0.9115 mM solution in 5.o mM BES, pH 7.2
E > 20 INDUSTRIAL POTENTIAL (Enantiomeric ratio)
e.e. > 70 INDUSTRIAL POTENTIAL (Enantiomeric excess)
ENANTIOMERIC RESOLUTION OF
1-PHENYLETHANOL
BY TRANSESTERIFICATION
WITH VINYL ACETATE
+
1 M 1-phenylethanol
1 M Vinylacetate
2 mL Iso-octane
20 mg E. coli esterase clones
Screening of hydrolytic activity: Conisma strains (the others to come)
Substrate: a-naphtylacetate, butyrate, laurate, palmitate, phosphate, glucoside, galactoside and Fast Blue RR
strain
identification
3A
7A
8A
14A
17A
18A
Alteromonas marina
Alteromonas macleodii
Pseudoalteromonas sp.
no identification
Alteromonas macleodii
Slope str., DIIII I c
98
99
92
1B
3B
4B
5B bis
6B
12B
17B
19B
Marinobacter hydrocarbonoclasticus
97
100
99
100
no identification
Bacillus licheniformis
Rhodospirilaceae bact
no identification
98
97
2D
4D
5D
Alteromonas macleodii
Alteromonas macleodii
Alteromonas macleodii
99
99
99
Marinobacter hydrocarbonoclasticus
Idiomarina baltica
Marinobacter hydrocarbonoclasticus
homology,% basin protease phosphatase esterase lipase glucosidase
99
97
A
A
A
A
A
A
B
B
B
B
B
B
B
B
D
D
D
3
3
3
3
1
3
2
1
3
3
2
2
3
3
2
2
3
3
3
2
2
3
3
3
3
3
3
3
ABSTRACT: enzyme properties
Here we describe a comparative analysis of ten novel esterases from a large library created from deep-sea hypersaline
anoxic basins of the Eastern Mediterranean.
A genomic DNA library of deep-sea hypersaline anoxic basins of the Eastern Mediterranean was established into
Escherichia coli, and screened for esterase activity by using a-naphtyl butyrate and Fast Blue RR. Eleven genes
(pBKOil2, pBKOil7, pBKOil8, pBKO.2, pBKO.4, pBKO.9, pBKO.12, pBKO.14, pBKO.16, pBKO.21) encoding eleven
esterases has been cloned and sequenced, and their protein products have been purified and characterized.
Sequence analysis revealed less than 40% identity to sequences of known esterases and lipases.
They preferred short chain water-soluble esters as substrates. The optimal pH and temperature of the purified enzymes
were in the range 8.0-9.5 and 40-60°C, respectively.
Purified esterases exhibited hydrolytic activity without addition of any metal ions. However they were optimally active
in the presence of different concentrations of Na+ or K+. Esterases from pBKO.4, pBKO.8, pBKO.12, pBKO13 were 2
times more active at concentration in the range from 25 to 75 mM. In contrast, esterases from pBKO.9, pBKO.14 and
pBKO.2 were inhibited above 25 mM. Some other esterases, i. e. from pBKO.16, pBKO.21, pBKOil2, pBKOil7 and
pBKOil8 showed a high activation at concentrations of Na+ or K+ 1.5-2.0, 1.0-1.5, 2.0-4.0, 3.5, 3.5, 0.8 and 4.0 M, which is
tipical for halophilic enzymes.
The positive clones, pBKO.2, pBKO.4, pBKO.21, were found to have 1(3) positional specificity against triacylglycerols.
Interestingly, pBKOil8 and pBKO.4 showed 2 positional specificity against triacylglycerols. The hydrolytic and synthetic
activities and estimated enantioselectivities towards chiral ester library was assayed. We found 4 lactone hydrolases
(pBKOil8, pBKO.2, pBKO.9 andpBKO.21), 6 highly specific for aromatic chiral compounds (pBKOil7, pBKO.2, pBKO.14
and pBKO.16). Esterases derived from pBKO.14, pBKO.16, pBKO.4, pBKO.21 and pBKOil8, were potentially used for
industrial resolution of chiral carboxylic acid with a stereocenter a to carbonyl.
pBKO.2, pBKO.14 pBKO.9 and pBKOil8 were the most stable against organic solvent and 1.5-fold times more active in
the presence of detergents. Taking the catalytic efficiency and the enantiomeric excess and ratio, we concluded that the
esterases produced by pBKOil8, pBKO.2, pBKO.4, pBKO.23 and pBKO.21 exhibited the best properties of all tested
esterases for application in the biotechnological resolution of chiral esters. We found that most of the esteraseencoding genes discovered are entirely new and the targeted activity and enantioselectivities test for the esterase
product revealed a unique phenotype for some of the new biocatalysts.
WHAT NEXT???
Check the esterase in unknown reactions.
Characterization of amylase: halophilic???
progress
Characterization of endoglucanase
Check for new enzymes: DNase, proteases,
oxidoreductases, alcohol dehydrogenases and
dehydratases, laccases, etc…
Crystallization
Surfactants – a big struggle…
Conclusions and outlook
Collection of hydrolytic enzymes from expression libraries obtained
after oil enrichment, have been characterised
they exhibit novel structures (low homology to the homologs), have
a good potential for industrial applications and “tell the stories” about the
environment and organisms they were derived from
Screening of “biosurfactant” producing isolates - finished
Nothing incredibly new or interesting obtained
Expression libraries are the best tool to exploit the diversity:
Work with separate isolates is less productive and time-consuming
Progress estimates:
DL29- M34 20 % “Structures of novel surfactants etc – (screening stage)”
DL30-M26 – 100 % “Clones, hyperexpression clones etc.”
DL31-M34 – 100 % * “ Data sets of activities of obtained compounds”
* all selected items characterized
Publications
Ready to publish after finishing sequences (2-3 months)
Env Microbiol Special Issue is planned
1. Env-specificity: bio(geo)chemistry
2. gradient Daniele etc
3. Misha etc
4. Henk etc
5. Marseille etc
6. Tassos etc
7. biotech applications (Peter and I), Proteus