the potential of marine/extremophilic microbes to explore novel · 2018. 8. 2. · atl11 ktl4 ktl7...

김상진김상진김상진김상진 Marine Biotechnology Research Centre

The potential of marine/extremophilic microbes to explore novel

biocatalysts using genomic approach

Sang-Jin Kim

KORDI

Global Forum On Biotechnology : Marine Biotechnology

Session 1: Productivity and Sustainability of the Oceanon 30 May, 2012, Vancouver, Canada


Contents

1. Introduction: Feature of marine environment, Status of arts of Marine products, Development of genome sequencing tech.

2. Diversity of epoxide hydrolases identified by homology-driven screening approach

3. DNA polymerase mined from whole genome sequence of an hyperthermophile

4. Diversity of Lipase/Esterase explored from marine metagenome libraries

5. Conclusion and perspectives


Features of marine environment

• Salinity: 3.5% Halophiles

• Temp.: -2 ~ 400°C

Psychrophiles and Hyperthermophiles

• Average depth: 3,800 m Piezophilies

• Photic zone: 0~200 m Oligotrophes


Cold-active, thermostable or any other biocatalysts representing unique characteristics could be preferentially obtained from the microorganisms or environmental DNA which were retrieved from the extreme marine habitats such as ocean trenches, deep-seas, polar seas, cold seeps, hydrothermal vents, etc (Bull et al., 2000, Microbiol. Mol. Biol. Rev. 64:573-606 ).


Products Application Source

Protein, Peptide & Amino acids

Vent TM DNA polymaerase Polymerase chain reaction (PCR)Deep-sea hydrothermalvent archaea

Green Fluorescent Protein Reporter gene Aequora victoria (jellyfish)

Aequorin Bioluminescent calcium indicator Aequora victoria (jellyfish)

Ziconotide (Prialt) Analgesic Conus magnus (Mollusc)

Hormones, cyclic peptidesAntioxidant, immunostimulants nutraceutical products

Fish hydrolysates

Kainic acid Anthelmintic insecticide Red algae

Saccharides

Carragreenans

Agars

Alginates

Cosmetrics, thickener, Pharmacy, mucoprotector,

Anti-coagulant, Antiviral

Red Algae

Chondroitin sulfateCosmetics, tissue replacement, anticoagulant

Fish

ChitosanCosmetics, colloids Pharmacy, icroencapsulation

Crustacean shells, fungi

Products derived from marine organisms


Fatty acids and miscellaneous

Long chain PUFA

(AA, EPA, DHA)Microalgae, seaweed, fish

Prevention of heart disease, mental development is premature children; antimoural; lipid metabolism

Formulaid Marine microalgaeFatty acids used as additive in infant formula nutritional supplement

ResiliencePseudompterogorgia elisabethae (Caribbean gorgonia)

Additive in skin creams

Phycoerythrin Red algaeConjugated antibodies used in ELISAs and flow cytometry

Manoalide Luffarella variabilis (sponge) Phospholipase- A inhibition

Cephalosporins Marine fungi Antibiotic

Spongoadenosine sponge Antiviral Herpes

Cytarabine (Ara-C) sponge Antitumoral (cytostatic)


2003 Human Genome Project 13 years 14X $2.7B

2007 Craig Venter 4 years 7.5X $100M

2008 James Watson 2 years 7.4X $2 M

2009 Sung-Jin Kim 6 months 29X $0.17M

2009 Every genome 30X $48,000

2013 ??? 30X $1,000

Development of Genome Sequencing


Genome Project(2012)

Total 17874 project

Archea

107 (13%)

Metagenome

72 (9%)

Eukarya

29 (3%)

Bacteria

628 (75%)

836 (5%) Marine genome project

836 Marine Genome Project

Complete12 (41%)Incomplete

17 (59%)Complete26 (36%)

Incomplete46 (64%)

Complete88 (82%)

Incomplete19 (18%)

Complete302 (48%)

Incomplete326 (52%)


Group 1

Group 2

Group 3

1a

1b

2a2b

2c2d

2e

2f

3a

3b

3c

Symbols :Blue line is isolated from marine sourceRed circle : Enzymes identified in this studyGreen circle: Enzymes with known EHase activity

Gene containing GXSXG, DXG, HGXP motif of Epoxide hydrolase

Phylogenetic tree of putative Epoxide Hydrolases


Roseovarius sp. HTCC2601

Oceanicaulis alexandrii HTCC2633

Janibacter sp. HTCC2649

Rhodobacterials sp. HTCC2654

Erythrobacter litoralis HTCC2594

Oceanicola batsensis HTCC2597

HTCC2143

Sphingopyxis alaskensis

Novosphingobium aromaticivorans

Oceanospirillum sp. MED92

Marinomonas sp. MED121

Roseobacter sp. MED193

Oceanobacter sp. RED65

Leeuwenhoekiella blandensis MED217

Vibrio sp. MED222

Reinekea sp. MED297

Limnobacter sp. MED105

Acquisition of genome sequenced marine microorganisms (15 st. from collaborator, 2 st. from ATCC )


M NEH

NEH

SEH

SEH

M REH

REH

M

EEH2EEH3EEH1

M EEH2,3

kDa

M MH M 1H M MH M 121 193

MH 1H 2H

MED121MED193

11 putative Epoxide hydrolase genes were expressed


Enzyme

Hydrolysis rate

(x 10-2) mg/min

SO GPE EX EB ECH TSO

(S) (R) (S) (R) (S) (R) (S) (R) (S) (R) (S) (R)

EEH1 9.00 28.00 20.00 11.00 8.00 7.00 15.00 15.00 1.05 7.79 NA NA

EEH2 0.06 0.05 0.07 0.06 0.07 0.07 0.08 0.08 0.23 0.23 ND ND

EEH3 0.14 0.10 0.10 0.10 0.09 0.17 0.11 0.08 0.28 0.26 ND ND

SEH 1.16 1.12 7.15 7.59 1.90 1.70 0.09 0.06 9.10 9.24 ND ND

NEH 4.13 6.22 15.34 12.26 13.35 13.82 8.77 12.11 6.68 10.95 NA NA

REH 1.15 7.29 32.70 1.39 2.86 6.02 0.86 0.60 2.71 2.37 0.01 0.07

MH 0.29 0.34 0.13 0.12 0.43 0.49 ND ND 0.64 0.64 ND ND

1H 0.45 0.85 0.67 0.32 0.65 2.31 ND ND 0.88 0.75 ND ND

2H 0.24 0.22 0.23 0.24 0.45 0.40 ND ND 0.52 0.52 ND ND

MED121 0.06 0.06 ND ND ND ND ND ND ND ND ND ND

MED193 0.08 0.07 ND ND ND ND ND ND ND ND ND ND

•ND: not determined, •NA: no activity (activity too low for determination)

Enantioselective activity of Ehases toward various epoxide substrates

TSOSO GPE ECHEX

CH3

EB

CH3


Epoxides Time (min) ee (%) c E Yield (%) Config.

5 mM SO 3 99.99 0.525 192.8 47.5 S

5 mM 3CSO 5 99.99 0.583 55.7 41.7 S

5 mM 4CSO 1 99.99 0.522 226.5 47.8 S

5 mM 4NSO 15 99.99 0.612 39.8 38.8 S

* The extent of conversion (c) [c={1−(ERs+ESs/ERso+ESso)}], where the initial epoxide of (R) and (S) was denoted as Eso, and theremaining epoxide of (R) and (S) was as Es* The enantiomeric ratio (E) is derived from the extent of conversion (c) and the enantiomeric excess of the remaining enantiomer of thesubstrate (ees) [E= In{(1−c) (1−ees)}/In{(1−c) (1+ees)}]

1, styrene oxide (SO)2, ortho-chlorostyrene oxide (2CSO)3, meta-chlorostyrene oxide (3CSO)4, para-chlorostyrene oxide (4CSO)

REH (20 ug) + SO 5 mM

Time (min)

0 2 4 6 8 10

Con

c. (

mM

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

En

anti

omer

ic e

xces

s (e

e, %

)

0

20

40

60

80

100

(R)-Styrene oxide(S)-Styrene oxide

ee (%)

diabetes careobesity care,

b3-adrenergic receptor agonistneuroprotective property

REH toward various epoxide substrates


Microorganism Name of polymerase Reference

Thermus aquaticus Taq polymerase Chien et al., (1976)

T. litoralis Vent polymerase Cariello et al., 1991

Mattila et al., 1991)

P. furiosus Pfu polymerase Lundberg et al., (1991)

P. woesi Pwo polymerase Frey and Suppmann, (1995)

Pyrococcus strain GB-D Deep Vent polymerase Perler et al., (1996)

Thermococcus sp. strain 9N-7

polymerase Southworth et al., 1996)

Thermococcus kodakaraensis KOD1

KOD polymerase Takagi et al., (1997)

DNA polymerase

The DNA polymerase world market is currently more than 350 million$ (282 million Euro) and growing. (2012) (www.in-pharmatechnologist.com)


Isolation, classification and genome sequencing of Thermococcus onnurineus NA1

PACMANUS Basin


Isolation, classification and genome sequencing of Thermococcus onnurineus NA1

0.01

Thermococcus kodakaraensis KOD1T (D38650)

Thermococcus peptonophilus JCM 9653T (AB055125)

Thermococcus stetteri DSM 5474T (Z75240)

Thermococcus profundus DT5342T (Z75233)Thermococcus acidaminovorans DSM 11906T (AB055120)

Thermococcus onnurineus NA1Thermococcus gorgonarius JCM 10552T (AB055123)

Thermococcus fumicolans JCM 10128T (AB055128)

Thermococcus guaymasensis DSMZ11113T (Y08385)

Thermococcus gammatolerans EJ3T (AF479014)

‘Thermococcus barossii’ (U76535)

Thermococcus celer DSM 2476T (M21529)

Thermococcus hydrothermalis AL662T (Z70244)

Thermococcus pacificus JCM 10553T (AB055124)

Thermococcus zilligii JCM 10554T (U76534)

Thermococcus siculi DSMZ 12349T (AJ298870)

Thermococcus atlanticus MA898T (AJ310754)

Thermococcus sibiricus DSM Z12597T (AJ238992)

Thermococcus alcaliphilus DSM 10322T (AB055121)

Thermococcus litoralis JCM 8560T (Z70252)

Thermococcus aegaeus DSMZ 12767T (AJ012643)Thermococcus aggregans DSM 10597T (Y08384)

Thermococcus barophilus DSM 11836T (U82237)

Pyrococcus sp. NA2Pyrococcus abyssi GE 5T (L19921)

Pyrococcus horikoshii JCM 9974T (D87344)Pyrococcus furiosus JCM 8422T (U20163)

Pyrococcus glycovornas CNCMI-2120T (Z70247)

Palaeococcus ferrophilus JCM 10246T (AB019239)

Methanocaldococcus jannaschii JAL-1 DSM2661T (M59126)

Methanopyrus kandleri av19 DSM 6324T (M59932)

100

97

100

9080

97

97

89

95


2-D Gel Electrophroesis of Thermostable Proteins of NA1

Identification of a novel dITPase (Kim et al. AMB. 79, 571-)Identification of a novel aminopeptidase (Lee et al. AEM. 72, 1886-) Characterization of Amylase (Lim et al. JMBiotechnol. 17, 1242-)Study on Deblocking aminopeptidase. (Lee et al. JBB. 104,188-)Identification of a dUTPase (Lee et al., Mar.Biotechnol. 9, 450-) Study on a Prolyl Oligopeptidase. (Lee et la., JBB. 103, 221-)Study on a DNA Polymerase (Kim et al. JMBiotechnol. 17, 1090-) Study on a Methionylaminopeptidase (Lee et al. Mar.Biotechnol. 8, 425-)Study on a carboxypeptidase (Lee et al. BBB 70, 1140-)

Study on a DNA ligase (Kim et al. Biotechnol. Lett. 28, 401-)

In silico analysis of novel biocatalysts and characterization of them


Source Expressed and purified from E. coliPurity >98% homogeneity by SDS-PAGE 5¢ exonuclease activity No detectionNicking activity No detection

λ DNA Genomic DNA

PCR using TNA1 DNA polymerase

M 2 5 8 10 M 2 4 8 (kb)12 15

Purified enzyme

TNA1 DNA polymerase

18


50 100 150 200 250 300 350 400 450 500

Number of bases added (nt)

Flu

ore

scen

ce s

ign

al TNA1

E7

Development of mutant TLA (E7) DNA polymerase

M 3 6 10 13.5 kb

Human DNA

Enzyme TNA1 E7 F7 P7 KOD Taq Pfu

Error rate(1) 1/4.5kb 1/4 kb ND 1/0.7kb ND

Processivity(2) 160bp 430 bp 210bp 180bp 60bp


Metagenomic screening of enzymatic activities have beenperformed to look for various enzymes (egesterases/lipases, β-Lactamases, Chitinases, amidases,cellulase, alkane hydroxylase and proteases) from marineenvironmental samples such as sea water, sediment andorganisms collected from various habitats (deep-sea,deep hyper saline basin, arctic, tidal flat, cold-seep, etc).Screening methods were functional, sequencing or PCRbased approaches (Lee et al., 2010, Current Opinion inBiotech. 21: 353-357).


Nebulizering

1.5 – 4 kb

Selection of positive clones(TBN plate)

Sequencing and primer working

T7 primer(Forward)

T3 primer(Reverse)

ori

pBluescript SK-

Insert DNA

Primer working

Open Reading FrameEnzyme site

lip

Forward primer

Reverse primer

Product

ori

Kan pET-24a(+)

(5369bp)

MCS

ori

Ka n

pET 24a(+) and inert DNA

Enzyme site

ORFEnzyme site Enzyme site

Open Reading FrameEnzyme site

lip

Forward primer

Reverse primer

Product

ori

Kan pET-24a(+)

(5369bp)

MCS

ori

Ka n

pET 24a(+) and inert DNA

Enzyme site

ORFEnzyme site Enzyme site

Construction of marine metagenomes and Screening esterase/lipase clones

Subcloning, expression, purification and application for biocatalysis

Ligation and Transformation (E.coli DH5α )

Expression (pET 24a(+) and E.coli BL21(DE3))

Purification

S

R

Ab

sorb

ance

at

33

0 n

m

EM: Edison Seamount sediment

KTL: Tidal flat

ATL: Arctic sediment


Burkholderia cepacia (M58494)Pseudomonas luteola (AF050153)

Burkholderia glumae (X70345)Pseudomonas fluorescens (AF031226)Pseudomonas fragi (X14033)

Staphylococcus epidermidis (AF090142)Staphylococcus aureus (M12715)EM2L7

Bacillus subtilis (M74010)Bacillus pumilus (A34992)

Streptomyces cinnamoneus (U80063)Propionibacterium acnes (X99255)

KTL3Moraxella sp. (X53869)Psychrobacter immobilis (X67712)

EM2L3Haemophilus influenzae (U32704)

Sulfolobus acidocaldarius (AF071233)EM2L8

EM2L1EM2L6

EM1L1EM2L4

Salmonella typhimurium (AF047014)Photorhabdus luminescens (X66379)

Pseudomonas aeruginosa (AF005091) Moraxella sp. (X53053)

Streptomyces exfoliatus (M86351)Streptomyces albus (U03114)

Arthrobacter oxydans (Q01470)Streptomyces coelicolor (CAA22794)

Bacillus subtilis (P37967)ATL7

Pseudomonas sp. B11-1 (AF034088)Alcaligenes eutrophus (L36817)

ATL5ATL1

ATL11KTL4

KTL7KTL9

Spirulina platensis (S70419)Pseudomonas fluorescens (S79600)

Rickettsia prowazekii (Y11778)Chlamydia trachomatis (AE001287)

ATL3ATL6

KTL1EM2L2

Arthrobacter globiformis (AAA99492)Streptomyces anulatus (CAA78842)

Pseudomonas fluorescens (AAC60471)

0.5

FamilyⅠ

FamilyⅤ

FamilyⅡ

FamilyⅢ

FamilyⅦ

FamilyⅣ

FamilyⅥ

FamilyⅧ

(HSL family)

New family type of lipasesEM: Edison Seamount sediment

ATL: Arctic sediment

KTL: Tidal flat

New group

New subfamily

23


Finding of Psychlophilic lipase/esterase

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

EM2L1

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e a

ctiv

ity

(%

)

0

20

40

60

80

100

Temperature (oC)

0 10 20 30 40 50 60 70

Rel

ati

ve

act

ivit

y (

%)

0

20

40

60

80

100

120

EstAT1

(A)

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

ATL6

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

KTL1

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

KTL4

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ati

ve

act

ivit

y (

%)

0

20

40

60

80

100

KTL7

Temperature (oC)

0 5 10 15 20 25 30 35 40 45 50 55

Rel

ati

ve

act

ivit

y (

%)

0

20

40

60

80

100

KTL9

Temperature (oC)

0 10 20 30 40 50 60 70

Rel

ativ

e ac

tivi

ty (

%)

0

20

40

60

80

100

120

EstAT11

(C) ATL11

EM2L2 EM2L3 ATL1

ATL3


Conclusion and Perspectives

- An enormous diversity in marine environment is still waiting to be discovered for novel biocatalysts.

- It is very promising to explore the novel and useful biocatalysts from marine microbes.

- Future new enzyme discoveries not only improve existing processes but also allow the design of entirely novel processes for making innovative products and high-value intermediates.


Thank you !

the potential of marine/extremophilic microbes to explore novel · 2018. 8. 2. · atl11 ktl4 ktl7...

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