Evidence for Styrene Oxidation in Evidence for Styrene Oxidation in Pseudomonas sp.Pseudomonas sp. via a via a metameta-fission -fission

PathwayPathway

Zhiguang Eric ZhangZhiguang Eric Zhang

Dr. Anthony HayDr. Anthony HayDepartment of MicrobiologyDepartment of Microbiology

Thursday, July 19, 2001Thursday, July 19, 2001

OverviewOverview

• IntroductionIntroduction– Significance of styreneSignificance of styrene

– Pathways for styrene degradationPathways for styrene degradation

• Methodology and experimental outlineMethodology and experimental outline– Isolation of organisms Isolation of organisms

– Molecular characterization of organismsMolecular characterization of organisms

– Preliminary findings: evidence for involvement of Preliminary findings: evidence for involvement of the the todtod pathway / homologous styrene pathway pathway / homologous styrene pathway

• ConclusionsConclusions• Future workFuture work

What is styrene?What is styrene?

– monosubstituted benzene (vinyl group)monosubstituted benzene (vinyl group)– strong odor, toxin, mutagen, and suspected strong odor, toxin, mutagen, and suspected

carcinogencarcinogen– starting material for industrial production of starting material for industrial production of

plastic and rubberplastic and rubber– primarily released to the environment through primarily released to the environment through

wastewater, evaporation, and pyrolysis of wastewater, evaporation, and pyrolysis of polystyrenepolystyrene

Photo:Marine Nationalé

• sinking of the sinking of the Ievoli SunIevoli Sun off the coast of France - off the coast of France - Oct. 31, 2000Oct. 31, 2000

• cargo included 4,000 tons cargo included 4,000 tons of styreneof styrene

Pathways for styrene oxidationPathways for styrene oxidation

• styABCD styABCD - monooxygenation of styrene and - monooxygenation of styrene and phenylacetate lower pathwayphenylacetate lower pathway– Pseudomonas putidaPseudomonas putida CA-3, CA-3, Pseudomonas sp. Pseudomonas sp.

VLB120VLB120– Exophiala jeanselmeiExophiala jeanselmei

• Dioxygenation of styrene and Dioxygenation of styrene and metameta-fission-fission– Rhodococcus rhodocrousRhodococcus rhodocrous NCIMB 13259 NCIMB 13259– Pseudomonas sp.Pseudomonas sp. s7, s15? s7, s15?

• closely studied at the closely studied at the molecular levelmolecular level

• regulatory genes regulatory genes stySRstySR• metabolic end products:metabolic end products:

styABCDstyABCD upper pathway upper pathway

styAB

styC

styD lower pathway

fumaratefumarate acetoacetateacetoacetate

The University of Minnesota Biocatalysis/Biodegradation Databasehttp://umbbd.ahc.umn.edu/

Styrene Styrene metameta-fission upper pathway - direct -fission upper pathway - direct aromatic attack through a catecholic intermediatearomatic attack through a catecholic intermediate

Enzymatic activity Enzymatic activity has been has been

identified but identified but there has been no there has been no molecular work molecular work

done to done to characterize the characterize the

genes or enzymes genes or enzymes involvedinvolved

The University of Minnesota Biocatalysis/Biodegradation Databasehttp://umbbd.ahc.umn.edu/

meta-fission

• metabolic end products:metabolic end products:- acrylate- acrylate

- acetaldehyde- acetaldehyde- pyruvate- pyruvate

MethodsMethods• Identified a consortia that removes styrene Identified a consortia that removes styrene

from air via a biofilter systemfrom air via a biofilter system

• Isolation and characterization of organismsIsolation and characterization of organisms– grew consortia in enrichment media grew consortia in enrichment media

((MSM+500ppm styrene) MSM+500ppm styrene) to select for styrene to select for styrene degradersdegraders

– isolated several organisms capable of growing isolated several organisms capable of growing on styrene as the sole carbon sourceon styrene as the sole carbon source

– characterized using RISA and BOXA1R characterized using RISA and BOXA1R genomic fingerprinting, 16s rRNA sequencing, genomic fingerprinting, 16s rRNA sequencing, and substrate utilization and substrate utilization

Characterization of isolates Characterization of isolates s7 and s15s7 and s15

• Identical RISA and BOXA1R banding patternsIdentical RISA and BOXA1R banding patterns• Same 16S rRNA sequence hit using BLAST Same 16S rRNA sequence hit using BLAST

search algorithmsearch algorithm

• Able to grow on the same substratesAble to grow on the same substrates • s7 grows faster on liquid and solid media than s15s7 grows faster on liquid and solid media than s15• s7 and s15 accumulate different colored s7 and s15 accumulate different colored

metabolites when grown with styrene as the sole metabolites when grown with styrene as the sole carbon sourcecarbon source

• One a derivative of the other?One a derivative of the other?

Growth substrates utilized by s7 and s15Growth substrates utilized by s7 and s15

Liquid Media s7 s15S+mandelic acid - -R-mandelic acid - -

S+phenylethanediol - -R-phenylethanediol - -phenylacetic acid + +

benzoic acid + +MSM + +

MSM-ethylbenzene - -MSM-toluene + +MSM-styrene + +

MSM-D-glucose + +MSM-D-lactose - -MSM-acetate + +

LB + +

Same strain - different metabolite Same strain - different metabolite accumulationaccumulation

- the same- the same Pseudomonas sp. Pseudomonas sp.

s7s7 s15s15MSM + Styrene atmosphere

How to explain different colored How to explain different colored metabolites?metabolites?

• Brown is characteristic of catecholic Brown is characteristic of catecholic polymerization productspolymerization products

• Yellow is characteristic of Yellow is characteristic of metameta-fission -fission productsproducts

• Involvement of Involvement of metameta-fission pathway via a -fission pathway via a catecholic intermediate?catecholic intermediate?

The University of Minnesota Biocatalysis/Biodegradation Databasehttp://umbbd.ahc.umn.edu/

todC1C2BA

todE

todD

The styrene The styrene metameta-fission -fission pathway vs. the pathway vs. the todtod pathway for toluene pathway for toluene

oxidationoxidation

Evidence for Evidence for tod tod / homologous styrene pathway:/ homologous styrene pathway:

• PCR using degenerate aromatic PCR using degenerate aromatic dioxygenase primers gave a ~450bp dioxygenase primers gave a ~450bp fragmentfragment

• BLASTBLAST results indicate region of results indicate region of sequence identity:sequence identity:– todC1todC1: : toluene dioxygenasetoluene dioxygenase iron-sulfur iron-sulfur

protein component - large subunitprotein component - large subunit

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi

506bp506bp396bp396bp

1% agarose1% agarose

s15s15s7s7

Evidence for Evidence for metameta-fission I:-fission I:

• Yellow metabolite (s7) - Yellow metabolite (s7) - metameta-fission product-fission product• Yellow color is lost when acidified and is restored Yellow color is lost when acidified and is restored

upon neutralizationupon neutralization• Brown metabolite (s15) is characteristic of Brown metabolite (s15) is characteristic of

catecholic polymerization productscatecholic polymerization products• Possible structure of brown polymerization Possible structure of brown polymerization

product (GC-MS)product (GC-MS)

O

OH

OH

Evidence for Evidence for metameta-fission-fission II:II:- GC-MS metabolite derivitization: detection of - GC-MS metabolite derivitization: detection of the the metameta-fission intermediate 3-vinylcatechol-fission intermediate 3-vinylcatechol

Mass Spectrum of Acylated Vinylcatechol Intermediate

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

50.1 56.1 64.1 70 77.1 83 89.1 95 101 107 115 121 134 145 160 176 192 222

M/Z

Inte

nsi

ty a

136

178

220

Vinylcatechol (m/z=136)

OH

OH

Use of 3-fluorocatechol - a Use of 3-fluorocatechol - a metameta-fission inhibitor-fission inhibitor

Polymerizationproduct

3-Vinylcatechol 3-Vinylcatecholwhen treated with 3-

FluorocatecholS7 + - ++

S15 ++ + ++

OH

OH

3-vinylcatechol3-vinylcatechol

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi

OH

OH

F

3-fluorocatechol3-fluorocatechol

?

O

OH

COOH

2-hydroxy-6-oxo-2-hydroxy-6-oxo-octa-2,4,7-trienoateocta-2,4,7-trienoate

Enzymatic activity?Enzymatic activity?

• Resting cell assay Resting cell assay – indicates that s7 cells induced with styrene are indicates that s7 cells induced with styrene are

capable of:capable of:• using styreneusing styrene

• using tolueneusing toluene

• using catecholusing catechol

...whereas cells that were grown on glucose were ...whereas cells that were grown on glucose were notnot

• This is evidence that…This is evidence that…– enzymes capable of utilizing styrene, toluene, and enzymes capable of utilizing styrene, toluene, and

catechol as substrates were produced when s7 cells catechol as substrates were produced when s7 cells were induced with styrenewere induced with styrene

ConclusionsConclusions• PCR, sequencing, resting cell assays, and GC-PCR, sequencing, resting cell assays, and GC-

MS indicate that MS indicate that Pseudomonas sp.Pseudomonas sp. s7 and s15 s7 and s15 utilize the upper part of the utilize the upper part of the todtod pathway or a pathway or a homologous styrene pathwayhomologous styrene pathway

• s7 and s15 appear to differ only in catechol 2,3-s7 and s15 appear to differ only in catechol 2,3-dioxygenase (dioxygenase (todEtodE) activity - one a mutant of ) activity - one a mutant of the other?the other?

• s7 and s15 provide a glimpse at an adaptation s7 and s15 provide a glimpse at an adaptation that has allowed for relaxed substrate that has allowed for relaxed substrate specificity in the specificity in the todtod pathway pathway

FutureFuture workwork

• todC1todC1 knockout strains for s7 and s15 knockout strains for s7 and s15

• todEtodE knockout strains for s7 and s15 knockout strains for s7 and s15

• Complementation of s15 Complementation of s15 todEtodE knockout with knockout with s7 s7 todEtodE gene gene– see accumulation of yellow see accumulation of yellow metameta-fission product -fission product

instead of brown 3-vinylcatechol polymer?instead of brown 3-vinylcatechol polymer?

• Sequence s7 and s15 Sequence s7 and s15 todEtodE genes - mutation? genes - mutation?

AcknowledgementsAcknowledgements– Dr. Anthony HayDr. Anthony Hay

– Lauren JunkerLauren Junker

– Tammy ArbogastTammy Arbogast

– Jeanne KagelJeanne Kagel

– Serena MakSerena Mak

– Paul Taylor at U. Melbourne for Ado Paul Taylor at U. Melbourne for Ado primersprimers

– Lew Daly at Triad Technologies - Lew Daly at Triad Technologies - Syracuse, NYSyracuse, NY

– Cornell Center for Advanced TechnologyCornell Center for Advanced Technology

– Develop Your Own Internship ProgramDevelop Your Own Internship Program

– Laurel, Pam, and the Hughes ProgramLaurel, Pam, and the Hughes Program

Questions?Questions?