UIowa 2005 - Iowa City, IA

Development of a Large Scale Coenzyme F420 Production Process and

Molecular Tools for Investigation of Related Gene Systems in

Rhodococcus and Mycobacterium

Randy SimpsonDaniels Lab

Microbiology

Genus Rhodococcus

• aerobic, G+, nocardioform, actinomycete• a genome has not been sequenced in this genus• ubiquitous in nature• biodegradation of environmental pollutants

– aromatic hydrocarbons, chlorinated hydrocarbons and aromatics, nitroaromatics

• industrial biotechnology– surfactants, steroids

• few molecular tools for genomic study to date

Derbyshire et al. (2000)

KmR

LB/Km

EZ::TN Electrotransformation and Insertion Data for R. opacus RB1

Selective Agar Type Cell Type Selected cfu/100L cfu/100L cfu/100LAverage

cfu/100LPre-Electroporation

LB wt 3.6*109 1.7*109 2.0*109 2.4*109

LB/Km spontaneous mutant 40 40 20 33Post Electroporation

LB survivors 6.0*108 2.0*108 4.4*108 4.1*108

LB/Km transformants 400 900 350 550

Time Constant (ms) 6.9 7 6.8 6.9Amino Acid Auxotrophs 2%Transformation Efficiency (transformants/survivors)

1.3*10-6

Transformation Efficiency (cfu/g*EZ:TN) 3.9*105

[Km]=40g/mL

Electroporation Parameters:2.5kV/ 400/ 0.2cm gap/ 400L/ 0.02g*EZ:TN

Negative Selection for Amino Acid Auxotrophy

LB/KmSuc/Km/NH3NO3

Proposed TNP and DNP Degradation Pathway inR. opacus HL PM-1

Hofmann et al. (2004)

NO2

NO2O2N

O

NO2-

NO2-

O

O2N

O

NO2--O2N

NOHO

O

NO2--O2N

H NO2

NO2

NO2

ONO2-

NO2-

O

O

H NO2

NO2

HNO2NO2

HOOCmineralization

NpdI

F420-H2

NpdI

NpdC

NpdC

tautomerase

hydrolase

F420-H2

F420-H2

F420-H2

TNP

DNP

H--DNP

orfA orfB npdC orfD orfD

orfE

npdR npdG

npdH

npdI orfJ orfJ

IGRI IGRII IGRIII IGRIV

Heiss et al. (2002)

96-Well Plate Screen for DNP- Mutants

LB/Km/DNP

Before Inoculation

LB/Km/DNP

After 48 h of growth

DNP- Mutants Summary

Colony PCR for npd+ Genotype in Mutants of Interest

Ladde

rE2

9

I5

wt G2

7F

8

S. av

ermiti

lisLadde

rK23

AD3

R45

fgd

npdInpdG

E32

(+) (-)

P14

Mutant Plasmid Profile using PFGE

680 Kb

610 Kb565 Kb

450 Kb

365 Kb

825 Kb

750 Kb

285 Kb

Ladde

r wtE29 R45 P14 G27

K23a

E32

E. col

i DH5

Ladde

r

225 Kb

436.5 Kb

485 Kb

388 Kb

679 Kb630.5 Kb582 Kb533.5 Kb

npd+npd-

(-)(+)

Rhodoccus Instabilityin Biodegradation Phenotypes

• ``...rhodococci often exhibit considerable genomic instability at many loci...´´

• ``... mobile elements possibly contribute to the acquisition of biodegradation phenotypes by promoting mutations and rearrangements in both the chromosome and transmissible plasmids.´´

• ``...transposable elements of Rhodococcus were discovered accidentally in DNA regions flanking biodegradation genes.´´

Larkin et al. 1998.

Mutant Phenotypes on NaAc/DNP/Km

npd+

npd-

wt wt

G27 K23a E32

E29 P14 R45

pRS6700 Construction from pSMT3 and pEP7000

HygR

Phsp60 fgdM

colE1 ori

AL ori6.7 kb

BamHI EcoRV

Restriction Digest Pattern for Replication of pRS6700 in Rhodococcus opacus RB1

R. opa

cus R

B1/pR

S6700

pSM

T3fg

dM

pRS67

00

E. col

i JM

109/

pRS67

00

R. opa

cus R

B1

E. col

i JM

109

5.7 Kb

1.0 Kb

(+) (+) (-) (-)

Ladde

r

Ladde

r

F420-H2

F420

D-GLP

D-G6P

F420-dependent glucose-6-phosphate dehydrogenase (fgd)

In -vitro Effect of pRS6700 Expression on Fgd Activity

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

R. opacusRB1

pRS6700

R. opacusRB1

R. opacus HLPM-1

M. smegmatis

Sp

ecif

ic A

ctiv

ity

(m

ol/

min

-mg

)

NCBI BlastX of E29 Insertion Site Sequence

NCBI ORF Finder Analysis of E29 Insertion Sequence

3660 bp

HygR

Phsp60 E29

colE1 ori

AL ori9.3 kb

PstI EcoRV

pE29 Construction from pSMT3 and E29

E29 Complement Phenotype on NaAc/DNP/Km/Hyg

E29C (-)

Construction of pRS10500 for E29 Gene Replacement

KmR

5.0 kb

lacZ

E29

3.0 kbcolE1

ori

pGEM

traJ

5.4 kbP15A

ori

oriT

sacB

pJQ200SK

GmR

RP4 mob

lacZ

AmpR

DNA Polymerase T4 DNA ligase

traJ

10.5 kbP15A

ori

oriT

sacB

pRS10500

GmR

RP4 mob

lacZ

NotI T4 DNA ligase

NotIE29

KmR

CIAP

TT

AA

lacZ

8.0 kbcolE1

ori

pRS8000

AmpR

KmR

E29

tra mob

E29::KmR

Escherichia coli S17.1 R. opacus RB1

E29 wt

R. opacus RB1 (E29::KmR)

E29::KmR

Schematic Diagram of E29 Mutant Re-creationvia Conjugation

pRS10500

2-Step Selection for Gene Replacement of E29

LB/Nal30/Km100/Gm10 LB/Suc10 LB/Km100/Suc10 LB/Km100/Suc10 LB/Km100/Suc10/Gm10

Integration Resolution

Integration 10.8 kb

KmR

E29traJ GmR sacB

3.7 kb

E29KmR/SucS/GmR

KmR

KmR/SucR/GmSE29

mutantstrain

E29

Resolution1.3 kb1.85 kb 1.85 kb

KmR

KmR/SucR/GmS

P1

P2

2-Primer PCR Verification of E29 Gene Replacement

wild typestrain

E29

E29 mutantstrain

KmS/SucR/GmS

P1

P2

(-)

wtE29

RCE29A

S. av

ermiti

lis

Ladde

r

Ladde

r

E29

RCE29B

Conclusions• npd cluster appears unstable, limiting the effectiveness of the

DNP bleaching screen to identify mutants of interest

• EZ::TN provides adequate transformation efficiency (3.9 x 105 cfu/g) and randomness of insertion (2% amino acid auxotrophs)

• pSMT3 is an effective expression vector

• pJQ200KS is a useful tool for gene replacement

• tools and methods developed in this work led to successful cloning of GTP Cyclohydrolase I in Nocardia and fah knockout in Mycobacterium smegmatis

Functions of Fah (fgd associated hydrolase)

in Mycobacterium smegmatis

Genus Mycobacterium

• aerobic, G+, mycolate containing, nocardioform, actinomycete

• pathogens such as M. tuberculosis, M. leprae and M. avium

• also hydrocarbon oxidizers in soil• few molecular tools for genomic study to date

Function and Importance of Fgd

Mycobacterium tuberculosis

*M. smegmatis is not sensitive to PA-824

F420-H2

F420

D-GLP

D-G6P

F420-dependentG6P dehydrogenase

Fgd Protein X

PA-824

PA-824-H2

fah and fgd in Mycobacterium

Ml mp fah fgd "p- pta " "p- ack "

Ma hp at fah fgd pta ack hp

M. bovis & Mtb pks6 fah fgd pta ack pknG

Mc fah fgd hp -man

Mf fah fgd mp tr ap

Mp hp fah fgd mp mp

Ms am hp fah fgd tr ar pt ap

fgd associated hydrolase (Fah)

• Fah has highly conserved regions that correspond with the locations of metal-binding ligands in -lactamases and hydrolases

• Fah could serve as a regulatory protein for transcription of fgd because of its location, and as a metal binding protein that could sense redox potential in the cell

• the phenotype of a fah- mutant is unknown; a targeted gene replacement of fah in M. smegmatis (not PA-824 sensitive) and the resulting effects on Fgd activity could provide insight

• downregulation of Fgd activity by mutation of fah could result in increased resistance to PA-824 in M. tuberculosis.

AmpR

9.9 kbcolE1

ori

f1 ori

lacZfah/fgd

pEP7000

KmR

5.3 kbP15A

ori

pACYC184

traJ

5.4 kbP15A

ori

oriT

sacB

pJQ200SK

AmpR

10.4 kbcolE1

ori

f1 ori

lacZ

fah

pRS11200

fgd

KmR

GmR

Rp4 mob

lacZ

TcR

BamHI

EcoNI

T4 DNA polymerase

T4 DNA polymerase

CIAP

T4 DNA ligase

traJ

13.7 kbP15A

ori

oriT

sacB

pRS13700

GmR

RP4 mob

lacZ

BamHI T4 DNA ligase

BamHI fgdfah KmR

CIAP

Construction of RS13700 for fah Replacement

Electrotransformation of pRS13700 into M. smegmatis

traJ

13.7 kbP15A ori

oriT

sacB

pRS13700

GmR

RP4 mob

lacZ

fgdfah KmR

M. smegmatis mc2 155

fah

M. smegmatis mc2 155 (fah-/KmR)

fah

KmR

LB/Km40/Gm10 LB/Suc10 LB/Km40/Suc10 LB/Km40/Suc10 LB/Km40/Suc10/Gm10

Integration Resolution

1.8 kb 1.3 kb 5.2 kb

fah mutant

strainfah KmR fgd

Resolution

KmR/SucR/GmS

Integration 13.7 kb

fah

KmR

fgdtraJ GmR sacB

7.3 kb

fgdfahKmR/SucS/GmR

2-Step Selection for Gene Replacement of fah

fah KmR fgd

KmR/SucR/GmS

FAHL

FAHR2 FAHR1

3-Primer PCR Verification of fah Gene Replacement

fah mutantstrain

wild typestrain

fah fgd

KmS/SucR/GmS

FAHL

FAHR2

Ladde

rwt

1 Step

2 Step

Ladde

r

S. av

ermiti

lis

(-)

Effect of Shaking Speed on Fgd Activity for wt versus fah::KmR

24 Hours

0

0.01

0.02

0.03

0.04

0.05

0.06

200 rpm 50 rpm

Sp

ecif

ic A

ctiv

ity

(m

ol/(

min

-mg)

)

wt fah::KmR

Effect of Culture Age on Fgd Activity for wtversus fah::KmR Mutant

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

11 20 24 35 45Culture Age (hr)

Fgd

Act

ivit

y (

mol

/(m

in-m

g))

wt fah::KmR

AmpR

3.5 kbpUC

ori

fah

pJP3600

KmR

5.3 kbP15A

ori

pACYC184

TcR

BamHI

XmnI

T4 DNA polymerase

T4 DNA polymerase

CIAP

T4 DNA ligase

Plac

AmpR

4.8 kbpUC

ori

fah

pRS4900

Plac

KmR

AmpR

4.1 kbpUC

ori

pRS4100

Plac

AmpR

2.8 kbpUC

ori

pHAT10

Plac

T4 DNA polymeraseBamHI

XmnI

T4 DNA polymerase

CIAP

T4 DNA ligase

pRS4800 Construction from pHAT/fah and KmR Cassette

fah+

construct

fah-

construct

Spectrophotometric Assay of -lactamase Activity in Cell Free Extract on Nitrocefin

pHAT/fah+/KmR pHAT/fah-/KmR pBluescript/AmpR

0.4 nmol/(min-mg) 0.15 nmol/(min-mg) 5.5 mol/(min-mg)

(-) (+)

Screening of Artificial Chromogenic NP Derivatives as Substrates for Fah

0

1

2

3

4

5

6

7

8

o-NP-β-Dgalactopyranoside

p-NP-β-Dgalactopyranoside

p-NP-β-Dglucopyranoside

p-NP-β-Dglucuronide

p-NP-β-Dgalacturonide

Sp

ecif

ic A

ctiv

ity

( n

mol

/(m

in*m

g))

fah+ fah-

Conclusions• pJQ200SK is an effective suicide vector for gene

inactivation in M. smegmatis• a 2-step process of integration then resolution is

useful using pJQ200SK • our lab is the first to successfully utilize pJQ200SK in

Actinomycetes (Mycobacterium and Rhodococcus)• fah knockout reduces Fgd activity by one half, but fah

is not essential for Fgd presence or growth• Fah exhibits 0.24 nmol/(min-mg) activity with

nitrocefin indicating slow -lactamase reaction• Fah exhibits 6.7 nmol/(min-mg) activity with ONPG

Scale-up of Coenzyme F420 Production

Why Develop a Large Scale F420 Recovery Process?

• there is no commercial source of coenzyme F420 and it is required for research by the Daniels Lab

• F420 is costly to obtain by present lab procedures using M. smegmatis

• the Daniels Lab receives frequent requests for the compound

• F420 is required for activation of the new anti-tuberculosis drug, PA-824

• obtaining a compound of medicinal value such as F420 from a waste product has attracted USDA interest

Coenzyme F420-2

OH N

CH2

CH

CH

CH

CH2 O P

O

O

O

CH

CH3

C

O

NH

CH

CH2

CH2

C

O

NH

CH

COO-

CH2

CH2

COO-

OH

OH

OH

COO-

NH

N

O

O

76

8 10

5

1 2

4

Municipal Anaerobic Digester Sludge Process

Coinjection of Coenzyme F420-2 Standard and DWS Extract via HPLC/Fluorescence Detector

F420-2

F420-2

F420-5,6

Mycobacterium smegmatis Extract Dewatered Sludge Extract

F420 Binding from DWS Extract using Mixed HiQ Bed

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

0 100 200 300 400 500

Elution Bed Volumes

5DF

(m

ol/L

)

F420-2 FO

C0F420-2= 1.8 mol/L

CF420-2= 0.09 mol/L

DWS= 225 gBed Volume= 0.5 mL

Mixing Time= 6 minRotation Speed= 5

Breakthrough Curve of F420 from Mixed HiQ Bed

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

8 16 24 32 40 48 56 64 72 80

Elution Bed Volumes

5DF

(m

ol/L

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

NH

4HC

O3

(M)

F420-2 F420-4,5,6 FO NH4HCO3


DWS= 0.4 kgBed Volume= 5mLExtract Volume= 400mL

0.86 mol F420-2/kg DWS

F420 Elution Profile with NH4HCO3 (pH7.8) and Mixed HiQ Bed

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

0 10 20 30 40 50

Exposure time (hr)

F4

20

-2 (

mol

/L)

200W Room Light Darkness

Photolability of F420 species in Eluentfrom Mixed HiQ Bed

Lab Scale Conclusions• HiQ strong anion exchange resin had a high capacity for

F420 binding (5 mL resin/ 1 kg DWS)• utilizing a mixed HiQ bed for sorption and elution

prevented column plugging• light exposure is a potential problem in later process

steps making shielding prudent• recovery of F420 bound HiQ resin by centrifugation would

present difficulties at large scale• 1M NH4HCO3 buffer is good for F420 elution from HiQ

resin• freeze drying HiQ eluate results in desalting of sample

by lyophilization of buffer to NH3 and CO2

Fermentor Scale Coenzyme F420-2 Production

75L Fermentor (15 kg DWS/ 65 L Water)

Fermentor Scale Coenzyme F420-2

Production Process Efficiency

0.00

0.20

0.40

0.60

0.80

1.00

1.20

FermentorExtraction

Filter Press Microfiltration Absorption

5DF

(m

ol/k

g D

WS

)

F420-2 FO15 kg DWS

Fermentor Scale Conclusions

• extraction in fermentor resulted in strong hydrogen sulfide and volatile fatty acid odor

• process recovers only about 10% of the F420 extracted from DWS due mainly to filtration losses

Radial Flow High Shear Impeller Mixing 5 kg DWS and 20 L of Water per Carboy

Carboy Scale Coenzyme F420-2 Production

Autoclaving 90 min Adding 20 L Water per Carboy and Re-Mixing DWS Slurry


Overnight Settling Basket Screening


Muck Layer

Sharples Centrifuge

Stage Comparison of Extract Clarification


Extraction Screening Centrifugation

Post Centrifugation Cake


Axial Flow Low Shear Impeller Extract Dilution (1:3 V/V)andMixed Bed HiQ Absorption of F420


HiQ Resin Recovery by Gravity Sedimentation Mixed Bed HiQ Elution of F420


0.00

0.50

1.00

1.50

2.00

2.50

2 4 6 8 10 12 14 16 18 20 22

Elution Bed Volumes

5DF

(m

ol/L

)

0

0.2

0.4

0.6

0.8

1

1.2

NH

4HC

O3

(mM

)

F420-4,5,6 F420-2 FO NH4HCO3


DWS= 10 kgBed Volume= 150 mLExtract Volume= 180 L

0.34 mol F420-2/ kg

DWS

F420 Elution Profile with NH4HCO3 (pH7.8)using Mixed HiQ Bed

0.0

0.5

1.0

1.5

2.0

2.5

3.0

5D

F (

mo

l/k

g D

WS

)

F420-2 FO10 kg DWS

Carboy Scale Coenzyme F420-2

Production Process Efficiency

Carboy Scale Conclusions

• mixing by high shear impeller and autoclaving provided adequate extraction of F420

• additional autoclaving time and/or two autoclave steps could enhance F420 extraction

• additional 1:3 dilution of extract with water after centrifugation reduced density of extract without filtration allowing HiQ beads to settle for recovery

Carboy Scale Conclusions

• HiQ strong anion exchange resin resulted in near 100% absorption of F420-2 from extract

• utilizing a mixed HiQ bed for binding and elution prevented any column plugging

• overall process recovers approximately 34% of the F420 extracted from DWS

M. smegmatis

1 kg preparation1 DWS

10 kg preparation

Total mol F420 in medium scale preparation2 NA 26.6

Total mol F420 in large scale extraction after centrifugation 74 6.0

Total mol F420 recovered from HiQ 58 3.3

Estimated extraction and HiQ labor costs3 $ 168 $ 252

Cost of HiQ used4 $ 11 $ 110

Cost of cells or DWS5 $ 1,061 $ 19

Sum of labor, feedstock and HiQ costs $ 1,240 $ 381

Cost per mol F420 $ 21 $ 116

Comparison of Laboratory Scale (1 kg) M. SmegmatisProcess and a Pilot Scale (10 kg) DWS Process

Process Modifications to EnhanceF420 Yield

• autoclaving longer (145 min) or autoclaving (90 min) then mixing and re-autoclaving could improve F420 extraction

• sludge from a high-starch agricultural processing wastes has been reported to contain 9 to 39 mol F420/kg compared to the 2.5 to 3.8 mol F420/kg for DWS

• we could reasonably increase the amount of F420 recovered by 2 to 3X with a new type of sludge and extraction modifications

• utilization of basket centrifuge could increase volume of extract recovered and possibly increase recovery efficiency 2X

• A 6X enhancement of F420 yield would make the DWS method more attractive than M. smegmatis cells.

Acknowledgments• Dr. Lacy Daniels

– Seong-Ae Kang• My dissertation committee

– Dr. David Gibson– Dr. Caroline Harwood– Dr. Michael Feiss– Dr. John Rosazza

• Dr. Linda McCarter• Dr. Gesche Heiss (University of Stuttgart)• Center for Biocatalysis and Bioprocessing• NSF Training Grant in Gene Expression and Bioremediation• USDA Grant to the Iowa Biotechnology Byproducts

Consortium

Freeze Dry/ Lyophilization

(2X)

NH3 and CO2

Mixed HiQ Bed Sorption

then Bead Recovery by Sedimentation

Fixed HiQ SupportSorption then

NH4HCO3 Elution

DI Water

Freeze Dry/ Lyophilization

(1X)

NH3 and CO2

DI Water

Fixed Florisil Support(60/100 PR mesh)

Semi-PreparativeC-18 HPLC/Fluorescence

DetectorF420-2

other F420 species and contaminants

Proposed Large Scale Coenzyme F420-2 Production

Mixed HiQ BedElution with NH4HCO3

Semi-Batch Basket

Centrifuge

Centrate

Cake

Mixing 5 kg DWS/ 25 L DI Water per

Carboy (2)

Autoclave(90 min)

Dilution with 20 L DI Water per Carboy (2)

then Mixing

Autoclave (90 min) then Mixing

UIowa 2005 - Iowa City, IA

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