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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
Mixing Time= 10 minRotation Speed= 3
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
Carboy Scale Coenzyme F420-2 Production
Overnight Settling Basket Screening
Carboy Scale Coenzyme F420-2 Production
Muck Layer
Sharples Centrifuge
Stage Comparison of Extract Clarification
Carboy Scale Coenzyme F420-2 Production
Extraction Screening Centrifugation
Post Centrifugation Cake
Carboy Scale Coenzyme F420-2 Production
Axial Flow Low Shear Impeller Extract Dilution (1:3 V/V)andMixed Bed HiQ Absorption of F420
Carboy Scale Coenzyme F420-2 Production
HiQ Resin Recovery by Gravity Sedimentation Mixed Bed HiQ Elution of F420
Carboy Scale Coenzyme F420-2 Production
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
Mixing Time= 20 minRotation Speed= 4
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