therapeutics from microbes: pathways and specific examples
DESCRIPTION
Therapeutics from Microbes: Pathways and Specific Examples. Blaine Pfeifer Department of Chemical & Biological Engineering Tufts University Medford, MA 02155, U.S.A. FMM Industry Day February 23, 2011. Motivation I – Range of therapeutics and activities. Penicillium chrysogenum. - PowerPoint PPT PresentationTRANSCRIPT
Blaine PfeiferBlaine PfeiferDepartment of Chemical & Biological EngineeringDepartment of Chemical & Biological Engineering
Tufts UniversityTufts UniversityMedford, MA 02155, U.S.A.Medford, MA 02155, U.S.A.
FMM Industry DayFMM Industry DayFebruary 23, 2011February 23, 2011
23Feb11
OH
CH3
H
O
O
H
O O
Penicillium chrysogenumPenicillium chrysogenum Artemisia annuaArtemisia annua Salinospora tropicaSalinospora tropica
ArtemisininArtemisinin
AntimalarialAntimalarial
LomaiviticinLomaiviticin
AnticancerAnticancer
PenicillinPenicillin
AntibioticAntibiotic
HN
O N
O
S
O
OH
OH
OH
O
ON2
O
O
OO OH
O
O N
O
OH
HO
O
O
N2
O
O
OO
HO
O
ON
O
2
• NCE’s between 1981 and 2006– 34% were natural
products or semi-synthetic derivatives
• Global antibacterial market generated $42 billion in sales in 2009– ~4% annual growth over
last five years
• MRSA, VRSA, other antibiotic resistant microbes in community and hospital environments– Now, MRSA kills more
U.S. citizens each year than AIDS
– Infectious Diseases Society of America “10 by ‘20” initiative
23Feb11 Hasmad B. Nat Rev Drug Discov. 2010 Sep;9(9):675-6.Newman DJ, Cragg GM. J Nat Prod. 2007 Mar;70(3):461-77.
3Clin Infect Dis. 2010 Apr 15;50(8):1081-3.Pollack A. New York Times. 2010 Nov 10.
23Feb11
Penicillium chrysogenumPenicillium chrysogenum Artemisia annuaArtemisia annua Salinospora tropicaSalinospora tropica
OH
CH3
H
O
O
H
O O
ArtemisininArtemisinin
AntimalarialAntimalarial
LomaiviticinLomaiviticin
AnticancerAnticancer
PenicillinPenicillin
AntibioticAntibiotic
HN
O N
O
S
O
OH
OH
OH
O
ON2
O
O
OO OH
O
O N
O
OH
HO
O
O
N2
O
O
OO
HO
O
ON
O
4
23Feb11
OH
CH3
H
O
O
H
O O
ArtemisininArtemisinin LomaiviticinLomaiviticinPenicillinPenicillin
Penicillium chrysogenumPenicillium chrysogenum Artemisia annuaArtemisia annua Salinospora tropicaSalinospora tropica
AntimalarialAntimalarial AnticancerAnticancerAntibioticAntibiotic
HN
O N
O
S
O
OH
OH
OH
O
ON2
O
O
OO OH
O
O N
O
OH
HO
O
O
N2
O
O
OO
HO
O
ON
O
5
23Feb11
GeneAGeneA
GeneBGeneB
GeneCGeneC
Identification & IsolationIdentification & Isolation
Escherichia coliEscherichia coli
GeneAGeneA GeneBGeneB GeneCGeneC
Heterologous ExpressionHeterologous Expression
Scientific Motivation:• Complex, slow-growing organisms• Complex chemistry• Vast knowledgeEventual Outcome:• Therapeutic access• Less expensive drugs• Widespread use
HN
O N
O
S
O
OH
Challenges:• Uncompetitive production titers6
23Feb11
Native Producer+ Produces natural product in large quantities
– Difficult or impossible to grow in the lab– Likely unable to genetically engineer
Heterologous Host+ Grows quickly+ Established genetic engineering protocols+ Extensive bioprocess knowledge– Does not produce natural product
Heterologous Producer+ Now produces natural product in small quantities
Genetic EngineeringMolecular Biology
Network& SystemsAnalysis
Pathway & Network
Engineering
TranscriptomicsProteomics
Metabolomics
StoichiometricModeling
Enzyme Engineering
Network Engineering
gTME
Signal Pathway Engineering
Improved Heterologous Producer+ Now produces natural product in large quantities
Scale-Up & Commercialization7
23Feb11
6 ×6 ×
PolyketidePolyketidePolyketidePolyketideSCoA
O
HOOCSCoA
O
O OH
OH
O
O OH
O O
OH
O
O O
OH OH
O
HON
O OH
O
6-Deoxyerythronolide B6-Deoxyerythronolide B ErythromycinErythromycin
HH
TaxadieneTaxadiene
O NH
O
OH
O
OH O
O
OH
O
O
HO
O
O
O
TaxolTaxol
IsoprenoidIsoprenoidIsoprenoidIsoprenoid
DMAPPDMAPP
(2(2SS)-Methymalonyl-CoA)-Methymalonyl-CoA
Propionyl-CoAPropionyl-CoA
CarbonCarbonSource(s)Source(s)
OPP
OPP
IPPIPP
3 ×3 ×
TaxusTaxusbrevifoliabrevifolia
SaccharopolysporaSaccharopolysporaerythraeaerythraea
8
23Feb11
O
O
OH
O
OO
OH
O
O
OH
OHO
N
HO
O
O
OH
O
OO
OH
O
O
OH
OHO
N
PCC EryCI CII CIII CIV CV CVI EryF
EryBI BII BIII BIV BV BVI BVII
Sfp PrpE
DEBS3
DEBS2
DEBS1
O
O
OH
OH
OHO
Erythromycin AErythromycin B
Erythromycin C
O
O
OH
O
OO
OH
O
OH
OH
OHO
N
HO
Erythromycin D
O
O
OH
O
OO
OH
O
OH
OH
OHO
N
EryK
EryGEryG
EryK
Propionate
6-deoxyerythronolide B (6dEB)
O-
O
Peiru S et al. Appl Environ Microbiol 2005 71:2539; Lee HY et al. PLoS Biol. 2007 5(2):e45.
His-tag sequence
Erythromycin tailoring and resistance genes
lac operatorT7 promoter
T7 terminator
Kanamycin resistance geneCarbenicillin resistance gene
eryBI BII BIII BIV BV BVI BVII ermE eryCI CII CIII CIV CV CVI eryF eryG eryK
pHZT1 pHZT2
Pfeifer BA, et al. Science. 2001 Mar;291(5509):1790-2.
0
2
4
6
8
10
12
14
Erythromycin D Erythromycin C Erythromycin B Erythromycin A
Co
nce
ntr
atio
n (
mg
/L)
Zhang H, et al. Chem Biol. 2010 Nov;17(11):1232-40.9
23Feb11
IPP
OPP
H
HOH
H
H
GGPPSTXS5α
Pyruvate
Glyceraldehyde 3-phosphate
1-deoxy-D-xylulose-5-phosphate (DXP)
2C-methyl-D-erythritol-4-phosphate (MEP) CDP-ME
MECHMBPP
CDP-MEP
DMAPP
DXS DXR IspD IspE
IspF
IspGIspH
IDI
Plasmid, promoter, strain combinations
Ajikumar PK, et al. Science. 2010 Oct 1;330(6000):70-4.10
• 25 predicted polyketide, nonribosomal peptide, and terpene gene clusters
• No products derived from these clusters (besides erythromycin) were indentified on 50 different types of solid and liquid media
23Feb11Oliynyk M, et al. Nat Biotechnol. 2007 Apr;25(4):447-53.
Boakes S, et al. J Mol Microbiol Biotechnol. 2004;8(2):73-80.11
23Feb11
O
O
OH
O
OO
OH
O
O
OH
OHO
N
HO
DEBS3
DEBS2
DEBS1
O
O
OH
OH
OHO
PropionateTailoring Enzymes
PCC Sfp PrpEO
-
O
PCC Sfp PrpE
DEBS3
DEBS2
DEBS1
O
O
OH
OH
OHO
O
O
OH
O
OO
OH
O
OH
OH
OHO
NTailoring Enzymes
Benzoate
O-
O
PropionateO
-
O
C:\Xcalibur\...\bene-6dEB\050310\ben5 5/3/2010 6:02:25 PM ben5
RT: 0.00 - 15.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e Ab
unda
nce
9.91
10.08
10.1310.28
10.659.24 14.9013.4211.649.190.53 8.481.77 2.51 7.452.94 5.855.49 6.584.55
NL:8.74E4m/z= 751.50-752.50 F: MS ben5
ben5 #2629 RT: 9.92 AV: 1 NL: 5.71E4T: ITMS + c ESI Full ms [ 300.00-2000.00]
400 600 800 1000 1200 1400 1600 1800 2000m/z
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
Inte
nsity
752.35
317.29 845.56
356.30 477.36521.37
433.37 565.44
609.42625.40 861.55 1012.82 1526.571127.16 1250.18 1344.47 1626.47 1827.33 1930.37
O
O
OH
O
OO
OH
O
OH
OH
OHO
N
Zhang H, et al. 2011. In preparation.12
23Feb11
T. brevifoliaExtraction
T. canadensisCell-Culture
A. thalianaHeterologous
Tomato Heterologous
S. cerevisiae Heterologous
(Horwitz 1994 Nature) (Ketchum, et al.1998 B&B)
(Besumbes, et al.2004 B&B)
(Kovacs, et al. 2007 Transgenic Res)
(Engels, et al. 2008Metab Eng)
HH
Process Time:Process Time: ~100 years~100 years ~Weeks~Weeks ~Weeks~Weeks ~Weeks~Weeks ~Days~DaysO NH
O
OH
O
OH O
O
OH
O
O
HO
O
O
O
13
~Days~Days
E. coli Heterologous(Ajikumar, et al.
2010 Science)
100 year old tree → 3 kg bark →300 mg Taxol → 1 dose!
100 year old tree → 3 kg bark →300 mg Taxol → 1 dose!
23Feb11 14
Defined Medium: -Potassium Phosphates -Ammonium Sulfate -Glycerol -Magnesium Sulfate -Vitamins
-Media optimization through 96-well format
HPLC-ELSD Analysis
96-well format
LB Medium: -Yeast Extract -NaCl -Tryptone
Defined Medium: -Potassium Phosphates -Ammonium Sulfate -Glycerol -Magnesium Sulfate -Vitamins -Trace Metals
Plackett-Burman Screening:
LB Medium: -Yeast Extract -NaCl -Tryptone
-Trace MetalsTryptone
Trace Metals
6dEB (mg/L)
Enhanced Medium: -Yeast Extract -Tryptone -Glycerol -NaCl -Vitamins -Trace Metals
Pistorino M, Pfeifer BA. Biotechnol Prog. 2009 Sep-Oct;25(5):1364-71.
23Feb11
• Students:– Haoran Zhang– Brett Boghigian– Jiequn Wu– Karin Skalina– Yong Wang
• Funding/Collaborators:– NIH (AI074224; GM085323)– NSF (0712019; 0924699)– Milheim Foundation– Greg Stephanopoulos (MIT)
15