Dipartimento di Biotecnologie e Scienze della Vita Università degli Studi dell’Insubria Varese

Interuniversitary Research Center of Protein Biotechnology “The Protein Factory”,

Politecnico of Milano and University of Insubria

Industrial Fermentation and Strain Improvement of

Producing Microorganisms

Flavia MarinelliFlavia Marinelli

Dubrovnik Summer School 31-8-2012

ExtractBankExtractBank

HTS

Strain Collection

Strain Collection

Enviromental samples

Selected strains

Flask fermentationMicrobial population

AssayAssay

TargetTarget

Optimized LeadOptimized Lead

TargetTarget

AssayAssay

Extract BankExtract Bank

HITHIT

LeadLead

Screening

Strain CollectionStrain Collection

Biological & ChemicalProfiling, Patent

Clinical candidateClinical candidate

Supply by fermentationIn vivo efficacy, PK, Tox

Analytical or biologicalquantification

Strain preservation

Strain taxonomyand maintenance

Vegetative and fermentation medium optimization

Isolation of spontaneous

mutants & strain purification

Fermentationprocessdefinition

Mutagenesisand selection

ProcessScale-up

Recombination byprotoplast fusion

Metabolic engineering

Strain maintenance

Filamentous actinomycetes and fungi survive aslyophilized for 25 years (MCB)

Good viability and stability at – 80°C (WCB)

Myxobacteria very often do not survive aslyophilized and/or cryopreserved

Cyanobacteria have specific requests of light and air bubling and have to be subcloned each month

Fingerprinting of the producing organism and quality of culture collection

Identity, stability, vitality, reproducibility,

safety and security

Patent, IPR protection, compliance with the regulatoryauthorities, drug master files, FDA approval etc.

Concept of Working Cell Bank and Master Cell Bank

Strain taxonomyand maintenance

Vegetative and fermentation medium optimization

Isolation of spontaneous

mutants & strain purification

Fermentationprocessdefinition

Mutagenesisand selection

ProcessScale-up

Recombination byprotoplast fusion

Metabolic engineering

First Screening of Culture Collection Strains and Literature Media

First Screening of Culture Collection Strains and Literature Media

Black box model of cell metabolism: “secondary metabolite" producers are generally

chemoorganotrophes

Our pannel of media for actinomycetes: rich complex industrial media

Selection of Nutrient SourcesSelection of Nutrient Sources

- One by one change

- Experimental design with the help of statisticalsoftwares

- Biochemical methods: precursors or effectors

Methods to optimize medium composition in fermentation processes

Methods to optimize medium composition in fermentation processes

Simplified Plackett-Burman factorial design experiments: the set up and data analysis was performed using Statgraphics plus 4.1 software

(Statistical Graphics Corp, Herndon, USA)

Volontè, Protein Expression and Purification, 2008

E26 [ g / l ]Yeast autolisate 4NaCl 1.25SBM 20Dextrose 25CaCO3 (B) 5Antifoam (Hodag) 0.3H2O demi

WCB -80°C Reactivation

cultureE26

1 % (v/v)

Vegetative seed

E26

3 % (v/v)

72-96h, 28°C, 200 rpm

Production,

AF3

10 % (v/v)

72-96 h, 28°C

g/L AF3Dextrose 11Sucrose 10Malt extract 25Yeast autolysate 12SBM 30NaCl 0,3Valine 1CaCO3 -Antifoam (Hodag) 0,3H2O demi

BATCH o FED-BATCH fermentationprocess to produce an antibiotic from an

actinomycete FeedingGlucose and precursors

SIROLIMUSChemical structure C51H79NO13

Molecular Weight 914.172 g/mol

Synonyms Rapamycine

Brand Names Rapamune

Producing strains S. hygroscopicus

Clinical Uses

Sirolimus in transplantation

• Kidney

Sirolimus non-transplantation uses

• Anti cancer activity (kidney, brain)

• Antifungal agent

• Starting compound for derivatives

Uni

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ity o

f Ins

ubria

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S

May 2007 - Temsirolimus (trade name Torisel ® - Wyeth Pharmaceutical) was approved bythe US FDA for the treatment of of patients with advanced kidney cancer.

March 2009 - Everolimus (trade name Afinitor ® - Novartis) was approved by the US FDA for the treatment of patients with advanced kidney cancer.

Uni

vers

ity o

f Ins

ubria

-D

BM

SIncrease in Sirolimus production

SIROLIMUS FERMENTATION PROCESS

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d (m

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C.Taurino et al. unpublished results

Free pHpH control at 4.3 + glucose feeding 10 g/(L d)

Free pH pH controlPMVpO2

pH controlledfed-batchfermentationtime course

Precursors & effectors:

Phenyl acetic acid in Penicillin G Phenoxy acetic acid in Penicillin VAminoacids in peptide antibioticsFatty acids or their aminoacidic precusors in lipopeptidessuch as teicoplanin, A40926 and ramoplaninPropanol in erythromycin and spinosynMethionine in cephalosporinBenzyl thiocyanate in tetracyclineDiethylbarbituric acid in rifamycin

Glycopeptide AntibioticTeicoplanin active against

multiresistant gram positive infections

5 µm

Actinoplanesteichomyceticus

Teicoplanin TA2 complex

Targosid:T-A2-1, T-A2-2, T-A2-3, T-A2-4 and T-A 2-5 represent6, 58.3, 7.3, 14.4 and 14 % of the total T-A2

Minutes

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DAD-CH1 254 nmstandard teico 250mg-lstandard teico 250mg-l.dat

TA2-1

T-A2-2

T-A2-3

T-A2-4T-A2-5

Targosid250mg/L

European Pharmacopoeia

Growth and teicoplanin production in 3 L-batch fermentations of A. teichomyceticus ATCC 31121 in TM1

The pH value was naturally self-regulated, whereas the pO2 was controlled over the 20% of saturation by adjusting agitation speed.

In (A), time courses of pH (●, solid line), pO2 (□, dashed line), glucose (▲, solid line), and growth curve measured as dry weight (Δ, dashed line) and PMV (■, solid line). In (B), production of T-A2 measured by HPLC analysis as mg/L (filled bars).

C.Taurino et al. Microbial Cell Factories 2011

Teicoplanin fermentation is reproducible fromminiaturized Duetz System to bioreactor scale

HPLC profile of 120 hour sample showing the following complex composition:

T-A2-1, T-A2-2, T-A2-3, T-A2-4 and T-A 2-5represent 7.3, 60.2, 13.1, 9.1 and 10.3 %of the total T-A2

Control fermentation

2.5 g/L corn oil

2.5 g/L olive oil

2.5 g/L sesame

oil

β -oxidation

Oleic acid C18H34O2Oleic acid C18H34O2 T-A2-3

n C:10:0

T-A2-3

n C:10:0

β -oxidation

Linoleic acid C18H32O2Linoleic acid C18H32O2 T-A2-1

n C:10:1

T-A2-1

n C:10:1

I. Biosynthetic tool : addition of fatty acid as precursors of linear acyl moieties in teicoplanin complex

T-A2-2T-A2-2

T-A2-4

T-A2-5T-A2-5

II. Biosynthetic tool : addition of amino acids as precursors of branched acyl moieties in teicoplanin complex

iso C:10:0

anteiso C:11:0

iso C:11:0

Growth and teicoplanin production in 3 L-batch fermentations of A. teichomyceticus ATCC 31121 in TM1

added with L-valine

The pH value was naturally self-regulated, whereas the pO2 was controlled over the 20% of saturation by adjusting agitation speed.

In (A), time courses of pH (●, solid line), pO2 (□, dashed line), glucose (▲, solid line), and growth curve measured as dry weight (Δ, dashed line) and PMV (■, solid line). In (B), production of T-A2 measured by HPLC analysis as mg/L (filled bars).

C.Taurino et al. Microbial Cell Factories 2011

HPLC profile of 120 hour sample showing the following complex composition:

T-A2-1, T-A2-2, T-A2-3, T-A2-4 and T-A2-5represent 7.3, 73.4 ,10.5 ,2.0 and 6.8 % of the

total T-A2.

Addition of L-valine increases T-A2-2and total productivity

Addition of L-valine increases T-A2-2and total productivity

CombinationCombination of oil and of oil and LL--valinevaline effecteffecttoto modulate modulate complexcomplex compositioncomposition

Control

Corn oil 2,5 g/L

Corn oil 2,5 g/L + L-Val 1 g/L

Minutes

8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0 14,5 15,0 15,5 16,0 16,5

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450DAD-CH1 236 nmWT controllo1 144h 22-07-10WT controllo1 144h 22-07-10

DAD-CH1 236 nmWT + Corn oil 2,5gL 22-07-10WT + Corn oil 2,5gL 22-07-10

DAD-CH1 236 nmWT + Corn oil 2,5gL + Val 1gLWT + Corn oil 2,5gL + Val 1gL

C.Taurino et al. Microbial Cell Factories 2011

Elicitors:

Heavy metals

Rare earth elements(17 elements including scandium, yttrium and lanthanides)

Oils

Microbial cell wall components

Dimethylsulfoxide

S-Adenosylmethionine

N-acetylglucosamine

Stirred tank fermentors up to 200 cubicmeters for antibiotic production

On-line and off-line analyses: analytical tools are extremely

important for the “metabolome” and for the flux analysis

Lab scale

Pilot plant scale