Natural Product Biotechnology - TU Dortmund Bio-Engineering... · 2014-03-26 · 04.03.2014 1 Pharmaceutical Bio-Engineering Dortmund, 04.03.2014 Laboratory of Chemical Biotechnology

04.03.2014

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Pharmaceutical Bio-Engineering

Dortmund, 04.03.2014

Laboratory of Chemical Biotechnology

Natural Product Biotechnology

Mattijs Julsing

Laboratory of Chemical Biotechnology

Construct, develop, and understand whole microbial cells as productive biocatalysts

Single step/multi step biocatalaysis , pathway engineering, cellular metabolism, process setup

Laboratory of Chemical Biotechnology(Prof. Andreas Schmid)

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Outline

Introduction natural products and their relevance in pharmacy

Introduction in some basic biotechnological concepts

Natural product biotechnology

applying the concepts for terpenoid synthesis / production

examples:artemisininlimonene and perillyl alcohol

How to develop a productive process for the biotechnological production of natural products?

Natural Products

natural product:

a chemical substance produced by a living organism:

a term commonly used for small molecules

a term commonly used in reference to chemical substances found in nature that have distinctive pharmacological effects.

such a substance is considered a natural product even if it can be prepared by total synthesis. (natural product synthesis is an important field in organic chemistry)

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Nature as source for medicines

Throughout history nature has inspired humans and served as a source formedicines

In our society, the relation between medicines and natural products is oftennot visible.

The use of natural products ≠ alternative medicine or homeopathy

…. but do realize

A large fraction of the world population still depends on medicines directly derived from natural sources

In many cultures traditional health care is accepted next to modern health care

Where can we (in western health care) find natural products… even today?

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Papaver somniverum

morfineHO

HO

N

O

H H

codeineHO

N

O

H H

O

Cinchona officinalis (bark)

N

N

CH3OHO H

H

kininequinine

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Cannabis sativa

OH

O

tetrahydrocannabinol (THC)

Natural products in pharmacy

In our society, the relation between medicines and natural products isoften not visible.

Why?

Major developments in organic synthesis during 20st century:chemical industry, natural product synthesis

organic synthesis replaced isolation from natural sources: aspirin

organic synthesis was used to make new semi-synthetic compounds

screening programs to find fully synthetic lead compounds

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Salix alba (white willow) chemical synthesis

OH

O

OH

OH

O

O

O

salicylic acid acetylsalicylic acid

Chemical analogues…. semi-synthetics

Morfine heroine

Penicillin numerous antibiotics

Quinine mefloquine, chloroquine

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New drugs (1981-2006)

Newman and Cragg, J Nat Prod (2007) 70, 461-477

N: natural productND: derived from natural product (semi-synthetic)S: synthetic S*: synthetic, but natural pharmacophoreNM: natural product mimic

What is with more complex and chiral structures?

Plant (natural source): limited access (low content, slow growth, ...)

Organic synthesis often possible, but time-consuming, side-products, expensive, production needs harsh chemicals, etc.

O

O

O

O

O

H3C

CH3

CH3

H H

H

H3CO OCH3

OCH3

O

O

OH

O

O

OO

OH

OOH

O O

O

O

O

OOH

NH

O

ONH

N

N

N

OH

R2H3CO

R1

OCOCH3OHH3COOC

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Biotechnology…. the use of enzymes.

Enzymes can be a good alternative (biotechnological approaches)

The use enzymes for chemical synthesis is called biocatalysis

Enzymes typically catalyze the conversion of specific substrates

highly substrate specific

highly regioselective

highly enantioselective

applied under mild reaction conditions

A need for chemical industries for more environmental friendly production processes:

green chemistry: process designthe use of sustainable substrates‚eco‘-efficiency

Biocatalytic processes: a continuum

Schrewe et al., 2013, Chem Soc Rev

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Isolated enzymes (cell-free systems)

indole oxidation to 2-oxoindole by chloroperoxidase (4.6 g L-1 ; 1h)

Van de Velde et al., 2000, B&B

• stable enzymes• cheap to buy (comercially

available) However, what to do when enzymes :

• have low stability in isolated form• are multicomponent • membrane-bound• are part of multi-step

pathways

Biocatalysis: Free enzymes or whole cells

e.g. lipases

• stable enzymes• easy to produce and

isolate• cheap to buy (comercially

available)

• cells produce the enzyme• enzymes with low stability

in isolated form• multicomponent enzymes• membrane-bound

enzymes• multi-step (or pathways)• cheap substrates

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Advantages Disadvantages

Self-generating: catalyst synthesis and maintenance

Side product formation (overoxidation)

Higher stabilityNo purification required

Product degradationTransport limitations

Co-factor regeneration Product toxicity

Co-expression of redox partners(oxidoreductases)

Whole-cell biocatalysis

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Cofactor regeneration from host cell

hydroxylation of limonene into perillyl alcohol

recombinant E. coli containing CYP153A6

multicomponent enzyme

NADH-dependent: metabolic active cells

O2

OH

NADH H2O NAD+

CYP153A6

Cornelissen et al., 2011, J Ind Microb Biotech

Biotransformation directly linked to metabolism

proline hydroxylation

recombinant E. coli containing proline 4-hydroxylase

2-oxoglutatare as co-substrate directly derived from TCA cylcus

Shibasakic et al., 2000, Biosci Biotech Biochem

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Fermentative processes

synthesis from cheap carbon sources (e.g. glucose)

multi-step

(artificial) biosynthetic pathways

Shibasaki et al., 2000, J Biosci BioengSzczebara et al., 2003, Nat BiotechRo et al., 2006, Nature

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Whole-cell biocatalysis

enzyme

Inhibitions Redox cofactors

Oxygen availability

Substrate uptake

Process setup

Enzyme kineticsSide reactions

Product excretion

Expression

Reaction equilibria

Toxicities

Intermediates

Overoxidation

Whole-cell biocatalysis (II)

enzyme

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Towards a productive process...

Enzyme Engineering

Cell Engineering

Reaction Engineering

http://opm.phar.umich.edu http://www.microfiltindia.com/

Process Engineering

Natural Product Biotechnology

terpenoid bioconversion/production as a well-investigated example

• diverse class of natural products built from isoprene units

• found in (almost) all living species

• >30,000 structures known

• pharmacologically relevant as anticancer, antimalarial and antimicrobialdrugs

• ingredients of plant essential oils, flavours , odors

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Terpene biosynthesis

Ajikumar et al., 2008, Mol. Pharmaceutics

C10

C15

C20

…. + C30, C40,…

C5 + C5

Artemisinin

sesquiterpene lactone

antimalarial compound

Artemisia annua (Chinese plant: quinhao)

the content of artemisinin in the leaves is influenced by many genetic, agricultural and environmental factors (Harvesting season, drying procedures and storage conditions)

total recovery of artemisinin can vary from 0.01 to 1.4% weight% of dry leaf mass

production in S. cerevisiae/E. coli possible?

Delabays et al., 2001, Curr Med Chem Ferreira et al., 2005, Plant Gen Res

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Terpene biosynthesis (II)

C10

C15

C20

…. + C30, C40,…

C5 + C5

synthesized in yeast


Artemisinin (II): metabolic engineering (cell eng.)

sesquiterpene lactone

antimalarial compound

Artemisia annua (Chinese plant: quinhao)

production in S. cerevisiae

Keasling, 2012, Metabolic Engineering

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Artemisinin (III)

recombinant S. cerevisiae

Keasling, 2012, Metabolic EngineeringNewman et al., 2006 B&BRo et al., 2006, Nature

amorphadiene: 27 g L-1

artemisinic acid: 2.5 g L-1

Productive process!!! (not yet artemisinin)

shorter process time compared to plant!!

artemisinic acid: bioprocess

artemisinin: photooxidation

2014: 60 tonnes (33% of market)

400 $ per kg

Nachrichten aus der Chemie, Februar 2014Nature, Vol. 494, February 2013

Artemisinin: the Sanofi-process

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Towards a productive process...

Enzyme Engineering

Cell Engineering



Process Engineering

Perillyl alcohol

in phase II clinical trials as an anti-tumor drug

oxygenated monoterpenoid (C10)

expensive compound

precursor limonene is cheap

OH

perillyl alcohol

O2

OH

NADH H2O NAD+

CYP153A6

limonene

Limonene: oil destillation from orange peals

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whole-cell biocatalyst

cytoplasmatic alkane monooxygenase

three component enzyme

originates from Mycobacterium sp. strain HXN-1500

NADH-dependent: regeneration by bacterial strain

living cells were applied

Biotransformation:

- Pseudomonas putida KT2440

growing on either citrate or glycerol

Figures adapted from: van Beilen and Funhoff, 2005, Curr. Opin. Biotechnol

OH

O2H2O

CYP153A6

Ferredoxin Ferredoxin reductase

2e-

.

NAD+

NADH + H+

2e-

.

Bacterial cytochrome P450 CYP153A6

Biotransformation with growing Pseudomonas cells:

a + b: (S)-limonene (□), (S)-perillyl alcohol (●), perillyl aldehyde (▲), perillic acid (♦), sum of terpenes (×), and biomass ()

c + d: productivity (○) and specific activity (●) profiles

citrate

glycerol

Selection of carbon- and energy source influences biocatalysis

but: side product formationCornelissen et al., 2011, J Ind Microb Biotech

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OHH H O OH O

7% 20%73%

Alcohol Aldehyde Acid

Side products are undesired

- Lower productivity

- Downstream processing

Pseudomonas dehydrogenases

Side product formation – overoxidation by Pseudomonas dehydrogenases

Cornelissen et al., 2011, J Ind Microb Biotech

Escherichia coli W3110

alcohol aldehyde

Change of bacterial host

E. coli: promising enzymatic background

no overoxidation by E. coli

OHH H O OH O

7% 20%73%

Alcohol Aldehyde Acid

Cornelissen et al., 2013, B&B

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E. coli W3110 (pCom8-PFR1500)

CYP153A6 in E. coli in a 2-LP setup


Improving substrate uptake into E. coli

Hydrophobicity of the substrate

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The outer membrane of gram-negative bacteria constitutes an efficient barrier for hydrophobic molecules

known strategy: destabilizing the outer membrane by chemical treatment or permeabilization (unwanted: NADH needed)

Improving substrate uptake into E. coli

Modified from: van den Berg (2010) ChemBioChem, 11, 1339

Facilitated substrate uptake

AlkL:

• hypothetical role in substrate uptake

• direct evidence for alkane substrates

• eight-stranded -barrel protein

• proposed mechanism: bypassing the LPS layer by lateral diffusion of hydrophobic substrates into outer membrane

Model structure based on OmpW from E. coli

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outer membrane

periplasmic space

cytoplasmic membrane

AlkL

O2

OH

NADH H2O NAD+

CYP153A6

Facilitated substrate uptake (II)

E. coli W3110 (pCom8-PFR1500) and AlkL

Effect of AlkL on limonene biotransformation

AlkL improves limonene biotransformation

Improving the CYP153A6 would not make sense: substrate uptake limitation


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Limonene Production E. coli

Can we produce limonene (and perillyl alcohol) in a fermentative process in E. coli?

Terpene biosynthesis (III)

C10

C15

C20

…. + C30, C40,…

C5 + C5

synthesized in yeastand E. coli


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Ajikumar et. al., Mol. Pharmaceutics ,2008

C10

C15

C20

…. + C30, C40,…

C5 + C5


Introducing 2 plant genes: enzymes active?

Enough C5-precursor in E. coli?

Is the volatility of limonene a problem?

Molecular Biology

P

atoB HMGS tHMGR

P

ERG12 ERG8 MVD1 idi pMBI

P

GPPS pET24a:AGPPS2

pBAD/TAC:LS

P

LS

pMevT

Sub-cloned into one single vector

+IPP

DMAPPAcetyl-CoA

Upstream Pathway Downstream Pathway

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Introduction of plant genes: active enzymes

in vitro assay: 0.5 g L-1 protein, 200 µM IPP/DMAPP

+IPP

DMAPP



C10

C15

C20

…. + C30, C40,…

C5 + C5


Introducing 2 plant genes: enzymes active? YES

Enough C5-precursor in E. coli?

Is the volatility of limonene a problem?

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Molecular Biology

P

atoB HMGS tHMGR

P

ERG12 ERG8 MVD1 idi pMBI

P

GPPS pET24a:AGPPS2

pBAD/TAC:LS

P

LS

pMevT

Sub-cloned into one single vector

+IPP

DMAPPAcetyl-CoA

Upstream Pathway Downstream Pathway

Limonene Production E. coli

in vivo experimental setup (screw-capped baffeled shaking flasks)

• 40 ml M9* medium, 1 % (w/v) glucose, 24 h

Genetic and reaction engineering

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C10

C15

C20

…. + C30, C40,…

C5 + C5


Introducing 2 plant genes: enzymes active? YES

Enough C5-precursor in E. coli? NO: YES (increased by genetic eng)

Is the volatity of limonene a problem? YES (reaction engineering)

Advantages:

limonene is volatile: circumvent evaporation

in situ product extraction in organic phase

controlled process

larger scale (in lab 2L, but …..)

decreasing product toxicity

application of high biomass concentrations possible

2-Liquid phase (2-LP) fermentation on reactor scale

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Limonene production

Up to 2.7 g L-1 produced in 42 h: productive process

Aim: Couple with CYP153A6 to produce perillyl alcohol value-added compound

Take home message

Natural products are still of interest for pharmacy

Nature products as source for new bioactive compounds

Derivatization of natural products delivers new drugs

Biotechnological process can be applied to:

produce natural products: e.g. as alternative source

convert natural products (biotransformation)

synthesize new non-natural natural products

by constructing artificial biosynthetic pathways

robust host organisms are a must

a biotechnological process is more than the introduction ofbiosynthetic genes

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Enzyme Engineering

Cell Engineering



Process Engineering

Natural scientist (pharmacists, biologists, biotechnologists), AND bio-engineers

Documents

Natural Product Biotechnology - TU Dortmund Bio-Engineering... · 2014-03-26 · 04.03.2014 1 Pharmaceutical Bio-Engineering Dortmund, 04.03.2014 Laboratory of Chemical Biotechnology