Mattijs Julsing Laboratory of Chemical Biotechnology

Biocatalyst and process development for the biotechnological synthesis of natural products

Natural Product Biotechnology

Laboratory of Chemical Biotechnology

Construct, develop, and understand productive biocatalysts

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

Laboratory of Chemical Biotechnology (Prof. Andreas Schmid)

Construct, develop, and understand productive biocatalysts

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

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)

Papaver somniverum

morfineHO

HO

N

O

H H

codeineHO

N

O

H H

O

Cinchona officinalis (bark)

N

N

CH3OHO H

H

kininequinine

Cannabis sativa

OH

O

tetrahydrocannabinol (THC)

Salix alba (white willow) chemical synthesis

OH

O

OH

OH

O

O

Osalicylic acid acetylsalicylic acid

Chemical analogues…. semi-synthetics

Morfine heroine Penicillin numerous antibiotics Quinine mefloquine, chloroquine

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

OO

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

NOH

R2H3CO

R1

OCOCH3OHH3COOC

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: Bioeconomy: green process design the use of sustainable substrates ‚eco‘-efficiency

The circular economy …. Biobased economy

Enormous challenge and potential for biotechnology

Traditional biotechnology

Fermentation for the production of ‚natural‘ compounds amino acids, organic acids, alcohols, proteins

Metabolic engineering and synthetic biology

Design of cell factories

Biotechnology

Design of cell factories

Microbial life and industrial demands – a happy couple? Many microorganisms are not evolved by nature to convert or produce

volatile, non-charged, hydrophobic or toxic reactants

Willrodt et al., Curr Opin Biotechnol 2015, 31:52–62

Bridging evolution and industrial applicability of microorganisms

Willrodt et al., Curr Opin Biotechnol 2015, 31:52–62

limonene

• toxic to microbial cells (1.8 mmol L-1)

• highly volatile

• insoluble (~45 µmol L-1) plethora of industrial applications

• household chemicals

• lubricants

• biogenic solvents

• jet fuels

• food and fragrance additives oxygenated derivatives

• antiproliferative, anticarcinogenic, antimicrobial properties

Seite 16

OH

O

OHO

OH

carvone

perillic acid menthol

perillyl alcohol

(S)-(-)-limonene (R)-(+)-limonene

Natural product biotechnology: engineering approaches for microbial monoterpenoid production

Schrewe et al. (2013) Chem. Soc. Rev., 42, 6346

Biocatalysis as a continuum

Substrate Product Carbon source Product

Whole cells

Cofactor regeneration via metabolism

CYP153A6:

- cytoplasmatic alkane monooxygenase

- three component enzyme, NADH-dependent

- originates from Mycobacterium sp. strain HXN-1500

- (S)-limonene (S)-perillyl alcohol

Biotransformation using recombinant:

- Pseudomonas putida

- Escherichia coli

O2

OH

NADH H2O NAD+

CYP153A6

Funhoff et al. (2006) J. Bacteriol, 188, 5520-7

Cofactor regeneration via metabolism

Cornelissen et al. (2013) Biotechnol. Bioeng., 110, 1282

• Side product formation by host intrinsic enzymes • Less side products in E. coli • Optimization of enzyme levels could not increase

activities: mass transfer of substrate?

OHH H O OH O

7% 20%73%

Alcohol Aldehyde Acid

Host selection

Mass transfer – substrate uptake

Hydrophobicity of the substrate?

Mass transfer

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)

Schrewe et al. (2013) Chem. Soc. Rev., 42, 6346

hydrophobic

hydrophobic

hydrophilic

Mass transfer – substrate uptake

AlkL

O2

OH

NADH H2O NAD+

CYP153A6

Introduction of outer membrane protein AlkL: • from a specific P. putida strain growing on alkanes • AlkL facilitates alkane uptake

Increased substrate availability at enzyme: – higher productiviy – less side-products

Cornelissen et al. (2013) Biotechnol. Bioeng., 110, 1282 Julsing et al. (2012) AMB, 78, 5724

Facilitated substrate uptake: cell engineering

Fermentative processes

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

Terpene biosynthesis

C10

C15

C20

…. + C30, C40,…

C5 + C5

synthesized in yeast and E. coli

adapted from: Ajikumar et al., (2008), Mol. Pharmaceutics, 5, 167

Willrodt et al., Biotechnol J 2014, 9:1000–1012

E. coli does not natively produce limonene

Low IPP/DMAPP availability via the DOXP pathway

Volatility, toxicity, low solubility

Fermentative limonene production

Willrodt et al., Biotechnol J 2014, 9:1000–1012

E. coli does not natively produce limonene • Synthesis of plant enzymes

In vitro: cell extracts

20°C

25°C

In vivo: No limonene detected

Fermentative limonene production

Willrodt et al., Biotechnol J 2014, 9:1000–1012

E. coli does not natively produce limonene

• Synthesis of plant enzymes

Low IPP/DMAPP availability via the DOXP pathway

• Expression of the yeast mevalonate pathway1

Volatility, toxicity, low solubility

1Martin et al., Nat Biotchnol 2003, 21:796–802

Fermentative limonene production

E. coli does not natively produce limonene

• Synthesis of plant enzymes

Low IPP/DMAPP availability via the DOXP pathway

• Expression of the yeast mevalonate pathway1

Volatility, toxicity, low solubility

• 2-Liquid-phase fermentation

Willrodt et al., Biotechnol J 2014, 9:1000–1012 1Martin et al., Nat Biotchnol 2003, 21:796–802

Diisononyl phtalate

>60-fold increase in limonene production by genetic engineering!

Fermentative limonene production

• Biomass formation as major competitive reaction

• Carbon and energy drain for cellular/redox-equivalent/energy equivalent

regeneration

• Decoupling limonene production from growth?

The idea: Resting cell fermentation • Prohibit microbial growth by omitting essential nutrients

• Resting cells: non growing, but metabolically active

Experimental setup • Normal batch-growth

• wash and resuspend in deficient medium, 1% glucose, 2-LP, 30°C

Resting cell setup: reaction engineering

condition

clim/

µmol Laq-1

clim/

mg Laq-1

Yp/s/

mglim gglc-1

Yp/x/

mglim gcdw-1

growing cells 111.5 ± 17.7 15.2 ± 2.4 1.5 ± 0.2 9.5 ± 1.5

resting cells (KPi) 164.8 ± 18.6 22.4 ± 2.5 11.6 ± 1.3 29.7 ± 3.4

resting cells (M9*-N) 170.1 ± 0.1 23.2 ± 0.0 10.6 ± 1.2 30.7 ± 0.0

resting cells (M9*MgSO4) 277.1 ± 2.4 37.8 ± 0.3 8.0 ± 0.1 50.0 ± 0.4

− Experimentally elaborate,

washing steps

− Limited stability of resting cells

− No cellular regeneration

Reduced competition between biomass

and limonene formation

Individual optimization of growth and

limonene formation

Temperature, medium, biomass

concentration, “non-natural“ conditions

Willrodt et al., Biotechnol Bioeng 2015, submitted

Resting cell setup: reaction engineering

Co-expression of a bacterial CYP450 for selective limonene functionalization Limonene: bulk Perillyl alcohol: fine chemical

POH

HO

perillyl alcohol

HO

HO

• Low CYP expression levels in combination with limonene pathway (<50 nmolCYP gcdw

-1) • No perillyl alcohol formation in

2-LP or monophasic cultivation • Hydroxylation of exogeneously added

limonene

From bulk to fine chemical

Enabling POH production in a 2-LP by genetic engineering?

Willrodt et al., Biotechnol Bioeng 2015, 112:1738-1750

1. Simultaneous expression of limonene pathway and high levels of P450

2. Establish spatial proximity between limonene production and its oxygenation

3. Application of membrane-bound limonene hydroxylase from P. putida1

1Speelmans et al., Appl Microbiol Biotechnol 1998, 50:538-544

E. coli MG1655 with limonene pathway and monooxygenase • M9* minimal medium, 1% glucose, 2-LP, sampling at glucose depletion

Production of perillyl acetate (POHAc) (acetylation of perillyl alcohol by CAT)1

Willrodt et al., Biotechnol Bioeng 2015, 112:1738-1750

Oxygenation is still limited by intracellular limonene availability! 1 Alonso-Gutierrez et al., Metab Eng 2013, 19:33-41

Enabling POH production in a 2-LP by genetic engineering?

Limonene extraction only partially relieved by genetic engineering

New concept: mixed-strain resting cell fermentation

Individual optimization of expression (e.g, strain, temperature)

Controllable stoichiometry

Reduced burden to individual cell

Modular, expandable

Willrodt et al., Biotechnol Bioeng 2015, 112:1738-1750

Mixed-strain setup: reaction engineering

Oxygenation by mixed-culture resting cell fermentation

Willrodt et al., Biotechnol Bioeng 2015, 112:1738-1750

Mixed-strain setup: reaction engineering

Patwhay and cellular engineering • AlkL improved mass transfer • 2 plant enzymes

• C5 precursors: MVA pathway

• >60x increase by genetic engineering Reaction engineering

• 2-LP concept • 2.7 g L-1 with growing cells in bioreactor

• >5x increased yields with resting cells under optimized conditions

• Limonene oxygenation by mixed strain-resting cell fermentation

Process engineering • >150 mg limonene (>96%) isolated

from 2-LP fermentation

Summary

38

Artemisinin: metabolic engineering (cell eng.)

sesquiterpene lactone antimalarial compound Artemisia annua (Chinese plant: quinhao) production in S. cerevisiae

Keasling, 2012, Metabolic Engineering

artemisinic acid: bioprocess artemisinin: photooxidation 2014: 60 tonnes (33% of market) 400 $ per kg

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

Artemisinin: the Sanofi-process

….. Expansion to other terpenes

41

Farnesene as sustainable additive for jet-fuel: 1.75 USD (aim < 1 USD)

Take home messages... Biotechnological approaches have high potential for the production of

natural products

An enzyme catalyzing a specific reaction can be found, but...

Genome sequencing, genetic engineering, and DNA synthesis resulted in a tremendous amount of knowledge and possibilities to develop biocatalytic strategies for chemical synthesis, but...

... the development of a productive biocatalytic process depends on more than genetic engineering only.

Integrated approach on the level of: - genetic engineering - (protein engineering) - metabolic/cellular engineering - reaction engineering - process engineering