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)
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
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
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
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
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
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