Engineering of Biosynthetic Assembly Lines

Prof. Dr. Markus Nett

Structurally diverse

Highly bioactive

Evolutionarily validated

Natural Products as Sources of Medicinal Drugs

Lovastatin Hypolipidemic

Vancomycin Antibiotic

Morphin Analgesic

Paclitaxel Anti-Cancer

Artemisinin Anti-Malaria

Acarbose Anti-Diabetic

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© Ada Yonath, Weizmann Institute

Streptogramin Antibiotics – A Perfect Fit

Pristinamycin IA Pristinamycin IIA

each compounds binds to the 50S subunit of the bacterial subunit

single agent => moderate bacteriostatic effect

combination => potent bactericidal activity 3

Derivatives Often Make the Better Drugs…

1. (Semi-)Synthetic derivatives

Docetaxel

derived from paclitaxel

improved water solubility

Amoxicillin

derived from benzylpenicillin

extended activity spectrum

oral administration possible

Chloroquine

inspired by quinine

less side-effects

Cheaper production

Simvastatin

inspired by lovastatin

More potent 4

2. Antibody-drug conjugates (ADCs)

Trastuzumab emtansine

derived from maytansinoids

reduced toxicity

treatment of HEr2-positive

metastatic breast cancer

Derivatives Often Make the Better Drugs…

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3. Unnatural natural products

Derivatives Often Make the Better Drugs…

Precursor-directed

biosynthesis Mutasynthesis

Pathway engineering

Combinatorial biosynthesis

?

Hijacking biosynthetic machinery

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DNA mRNA protein natural product

Informational Flow in Biological Systems

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Blueprints for natural product biosynthesis are genetically fixed

Biosynthetic enzymes select and link the building blocks

Building blocks can be small carboxylic acids, amino acids, sugars,…

Clustering of biosynthesis genes (in bacteria and fungi!)

biosynthesis gene cluster

Principles of Natural Product Biosynthesis

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Assembly-Line Enzymology

Polyketide Synthases (PKS)

Nonribosomal Peptide Synthetases (NRPS)

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genes

proteins

modules

domains

eryAI eryAII eryAIII

DEBSI DEBSII DEBSIII

Organization of Biosynthetic Assembly Lines

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Selection and Loading of PKS Building Blocks

building blocks are acyl compounds

the carboxyl group is activated for capture by nucleophiles

the acyl transfer (AT) domain acts as a gatekeeper:

it selects the correct acyl unit for incorporation

it catalyzes a transthiolation reaction

an acyl carrier protein (ACP) domain holds the thioesterified building block

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PKS Chain Extension

a ketoacyl synthase (KS) domain forms a C-C bond via Claisen condensation

afterwards a chain translocation to the KS domain of the next module occurs

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PKS Reductive Chemistry

ketoreductase (KR) domains reduce the β-keto function

dehydratase (DH) domains eliminate water

enoylreductase (ER) domains generate a fully saturated acyl backbone

in polyketide biosynthesis the reductive steps are optional!

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Assembly of the Macrolide Core in Erythromycin

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Nonribosomal Peptide Synthetases

adenylation (A) select and activate amino acids (or similar substrates)

peptidyl carrier protein (PCP) domains hold the thioesterified building blocks

condensation (C) domains catalyze amide bond formations

T. W

eber

& M

. A. M

ahar

iel,

Stru

ctu

re 2

00

1, 9

, R3

-R9

Bleomycin A2

Ciclosporin

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Example: Biosynthesis of Penicillin G

ACV isopenicillin N (IPN)

6-aminopenicillic acid (6-APA)

phenylacetic acid

penicillin G (benzylpenicillin)

❶ ❷

❸

❸

❶ ACV synthetase

❷ IPN synthase

❸ N-acyl transferase

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Precursor-Directed Biosynthesis (PDB)

Advantages Disadvantages

no genetic manipulation required

broad applicability

simple implementation

empiric method

parental compound is produced

alongside any derivative

=> separation needed

figure taken from: A. Kirschning, F. Taft, T. Knobloch, Org Biomol Chem 2007, 5, 3245-3259

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Proof of Concept: Penicillin V

first made in 1951

feeding of Penicillium chrysogenum with 2-phenoxyethanol

more acid-stable than benzylpenicillin

can be given orally

penicillin V penicillin G

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Generation of Unnatural Myxochelins

Myxochelin A is a potent inhibitor of 5-lipoxygenase:

Schieferdecker, König, Koeberle, Dahse, Werz & Nett, J Nat Prod 2015, 78, 335-338

Korp, König, Schieferdecker, Dahse, König, Werz & Nett, ChemBioChem 2015, 16, 2445-2450

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Mutasynthesis

figure taken from: A. Kirschning, F. Taft, T. Knobloch, Org Biomol Chem 2007, 5, 3245-3259

Advantages Disadvantages

no competition with natural precursors

substrate tolerance can be expanded

more predictable than PDB

producing organism must be

amenable to genetic manipulation

biosynthesis genes must have been

identified

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Proof of Concept: Doramectin

avermectins = anthelmintic macrolides produced by Streptomyces avermitilis

feeding of carboxylic acids to a 2-methylbutyrate-deficient mutant yielded

doramectin, being the first commercial drug made by mutasynthesis

later cyclohexylcarboxyl-CoA biosynthesis genes were introduced from S. collinus

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Limitations of PDB and Mutasynthesis

substrate flexibility of biosynthetic enzymes

PKS discriminate malonyl-CoA and methylmalonyl-CoA

=> extender units in polyketide biosynthesis can hardly be replaced

=> only variation of non-acetate/non-propionate starter units

NRPS are in general a little bit more promiscuous,

but they also tolerate only minor substrate changes, e.g. Ile -> Val

Precursor-directed biosynthesis and mutasynthesis

enable only modest degrees of variation.

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Pathway Engineering

X

selective gene or domain inactivations

X

Advantages Disadvantages

may expose functional groups for

further semisynthetic modifications

reduces the number of compounds

produced

same as mutasynthesis

elimination of biosynthetic functions

often affects yield

only few interventions possible

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0.5 μm

Micacocidin – an Antibiotic Against Mycoplasma spp.

mycoplasmas possess no cell wall

M. peumoniae as leading cause of pneumonia in children

2 million cases/year in the US, including 100,000 hospitalizations

macrolide resistance is on the rise

resistance development under therapy

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A Non-Canonical Assembly

involves a hybrid PKS/NRPS assembly line

features an iteratively acting, partially reducing PKS

M. F. Kreutzer, H. Kage, P. Gebhardt, B. Wackler, H. P. Saluz, D. Hoffmeister, M. Nett, Appl. Environ. Microbiol. 2011, 77, 6117-6124

H. Kage, M. F. Kreutzer, B. Wackler, D. Hoffmeister, M. Nett, Chem. Biol. 2013, 20, 764-771

H. Kage, E. Riva, J. S. Parascandolo, M. F. Kreutzer, M. Tosin, M. Nett, Org. Biomol. Chem. 2015, 13, 11414-11417 25

Reconstitution and Minimally Inavsive Mutagenesis

Programming of biosynthetic assembly lines

is highly complex! 26

Combinatorial Biosynthesis

gene cluster A

gene cluster B

Advantages Disadvantages

allows significant structural variation same as mutasynthesis

insufficient understanding of

functional interactions

low success rate

low yields 27

The Story of Daptomycin

lipopeptide antibiotic from Streptomyces roseosporus

MOA: interference with bacterial cell membrane function

discovered at Eli Lilly in the late 1980s

early clinical trials were discontinued due to muscle toxicity

licensing to Cubist Pharmaceuticals in 1997

altering of the dosing regime minimized muscle toxicity

FDA approval in 2003 for the treatment of skin and skin structure infections

caused by Gram-positive bacteria

R. M. Humphries et al., Clin. Microbiol. Rev. 2013, 26, 759-780

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Daptomycin Biosynthesis

the lipopeptide is the product of a giant assembly line

featuring 13 NRPS modules

a related megasynthetase is found in Streptomyces fradiae

figures taken from: R. H. Baltz, ACS Synth Biol 2014, 3, 748-758 and R. H. Baltz, V. Miao, S. K. Wrigley, Nat Prod Rep 2005, 22, 717-741

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Programming of NRPS – Key Insights

substrate specificity is not only encoded in A domains (!)

C and PCP domains are essential for turnover of intermediates

C-A bidomains have co-evolved as functional units

daptomycin NRPS feature 3 subtypes of C domains: LCL – DCL – Starter-C

PCP domains interact with upstream C and A domains

PCP domains interact with downstream (i) C, (ii) E and C, or (iii) TE domains

figure taken from: K. T. Nguyen et al., Proc Natl Acad Sci USA 2006, 103, 17462-17467

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New Antibiotics from Combinatorial Biosynthesis

table taken from: R. H. Baltz, ACS Synth Biol 2014, 3, 748-758

transplanting of individual A

domains was not successful

bi- and tridomain exchanges

are productive

more than 40 novel

lipopeptide antibiotics

derivatives that are barely

affected by surfactant

reduced mammalian toxicity

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Engineering of biosynthetic assembly lines has evolved from feeding experiments

to precise genetic interventions

These techniques provide access to new drugs

Understanding of the underlying biosynthetic logic is instrumental

Summary

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