Reading “Harnessing the biosynthetic code...” pp 63- 68 “Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “un-natural” natural products” pp 1846-1851

Polyketide Biosynthesis Many bioactive natural products are polyketides, polymers of acetate or other small, oxygenated organic molecules (like propionate, a C3) Includes antibiotics (erythromycin, tetracycline), anticancer drugs (daunomycin), immunosuppresants used after transplants (rapamycin) polypropionate chain (imaginary precursor) the erythromycin aglycone core (missing attached sugars)

Polyketide Biosynthetic Enzymes Polyketides are made by polyketide synthases (PKS’s), huge multi-functional enzymes that act like production lines PKS proteins are organized into linear modules In turn, each successive module: - adds another unit to the chain (elongation) - makes modifications to that piece of the chain - hands the chain off to the next module Function like big molecular assembly lines

Erythromycin Biosynthesis The macrocyclic core of erythromycin is made by a 6-module polyketide synthase called DEBS, producing the lactone 6-dEB Complete synthase is 10,283 amino acids long ! - 3 huge subunits, each containing 2 modules - each module has 3-6 domains, or catalytic sites

A Word About Precursors either can be the initial piece, or primer can be decarboxylated adds 2-carbon units to skeleton plus methyl groups methyl can have either stereochemistry

PKS enzymes: Modularity + Domains Each module consists of a string of catalytic domains - different domains carry out distinct types of reactions EVERY module has 3 core domains (1) ketosynthase (KS) (2) acyl transferase (AT) (3) acyl carrier protein (ACP) together, add 1 block to the growing chain

PKS enzymes I: 3 core domains EVERY module has 3 core domains (1) Ketosynthase (KS) - Accepts polyketide from ACP domain of previous module - Polyketide chain is bound via thioester to Cys -S- (2) Acyl transferase (AT) - Determines which extender unit gets incorporated next (acetate C2, propionate C3, methyl malonate C4) (3) Acyl carrier protein (ACP) - Condenses chain w/ next extender unit, bound as a thioester - Chain is attached via flexible phosphopantetheine linker, ready for the hand-off to the next module

decarboxylation makes the a-carbon of malonate a good nucleophile thioester decarboxylation makes the a-carbon of malonate a good nucleophile the flexible linker phosphopantetheine allows one ACP to pass growing chain on to the next module

skeleton extended by 2-carbon backbone unit (plus -CH3) decarboxylation makes the a-carbon of malonate a good nucleophile the flexible linker phosphopantetheine allows one ACP to pass growing chain on to the next module skeleton extended by 2-carbon backbone unit (plus -CH3)

Polyketide Biosynthetic Enzymes Each cycle adds a 2-carbon extension to chain, introducing a b-keto group and a possible side chain (depending on choice of extender unit by AT domain) Each b-keto group then undergoes none, some, or all in a series of optional reduction steps

PKS enzymes II: optional domains In addition to 3 core domains, each module contains 0-3 optional domains that determine how much the b-keto group added by the previous module gets reduced (4) Ketoreductase (KR) - reduces (5) Dehydratase (DH) - reduces (6) Enoyl reductase (ER) - reduces

Polyketide Biosynthetic Enzymes In addition to 3 core domains, each module contains 0-3 optional domains that determine how much the b-keto group added by the previous module gets reduced A given module will have: none KR KR + DH KR + DH + ER - optional domains control the extent of oxidation throughout the mature polyketide - reductions are done as you go, not after the chain is complete

Closing the Macrocycle Ring Final cyclization is done by the terminal thioesterase domain (TE) - Catalyzes formation of the lactone ring of erythromycin This reaction also proceeds spontaneously, but very slowly

Post-cyclization Modifications Final modifications of 6-dEB are made by downstream enzymes that oxidize C6 and glycosylate (add sugars to) C3 + C5 6-deoxy-erythronolide B (6-dEB) Erythromycin Such post-PKS enzymes are often found in nearby gene clusters

Polyketide Diversity & Biosynthesis The tremendous structural variation found among natural polyketides stems from differences in: (1) choice of starter unit (the “handle” at 1 end of the molecule) (2) choice of extender units (structure + stereochemistry) (3) overall chain length (# of modules) (4) extent of b-keto modification (type of optional domains) (5) regiospecific cyclizations (action of terminal TE domain) (6) downstream (post-PKS) enzymatic modifications [for example, adding sugars]

Engineered Biosynthesis Knowing the function of domains from different modules, and from entirely different organisms, can we use genetic tools to engineer new PKS enzymes? - How much can you alter the sequence of domains & modules and still have a functional enzyme? Are domains really independent of each other? Can we now custom-tailor new polyketides, built to order, by putting together the correct sequence of domains into a recombinant PKS enzyme?

Deletion of Modules from DEBS add TE = more product

Deletion of Modules from DEBS Results showed that domains from 1 module could be fused to domains from another module and produce a functional PKS - TE domain is “flexible” enough to recognize much shorter chains than its normal substrate - TE domain improves enzyme turnover (rate of production)

Deletion of Modules from DEBS Results showed that domains from 1 module could be fused to domains from another module and produce a functional PKS - TE domain is “flexible” enough to recognize much shorter chains than its normal substrate - Domains can be rearranged without loss of activity Next: can you tack on domains from other modular enzymes?

Module Swapping Replaced DEBS “loading” module (which uses propionate) with Non-Ribosomal Peptide loading domain of rifamycin synthase

NRPS loading domain uses benzoic acid as a starting block to prime rifamycin synthesis, not propionate - the fusion protein incorporated benzoate into the expected derivative of 6-dEB, w/ benzene ring in place of ethyl chain

Engineering 6-dEB Derivatives Alter the domains in DEBS Module 2, which controls blue area: 1 2 3 (1) replace DEBS AT domain w/ the AT from rapamycin PKS module 2, which uses malonyl-CoA instead of methyl-malonyl - as predicted, product is missing the methyl group normally found at this position 6-dEB

Engineering 6-dEB Derivatives Alter the domains in DEBS Module 2, which controls blue area: 1 2 3 (2) replace KR domain w/ rap KR/DH from module 4, to reduce the -OH - alcohol moiety replaced w/ alkene carbon 6-dEB

Engineering 6-dEB Derivatives Alter the domains in DEBS Module 2, which controls blue area: 1 2 3 (3) replace KR domain w/ rap KR/DH/ER from module 4, to fully reduce the alkene - alcohol in 6-dEB replaced w/ alkane carbon in the engineered product 6-dEB

Engineering 6-dEB Derivatives 1 2 3 4 5 (4) combinatorial replacement: - replace AT domain w/ rap AT from module 2 - replace KR domain w/ rap KR/DH from module 4 - product is missing the methyl group and has the alkene

Engineering 6-dEB Derivatives 1 2 3 4 5 (5) combinatorial replacement: - replace AT domain w/ rap AT from module 2 - replace KR domain w/ rap KR/DH/ER from module 4 - product is missing the methyl group and has the alkane

Library of “unnatural” natural products made by combinatorial biosynthesis By subbing 5 alternative cassettes into a scaffold of 6 modules, produced >100 macrolides Most could be converted to erythromycin analogs by post-PKS modifier enzymes McDaniel et al. 1999, PNAS 1846-1851

Problems for Genetic Manipulation The DEBS PKS is made by Saccharopolyspora erthraea, which is not a genetically well-understood or easily cultured bacteria - genes are typically cloned onto plasmids in E. coli, DNA circles made for easy insertion of pieces of DNA, that will express cloned protein in bacterial cultures Problems for working with PKS genes: (1) DEBS proteins are so huge, they don’t always fold correctly in E. coli (2) E. coli lacks the appropriate accessory enzymes - missing metabolic precursors (2-methyl-malonate) - no downstream modifying enzymes (glycosylases)

Problems for Genetic Manipulation How do you perform complex genetic rearrangements and end up with an easily cultured organism to grow in mass quantities? Is there a way to incorporate genes from bacteria that have never been cultivated or studied genetically? - Many bacteria, especially marine species and “extreme-ophiles”, cannot be cultured

Host Issues: Solution 1 1994 (Science 265: 509-512) - Make plasmids in E. coli, where genetic manipulation is easy - Then move plasmids into genetically less tractable host, a strain of the PK-producing Streptomyces coelicolor - Not nearly as handy as E. coli, but possesses the accessory enzymes and precursors needed for macrolactone biosynthesis - Produces sizeable amounts of polyketides according to the PKS sequences found on the engineered plasmids

Host Issues: Solution 2 1995 (Nature 378: 263-266) - Cell-free expression system: purify high-mol. weight PKS enzymes from S. coelicolor homogenates - Shown in vitro that the enzymes carry out polyketide synthesis - In fact, proteins were able to incorporate various unnatural substrates into polyketide chains, suggesting great flexibility of these enzymes for substrate recognition

Host Issues: Solution 3 2001 (Science 291: 1790-1792) - Made a metabolically engineered strain of E. coli that can express and fold PKS proteins + produce correct precursors - Move PKS genes off of plasmids, onto the E. coli chromosome - Engineered E. coli produced amounts of 6-dEB comparable to the native bacterium - Shows sophistication of genetic control, flexibility of natural biosynthetic pathways to rational manipulation