Polyketide Synthase PKS Engineering

In the case of polyketide-derived compounds, the biosynthetic modules that are responsible for the iterative addition of two-carbon units to the nascent chain can be exchanged within sequences to alter both the substitution and oxidation state of the resultant unit. The most studied case of this biosynthetic class is erythromycin. The polyketide assembly of this macrolide antibiotic is illustrated in Figure 6.12, where three multifunctional enzymes DEBS 1, 2, and 3, encoded by eryAl, ll, and Ill genes, assemble a starter unit propionate residue with six propionate extender units to produce the substituted poyketide chain. The chain is indicated in Figure 6.12, growing as the successive condensations add the propionate units. The resulting keto groups are reduced to alcohols by keto-reductase functions (KR, modules 1, 2, 5, and 6), not reduced at all as in module 3 (note the lack of a reductive loop), or fully reduced to the bare methylene by ketone reduction, enolization, and hydrogenation (KR, ER, DH, and module 4). The chain is terminated and cyclized through the action of the final active site in DEBS 3, the thioesterase (TE). Additional tailoring enzymes further modify 6-deoxyerythronolide B, by oxidation, glycosylation, and methylation processes to yield the fully functionalized erythromycin A.

H+ NH+

NAD(P)+

* N^"^COOH H

rapL

L-Pipecolate

Control rapL knockout

FIGURE 6.11 Mutasynthesis of rapamycin analogs. (a) Conversion of lysine to pipecolic acid mediated by rapL. (b) Incorporation of pipecolate in control strain leads to rapamycin. (c) Feeding 4-hydroxyproline to the rapL knockout stain yields a novel analog.

eryAI, ORF C

eryAII, ORF B

eryAIII, ORF A

DEBS1

DEBS2

DEBS3

Load Module 2 Module 4 Module 6

DEBS1

DEBS2

DEBS3

Load Module 2 Module 4 Module 6

Proteins

TE = Thioesterase

6-Deoxyerythronolide B FIGURE 6.12 Polyketide pathway for the biosynthesis of erythromycin.

TE = Thioesterase

6-Deoxyerythronolide B FIGURE 6.12 Polyketide pathway for the biosynthesis of erythromycin.

Genes

Proteins

Poly-ketides

In order to modify the alkyl substitution of the macrocycle, one would alter the specificity of the acyltransferase moiety (AT) that is responsible for recruiting these units to join the growing chain. As shown in Figure 6.13, by swapping the genes that encode for the propionyl group to be added in module 4 for another AT domain that is specific for an acetyl group, it is possible to encode for an erythromycin analog that lacks methyl substitution at position 6. In theory, similar modifications in alkyl substitution and oxidation state can be engineered for the other positions on the macrocycle.

AT module swap

DEBS module 4 ^^ Propionate-specific

AT module swap

DEBS module 4 ^^ Propionate-specific

iERl

DEBS module 4 (A^ Acetate-specific

DEBS module 4 (A^ Acetate-specific

Erythromycin A

6-Desmethyl-erythromycin A

FIGURE 6.13 Substitution of an acetate-specific AT for the propionate-specific AT in DEBS module 4 leads to 6-desmethyl analogs.

Erythromycin A

6-Desmethyl-erythromycin A

FIGURE 6.13 Substitution of an acetate-specific AT for the propionate-specific AT in DEBS module 4 leads to 6-desmethyl analogs.

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