A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

Abstract

Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.

Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I modular polyketide synthases: opportunities and challenges

“…The exchange of ketoreductase (KR) domains in model PKSs has been shown to predictably alter the stereochemistry of both the β-hydroxyl and α-methyl groups. [18][19][20] Using LipPks1+TE as a model, we could successfully alter the 2-methyl stereochemistry from S to R that provided the first experimental evidence of stereochemical conversion of polyketide products from anti to syn in PKS engineering experiments. 20 Although we could not change the β-hydroxy stereochemistry by KR swapping in our model PKS, Leadlay and co-workers 19 had successfully altered the stereochemistry of a β-hydroxy group from S to R by exchanging a KR domain in a different model PKS.…”

Insights into polyketide biosynthesis gained from repurposing antibiotic-producing polyketide synthases to produce fuels and chemicals

“…The exchange of KR domains in model PKSs has been shown to predictably alter the stereochemistry of both the β-hydroxyl and α-methyl groups. 20,21 A naming convention has been established to describe the β-hydroxy (A or B) and α-substituent (1 = nonepimerized, 2 = epimerized) sterochemical outcomes, as shown in Figure 4a. In the aforementioned lipomycin LipPks1+TE studies, we could successfully produce various 3-hydroxy carboxylic acids in vitro (Figure 1b).…”

“…23,24 Importantly, these products could potentially be used to produce bio-plastics in the presence of polyhydroxyalkanoate synthases if the stereochemistry of the hydroxyl group were converted from S to R. 25 To accomplish this, we exchanged the original A2-type KR domain with three different B-type KR domains (B, B1, and B2) that were inferred to yield 3 R-OH groups but, although all of the engineered PKSs were competent in condensation, none of these attempts were successful in producing a (3 R)-hydroxycarboxylic acid. 22 The reason is still unclear, particularly in light of the finding that Leadlay and co-workers 21 had successfully altered the stereochemistry of the 3-OH group by exchanging an A1-type KR with a B1-type KR. We did, however, alter the 2-methyl stereochemistry in the 3-hydroxy acid by employing an A-1 type KR domain in a KR swapping experiment.…”

Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

“…[2][3][4] However, the mechanisms by which certain structural features arise remain poorly understood. For example, most double bonds in these compounds (whether retained or further reduced) have trans (E) geometry, arising from the syn dehydration of 3R alcohols (as in the analogous process in fatty acid synthesis) [5,6] but a significant fraction of polyketides have double bonds with the alternative cis (Z) configuration. In some cases, cis double bonds have been shown to arise from the action of exogenous enzymes, [7][8][9] but for other modular PKSs it has been proposed that cis double bonds arise by syn dehydration of a 3S alcohol by the DH domain.…”