Functional Modular Dissection of DEBS1-TE Changes Triketide Lactone Ratios and Provides Insight into Acyl Group Loading, Hydrolysis, and ACP Transfer

Abstract

The DEBS1-TE fusion protein is comprised of the loading module, the first two extension modules, and the terminal TE domain of the Saccharopolyspora erythraea 6-deoxyerythronolide B synthase. DEBS1-TE produces triketide lactones that differ on the basis of the starter unit selected by the loading module. Typical fermentations with plasmid-based expression of DEBS1-TE produce a 6:1 ratio of propionate to isobutyrate-derived triketide lactones. Functional dissection of the loading module from the remainder of DEBS1-TE results in 50% lower titers of triketide lactone and a dramatic shift in the production to a 1:4 ratio of propionate to isobutyrate-derived products. A series of radiolabeling studies of the loading module has shown that transfer from the AT to the ACP occurs much faster for propionate than for isobutyrate. However, the equilibrium occupancy of the AT favors isobutyrate such that propionate is outcompeted for ACP occupancy. Thus, propionyl-ACP is the kinetic product, while isobutyryl-ACP is the thermodynamic product. A slowed transfer from the loading domain ACP to first-extension module KS due to functional dissection of DEBS1-TE allows this isobutyryl-ACP-favored equilibrium to be realized and likely accounts for the observed shift in triketide lactone products.

Accessing Nature’s diversity through metabolic engineering and synthetic biology

“…Combinatorial NRPS/PKS systems have enabled predictable changes to the scaffold core, derived from three programmable inputs into the biosynthesis. The inputs include the following: 1) variable use of organic building blocks such as short-chained acyl-coenzyme A (CoA) molecules or amino acids for the chain elongation step of scaffold synthesis 61 , 63 – 68 ; 2) chain length variations originating from KS and TE engineering 69 – 70 ; and 3) alterations in the reduction program of the scaffold as a result of DH, KR, and ER engineering 71 – 72 . Yan et al exemplified the biosynthetic potential to diversify antimycin (ANT) scaffolds through the metabolic engineering of promiscuous NRPS/PKS enzymes in the ANT-producing Streptomyces sp.…”

Chemoinformatic-guided engineering of polyketide synthases

“…With the introduction of a reductive loop swap, the chimeric enzymes would programmatically produce 2,4-dimethyl pentanoic acid. As in vitro PKS studies have shown divergence from in vivo results 24,25 due to underestimation of factors including limiting substrate, crowding, and solubility, 26 we cloned ten chimeric modules into an E. coli -Streptomyces albus shuttle vector and conjugated it into Streptomyces albus J1074 ( Table S1 ). 27 Following ten-day production runs in a rich medium, cultures of Streptomyces albus harboring each of the constructs were harvested and the supernatants were analyzed with LC-MS for product levels.…”

Engineered polyketides: Synergy between protein and host level engineering

“…While in the native Saccharopolyspora erythrea DEBS is primed exclusively by propionyl-CoA [196] , the LM of DEBS incorporates other priming units in heterologous systems [197] . Depending on the host, TKLs from DEBS1+TE can form via priming by acetyl-CoA (in S. coelicolor [198] and S. venezuelae [197] ) or isobutyryl-CoA (in S. venezuelae [197] ), albeit as minor products. Reynolds and coworkers interrogated the relationship between PKS architecture and starter unit incorporation in variations of DEBS1+TE expressed in S. venezuelae .…”

“…When the loading domain is placed on a separate polypeptide from module 1, there is a resulting kinetic stall. This kinetic stall shifts the product formation towards thermodynamic control, especially when higher levels of isobutyl-CoA are present [197] . This example demonstrates that the apparent selectivity of PKS domains can be altered depending on the architecture of the PKS in concert with the precursor availability.…”