Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase

Abstract

Functional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide in Chromobacterium violaceum strain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway in Escherichia coli cells, two essential genes, fabD1 and fabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineered E. coli strains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of the trans complementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.Polyketide synthases (PKSs) share many similarities with fatty acid synthases (FASs) and can be largely classified as type I, II, and III, according to their architecture and mode of catalysis, yet many variants of PKSs exist in nature (5, 27, 29). Type I PKSs are multifunctional enzymes organized into modules, each of which harbors a set of distinct domains responsible for the catalysis of one cycle of polyketide chain elongation. A prototypical type I PKS elongation module contains minimally three integral domains-a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP)-that together catalyze one round of polyketide chain elongation. Optional domains (such as ketoreductase [KR], dehydratase [DH], and enoylreductase [ER]) are found between the AT and ACP domains, which carry out variable steps of reductive modifications on polyketide intermediates. Type II PKSs are multienzyme complexes that carry a single set of catalytic domains acting iteratively. Type III PKSs are chalcone synthase (CHS)-like enzymes that essentially are iteratively acting condensing enzymes. FASs and type I and type II PKSs all use ACPs to tether acyl coenzyme A (CoA) substrates during fatty acid or polyketide biosynthesis, whereas type III PKSs directly condense acyl-CoA substrates without carrier proteins. Prior to biosynthesis, the apo form of ACPs must be activated to the hol...

Identification and characterization of the spiruchostatin biosynthetic gene cluster enable yield improvement by overexpressing a transcriptional activator

“…In particular, the deduced products of eight genes ( spiA , spiB , spiC1 , spiF , spiG , spiH , spiI , and spiJ ) share 57%/74% or higher sequence identity/similarity with their respective counterparts from the FK228 biosynthetic pathway. The dep gene cluster does not contain a gene encoding the necessary phosphopantetheinyl transferase activity for posttranslational modification of carrier proteins [11]; instead, a discrete sfp gene, physically detached from the dep gene cluster, encodes an Sfp-type phosphopantetheinyl transferase for the biosynthesis of FK228 [26]. Similarly, the spi gene cluster does not contain a phosphopantetheinyl transferase-encoding gene either; a search of the draft genome of PsWT identified a discrete candidate pcpS gene that encodes a sole Pseudomonas -type carrier protein synthetase (PCPS) [8] which may provide the missing phosphopantetheinyl transferase activity for the biosynthesis of spiruchostatins.…”

“…Similarly, the spi gene cluster does not contain a phosphopantetheinyl transferase-encoding gene either; a search of the draft genome of PsWT identified a discrete candidate pcpS gene that encodes a sole Pseudomonas -type carrier protein synthetase (PCPS) [8] which may provide the missing phosphopantetheinyl transferase activity for the biosynthesis of spiruchostatins. Additional differences between the two parallel gene clusters or biosynthetic pathways were identified as follows: (i) unlike the FK228 biosynthetic pathway which employs a discrete acyltransferase encoded by either one of the two functionally overlapping house-keeping genes fabD1 and fabD2 located separately from the dep gene cluster [26], spiP appears to encode the acyltransferase activity necessary for in trans complementing the three “AT-less” PKS modules on SpiB, SpiC1 and SpiC2 proteins; (ii) unlike depR which is located downstream of the dep gene cluster and encodes an OxyR-type transcriptional activator [15], spiR is located upstream of the spi gene cluster and encodes an LysR-type transcriptional activator; these two deduced regulatory proteins only share weak sequence homology (25% identity/44% similarity); (iii) there is no depM -equivalent in the spi gene cluster; (iv) there are two copies of depC -like gene in the spi gene cluster, the second copy is fused to DNA encoding a likely inactive epimerase (E) domain and is located after spiDE1 ; (v) a depE -like gene in the spi gene cluster is split into two parts, the first part is fused to the end of spiD , and the second part is transposed to a downstream location between spiG and spiH ; (vi) unlike the pseudogene “ depN” , the deduced protein of spiN appears to be a functional peptidyl carrier protein (PCP) containing a critical serine residue for phosphopantetheinylation, but the role of this PCP is yet to be defined.…”

“…Dried pellet was eluted with 5 ml of ethyl acetate with agitation for 1 hr at 30 °C. One milliliter of the extract was centrifuged at 17,000 × g for 5 min, and 20 μl of the supernatant was injected into a liquid chromatography-mass spectrometer (LC-MSD Trap from Agilent Technologies, Santa Clara, CA) for the identification and quantification of spiruchostatins, similarly as the identification and quantification of FK228 described elsewhere [26]. …”

“…Biosynthesis of spiruchostatins in Pseudomonas sp. Q71576 is thus predicted to be catalyzed by a hybrid nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway similar to that of FK228 [3,15,26]. …”

Genetic Determinants of Reutericyclin Biosynthesis in Lactobacillus reuteri

“…The reutericyclin genomic island does not code for enzymes related to lipid metabolism; the C 10 fatty acid in reutericyclin thus likely originates from the general metabolism. Cross talk between polyketide synthases and fatty acid synthases has been reported in Escherichia coli (47). In bacteria, the 2-decenoic acid component can be synthesized by ␤-hydroxydecanoyl-ACP dehydratase (FabA) or a more common enzyme, hydroxyacyl-ACP dehydratase (FabZ).…”