Engineered Fatty Acid Biosynthesis in
 Streptomyces
 by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

Smirnova, Natalia; Reynolds, Kevin A.

doi:10.1128/jb.183.7.2335-2342.2001

Cited by 28 publications

(21 citation statements)

References 31 publications

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“…This observation is in contrast to the data with the E. coli FabH, which has been shown to have a strong preference for straight-chain substrates such as acetyl and propionyl-CoA [57]. While E. coli does not generate branched-chain fatty acids, production of small levels of them has been observed when the natural FabH is replaced with FabH which can process branched acyl-CoA starter units [58,65]. This production presumably is dependent upon the relaxed acyl group specificity of the E. coli FabG.…”

Section: For the Streptomycetes Fabg Homologs This Results Is Consistecontrasting

confidence: 54%

Enzymatic Control of the Related Pathways of Fatty Acid and Undecylprodiginine Biosynthesis in Streptomyces coelicolor

Singh¹

2000

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Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH- Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonylFabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyliii FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyrylCoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines.The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-

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Section: For the Streptomycetes Fabg Homologs This Results Is Consistecontrasting

confidence: 54%

Enzymatic Control of the Related Pathways of Fatty Acid and Undecylprodiginine Biosynthesis in Streptomyces coelicolor

Singh¹

2000

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“…Despite the large increase in FabH activity in YL/sgFabH relative to M511/pSE4, this phenomenon was not observed. We have previously noted that this phenomenon was also not observed with expression of this S. coelicolor FabH or S. glaucescens FabH in E. coli (6,19).…”

Section: Resultsmentioning

confidence: 94%

“…Streptomyces coelicolor M511 (3) was provided by Greg Challis (University of Warwick, England). The plasmid pSW7, expressing the Streptomyces glaucescens fabH from P ermE* , has been described previously and was generated by inserting the corresponding fabH gene as a BglII fragment into the Streptomyces expression plasmid pSE34 (19). The plasmid pSE4, expressing the E. coli fabH under P ermE* control, was similarly constructed.…”

Section: Methodsmentioning

confidence: 99%

“…Contributing reasons for the lack of significant change in these experiments include the lack of appro-priate precursors (particularly for BCFA biosynthesis in E. coli) and competition with the endogenous FabH. A more significant change in fatty acid profiles was accomplished by plasmid-based expression of an sgFabH mutant bearing a Cys122Gln mutation in Streptomyces glaucescens, where a fivefold increase in SCFAs was observed (19). This change was attributed to the sgFabH mutant being unable to catalyze the normal decarboxylative condensation reaction yet retaining the ability to decarboxylate malonyl-ACP to acetyl-ACP (a presumed precursor for SCFA biosynthesis).…”

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confidence: 99%

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Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

Florova

Reynolds

2005

J Bacteriol

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The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight-and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (ϳ70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a ⌬fabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.

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“…Acetyl-FabC (ACP) was generated by enzymatic decarboxylation of malonyl-FabC using E. coli ␤-ketoacyl acyl carrier protein synthase III (FabH) as described previously (29). Briefly, malonyl-FabC was incubated with 10 nM FabH, and decarboxylation was monitored using ESI-LC-MS as described below.…”

Section: Methodsmentioning

confidence: 99%

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

Whicher

Florova

Sydor

et al. 2011

Journal of Biological Chemistry

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Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a ␤-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.Modular polyketide synthases (PKSs) 5 and non-ribosomal peptide synthetases (NRPSs) are large multienzymes that catalyze the production of a wide range of biologically active compounds currently used as therapies for human diseases (1). Most PKSs and NRPSs are organized as assembly lines in which specific enzymatic domains catalyze elongation or modification of specific pathway intermediates. One key difference between these two types of assembly lines is that PKSs use acyl thioesters as substrates, whereas NRPSs use amino acids. Both PKS and NRPS pathway intermediates are tethered, via a thioester bond, to the phosphopantetheine (Ppant) arms of acyl/ peptidyl carrier protein (ACP/PCP) domains throughout chain assembly. Thioester cleavage, most often catalyzed by a terminating or type I thioesterase (TE I), releases the fully assembled polyketide/peptide from the carrier domain.Prodiginine biosynthesis is directed by the red cluster in Streptomyces coelicolor and involves hybrid NRPS/PKS multienzymes employing an ␣-oxoamine synthase (OAS) in a highly unusual chain release mechanism to assemble undecylprodiginine (1) and its carbocyclic derivative streptorubin B (2) (Fig. 1A) (2-5). The therapeutic potential of prodiginines was demonstrated in prior studies in which analogues of undecylprod...

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Engineered Fatty Acid Biosynthesis in Streptomyces by Altered Catalytic Function of β-Ketoacyl-Acyl Carrier Protein Synthase III

Cited by 28 publications

References 31 publications

Enzymatic Control of the Related Pathways of Fatty Acid and Undecylprodiginine Biosynthesis in <i>Streptomyces coelicolor</i>

Enzymatic Control of the Related Pathways of Fatty Acid and Undecylprodiginine Biosynthesis in <i>Streptomyces coelicolor</i>

Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

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