Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase

BACKGROUND: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system. RESULTS: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate. CONCLUSIONS: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.     

Genetic analysis of wild-isolated Neurospora crassa strains identified as dominant suppressors of repeat-induced point mutation

Repeat-induced point mutation (RIP) in Neurospora results in inactivation of duplicated DNA sequences. RIP is thought to provide protection against foreign elements such as retrotransposons, only one of which has been found in N. crassa. To examine the role of RIP in nature, we have examined seven N. crassa strains, identified among 446 wild isolates scored for dominant suppression of RIP. The test system involved a small duplication that targets RIP to the easily scorable gene erg-3. We previously showed that RIP in a small duplication is suppressed if another, larger duplication is present in the cross, as expected if the large duplication competes for the RIP machinery. In two of the strains, RIP suppression was associated with a barren phenotype--a characteristic of Neurospora duplications that is thought to result in part from a gene-silencing process called meiotic silencing by unpaired DNA (MSUD). A suppressor of MSUD (Sad-1) was shown not to prevent known large duplications from impairing RIP. Single-gene duplications also can be barren but are too short to suppress RIP. RIP suppression in strains that were not barren showed inheritance that was either simple Mendelian or complex. Adding copies of the LINE-like retrotransposon Tad did not affect RIP efficiency.     

Biosynthesis of Yersiniabactin, a complex polyketide-nonribosomal peptide, using Escherichia coli as a heterologous host

The medicinal value associated with complex polyketide and nonribosomal peptide natural products has prompted biosynthetic schemes dependent upon heterologous microbial hosts. Here we report the successful biosynthesis of yersiniabactin (Ybt), a model polyketide-nonribosomal peptide hybrid natural product, using Escherichia coli as a heterologous host. After introducing the biochemical pathway for Ybt into E. coli, biosynthesis was initially monitored qualitatively by mass spectrometry. Next, production of Ybt was quantified in a high-cell-density fermentation environment with titers reaching 67 +/- 21 (mean +/- standard deviation) mg/liter and a volumetric productivity of 1.1 +/- 0.3 mg/liter-h. This success has implications for basic and applied studies on Ybt biosynthesis and also, more generally, for future production of polyketide, nonribosomal peptide, and mixed polyketide-nonribosomal peptide natural products using E. coli.     

Structure-based mutagenesis of the malonyl-CoA:acyl carrier protein transacylase from Streptomyces coelicolor

Malonyl-CoA:acyl carrier protein transacylase (MAT) provides acyl-ACP thioesters for the biosynthesis of aromatic polyketides, and thus is the primary gatekeeper of substrate specificity in type II PKS. A recent report described the X-ray crystal structure of the Streptomyces coelicolor MAT and suggested active site residues which may be important for substrate selectivity [Keatinge-Clay, A. T., et al. (2003) Structure 11, 147-154]. Mutants were made to test the proposed roles of these residues, and the enzymes were characterized kinetically with respect to native and non-native substrates. The activity of the MAT was observed to be greatly attenuated in many of the observed mutants; however, the K(m) for malonyl-CoA was only modestly affected. Our results suggest the MAT uses an active site that is rigorously ordered around the acyl-thioester moiety of the acyl-CoA to facilitate rapid and efficient transacylation to an ACP. Our results also suggest that the MAT does not discriminate against alpha-substituted acyl-CoA thioesters solely on the basis of substrate size.     

Catalysis,specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.     

Measurement of gene expression following cryogenic mu-CT scanning of human iliac crest biopsies

An important consideration in interpreting indices of gene expression in human bone is relating mRNA levels to functional endpoints such as bone architecture. In the present study, a method was developed for quantitative measurement of gene expression and bone morphology in the same specimen. Three-dimensional images of iliac crest bone biopsies from healthy premenopausal women were obtained using a novel high resolution cryogenic mu-CT scanner. RNA was isolated from the biopsies and mRNA levels were measured for genes related to bone metabolism. The gene expression profile and variability of expression within iliac crest biopsies of women was similar to human osteoblastic cell lines and rat long bones. mRNA for alkaline phosphatase, bone matrix proteins, and selected cytokines and cytokine receptors were consistently detected in biopsies. As previously shown in rat bone, there was a tight correlation between mRNA levels for type 1 collagen and osteonectin, a weaker correlation between type 1 collagen and osteocalcin and no correlation between bone matrix proteins and alkaline phosphatase. The relative abundance of the mRNA for the three most prevalent transforming growth factor-beta (TGF-beta) isoforms in bone (TGF-beta(1)> TGF-beta(3)> TGF-beta(2)) was the same as the known abundance of the corresponding TGF-beta peptides in bone matrix. The results demonstrate the feasibility of analyzing the three-dimensional architecture of a bone biopsy using cryogenic mu-CT imaging and then measuring expression of genes related to bone cell function within the same specimen following RNA extraction and analysis.     

Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.     

Engineering of molecular and cellular biocatalysts: selected contributions by James E. Bailey

James (Jay) E. Bailey was a pioneer in biotechnology and biochemical engineering. During his 30 years in academia he made seminal contributions to many fields of chemical engineering science, including catalysis and reaction engineering, bioprocess engineering, mathematical modeling of cellular processes, recombinant DNA technology, enzyme engineering, and metabolic engineering. This article celebrates some of his contributions to the engineering of molecular and cellular biocatalysts, and identifies the influence he had on current and future research in biotechnology.     

The loading and initial elongation modules of rifamycin synthetase collaborate to produce mixed aryl ketide products

Rifamycin synthetase assembles the chemical backbone that members of the rifamycin family of antibiotics have in common. The synthetase contains a mixed biosynthetic interface between its loading module, which uses a nonribosomal peptide synthetase mechanism, and its initial elongation module, which uses a polyketide synthase mechanism. Biochemical studies of the loading and initial elongation modules of rifamycin synthetase reveal that this bimodular protein (LM-M1) catalyzes the formation of the phenyl ketide 3-hydroxy-2-methyl-3-phenylpropionate via a series of reactions that require benzoate, Mg.ATP, methylmalonyl-CoA, and NADPH. The overall rate of phenyl ketide production appears to be determined by the covalent loading of benzoate onto LM-M1, rather than by subsequent steps such as intermodular transfer of benzoate or condensation of benzoate and methylmalonate. Substituted benzoates that have previously been shown to be substrates for the loading module alone can also be incorporated into the corresponding aryl ketides by LM-M1, suggesting that the bimodular protein has a broad substrate tolerance. Discrimination between the substituted benzoates appears to reside in the benzoate loading reaction, and preincubation of LM-M1 with substituted benzoates and Mg.ATP allows faster downstream reactions to be unmasked. LM-M1 may be a useful biochemical system for exploring interactions between nonribosomal peptide synthetase and polyketide synthase modules.     

Hemoglobin Biosynthesis in Vitreoscilla stercoraria DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene

In the strictly aerobic, gram-negative bacterium Vitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure of Vitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla, V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation of V. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within the V. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscilla strain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species of Vitreoscilla. The relatively slower autooxidation rate of V. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercoraria DW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.