Biomedical subjects

C Khosla

Publications and source records attributed to C Khosla.

At least 73 records · Page 4Linked to original sources

PubMed1996

Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution.

A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria. A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified. Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized. When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively. We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways. If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation. Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules. They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.

Anti-Bacterial Agents↗

PubMed1996

Generation of polyketide libraries via combinatorial biosynthesis.

Polyketides are a family of structurally complex natural products that include a number of important pharmaceuticals. Motivated by the value of these natural products, there has been much research focused on developing guidelines for engineering polyketide synthases (PKSs) to generate novel polyketides. Recent studies have provided interesting insights into the enzymatic specificity of the polyketide synthesis pathway, and have demonstrated that various PKSs can be genetically manipulated to synthesize 'unnatural' polyketide natural products. In this article, we discuss the synthesis of polyketides and polyketide libraries by combinatorial biosynthesis.

Anti-Bacterial Agents↗

PubMed1996

Erythromycin biosynthesis: exploiting the catalytic versatility of the modular polyketide synthase.

DEBS 1 + TE is a recombinant modular polyketide synthase (PKS) in which the first two biosynthetic modules of the 6-deoxyerythronolide B synthase are linked to the thioesterase domain normally found at the C-terminus of DEBS 3. Incubation of DEBS 1 + TE with propionyl-CoA, methylamalonyl-CoA, and NADPH gives the triketide lactone (2R,3S,4S,5R)-2,4-dimethyl-3, 5-dihydroxy-n-heptanoic acid delta-lactone (2), the cyclized form of the normal triketide chain elongation product of DEBS 1. In order to probe the molecular recognition features of the PKS and to explore its synthetic versatility, [2,3-13C2]-(2S,3R)-2-methyl-3-hydroxypentanoyl-NAC thioester (3), an analogue of the normal diketide chain elongation intermediate, and (2RS)-methyl-malonyl-CoA were incubated with DEBS 1 + TE, leading to the formation of the predicted labeled triketide ketolactone [4,5-13C2]-8, as established by 13C NMR analysis and comparison with spectra of synthetic 8. This stereoselective conversion illustrates the potential of using modular PKSs as multifunctional catalysts for the enzymatic synthesis of novel polyketides.

Erythromycin↗

PubMed1996

Combinatorial chemistry and biology: an opportunity for engineers.

Within the past two years, the burgeoning field of combinatorial chemistry and biology has witnessed major advances in both technologies and applications. New ideas have emerged, and continue to be sought, with regard to library design, construction, and analysis. The highly multi-disciplinary nature of the field, together with its need for a systems-based view of pertinent challenges and problems, makes it an ideal area for biochemical engineers.

Biotechnology↗

PubMed1996

A functional chimeric modular polyketide synthase generated via domain replacement.

BACKGROUND: Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzymes that catalyze the biosynthesis of structurally complex and medically important natural products. Active sites within these assemblies are organized into 'modules', such that each module catalyzes the stereospecific addition of a new monomer onto a growing polyketide chain and also sets the reduction level of the beta-carbon atom of the resulting intermediate. The core of each module is made up of a 'reductive segment', which includes all, some, or none of a set of ketoreductase (KR), dehydratase, and enoylreductase domains, in addition to a large interdomain region which lacks overt function but may contribute to structural stability and inter-domain dynamics within modules. The highly conserved organization of reductive segments within modules suggests that they might be able to function in unnatural contexts to generate novel organic molecules. RESULTS: To investigate domain substitution as a method for altering PKS function, a chimeric enzyme was engineered. Using a bimodular derivative of DEBS (DEBS1+TE), the reductive segment of module 2, which includes a functional KR, was replaced with its homolog from module 3 of DEBS, which contains a (naturally occurring) nonfunctional KR. A recombinant strain expressing the chimeric gene produced the predicted ketolactone with a yield (35 %) comparable to that of a control strain in which the KR2 domain was retained but mutationally inactivated. CONCLUSIONS: These results demonstrate considerable structural tolerance within an important segment found in virtually every PKS module. The domain boundaries defined here could be exploited for the construction of numerous loss-of-function and possibly even gain-of-function mutants within this remarkable family of multifunctional enzymes.

Multienzyme Complexes↗

PubMed1996

A new enzyme superfamily - the phosphopantetheinyl transferases.

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

Amino Acid Sequence↗

PubMed1996

Deciphering the biosynthetic origin of the aglycone of the aureolic acid group of anti-tumor agents.

BACKGROUND: Mithramycin, chromomycin, and olivomycin belong to the aureolic acid family of clinically important anti-tumor agents. These natural products share a common aromatic aglycone. Although isotope labeling studies have firmly established the polyketide origin of this aglycone, they do not distinguish between alternative biosynthetic models in which the aglycone is derived from one, two or three distinct polyketide moieties. We set out to determine the biosynthetic origin of this moiety using a recombinant approach in which the ketosynthase and chain-length factor proteins from the antibiotic-producer strain, which determine the chain length of a polyketide, are produced in a heterologous bacterial host. RESULTS: The ketosynthase and chain-length factor genes from the polyketide synthase gene cluster from the mithramycin producer, Streptomyces argillaceus ATCC 12956, and the acyl carrier protein and ketoreductase genes from the actinorhodin polyketide synthase were expressed in Streptomyces coelicolor CH999. The recombinant strain produced a 20-carbon polyketide, comprising the complete backbone of the aglycone of mithramycin. CONCLUSIONS: The aglycone moieties of mithramycin, chromomycin, and olivomycin are derived from a single polyketide backbone. The nascent polyketide backbone must undergo a series of regiospecific cyclizations to form a tetracenomycin-like tetracyclic intermediate. The final steps in the aglycone biosynthetic pathway presumably involve decarboxylation and oxidative cleavage between C-18 and C-19, followed by additional oxidation, reduction, and methylation reactions.

Antibiotics, Antineoplastic↗

PubMed1996

Engineered biosynthesis of novel polyketides: properties of the whiE aromatase/cyclase.

The ORFVI from the cluster of genes, which is responsible for the biosynthesis of the Streptomyces coelicolor spore pigment, the whiE cluster, has been described as a bifunctional aromatase/cyclase. In order to evaluate its potential use for generating novel polyketides, combinations of this gene with those encoding minimal polyketide synthase enzymes with or without a ketoreductase from S. coelicolor A3(2) were constructed and analyzed in vivo. Analysis of the polyketide products generated from these constructs indicates that the whiE-ORFVI enzyme has properties similar to those of TcmN, although the whiE aromatase/cyclase normally acts on a polyketide intermediate that is four carbons longer than the TcmN substrate. The whiE aromatase/cyclase can influence the regiospecificity of the first cyclization of unreduced, but not reduced, backbones and is also responsible for the second ring aromatization. An unusual new polyketide, EM18, was identified which is not seen in equivalent strains expressing the tcmN aromatase/cyclase or the act aromatase genes. The structure of EM18 suggests that the WhiE-ORFVI product might have some unique properties within this family of polyketide synthase subunits, and may therefore be useful in the design of combinatorial biosynthetic strategies.

Anti-Bacterial Agents↗

PubMed1995

Cell-free synthesis of polyketides by recombinant erythromycin polyketide synthases.

Modular polyketide synthases (PKSs) are complex multi-enzyme proteins that catalyse the bacterial biosynthesis of many pharmaceutically useful polyketides. The PKSs are organized into a series of modules, each containing the active catalytic sites required for one step in the synthesis process. Here we report a method for cell-free enzymatic synthesis of 6-deoxyerythronolide B (6-dEB), the parent molecule of the antibiotic erythromycin A, using recombinant 6-deoxyerythronolide B synthase (DEBS), a modular PKS with at least 28 distinct active sites. We have also synthesized in vitro a triketide lactone by using a truncated mutant of DEBS. The availability of such cell-free synthetic routes will allow direct investigation of the structural and mechanistic basis for the unusual combination of high substrate specificity and tolerance to genetic reprogramming found in this enzyme family.

Amino Acid Sequence↗

PubMed1995

Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits.

Recent advances in understanding of bacterial aromatic polyketide biosynthesis allow the development of a set of design rules for the rational manipulation of chain synthesis, reduction of keto groups and early cyclization steps by genetic engineering. The concept of rational design is illustrated by the preparation of Streptomyces strains that produce two new polyketides by expression of combinations of appropriate enzymatic subunits from naturally occurring polyketide synthases. The potential for generating molecular diversity within this class of molecules by genetic engineering is enormous.

Genetic Engineering↗

PubMed1995

Combinatorial biosynthesis of 'unnatural' natural products: the polyketide example.

Multi-enzyme systems, such as those involved in the biosynthesis of polyketides, typically catalyze several distinct reactions that are combined in different ways to generate diverse natural products. The variability available in such systems has not been fully harnessed from nature. It may therefore be possible to create 'unnatural' natural products, which may be as structurally diverse and medicinally valuable as existing natural products, using combinatorial biosynthesis.

Multienzyme Complexes↗

PubMed1995

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2).

The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.

Amino Acid Sequence↗

PubMed1995

Erythromycin biosynthesis. Highly efficient incorporation of polyketide chain elongation intermediates into 6-deoxyerythronolide B in an engineered Streptomyces host.

Feeding of (2S,3R)-[2,3-13C2]-2-methyl-3-hydroxypentanoyl NAC thioester (1a) to the recombinant organism Streptomyces coelicolor CH999/pCK7 harboring the complete set of eryA genes from Saccharopolyspora erythraea encoding the 6-deoxyerythronolide B synthase (DEBS) resulted in the formation of 6-deoxyerythronolide B (2a) labeled with 13C at C-12 and C-13, as evidenced by the appearance of a pair of enhanced and coupled doublets in the 13C NMR spectrum. The level of 13C enrichment was 15-20 atom% 13C, as much as 100 times higher than the usually observed efficiency of incorporation of NAC thioesters into polyketide metabolites. Similar incorporation of (2S,3R)-[3-2H,3-13C]-2-methyl-3-hydroxypentanoyl NAC thioester (1b) gave 6-deoxyerythronolide B (2b) labeled with both 13C and deuterium at C-13. The intact incorporation of both precursors confirms the normal functioning of the recombinant DEBS proteins in the heterologous host.

DNA, Recombinant↗

PubMed1994

Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase.

To identify the minimum set of polyketide synthase (PKS) components required for in vivo biosynthesis of aromatic polyketides, combinations of genes encoding subunits of three different aromatic PKSs--act from Streptomyces coelicolor A3(2) (an actinorhodin producer), fren from Streptomyces roseofulvus (a frenolicin and nanaomycin producer), and tcm from Streptomyces glaucescens (a tetracenomycin producer)--were expressed in a recently developed Streptomyces host-vector system. The "minimal" components (ketosynthase/putative acyltransferase, chain length-determining factor, and acyl carrier protein) were produced with and without a functional polyketide ketoreductase and/or cyclase, and the polyketide products of these recombinant strains were structurally characterized. Several previously identified polyketides were isolated in addition to two previously unidentified polyketides, dehydromutactin and SEK 15b, described here. The results proved that the act cyclase is not required for the biosynthesis of several aberrantly cyclized products that have been previously reported. They are also consistent with earlier conclusions that the minimal PKS controls chain length as well as the regiospecificity of the first cyclization and that it can do so in the absence of both a ketoreductase and a cyclase. However, the ability of the minimal tcm PKS to synthesize two different singly cyclized intermediates suggests that it is unable to accurately control the course of this reaction by itself. In the presence of a downstream enzyme, the flux through one branch of the cyclization pathway increases relative to the other. We propose that these alternative specificities may be due to the ability of downstream enzymes to associate with the minimal PKS and to selectively inhibit a particular branch of the cyclization pathway.

DNA Mutational Analysis↗

PubMed1994

Engineered biosynthesis of novel polyketides: stereochemical course of two reactions catalyzed by a polyketide synthase.

A genetically engineered strain expressing the essential components of the tetracenomycin polyketide synthase (tcm PKS) along with the actinorhodin ketoreductase (act KR) was found to produce two new (diastereomeric) aromatic polyketides, designated RM20b and RM20c, in addition to RM20, whose structure was reported earlier [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550]. Spectroscopic and in vivo isotopic labeling analysis of RM20b and RM20c revealed that their polyketide backbones were identical to that of RM20 with respect to chain length, regiospecificity of ketoreduction, and regiospecificity of the first intramolecular aldol condensation. This is consistent with earlier predictions that the essential components of the PKS--a bifunctional ketosynthase/acyltransferase, a chain length determining factor, and an acyl carrier protein--are responsible for controlling each of these features of the polyketide backbone [McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) Science 262, 1546-1550; McDaniel, R., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1993) J. Am. Chem. Soc. 115, 11671-11675; Fu, H., Ebert-Khosla, S., Hopwood, D. A., & Khosla, C. (1994) J. Am. Chem. Soc. 116, 4166-4170]. In addition, however, RM20b and RM20c possess two unusual features. In both molecules the hydroxyls on sp3 C-9 and C-7 of the first six-membered ring, which arise as a result of ketoreduction and aldol condensation, respectively, are intact, rather than being lost via dehydration. Furthermore, the relative yield of RM20b (in which these hydroxyls are syn) is 7-fold greater than that of RM20c (in which they are anti).(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗

PubMed1994

Engineered biosynthesis of a complete macrolactone in a heterologous host.

Macrocyclic polyketides have been subjects of great interest in synthetic and biosynthetic chemistry because of their structural complexity and medicinal activities. With expression of the entire 6-deoxyerythronolide B synthase (DEBS) (10,283 amino acids) in a heterologous host, substantial quantities of 6-deoxyerythronolide B (6dEB), the aglycone of the macrolide antibiotic erythromycin, and 8,8a-deoxyoleandolide, a 14-membered lactone ring identical to 6dEB except for a methyl group side chain in place of an ethyl unit, were synthesized in Streptomyces coelicolor. The biosynthetic strategy utilizes a genetic approach that facilitates rapid structural manipulation of DEBS or other modular polyketide synthases (PKSs), including those found in actinomycetes with poorly developed genetic methods. From a technological viewpoint, this approach should allow the rational design of biosynthetic products and may eventually lead to the generation of diverse polyketide libraries by means of combinatorial cloning of naturally occurring and mutant PKS modules.

Acyl Coenzyme A↗

PubMed1994

Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific, control of cyclization of an aromatic polyketide precursor.

BACKGROUND: Aromatic polyketide synthases (PKSs) catalyze the formation and cyclization of polyketide chains of variable lengths, generating a family of compounds of proven medical significance. Initial control over the regiospecificity of cyclization is believed to be exercised by the minimal PKS, composed of the three essential components for polyketide biosynthesis, which catalyzes an intramolecular aldol condensation towards the middle of the chain. Subsequent cyclization reactions are either catalyzed by additional components of the PKS, or occur in the absence of specific catalysts. RESULTS: Structural and biosynthetic studies on SEK4b, a novel octaketide product of a minimal PKS, revealed an unusual cyclization pattern. The first cyclization (an aldol condensation) occurs at the methyl end of the unreduced polyketide backbone precursor. This is followed by hemiketal formation and lactonization. The overall structure of SEK4b is similar to that of SEK4, a previously-identified product of the same genetically-engineered strain, differing only in the positions of a methyl and a pyrone group around a common fused-ring system. The biosynthetic pathways of the two molecules are quite different, however. The yield of SEK4b relative to SEK4 is much higher in the absence of PKS components (aromatases and cyclases) acting later in the pathway. CONCLUSIONS: In this cyclization pathway, the regiospecificity of cyclization is not directly controlled by the minimal PKS. Instead, we propose that the enzyme influences cyclization by controlling the timing of chain release. Chain release and cyclization may be concurrent with synthesis. Other PKS subunits appear to stabilize the complex of the PKS with the nascent chain, preventing premature release.

Catalysis↗

PubMed1994

Efficient sampling of protein sequence space for multiple mutants.

We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints. This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems. The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids. Up to 10(8) independent recombinants were generated in 1 ml of an E. coli culture, and this number scales linearly (or better) with increasing volume. To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone. Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes.

Animals↗