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C Khosla

Publications and source records attributed to C Khosla.

85 records · Page 5Linked to original sources
PubMed1993

Engineered biosynthesis of novel polyketides.

Polyketide synthases (PKSs) are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in their length and patterns of functionality and cyclization. Many polyketides are valuable therapeutic agents. A Streptomyces host-vector system has been developed for efficient construction and expression of recombinant PKSs. Using this expression system, several novel compounds have been synthesized in vivo in significant quantities. Characterization of these metabolites has provided new insights into key features of actinomycete aromatic PKS specificity. Thus, carbon chain length is dictated, at least in part, by a protein that appears to be distinctive to this family of PKSs, whereas the acyl carrier proteins of different PKSs can be interchanged without affecting product structure. A given ketoreductase can recognize and reduce polyketide chains of different length; this ketoreduction always occurs at the C-9 position. The regiospecificity of the first cyclization of the nascent polyketide chain is either determined by the ketoreductase, or the chain-extending enzymes themselves. However, the regiospecificity of the second cyclization is determined by a distinct cyclase, which can discriminate between substrates of different chain lengths.

Base Sequence↗
PubMed1993

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins.

The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.

Acyl Carrier Protein↗
PubMed1992

Genes for polyketide secondary metabolic pathways in microorganisms and plants.

Recent advances in molecular genetics have led to the isolation, sequencing and functional analysis of genes encoding synthases that catalyse the formation of several classes of polyketides. The structures of the genes and their protein products differ strikingly in the various examples. For Streptomyces aromatic polyketides, exemplified by granaticin and tetracenomycin, the synthases correspond to Type II (bacterial and plant) fatty acid synthases in consisting of distinct proteins for such processes as condensation, acyl carrier function and ketoreduction. In contrast, for actinomycete macrolides such as erythromycin, similar catalytic functions are performed by a set of multifunctional proteins resembling Type I (animal) fatty acid synthases, but with every step in chain-building being catalysed by a different enzymic domain. Penicillium patulum has a simple Type I synthase for 6-methylsalicylic acid. For plant chalcones and stilbenes, a single small polypeptide acts as a condensing enzyme for carbon chain-building and may be unrelated to any of the other polyketide and fatty acid synthases. Thus, although these systems share a common general mechanism of chain assembly, they must differ in the ways that synthase 'programming' has evolved to determine chain length, choice of chain starter and extender units, and handling of successive keto groups during chain assembly, and so control the great diversity of possible chemical products.

Actinomycetaceae↗
PubMed1992

Targeted gene replacements in a Streptomyces polyketide synthase gene cluster: role for the acyl carrier protein.

A methodology was developed to construct any desired chromosomal mutation in the gene cluster that encodes the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2). A positive selection marker (resistance gene) is first introduced by double crossing-over into the chromosomal site of interest by use of an unstable delivery plasmid. This marker is subsequently replaced by the desired mutant allele via a second high-frequency double recombination event. The technology has been used to: (i) explore the significance of translational coupling between two adjacent PKS genes; (ii) prove that the acyl carrier protein (ACP) encoded by a gene in the cluster is necessary for the function of the actinorhodin PKS; (iii) provide genetic evidence supporting the hypothesis that serine 42 is the site of phosphopantetheinylation in the ACP of the actinorhodin PKS; and (iv) demonstrate that this ACP can be replaced by a Saccharopolyspora fatty acid synthase ACP to generate an active hybrid PKS.

Acyl Carrier Protein↗
PubMed1990

Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter.

The oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.

Bacteria↗
PubMed1990

Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli.

We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form. Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid. Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged. This suggests that the net effect of VHb in E. coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism. We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis. These suggest other systems in which cloned VHb may enhance bioprocess productivity.

Bacterial Proteins↗
PubMed1989

Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli. Implications for protein function.

The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.

Amino Acid Sequence↗
PubMed1989

Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli.

The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of significant homology to the catabolite activator protein-binding site near the E. coli lac promoter.

Aerobiosis↗
PubMed1989

A new oxygen-regulated promoter for the expression of proteins in Escherichia coli.

Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.

Bacterial Proteins↗
PubMed1988

Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli.

Rational design of novel as well as improved cellular biocatalysts by genetic manipulation of cellular metabolism has recently attracted considerable interest. A wide range of bacteria have been genetically modified by integrating new enzymatic functions into their metabolic network. A central problem in the aerobic growth of any cell culture is the maintenance of dissolved oxygen (DO) concentrations above growth-limiting levels especially in high cell-density fermentations which are usually of a fed-batch type. The optimal rate of nutrient addition (and consequently the productivity) is ultimately limited by the rate at which cells can aerobically catabolize the carbon source without generating growth-inhibitory metabolites such as lactate and acetate. All approaches thus far have concentrated on improving the oxygen mass transfer rates by manipulating various environmental parameters. We have isolated the gene for a haemoglobin-like molecule, expressed by the aerobic bacterium Vitreoscilla in poorly-oxygenated environments, and expressed it in Escherichia coli. The recombinant cells contain enhanced haem as well as active haemoglobin, and they grow faster and to considerably greater cell densities than comparable plasmid-containing cells which do not express haemoglobin. This haemoglobin increases the rate of oxygen use, especially when dissolved oxygen is less than 5% of air saturation.

Aerobiosis↗
PubMed1988

The Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli.

Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agreement with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The downstream sequence exhibits homology with the 3' end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.

Amino Acid Sequence↗
PubMedDate not supplied

Enhancing the atom economy of polyketide biosynthetic processes through metabolic engineering.

Polyketides, a large family of bioactive natural products, are synthesized from building blocks derived from alpha-carboxylated Coenzyme A thioesters such as malonyl-CoA and (2S)-methylmalonyl-CoA. The productivity of polyketide fermentation processes in natural and heterologous hosts is frequently limited by the availability of these precursors in vivo. We describe a metabolic engineering strategy to enhance both the yield and volumetric productivity of polyketide biosynthesis. The genes matB and matC from Rhizobium trifolii encode a malonyl-CoA synthetase and a putative dicarboxylate transport protein, respectively. These proteins can directly convert exogenous malonate and methylmalonate into their corresponding CoA thioesters with an ATP requirement of 2 mol per mol of acyl-CoA produced. Heterologous expression of matBC in a recombinant strain of Streptomyces coelicolor that produces the macrolactone 6-deoxyerythronolide B results in a 300% enhancement of macrolactone titers. The unusual efficiency of the bioconversion is illustrated by the fact that approximately one-third of the methylmalonate units added to the fermentation medium are converted into macrolactones. The direct conversion of inexpensive feedstocks such as malonate and methylmalonate into polyketides represents the most carbon- and energy-efficient route to these high value natural products and has implications for cost-effective fermentation of numerous commercial and development-stage small molecules.

Acyl Coenzyme A↗