Bioengineering natural product biosynthetic pathways for therapeutic applications

1. Download : Download high-res image (124KB)
2. Download : Download full-size image

Highlights? Highlights of recent methods for enhancing natural product yields, activating cryptic clusters, and biosynthetic engineering of natural products. ? Advances in genomics have allowed identification of numerous cryptic biosynthetic clusters. ? Exploitation of regulatory pathways has led to cryptic cluster activation and increased natural product titres. ? Combinatorial biosynthesis, mutasynthesis and protein engineering have led to new derivatives of natural products with modulated biological activity.     

Discovery and molecular engineering of sugar-containing natural product biosynthetic pathways in actinomycetes

Significant progress has recently been made concerning the engineering of deoxysugar biosynthesis. The biosynthetic gene clusters of several deoxysugars from various polyketides and aminoglycosides-producing microorganisms have been cloned and studied. This review introduces the biosynthetic pathways of several deoxysugars and the generation of novel hybrid macrolide antibiotics via the coexpression of deoxysugar biosynthetic gene cassettes and the substrateflexible glycosyltransferases in a host organism as well as the production of TDP-deoxysugar derivatives via one-pot enzymatic reactions with the identified enzymes. These recent developments in the engineering of deoxysugars biosynthesis may pave the way to create novel secondary metabolites with potential biological activities.     

Strain-specific proteogenomics accelerates the discovery of natural products via their biosynthetic pathways

The use of proteomics for direct detection of expressed pathways producing natural products has yielded many new compounds, even when used in a screening mode without a bacterial genome sequence available. Here we quantify the advantages of having draft DNA-sequence available for strain-specific proteomics using the latest in ultrahigh-resolution mass spectrometry for both proteins and the small molecules they generate. Using the draft sequence of Streptomyces lilacinus NRRL B-1968, we show a >tenfold increase in the number of peptide identifications vs. using publicly available databases. Detected in this strain were six expressed gene clusters with varying homology to those known. To date, we have identified three of these clusters as encoding for the production of griseobactin (known), rakicidin D (an orphan NRPS/PKS hybrid cluster), and a putative thr and DHB-containing siderophore produced by a new non-ribosomal peptide sythetase gene cluster. The remaining three clusters show lower homology to those known, and likely encode enzymes for production of novel compounds. Using an interpreted strain-specific DNA sequence enables deep proteomics for the detection of multiple pathways and their encoded natural products in a single cultured bacterium.     

Rapid characterization and engineering of natural product biosynthetic pathways via DNA assembler

We report a synthetic biology strategy for rapid genetic manipulation of natural product biosynthetic pathways. Based on DNA assembler, this method synthesizes the entire expression vector containing the target biosynthetic pathway and the genetic elements required for DNA maintenance and replication in various hosts in a single-step manner through yeast homologous recombination, offering unprecedented flexibility and versatility in pathway manipulations.     

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.     

High-field 1H NMR studies of prostaglandin H2 and its decomposition pathways

Prostaglandin H2 displays at 500 MHz a detailed 1H-NMR in which all methylene groups are non-equivalent in C6D6 solution. The spectrum was assigned by analogy to isosteric structures. The dissymmetric perturbation and steric hindrance of the bicyclo [2.2.1] core caused by the side-chains provides a rationale for the selective fragmentations which PGH2 undergoes. Purified PGH2 is considerably more robust than previous literature accounts suggest. The following transformations were monitored by 1H-NMR: 1) O-O bond cleavage by Ph3P , 2) aqueous media fragmentation to PGE2 and PGD2, 3) base catalyzed fragmentation to ketoaldehydes , and 4) thermolysis attempts.     

NanoSIMS 50 elucidation of the natural element composition in structures of cyanobacteria and their exposure to halogen compounds

Aims: NanoSIMS (secondary ion mass spectrometry) is a powerful technique for mapping the elemental composition of a variety of small-scale samples (e.g. in Material Research, Cosmochemistry and Geology). However, its analytical features are making it also valuable to address biological questions. We demonstrate the ability of the NanoSIMS 50 to map elements at subcellular lateral resolution (approx. 50 nm) within cyanobacteria (Anabaena sp. and Cylindrospermum alatosporum) and its feasibility to investigate the uptake of bromine-containing substances (NaBr and deltamethrin). Methods and Results: Elemental maps of O, N, P and S were obtained from semi-thin sections of different cell types (chemically fixed and resin-embedded heterocysts, akinetes and vegetative cells). NanoSIMS enabled the detection of various characteristic cell sub-structures and inclusions. A homogenous bromine distribution was detected following NaBr and deltamethrin exposure, at Br-concentrations of 0·05, 0·5 (NaBr) and 0·0025 mmol l⁻¹ (deltamethrin). Conclusions: NanoSIMS allowed study of the mapping of common elements in cyanobacterial cells and the uptake of NaBr and deltamethrin. Significance and Impact of the Study: These results highlight the potential usefulness of NanoSIMS analysis for tracking elements within cell structures at the nanoscale and the ability to detect marker elements of xenobiotic compounds within exposed organisms.     

Directed evolution of enzymes and biosynthetic pathways

Directed evolution is an important tool for overcoming the limitations of natural enzymes as biocatalysts. Recent advances have focused on applying directed evolution to a variety of enzymes, such as epoxide hydrolase, glyphosate N-acetyltransferase, xylanase and phosphotriesterase, in order to improve their activity, selectivity, stability and solubility. The focus has also shifted to manipulating biosynthetic pathways for the production of many naturally synthesized compounds, as well as the production of novel 'unnatural' compounds. A combined directed evolution and computational design approach is becoming increasingly important in exploring enzyme sequence-space and creating improved or novel enzymes. Fueled by recent breakthroughs in genomics and metagenomics, these developments should help expand the use of biocatalysts in industry.     

Applications of genetically-encoded biosensors for the construction and control of biosynthetic pathways

Cells are filled with biosensors, molecular systems that measure the state of the cell and respond by regulating host processes. In much the same way that an engineer would monitor a chemical reactor, the cell uses these sensors to monitor changing intracellular environments and produce consistent behavior despite the variable environment. While natural systems derive a clear benefit from pathway regulation, past research efforts in engineering cellular metabolism have focused on introducing new pathways and removing existing pathway regulation. Synthetic biology is a rapidly growing field that focuses on the development of new tools that support the design, construction, and optimization of biological systems. Recent advances have been made in the design of genetically-encoded biosensors and the application of this class of molecular tools for optimizing and regulating heterologous pathways. Biosensors to cellular metabolites can be taken directly from natural systems, engineered from natural sensors, or constructed entirely in vitro. When linked to reporters, such as antibiotic resistance markers, these metabolite sensors can be used to report on pathway productivity, allowing high-throughput screening for pathway optimization. Future directions will focus on the application of biosensors to introduce feedback control into metabolic pathways, providing dynamic control strategies to increase the efficient use of cellular resources and pathway reliability.     

Microscale methodology for structure elucidation of natural products

Advances in microscale spectroscopic techniques, particularly microcryoprobe NMR, allow discovery and structure elucidation of new molecules down to only a few nanomole. Newer methods for utilizing circular dichroism (CD) have pushed the limits of detection to picomole levels. NMR and CD methods are complementary to the task of elucidation of complete stereostructures of complex natural products. Together, integrated microprobe NMR spectroscopy, microscale degradation and synthesis, are synergistic tools for the discovery of bioactive natural products and have opened new realms for discovery among extreme sources including compounds from uncultured microbes, rare invertebrates and environmental samples.     

Large-scale elucidation of drug response pathways in humans

Elucidating signaling pathways is a fundamental step in understanding cellular processes and developing new therapeutic strategies. Here we introduce a method for the large-scale elucidation of signaling pathways involved in cellular response to drugs. Combining drug targets, drug response expression profiles, and the human physical interaction network, we infer 99 human drug response pathways and study their properties. Based on the newly inferred pathways, we develop a pathway-based drug-drug similarity measure and compare it to two common, gold standard drug-drug similarity measures. Remarkably, our measure provides better correspondence to these gold standards than similarity measures that are based on associations between drugs and known pathways, or on drug-specific gene expression profiles. It further improves the prediction of drug side effects and indications, elucidating specific response pathways that may be associated with these drug properties. Supplementary Material for this article is available at www.liebertonline.com/cmb.     

19F NMR metabolomics for the elucidation of microbial degradation pathways of fluorophenols

Of all NMR-observable isotopes ¹⁹F is the one most convenient for studies on the biodegradation of environmental pollutants and especially for fast initial metabolic screening of newly isolated organisms. In the past decade we have identified the ¹⁹F NMR characteristics of many fluorinated intermediates in the microbial degradation of fluoroaromatics including especially fluorophenols. In the present paper we give an overview of results obtained for the initial steps in the aerobic microbial degradation of fluorophenols, i.e. the aromatic hydroxylation to di-, tri- or even tetrahydroxybenzenes ultimately suitable as substrates for the second step, ring cleavage by dioxygenases. In addition we present new results from studies on the identification of metabolites resulting from reaction steps following aromatic ring cleavage, i.e. resulting from the conversion of fluoromuconates by chloromuconate cycloisomerase. Together the presented data illustrate the potential of the ¹⁹F NMR technique for (1) fast initial screening of biodegradative pathways, i.e. for studies on metabolomics in newly isolated microorganisms, and (2) identification of relatively unstable pathway intermediates like fluoromuconolactones and fluoromaleylacetates. Journal of Industrial Microbiology & Biotechnology (2001) 26, 22–34. Received 20 April 2000/ Accepted in revised form 22 May 2000