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Results for “biosynthetic pathway engineering”

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PubMed2005

Chemoenzymatic synthesis of cryptophycin/arenastatin natural products.

Microbially derived modular polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways are a rich source of novel natural products. Development of these systems for the engineered biosynthesis of diverse secondary metabolites continues to progress as a robust source of chemical diversity. Recent efforts that employ individual enzymes and catalytic domains for the production or modification of small molecules have met with growing success. In this study, the thioesterase domain from the cryptophycin biosynthetic pathway was isolated and its function evaluated with a series of linear chain elongation intermediates in developing a novel chemoenzymatic synthesis of the cryptophycin/arenastatin class of antitumor agents. The results show the high efficiency of the thioesterase in generating the 16-membered depsipeptide ring of this important natural product system. Moreover, analysis of selected substrates revealed considerable tolerance for structural variation within the seco-cryptophycin unit C beta-alanine residue, but strict structural requirements at the phenyl group position of the unit A delta-hydroxy octadienoate chain elongation intermediates.

Amino Acid Sequence↗

PubMed1998

Quantification of metabolites in the indole alkaloid pathways of catharanthus roseus: implications for metabolic engineering

In this article, we present a review of the current state of metabolic engineering in Catharanthus roseus. A significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, knowledge of the regulation of these pathways is still sparse. Using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. Through precursor feeding studies, the putative rate-limiting steps of the terpenoid pathway in hairy root cultures also have been examined. Relating this knowledge to specific events at the molecular level, and the cloning of corresponding genes are the next key steps in metabolic engineering of the C. roseus alkaloids. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

PubMed1999

Combinatorial chemistry and its applications in agriculture and food.

Combinatorial chemistry has become a major focus of research activity in the pharmaceutical industry for development new therapeutic compounds. The same techniques could be potentially applied to benefit agricultural and food research. This article reviews the various procedures used in combinatorial chemistry, outlines some of the strengths and limitations of the various methods, and proposes potential areas in agriculture and food that could be benefited by this technology. These areas include developing new antimicrobial agents, antioxidants, and other additives, creating antigen-binding molecules for the detection or removal of food pathogens or toxicants, engineering food proteins and enzymes for specific functions, and modifying biosynthetic pathways for the production of novel natural products.

Chemistry, Agricultural↗

PubMed2006

Genetic localization and heterologous expression of validamycin biosynthetic gene cluster isolated from Streptomyces hygroscopicus var. limoneus KCCM 11405 (IFO 12704).

The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of alpha-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s(-1); K(m), 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.

Bacterial Proteins↗

PubMed2006

Combinatorial biosynthesis of novel antibiotics related to daptomycin.

Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. lambda-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.

Amino Acid Sequence↗

PubMed1999

Generation of a novel polysaccharide by inactivation of the aceP gene from the acetan biosynthetic pathway in Acetobacter xylinum.

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with beta-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.

Amino Acid Sequence↗

PubMed2003

Metabolite profiling as a functional genomics tool.

Plants accumulate a very large number of small molecules (phytochemicals) with important functions in the ecology of plants and in the protection against biotic and abiotic stress conditions. Little is known on how phytochemical biosynthetic pathways are regulated, which is a key step to successfully engineering plant metabolism. Plant natural products are usually not essential, and genetic analyses often fail to identify phenotypes associated with the absence of these compounds. We have investigated the use of metabolite profiling of plant cells in culture to establish the function of transcription factors suspected to control plant metabolic pathways.

Cell Culture Techniques↗

PubMed2002

[Gene and gene engineering of carotenoid biosynthesis].

Carotenoids have a range of diverse biological functions and actions, especially playing an important role in human health with provitamin A activity, anti-cancer activity, enhancing immune ability and so on. Human body can't synthesis carotenoids by itself and must absorb them from outside. However, carotenoid contents in many plant are very low, and many kinds of carotenoid are difficult to produce by chemical ways. With the elucidation of carotenoid biosynthetic pathway and cloning genes of relative enzymes from microorganisms and higher plants, it is possible to regulate carotenoid biosynthesis via genetic engineering. This article reviews gene cloning of carotenoid biosynthetic enzymes in microorganisms and higher plants, and advances in the studies of carotenoid production in heterologous microorganisms and crop plants using gene-manipulated carotenoid biosynthesis.

Candida albicans↗

PubMed1996

Production of a polyhydroxyalkanoate biopolymer in insect cells with a modified eucaryotic fatty acid synthase.

A novel pathway for the synthesis of poly-3-hydroxybutyrate has been engineered by simultaneous delivery of two genes into insect cells (Spodoptera frugiperda) by use of individual baculovirus vectors. This system includes expression of a dehydrase-domain mutant rat fatty acid synthase cDNA and the phbC gene encoding polyhydroxyalkanoate synthase from Alcaligenes eutrophus. The dehydrase-deficient fatty acid synthase provides de novo synthesis of R-(-)-3-hydroxybutyryl-coenzyme A as a premature termination product rather than palmityl-coenzyme A, the normal product of wild-type rat fatty acid synthase. High levels of this mutant multifunctional protein provide a suitable precursor pool of R-(-)-3-hydroxybutyryl-coenzyme A for conversion to poly-3-hydroxybutyrate in insect cells coexpressing the phbC gene product. This strategy for redesigning a poly-3-hydroxybutyrate biosynthetic pathway suggests a new method for generating structurally diverse polyhydroxyalkanoates by metabolic engineering.

Animals↗

PubMed2026

Engineering Bacillus Subtilis for Efficient Biosynthesis of Riboflavin: Current Knowledge and Future Perspectives.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Bacillus subtilis↗

PubMed2003

Engineering the aveC gene to enhance the ratio of doramectin to its CHC-B2 analogue produced in Streptomyces avermitilis.

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site-specific mutagenesis and error-prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC-B1, produced relative to the undesired CHC-B2 fermentation component. High-throughput (HTP) screening of cultures grown on solid-phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4-fold improvement in the ratio of doramectin to CHC-B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product.

Base Sequence↗

PubMed2002

The role of glycine betaine in the protection of plants from stress: clues from transgenic plants.

The acclimation of a plant to a constantly changing environment involves the accumulation of certain organic compounds of low molecular mass, known collectively as compatible solutes, in the cytoplasm. The evidence from numerous investigations of the physiology, genetics, biophysics and biochemistry of plants strongly suggests that glycine betaine (GB), an amphoteric quaternary amine, plays an important role as a compatible solute in plants under various types of environmental stress, such as high levels of salts and low temperature. Plant species vary in their capacity to synthesize GB and some plants, such as spinach and barley, accumulate relatively high levels of GB in their chloroplasts while others, such as Arabidopsis and tobacco, do not synthesize this compound. Genetic engineering has allowed the introduction into GB-deficient species of biosynthetic pathways to GB from both micro-organisms and higher plants; this approach has facilitated investigations of the importance of GB in stress protection. In this review, we summarize recent progress in the genetic manipulation of the synthesis of GB, with special emphasis on the relationship between the protective effects of GB in vivo and those documented in vitro.

Journal Article↗

PubMed2001

Combinatorial biosynthesis in microorganisms as a route to new antimicrobial, antitumor and neuroregenerative drugs.

Combinatorial biosynthesis utilizes the genes of biosynthetic pathways that produce microbial products to create novel chemical structures. The engineering of mondular polyketide synthase (PKS) genes has been the major focus of this effort and has led to the production of analogs of macrolide antibiotics like the erythromycins and their derived ketolides, and of the immunosuppressive macrolide FK-520 (Fujisawa Pharmaceutical Co Ltd). Approaches to making analogs of the promising antitumor compounds known as epothilones are also being explored. Lead compounds for further study have resulted and routes to analogs of other pharmacologically important compounds have been established. To facilitate this work, many new tools for manipulating and studying the multifunctional PKSs have been developed including the development of Escherichia coli as a PKS expression last. These developments have resulted in faster ways of engineering PKS to produce new compounds for the development of chemotherapeutic agents from natural products.

Animals↗

PubMed2006

Inactivation of aldehyde dehydrogenase: a key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae.

Production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is restrained by ethanol formation. The first step in the formation of ethanol from acetyl-CoA is catalyzed by aldehyde dehydrogenase (ALDH), an enzyme that competes with 1,3-PD oxidoreductase for the cofactor NADH. This study aimed to improve the production of 1,3-PD by engineering the ethanol formation pathway. An inactivation mutation of the aldA gene encoding ALDH in K. pneumoniae YMU2 was generated by insertion of a tetracycline resistance marker. Inactivation of ALDH resulted in a nearly abolished ethanol formation but a significantly improved 1,3-PD production. Metabolic flux analysis revealed that a pronounced redistribution of intracellular metabolic flux occurred. The final titer, the productivity of 1,3-PD and the yield of 1,3-PD relative to glycerol of the mutant strain reached 927.6 mmol L(-1), 14.05 mmol L(-1)h(-1) and 0.699 mol mol(-1), respectively, which were much higher than those of the parent strain. In addition, the specific 1,3-PD-producing capability (1,3-PD produced per gram of cells) of the mutant strain was 2-fold that of the parent strain due to a lower growth yield of the mutant. By increasing NADH availability, this study demonstrates an important metabolic engineering approach to improve the efficiency of oxidoreduction-coupled bioprocesses.

Aldehyde Dehydrogenase↗

PubMed2005

Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways.

The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery. It enables the (over)production of structurally complex substances through transfer of the biosynthetic genes from the original producer to more amenable heterologous hosts, and provides the basis to generate novel analogs through biosynthetic engineering. Furthermore, the lateral transfer of 'silent' (not expressed under standard laboratory conditions) secondary metabolite pathways or metagenomic DNA into surrogate host strains is expected to yield new, potentially bioactive compounds. This review discusses recent reports on the heterologous production of natural products with emphasis on polyketide and nonribosomally biosynthesized peptide compounds.

Bacteria↗

PubMed2006

Pathways and regulation of bacterial arginine metabolism and perspectives for obtaining arginine overproducing strains.

L-arginine is produced by bacterial fermentation and is consumed in food flavoring and pharmaceutical industries. A better understanding of arginine metabolism in bacteria could be beneficial for a rational design of recombinant L-arginine producers by genetic engineering. This mini-review illustrated the current status of genes and enzymes for arginine metabolism, including biosynthetic pathways, catabolic pathways, uptake and excretion systems, and regulation. The linkage of polyamine and glutamate metabolism to the arginine network was also discussed, followed by a perspective view on how to construct arginine overproducing strains of bacteria with increasing biosynthesis and excretion and decreasing catabolism and uptake.

Arginine↗

PubMed1998

Genetic manipulation of antitumor-agent biosynthesis to produce novel drugs.

Current methods of obtaining novel drugs may be complemented in the near future by the genetic engineering of antitumor-agent biosynthesis in microorganisms. Biosynthetic gene clusters from several antitumor pathways in actinomycetes are presently being characterized and expressed in order to generate novel drugs. Several novel hydroxylated and glycosylated antitumor-drug derivatives have been produced that show a relaxed substrate specificity for secondary-metabolic enzymes, which opens up the possibility of generating novel drugs by genetic manipulation.

Actinomycetales↗

PubMed2001

Disruption of a global regulatory gene to enhance central carbon flux into phenylalanine biosynthesis in Escherichia coli.

Genetic engineering of microbes for commercial metabolite production traditionally has sought to alter the levels and/or intrinsic activities of key enzymes in relevant biosynthetic pathway(s). Microorganisms exploit similar strategies for flux control, but also coordinate flux through sets of related pathways by using global regulatory circuits. We have engineered a global regulatory system of Escherichia coli, Csr (carbon storage regulator), to increase precursor for aromatic amino acid biosynthesis. Disruption of csrA increases gluconeogenesis, decreases glycolysis, and thus elevates phosphoenolpyruvate, a limiting precursor of aromatics. A strain in which the aromatic (shikimate) pathway had been optimized produced twofold more phenylalanine when csrA was disrupted. Overexpression of tktA (transketolase) to increase the other precursor, erythrose-4-phosphate, yielded approximately 1.4-fold enhancement, while both changes were additive. These effects of csrA were not mediated by increasing the regulatory enzymes of phenylalanine biosynthesis. This study introduces the concept of "global metabolic engineering" for second-generation strain improvement.

Bacterial Proteins↗