Current status, strategies, and potential for the metabolic engineering of heterologous polyketides in Escherichia coli

Heterologous natural product biosynthesis has emerged as a strategy to produce medicinal compounds that pose challenges to conventional production routes. Polyketide compounds, an important class of natural products with wide-ranging therapeutic value, have been heterologously produced through Escherichia coli, presenting new opportunities to realize the medicinal potential of polyketide natural products. However, current production levels are often suboptimal when compared to native strain producers or heterologous theoretical yields. This problem provides an excellent opportunity to apply and further develop current metabolic engineering tools.     

Features and applications of bacterial glycosyltransferases: current state and prospects

The bioactivity of many natural products including valuable antibiotics and anticancer therapeutics depends on their sugar moieties. Changes in the structures of these sugars can deeply influence the biological activity, specificity and pharmacological properties of the parent compounds. The chemical synthesis of such sugar ligands is exceedingly difficult to carry out and therefore impractical to establish on a large scale. Therefore, glycosyltransferases are essential tools for chemoenzymatic and in vivo approaches for the development of complex glycosylated natural products. In the last 10 years, several examples of successful alteration and diversification of natural product glycosylation patterns via metabolic pathway engineering and enzymatic glycodiversification have been described. Due to the relaxed substrate specificity of many sugar biosynthetic enzymes and glycosyltransferases involved in natural product biosynthesis, it is possible to obtain novel glycosylated compounds using different methods. In this review, we would like to provide an overview of recent advances in diversification of the glycosylated natural products and glycosyltransferase engineering.     

Drugs from the seas - current status and microbiological implications

The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.     

萜类合成酶定向进化的新思路

萜类化合物是天然产物中种类最多且主要存在于植物和微生物体内的一类化合物。随着越来越多具有应用价值的萜类化合物被挖掘,其应用前景引起了人们的关注,但由于含量低、提取成本高等缺点,因此制约了萜类化合物的广泛应用。合成生物学的兴起,为异源合成具有应用价值的萜类化合物提供了新思路,使构建定向、高效的微生物细胞工厂成为现实。萜类合成酶常作为萜类化合物异源合成代谢调控的靶酶,但天然的萜类合成酶存在催化效率低、底物专一性差、立体/区域选择性差、稳定性差等问题,严重影响萜类化合物的产量。萜类合成酶的定向进化可以有效地解决上述问题,为实现微生物细胞工厂异源、高效合成萜类化合物奠定基础。本文综述了近年来酶的定向进化技术的最新进展及应用,并提出了萜类合成酶定向进化的策略。     

Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli

Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli.     

Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.     

Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.     

Significant natural product biosynthetic potential of actinorhizal symbionts of the genus frankia, as revealed by comparative genomic and proteomic analyses

Bacteria of the genus Frankia are mycelium-forming actinomycetes that are found as nitrogen-fixing facultative symbionts of actinorhizal plants. Although soil-dwelling actinomycetes are well-known producers of bioactive compounds, the genus Frankia has largely gone uninvestigated for this potential. Bioinformatic analysis of the genome sequences of Frankia strains ACN14a, CcI3, and EAN1pec revealed an unexpected number of secondary metabolic biosynthesis gene clusters. Our analysis led to the identification of at least 65 biosynthetic gene clusters, the vast majority of which appear to be unique and for which products have not been observed or characterized. More than 25 secondary metabolite structures or structure fragments were predicted, and these are expected to include cyclic peptides, siderophores, pigments, signaling molecules, and specialized lipids. Outside the hopanoid gene locus, no cluster could be convincingly demonstrated to be responsible for the few secondary metabolites previously isolated from other Frankia strains. Few clusters were shared among the three species, demonstrating species-specific biosynthetic diversity. Proteomic analysis of Frankia sp. strains CcI3 and EAN1pec showed that significant and diverse secondary metabolic activity was expressed in laboratory cultures. In addition, several prominent signals in the mass range of peptide natural products were observed in Frankia sp. CcI3 by intact-cell matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This work supports the value of bioinformatic investigation in natural products biosynthesis using genomic information and presents a clear roadmap for natural products discovery in the Frankia genus.     

Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes: isolation, characterization and generation of novel glycosylated derivatives

Many bioactive natural products synthesized by actinomycetes are glycosylated compounds in which the appended sugars contribute to specific interactions with their biological target. Most of these sugars are 6-deoxyhexoses, of which more than 70 different forms have been identified, and an increasing number of gene clusters involved in 6-deoxyhexoses biosynthesis are being characterized from antibiotic-producing actinomycetes. Novel glycosylated compounds have been generated by modifying natural deoxysugar biosynthesis pathways in the producer organisms, and/or the simultaneous expression in these strains of selected deoxysugar biosynthesis genes from other strains. Non-producing strains endowed with the capacity to synthesize novel deoxysugars through the expression of engineered deoxysugar biosynthesis clusters can also be used as alternative hosts. Transfer of these deoxysugars to a multiplicity of aglycones relies upon the existence of glycosyltransferases with an inherent degree of 'relaxed substrate specificity'. In this review, we analyze how the knowledge coming out from isolation and characterization of deoxysugar biosynthesis pathways from actinomycetes is being used to produce novel glycosylated derivatives of natural products.     

Enzymes of secondary metabolism and the biosynthesis of macrolide antibiotics

Enzymes participating in the biosynthesis of macrolide antibiotics are reviewed. Enzyme activities are known to play a pivotal role in the formation of biologically active compounds. Hence it is essential to understand these enzymes, their properties and regulation. Macrolide antibiotics represent a relatively compact group of natural products and include several excellent model compounds suitable for enzyme studies that could be generalized to other oligoketide antibiotics.     

Combinatorial biosynthesis of medicinal plant secondary metabolites

Combinatorial biosynthesis is a new tool in the generation of novel natural products and for the production of rare and expensive natural products. The basic concept is combining metabolic pathways in different organisms on a genetic level. As a consequence heterologous organisms provide precursors from their own primary and secondary metabolism that are metabolised to the desired secondary product due to the expression of foreign genes. In this review we discuss the possibilities and limitations of combining genes from different organisms and the expression of heterologous genes. Major focuses are fundamentals of the genetic work, used expression systems and latest progress in this field. Combinatorial biosynthesis is discussed for important classes of natural products, including alkaloids (vinblastine, vincristine), terpenoids (artemisinin, paclitaxel) and flavonoids. The role and importance of today's used host organisms is critically described, and the latest approaches discussed to give an outlook for future trends and possibilities.     

TNT biotransformation: when chemistry confronts mineralization

The recent increase and availability of whole genome sequences have revised our view of the metabolic capabilities of microorganisms. From these data, a large number of orphan biosynthesis pathways have been identified by bio-informatics. Orphan biosynthetic pathways are gene clusters for which the encoded natural product is unknown. It is worthy to note that the number of orphan pathways coding for putative natural products outnumbers by far the number of currently known metabolites for a given organism. Whilst Streptomyces coelicolor was known to produce only 4 secondary metabolites, the genome analysis revealed 18 additional orphan biosynthetic pathways. It is intriguing to note that this is not a particular case because analysis of other microbial genomes originating from myxobacteria, cyanobacteria and filamentous fungi showed the presence of a comparable or even larger number of orphan pathways. The discovery of these numerous pathways represents a treasure trove, which is likely to grow exponentially in the future, uncovering many novel and possibly bio-active compounds. The few natural products that have been correlated with their orphan pathway are merely the tip of the iceberg, whilst plenty of metabolites await discovery. The recent strategies and methods to access these promising hidden natural products are discussed in this review.     

Combinatorial biosynthesis--potential and problems

Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author's laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations.     

Biomimetic syntheses of racemic natural products

Racemic natural products are rarely produced in plants and microorganisms and are thought to be the result of nonenzymatic, spontaneous reactions. These compounds are often highly complex with multiple contiguous chiral centers that present a challenge to organic synthesis. Formation of these racemates often occurs by cyclization reactions that can generate multiple stereocenters from achiral precursors. Biomimetic synthesis of these racemic natural products provides support for their proposed nonenzymatic spontaneous biosynthesis. These elegant syntheses also provide scalable and efficient routes to these complex natural products. Although the number of reported racemic natural products is relatively low, an isolated natural product that has a very small optical rotation has been shown to be a true racemate. Thus, the occurrence of racemic natural products could be more common than thought.     

Biosynthesis of nitrogen-containing natural products,C7N aminocyclitols and bis-indoles,from actinomycetes

Actinomycetes are a major source of bioactive natural products with important pharmaceutical properties. Understanding the natural enzymatic assembly of complex small molecules is important for rational metabolic pathway design to produce “artificial” natural products in bacterial cells. This review will highlight current research on the biosynthetic mechanisms of two classes of nitrogen-containing natural products, C₇N aminocyclitols and bis-indoles. Validamycin A is a member of C₇N aminocyclitol natural products from Streptomyces hygroscopicus. Here, two important biosynthetic steps, pseudoglycosyltranferase-catalyzed C–N bond formation, and C₇-sugar phosphate cyclase-catalyzed divergent carbasugar formation, will be reviewed. In addition, the bis-indolic natural products indolocarbazole, staurosporine from Streptomyces sp. TP-A0274, and rearranged bis-indole violacein from Chromobacterium violaceum are reviewed including the oxidative course of the assembly pathway for the bis-indolic scaffold. The identified biosynthesis mechanisms will be useful to generating new biocatalytic tools and bioactive compounds.     

Metabolic engineering of flavonoids in plants and microorganisms

Over 9,000 flavonoid compounds have been found in various plants, comprising one of the largest families of natural products. Flavonoids are an essential factor in plant interactions with the environment, often serving as the first line of defense against UV irradiation and pathogen attacks. Flavonoids are also major nutritional compounds in foods and beverages, with demonstrated health benefits. Some flavonoids are potent antioxidants, and specific flavonoid compounds are beneficial in many physiological and pharmacological processes. Therefore, engineering of flavonoid biosynthesis in plants or in microorganisms has significant scientific and economical importance. Construction of biosynthetic pathways in heterologous systems offers promising results for large-scale flavonoid production by fermentation or bioconversion. Genomics and metabolomics now offer unprecedented tools for detailed understanding of the engineered transgenic organism and for developing novel technologies to further increase flavonoid production yields. We summarize some of the recent metabolic engineering strategies in plants and microorganisms, with a focus on applications of metabolic flux analysis. We are confident that these engineering approaches will lead to successful industrial flavonoid production in the near future.     

A comprehensive and engaging overview of the type III family of polyketide synthases

Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds.     

Toward biosynthetic design and implementation of Escherichia coli-derived paclitaxel and other heterologous polyisoprene compounds

Escherichia coli offers unparalleled engineering capacity in the context of heterologous natural product biosynthesis. However, as with other heterologous hosts, cellular metabolism must be designed or redesigned to support final compound formation. This task is at once complicated and aided by the fact that the cell does not natively produce an abundance of natural products. As a result, the metabolic engineer avoids complicated interactions with native pathways closely associated with the outcome of interest, but this convenience is tempered by the need to implement the required metabolism to allow functional biosynthesis. This review focuses on engineering E. coli for the purpose of polyisoprene formation, as it is related to isoprenoid compounds currently being pursued through a heterologous approach. In particular, the review features the compound paclitaxel and early efforts to design and overproduce intermediates through E. coli.     

Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis

Alkaloids are a group of approximately 12,000 low molecular weight and nitrogenous secondary metabolites found in 20% of plant species. Their potent biological activity suggests that alkaloids function as defense compounds. Benzylisoquinoline alkaloids (BIAs) are derived from tyrosine and are diversified by an intricate biochemical network of intramolecular coupling, reduction, methylation, hydroxylation, and other reactions to generate the estimated 2500 known structures. Several BIAs are used directly as pharmaceuticals or serve as precursors for the synthesis of semi-synthetic drugs. Plants remain the only economical source for the production of compounds such as morphine and codeine owing to their chemical complexity, which makes de novo synthesis challenging and costly. Much research has been directed toward understanding the biosynthesis of the BIAs and manipulating source plants to increase production of key products and pathway intermediates. However, metabolic engineering experiments often yield unexpected results demonstrating the need for an improved perspective on the biochemistry, regulation, and cell biology of BIA pathways. This review summarizes recent advances in the establishment of predictive metabolic engineering within the context of plant alkaloid biosynthesis.     

Glycosyltransferases involved in the biosynthesis of biologically active natural products that contain oligosaccharides

A unique characteristic of carbohydrates is their structural diversity which is greater than that of many other classes of biological compounds. Carbohydrate-containing natural products show many different biological activities and some of them have been developed as drugs for medical use. The biosynthesis of carbohydrate-containing natural products is catalysed by glycosyltransferases. In this review we will present information on the function of glycosyltransferases involved in the biosynthesis of oligosaccharide antibiotics focusing especially on urdamycins and landomycins, two angucycline antibiotics with interesting antitumor activities. We will also discuss the use of glycosyltransferases in combinatorial biosynthesis to generate new "hybrid" antibiotics.