Engineering Of Biosynthetic Pathways Research Articles

Recent advances in natural products exploitation in Streptomyces via synthetic biology.

Natural products of microbial origin have proven to be the wellspring of clinically useful compounds for human therapeutics. Streptomyces species are predominant sources of bioactive compounds, most of which serve as potential drug candidates. While the exploitation of natural products has been severely reduced over the past two decades, the growing crisis of evolution and dissemination of drug resistant pathogens have again attracted great interest in this field. The emerging synthetic biology has been heralded as a new bioengineering platform to discover novel bioactive compounds and expand bioactive natural products diversity and production. Herein, we review recent advances in the natural products exploitation of Streptomyces with the applications of synthetic biology from three major aspects, including recently developed synthetic biology tools, natural products biosynthetic pathway engineering strategies as well as chassis host modifications.

Transcriptome analysis and targeted metabolic profiling for pathway elucidation and identification of a geraniol synthase involved in iridoid biosynthesis from Gardenia jasminoides

Iridoid compounds have been reported to be accumulated in the dried fruits of Gardenia jasminoides (Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.

The Effect of Divalent Cations on the Thermostability of Type II Polyketide Synthase Acyl Carrier Proteins.

The successful engineering of biosynthetic pathways hinges on understanding the factors that influence acyl carrier protein (ACP) stability and function. The stability and structure of ACPs can be influenced by the presence of divalent cations, but how this relates to primary sequence remains poorly understood. As part of a course-based undergraduate research experience, we investigated the thermostability of type II polyketide synthase (PKS) ACPs. We observed an approximate 40 °C range in the thermostability amongst the 14 ACPs studied, as well as an increase in stability (5 - 26 °C) of the ACPs in the presence of divalent cations. Distribution of charges in the helix II-loop-helix III region was found to impact the enthalpy of denaturation. Taken together, our results reveal clues as to how the sequence of type II PKS ACPs relates to their structural stability, information that can be used to study how ACP sequence relates to function.

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown. Based on gene inactivation and chemical investigation of three mutant strains, we investigated the tailoring steps catalyzed by two cytochromes P450 (P450s), LnmA and LnmZ, in LNM biosynthesis. Our studies revealed that (i) LnmA and LnmZ regio- and stereoselectively hydroxylate the C-8 and C-4' positions, respectively, on the scaffold of LNM; (ii) both LnmA and LnmZ exhibit substrate promiscuity, resulting in multiple LNM analogs from several shunt pathways; and (iii) the C-8 and C-4' hydroxyl groups play important roles in the cytotoxicity of LNM analogs against different cancer cell lines, shedding light on the structure-activity relationships of the LNM scaffold and the LNM-type natural products in general. These studies set the stage for future biosynthetic pathway engineering and combinatorial biosynthesis of the LNM family of natural products for structure diversity and drug discovery.

Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer

Flowers of the hop plant provide both bitterness and “hoppy” flavor to beer. Hops are, however, both a water and energy intensive crop and vary considerably in essential oil content, making it challenging to achieve a consistent hoppy taste in beer. Here, we report that brewer’s yeast can be engineered to biosynthesize aromatic monoterpene molecules that impart hoppy flavor to beer by incorporating recombinant DNA derived from yeast, mint, and basil. Whereas metabolic engineering of biosynthetic pathways is commonly enlisted to maximize product titers, tuning expression of pathway enzymes to affect target production levels of multiple commercially important metabolites without major collateral metabolic changes represents a unique challenge. By applying state-of-the-art engineering techniques and a framework to guide iterative improvement, strains are generated with target performance characteristics. Beers produced using these strains are perceived as hoppier than traditionally hopped beers by a sensory panel in a double-blind tasting.

Living GenoChemetics by hyphenating synthetic biology and synthetic chemistry in vivo

Marrying synthetic biology with synthetic chemistry provides a powerful approach toward natural product diversification, combining the best of both worlds: expediency and synthetic capability of biogenic pathways and chemical diversity enabled by organic synthesis. Biosynthetic pathway engineering can be employed to insert a chemically orthogonal tag into a complex natural scaffold affording the possibility of site-selective modification without employing protecting group strategies. Here we show that, by installing a sufficiently reactive handle (e.g., a C–Br bond) and developing compatible mild aqueous chemistries, synchronous biosynthesis of the tagged metabolite and its subsequent chemical modification in living culture can be achieved. This approach can potentially enable many new applications: for example, assay of directed evolution of enzymes catalyzing halo-metabolite biosynthesis in living cells or generating and following the fate of tagged metabolites and biomolecules in living systems. We report synthetic biological access to new-to-nature bromo-metabolites and the concomitant biorthogonal cross-coupling of halo-metabolites in living cultures.

How To Make a Glycopeptide: A Synthetic Biology Approach To Expand Antibiotic Chemical Diversity.

Modification of natural product backbones is a proven strategy for the development of clinically useful antibiotics. Such modifications have traditionally been achieved through medicinal chemistry strategies or via in vitro enzymatic activities. In an orthogonal approach, engineering of biosynthetic pathways using synthetic biology techniques can generate chemical diversity. Here we report the use of a minimal teicoplanin class glycopeptide antibiotic (GPA) scaffold expressed in a production-optimized Streptomyces coelicolor strain to expand GPA chemical diversity. Thirteen scaffold-modifying enzymes from 7 GPA biosynthetic gene clusters in different combinations were introduced into S. coelicolor, enabling us to explore the criteria for in-cell GPA modification. These include identifying specific isozymes that tolerate the unnatural GPA scaffold and modifications that prevent or allow further elaboration by other enzymes. Overall, 15 molecules were detected, 9 of which have not been reported previously. Some of these compounds showed activity against GPA-resistant bacteria. This system allows us to observe the complex interplay between substrates and both non-native and native tailoring enzymes in a cell-based system and establishes rules for GPA synthetic biology and subsequent expansion of GPA chemical diversity.

In silico methods for linking genes and secondary metabolites: The way forward

In silico methods for linking genomic space to chemical space have played a crucial role in genomics driven discovery of new natural products as well as biosynthesis of altered natural products by engineering of biosynthetic pathways. Here we give an overview of available computational tools and then briefly describe a novel computational framework, namely retro-biosynthetic enumeration of biosynthetic reactions, which can add to the repertoire of computational tools available for connecting natural products to their biosynthetic gene clusters. Most of the currently available bioinformatics tools for analysis of secondary metabolite biosynthetic gene clusters utilize the “Genes to Metabolites” approach. In contrast to the “Genes to Metabolites” approach, the “Metabolites to Genes” or retro-biosynthetic approach would involve enumerating the various biochemical transformations or enzymatic reactions which would generate the given chemical moiety starting from a set of precursor molecules and identifying enzymatic domains which can potentially catalyze the enumerated biochemical transformations. In this article, we first give a brief overview of the presently available in silico tools and approaches for analysis of secondary metabolite biosynthetic pathways. We also discuss our preliminary work on development of algorithms for retro-biosynthetic enumeration of biochemical transformations to formulate a novel computational method for identifying genes associated with biosynthesis of a given polyketide or nonribosomal peptide.

Modulation of FadR binding capacity for acyl-CoA fatty acids through structure-guided mutagenesis.

FadR is a versatile global regulator in Escherichia coli that controls fatty acid metabolism and thereby modulates the ability of this bacterium to grow using fatty acids or acetate as the sole carbon source. FadR regulates fatty acid metabolism in response to intra-cellular concentrations of acyl-CoA lipids. The ability of FadR to bind acyl-CoA fatty acids is thus of significant interest for the engineering of biosynthetic pathways for the production of lipid-based biofuels and commodity chemicals. Based on the available crystal structure of E. coli bound to myristoyl-CoA, we predicted amino acid positions within the effector binding pocket that would alter the ability of FadR to bind acyl-CoA fatty acids without affecting DNA binding. We utilized fluorescence polarization to characterize the in vitro binding properties of wild type and mutant FadR. We found that a Leu102Ala mutant enhanced binding of the effector, likely by increasing the size of the binding pocket for the acyl moiety of the molecule. Conversely, the elimination of the guanidine side chain (Arg213Ala and Arg213Met mutants) of the CoA moiety binding site severely diminished the ability of FadR to bind the acyl-CoA effector. These results demonstrate the ability to fine tune FadR binding capacity. The validation of an efficient method to fully characterize all the binding events involved in the specific activity (effector and DNA operator binding) of FadR has allowed us to increase our understanding of the role of specific amino acids in the binding and recognition of acyl-CoA fatty acids and will greatly facilitate efforts aimed at engineering tunable FadR regulators for synthetic biology.

De novo biosynthesis of terminal alkyne-labeled natural products.

The terminal alkyne is a functionality widely used in organic synthesis, pharmaceutical science, material science and bioorthogonal chemistry. This functionality is also found in acetylenic natural products, but the underlying biosynthetic pathways for its formation are not well understood. Here we report the characterization of what is to our knowledge the first carrier protein-dependent terminal alkyne biosynthetic machinery in microbes. We further demonstrate that this enzymatic machinery can be exploited for the in situ generation and incorporation of terminal alkynes into two natural product scaffolds in Escherichia coli. These results highlight the prospect for tagging major classes of natural products, including polyketides and polyketide/nonribosomal peptide hybrids, using biosynthetic pathway engineering.

Bacillus subtilis as heterologous host for the secretory production of the non-ribosomal cyclodepsipeptide enniatin.

The heterologous expression of genes or gene clusters in microbial hosts, followed by metabolic engineering of biosynthetic pathways, is key to access industrially and pharmaceutically relevant compounds in an economically affordable and sustainable manner. Therefore, platforms need to be developed, which provide tools for the controlled synthesis of bioactive compounds. The Gram-positive bacterium Bacillus subtilis is a promising candidate for such applications, as it is generally regarded as a safe production host, its physiology is well investigated and a variety of tools is available for its genetic manipulation. Furthermore, this industrially relevant bacterium provides a high secretory potential not only for enzymes but also for primary and secondary metabolites. In this study, we present the first heterologous expression of an eukaryotic non-ribosomal peptide synthetase gene (esyn) coding for the biosynthesis of the small molecule enniatin in B. subtilis. Enniatin is a pharmaceutically used cyclodepsipeptide for treatment of topical bacterial and fungal infections. We generated various enniatin-producing B. subtilis strains, allowing for either single chromosomal or plasmid-based multi-copy expression of the esyn cluster under the control of an acetoin-inducible promoter system. Optimization of cultivation conditions, combined with modifications of the genetic background and multi-copy plasmid-based esyn expression, resulted in a secretory production of enniatin B. This work presents B. subtilis as a suitable host for the expression of heterologous eukaryotic non-ribosomal peptide synthetases (NRPS) clusters.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-6199-0) contains supplementary material, which is available to authorized users.

Metabolic Engineering of Biosynthetic Pathway for Production of Renewable Biofuels

Metabolic engineering is an important area of research that involves editing genetic networks to overproduce a certain substance by the cells. Using a combination of genetic, metabolic, and modeling methods, useful substances have been synthesized in the past at industrial scale and in a cost-effective manner. Currently, metabolic engineering is being used to produce sufficient, economical, and eco-friendly biofuels. In the recent past, a number of efforts have been made towards engineering biosynthetic pathways for large scale and efficient production of biofuels from biomass. Given the adoption of metabolic engineering approaches by the biofuel industry, this paper reviews various approaches towards the production and enhancement of renewable biofuels such as ethanol, butanol, isopropanol, hydrogen, and biodiesel. We have also identified specific areas where more work needs to be done in the future.

Expanding nature's small molecule diversity via in vitro biosynthetic pathway engineering

The enormous pool of chemical diversity found in nature serves as an excellent inventory for accessing biologically active compounds. This chemical inventory, primarily found in microorganisms and plants, is generated by a broad range of enzymatic pathways under precise genetic and protein-level control. In vitro pathway reconstruction can be used to characterize individual pathway enzymes, identify pathway intermediates, and gain an increased understanding of how pathways can be manipulated to generate natural product analogs. Moreover, through in vitro approaches, it is possible to achieve a diversification that is not restricted by toxicity, limited availability of intracellular precursors, or preconceived (by nature) regulatory controls. Additionally, combinatorial biosynthesis and high-throughput techniques can be used to generate both known natural products and analogs that would not likely be generated naturally. This current opinion review will focus on recent advances made in performing in vitro pathway-driven natural product diversification and opportunities for exploiting this approach for elucidating and entering this new chemical biology space.

Engineering secondary metabolite production in hairy roots

Plants are a rich source of valuable secondary metabolites. Plant cell culture technologies developed in the past as possible tools for secondary metabolites production. In most cases, for the large scale production of these compounds, production is too low for commercialization. The cultured cell suspensions being undifferentiated have not gained momentum because of their instability and non-uniformity of the product formation. Secondary metabolite production is under strict metabolic regulation and tissue specific localization. Hence the differentiated cultures such as hairy root cultures are widely studied. Agrobacterium rhizogenes causes hairy root disease in plants. Transformed roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce amounts of secondary metabolites comparable to that of intact plants. Elicitation of hairy roots leads to increased production of secondary metabolites and also helps in designing of metabolic traps to allow adsorption of product, preventing feedback inhibition and protection of metabolites from degradation in the culture media. Permeabilization and in situ product adsorption result in many fold increase in product yield. T- DNA activation tagging allows overexpressing the respective gene and increasing the product formation in transformed hairy roots. Recent progress in transgenic research has opened up the possibility of the metabolic engineering of biosynthetic pathways to produce high-value secondary metabolites. Metabolic engineering offers promising perspectives to improve yields; however it requires the understanding of the regulation of the secondary metabolite pathways involved in the regulation of levels of product, enzymes and genes, including aspects as transport and compartmentation. This article reviews the recent advances in secondary metabolites production in transformed hairy roots.

Application of two bicistronic systems involving 2A and IRES sequences to the biosynthesis of carotenoids in rice endosperm

Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (Psy) from Capsicum and carotene desaturase (CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC (Psy-2A-CrtI) and PIC (Psy-IRES-CrtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.

Trends for diverse production strategies of plant medicinal alkaloids

Covering: 1999 to 2009

Exploration and engineering of biosynthetic pathways in the marine actinomycete Salinispora tropica

Abstract In recent years, members of the marine actinomycete genus Salinispora have proven to be a precious source of structurally diverse secondary metabolites, including the potent anticancer agent salinosporamide A and the enediyne-derived sporolides. The tremendous potential of these marine-dwelling microbes for natural products biosynthesis, however, was not fully realized until sequencing of the Salinispora tropica genome revealed the presence of numerous orphan biosynthetic loci besides a plethora of rare metabolic pathways. This contribution summarizes the biochemical exploration of this prolific organism, highlighting studies in which genome-based information was exploited for the discovery of new enzymatic processes and the engineering of unnatural natural products. Inactivation of key genes within the salinosporamide pathway has expanded its inherent metabolic plasticity and enabled access to various salinosporamide derivatives by mutasynthesis. New insights into the biosynthesis of the sporolides allowed us to increase production titers of these structurally complex molecules, thereby providing the means to search for the DNA cleaving presporolide enediyne.

DNA assembly: simple and super-sized

Researchers at the J. Craig Venter Institute have developed an in vitro method for assembling DNA molecules up to several hundred kilobases long in a single step. The approach could facilitate the engineering of biosynthetic pathways in microorganisms for research and manufacturing.

Opportunities for enzyme engineering in natural product biosynthesis

Organisms from all kingdoms of life produce a plethora of natural products that display a range of biological activities. One key limitation of developing these natural products into pharmaceuticals is the inability to perform effective, fast, and inexpensive structure-activity relationship studies (SAR). Recently, enzyme engineering strategies have allowed the exploration of metabolic engineering of biosynthetic pathways to create new 'natural' products that can be used for SAR. The enzymes that enable the biosynthesis of natural products represent a largely untapped resource of potential biocatalysts. A challenge for the field is how to harness the wealth of reaction types used for natural product metabolism to obtain useful biocatalysts for industrial biotransformations.

Metabolic pathway engineering by plastid transformation is a powerful tool for production of compounds in higher plants

Plastid transformation is a powerful tool for the production of useful compounds in higher plants through metabolic engineering, because it has many advantages over conventional nuclear transformation: high-level foreign protein accumulation, no need for a transit peptide, absence of gene silencing, and convenient transgene stacking in an operon. Plastid transformation has recently yielded remarkable results in the production of highly valued biopharmaceutical proteins and in conferring herbicide and insect resistance. Metabolic pathway engineering by plastid transformation has also produced higher levels of useful compounds than nuclear transformation. Furthermore, recent reports have shown the functional regulation of transgene expression from the plastid genome. In this review, we have focused on the progress of plastid transformation in material production from the aspect of biosynthetic pathway engineering, discussing the issues for future expansion of plastid transformation.