Metabolic engineering of isoprenoids

The metabolic engineering of natural products has begun to prosper in the past few years due to genomic research and the discovery of biosynthetic genes. While the biosynthetic pathways and genes for some isoprenoids have been known for many years, new pathways have been found and known pathways have been further investigated. In this article, we review the recent advances in metabolic engineering of isoprenoids, focusing on the molecular genetics that affects pathway engineering the most. Examples in mono- sequi-, and diterpenoid synthesis as well as carotenoid production are discussed.     

Yarn design for functional tissue engineering

Tissue engineering requires the ability to design scaffolds with mechanical properties similar to those of the native tissue. Here, B. mori silk yarns are used as a model system to demonstrate the potential benefits and drawbacks of several textile methods used to fabricate tissue engineering scaffolds. Fibers are plied, twisted, cabled, braided, and/or textured to form several geometries with a wide range of mechanical outcomes. Predictable changes in ultimate tensile strength and stiffness are demonstrated following processing and as a function of test environment. The mechanical effects of increasing turns per inch and combining groups of fibers into higher-order yarn structures are demonstrated. Braids, one of the most commonly used textile structures, are shown to be limited by a change in stiffness following the locking-angle and therefore, potentially not the ideal structure for tissue engineering. Cabled yarns appear to allow the most flexibility in mechanical outcomes with a highly organized geometry. Twisted yarns, while more economical than cabled yarns, result in a higher stiffness and lower percent elongation at break than cabled yarns.     

Mevalonate-suppressive dietary isoprenoids for bone health

Osteoclastogenesis and osteoblastogenesis, the balancing acts for optimal bone health, are under the regulation of small guanosine triphosphate-binding proteins (GTPases) including Ras, Rac, Rho and Rab. The activities of GTPases require post-translational modification with mevalonate-derived prenyl pyrophosphates. Mevalonate deprivation induced by competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (e.g., statins) prevents the activation of GTPases, suppresses the expression of the receptor for activation of nuclear factor kappa B (NFκB) ligand (RANKL) and activation of NFκB and, consequently, inhibits osteoclast differentiation and induces osteoclast apoptosis. In contrast, statin-mediated inactivation of GTPases enhances alkaline phosphatase activity and the expression of bone morphogenetic protein-2, vascular epithelial growth factor, and osteocalcin in osteoblasts and induces osteoblast proliferation and differentiation. Animal studies show that statins inhibit bone resorption and increase bone formation. The anabolic effect of statins and other mevalonate pathway-suppressive pharmaceuticals resembles the anti-osteoclastogenic and bone-protective activities conferred by dietary isoprenoids, secondary products of plant mevalonate metabolism. The tocotrienols, vitamin E molecules with HMG CoA reductase-suppressive activity, induce mevalonate deprivation and concomitantly suppress the expression of RANKL and cyclooxygenase-2, the production of prostaglandin E2 and the activation of NFκB. Accordingly, tocotrienols inhibit osteoclast differentiation and induce osteoclast apoptosis, impacts reminiscent of those of statins. In vivo studies confirm the bone protective activity of tocotrienols at nontoxic doses. Blends of tocotrienols, statins and isoprenoids widely found in fruits, vegetables, grains, herbs, spices, and essential oils may synergistically suppress osteoclastogenesis while promoting osteoblastogenesis, offering a novel approach to bone health that warrants clinical studies.     

Pathway engineering by designed divergent evolution

Designed divergent evolution is a proposed protein engineering methodology to redesign enzyme function. The methodology was developed on the basis of the theories of divergent molecular evolution: (i) enzymes with more active and specialized functions have evolved from ones with promiscuous functions; (ii) this process is driven by small numbers of amino acid substitutions (plasticity); and (iii) the effects of double or multiple mutations are often additive (quasi-additive assumption). Thus, in many cases the impact of multiple mutations can be calculated by first determining the effects of a mutation at a single position and subsequently summing these effects using the quasi-additive assumption. In this way, the shape of the fitness landscape of a particular enzyme function can be estimated. The combinations of mutations predicted to yield global optima for desired functions can then be selected and introduced into the enzymes. The methodology has been demonstrated to be very powerful to redesign enzyme function. The use of multiple redesigned enzymes in novel or reconstructed metabolic pathways will enable the production of natural and unnatural products that will find use as pharmaceuticals, agrochemicals and many other applications.     

Metabolic engineering for production of functional polysaccharides

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Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato

The genetic manipulation of both the mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3-hydroxymethylglutaryl CoA (hmgr-1) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) genes, respectively. Transgenic plants containing an additional hmgr-1 from Arabidopsis thaliana, under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4-fold), but IPP-derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6-fold), which was inherited in the next generation. Phytoene and beta-carotene exhibited the greatest increases (2.4- and 2.2-fold, respectively). Extra-plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.     

Pathway engineering and synthetic biology using acetogens

Acetogenic anaerobic bacteria are defined as organisms employing the Wood-Ljungdahl pathway to synthesize acetyl-CoA from CO(2) or CO. Their autotrophic mode of metabolism offers the biotechnological chance to combine use of abundantly available substrates with reduction of greenhouse gases. Several companies have already established pilot and demonstration plants for converting waste gases into ethanol, an important biofuel and a natural product of many acetogens. Recombinant DNA approaches now opened the door to construct acetogens, synthesizing important industrial bulk chemicals and biofuels such as acetone and butanol. Thus, novel microbial production platforms are available that no longer compete with nutritional feedstocks.     

Pathway engineering strategies for production of beneficial carotenoids in microbial hosts

Carotenoids, such as lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin have many benefits for human health. In addition to the functional role of carotenoids as vitamin A precursors, adequate consumption of carotenoids prevents the development of a variety of serious diseases. Biosynthesis of carotenoids is a complex process and it starts with the common isoprene precursors. Condensation of these precursors and subsequent modifications, by introducing hydroxyl- and keto-groups, leads to the generation of diversified carotenoid structures. To improve carotenoid production, metabolic engineering has been explored in bacteria, yeast, and algae. The success of the pathway engineering effort depends on the host metabolism, specific enzymes used, the enzyme expression levels, and the strategies employed. Despite the difficulty of pathway engineering for carotenoid production, great progress has been made over the past decade. We review metabolic engineering approaches used in a variety of microbial hosts for carotenoid biosynthesis. These advances will greatly expedite our efforts to bring the health benefits of carotenoids and other nutritional compounds to our diet.     

Metabolic engineering of biosynthetic pathways leading to isoprenoids: Mono- and sesquiterpenes in plastids and cytosol

Abstract

Isoprenoids are a large and structurally diverse family of compounds that play essential roles in plants as hormones, photosynthetic pigments, electron carriers, and membrane components as well as serving in communication and defense. Now it is unequivocally proved that two distinct and independent biosynthetic routes exist to isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), the two building blocks for isoprenoids in plants. The cytosolic pathway is triggered by Acetyl Coenzyme A where classical intermediate mevalonic acid is formed which, in turn, converts into IPP and DMAPP. These further combine to elongate into sesquiterpenes (C₁₅) and triterpenes (C₃₀); whereas the plastidial pathway provides precursors for the biosynthesis of isoprene (C₅), monoterpenes (C₁₀), diterpenes (C₂₀), and tetraterpenes (C₄₀). The pathway is initiated by the transketolase-type condensation of pyruvate (C-2 and C-3) and glyceraldehyde-3-phosphate to 1-deoxyxylulose-5-phosphate (DXP), followed by the isomerization and reduction of this intermediate to 2-C-methylerythritol-4-phosphate (MEP), formation of the cytidine 5'-diphosphate (CDP) derivative, phosphorylation at C2, and cyclization to 2-C-methylerythritol-2,4-cyclodiphosphate (CDP-Me2P as the last defined step). The genes encoding each enzyme of the plastid pathway up to formation of the cyclic diphosphate have been isolated from plants and from eubacteria where the pathway exists. Studies on the complete biosynthetic pathways using radio-labeled substrates will help in characterizing and identifying the enzymes involved in each and every step of cyclization, isomerization, chain elongation, hydrogen shifts, oxidation and hydroxylation during the formation of many isoprenoid compounds present in food and flavor substances and are highly useful to human beings.     

Pathway analysis and metabolic engineering in Corynebacterium glutamicum

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.     

Pathway analysis, engineering, and physiological considerations for redirecting central metabolism

The rate and yield of producing a metabolite is ultimately limited by the ability to channel metabolic fluxes from central metabolism to the desired biosynthesis pathway. Redirection of central metabolism thus is essential to high-efficiency production of biochemicals. This task begins with pathway analysis, which considers only the stoichiometry of the reaction networks but not the regulatory mechanisms. An approach extended from convex analysis is used to determine the basic reaction modes, which allows the determination of optimal and suboptimal flux distributions, yield, and the dispensable sets of reactions. Genes responsible for reactions in the same dispensable set can be deleted simultaneously. This analysis serves as an initial guideline for pathway engineering. Using this analysis, we successfully constructed an Escherichia coli strain that can channel the metabolic flow from carbohydrate to the aromatic pathway with theoretical yield. This analysis also predicts a novel cycle involving phosphoenolpyruvate (PEP) carboxykinase (Pck) and the glyoxylate shunt, which can substitute the tricarboxylic acid cycle with only slightly less efficiency. However, the full cycle could not be confirmed in vivo, possibly because of the regulatory mechanism not considered in the pathway analysis.In addition to the kinetic regulation, we have obtained evidence suggesting that central metabolites are involved in specific regulons in E. coli. Overexpression of PEP-forming enzymes (phosphoenolpyruvate synthase [Pps] and Pck) stimulates the glucose consumption rate, represses the heat shock response, and negatively regulates the Ntr regulon. These results suggest that some glycolytic intermediates may serve as a signal in the regulation of the phosphotransferase system, heat shock response, and nitrogen regulation. However, the role of central metabolites in these regulations has not been determined conclusively. (c) 1996 John Wiley & Sons, Inc.     

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Recent advances in structure-based enzyme engineering for functional reconstruction

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure–function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.     

3D printing of functional biomaterials for tissue engineering

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Opportunistic emissions of volatile isoprenoids

Isoprene, monoterpenes and sesquiterpenes are synthesized and emitted by some plant species, but not all plant species have this ability. These volatile, nonessential isoprenoid compounds share the same biochemical precursors as larger essential isoprenoids such as gibberellic acids and carotenoids. They have many protective and ecological functions for the plant species that produce them, but plant species that do not produce these compounds also grow and reproduce successfully. Here, we develop an 'opportunist hypothesis' suggesting that (i) volatile isoprenoid production takes advantage of dimethylallyl diphosphate (DMAPP) and its isomer isopentenyl diphosphate (IPP), which are synthesized primarily to produce essential isoprenoids, and (ii) conditions affecting synthesis of the higher isoprenoids will affect the production and emission of volatile isoprenoids.