The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid.Keywords: autophagosome; mannose-capped LAM; membrane proteome; mycobacterial lipoglycans; Phagosome;

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen possessing a complex mixture of cell wall lipids that are thought to modulate the activities of host macrophages. In this study, we employed two state-of-the-art quantitative proteomic approaches, metabolic labeling SILAC and chemical isobaric tagging iTRAQ, to study changes in macrophage protein expression in response to exposure to M. tuberculosis lipids. From a total of 1286 proteins identified, 463 were discovered by both isotope-labeling strategies at a high consistency, and the rest of proteins were detected by only one of the two approaches. Upon exposure to mycobacterial cell wall lipids, 166 macrophage proteins showed differential expression. These included proteins involved in the immune response, oxidation and reduction, and vesicle transport, as well as other cellular processes. The response of the macrophage proteome to M. tuberculosis lipids reflects the cell’s innate defense mechanisms as well as lipid-induced processes that may benefit the pathogen.Keywords: Cell wall lipids; differential expression; iTRAQ; macrophage; SILAC;

Streptomyces have a rich history as producers of important natural products and this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biology. However, the dearth of genetic tools available to control and monitor protein production precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein production in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate its use in producing the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts.Keywords: bisabolene; fluorescent protein; promoter; streptomyces;

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.Keywords: acyltransferase domain; protein engineering; substrate specificity; synthetic biology tool; Type I modular polyketide synthase;

Metabolic engineering efforts toward rewiring metabolism of cells to produce new compounds often require the utilization of non-native enzymatic machinery that is capable of producing a broad range of chemical functionalities. Polyketides encompass one of the largest classes of chemically diverse natural products. With thousands of known polyketides, modular polyketide synthases (PKSs) share a particularly attractive biosynthetic logic for generating chemical diversity. The engineering of modular PKSs could open access to the deliberate production of both existing and novel compounds. In this review, we discuss PKS engineering efforts applied at both the protein and cellular level for the generation of a diverse range of chemical structures, and we examine future applications of PKSs in the production of medicines, fuels and other industrially relevant chemicals.

Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design–build–test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS’ first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to “debug” PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry.Keywords: adipic acid; polyketide synthase; tandem mass-spectrometry;

Polyketide natural products have broad applications in medicine. Exploiting the modular nature of polyketide synthases to alter stereospecificity is an attractive strategy for obtaining natural product analogues with altered pharmaceutical properties. We demonstrate that by retaining a dimerization element present in LipPks1+TE, we are able to use a ketoreductase domain exchange to alter α-methyl group stereochemistry with unprecedented retention of activity and simultaneously achieve a novel alteration of polyketide product stereochemistry from anti to syn. The substrate promiscuity of LipPks1+TE further provided a unique opportunity to investigate the substrate dependence of ketoreductase activity in a polyketide synthase module context.

•Highest resolution and first cofactor-bound structure of a terminal reductase domain•Computational modeling advances hypotheses made from the crystal structure•Biochemical analysis defines residues critical for substrate specificity and catalysis•Result-based engineering enabled improved reduction of highly reduced substratesThe terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.Figure optionsDownload full-size imageDownload high-quality image (138 K)Download as PowerPoint slide

The borrelidin polyketide synthase (PKS) begins with a carboxylated substrate and, unlike typical decarboxylative loading PKSs, retains the carboxy group in the final product. The specificity and tolerance of incorporation of carboxyacyl substrate into type I PKSs have not been explored. Here, we show that the first extension module is promiscuous in its ability to extend both carboxyacyl and non-carboxyacyl substrates. However, the loading module has a requirement for substrates containing a carboxy moiety, which are not decarboxylated in situ. Thus, the loading module is the basis for the observed specific incorporation of carboxylated starter units by the borelidin PKS.

The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5′ of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.

LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic α-lipomycin in Streptomyces aureofaciens Tü117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.

Polyketides, an important class of natural products with complex chemical structures, are widely used as antibiotics and other pharmaceutical agents. A clear barrier to heterologous polyketide biosynthesis in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common substrate of multimodular polyketide synthases. Here we report a route for synthesizing (2S)-methylmalonyl-CoA from malonyl-CoA with a 3-hydroxypropionate cycle in thermoacidophilic crenarchaeon. The engineered E. coli strain produced both propionyl-CoA and methylmalonyl-CoA at intracellular levels similar to those of acetyl-CoA and succinyl-CoA, respectively. This approach may open a way to produce a variety of polyketide drugs in E. coli from renewable carbon sources.

Genome sequence analysis of Ricinus communis has indicated the presence of at least 22 putative terpene synthase (TPS) genes, 13 of which appear to encode sesquiterpene synthases (SeTPSs); however, no SeTPS genes have been isolated from this plant to date. cDNAs were recovered for six SeTPS candidates, and these were subjected to characterization in vivo and in vitro. The RcSeTPS candidates were expressed in either Escherichia coli or Saccharomyces cerevisiae strains with engineered sesquiterpene biosynthetic pathways, but only two (RcSeTPS1 and RcSeTPS7) produced detectable levels of product. In order to check whether the engineered microbial hosts were adequately engineered for sesquiterpene production, a selection of SeTPS genes was chosen from other plant species and demonstrated consistently high sesquiterpene titers. Activity could be demonstrated in vitro for two of the RcSeTPS candidates (RcSeTPS5 and RcSeTPS10) that were not observed to be functional in our microbial hosts. RcSeTPS1 produced two products, (−)-α-copaene and (+)-δ-cadinene, while RcSeTPS7 produced a single product, (E, E)-α-farnesene. Both RcSeTPS5 and RcSeTPS10 produced multiple sesquiterpenes.Graphical abstractFour sesquiterpene synthases (SeTPSs) from Ricinus communis were characterized and represent the first examples of SeTPSs from the Euphorbiaceae. Challenges faced in the isolation and functional expression of cDNAs for all putative SeTPSs in the R. communis genome are described.Highlights► Analysis of Ricinus communis establishes up to 13 putative sesquiterpene synthases (SeTPSs). ► Only four of the recovered cDNAs were found to produce sesquiterpene products. ► Production levels in engineered microbial hosts were low compared to other plant SeTPSs. ► Verified products include (−)-α-copaene, (+)-δ-cadinene, and (E, E)-α-farnesene.

The ability to compose biological systems from smaller elements that act independently of the other upon assembly may help make the forward engineering of biological systems practical. Engineering biology in this manner is made difficult by the inherent nonlinear response of organisms to genetic devices. Devices are inevitably coupled to one another in the cell because they share the same transcriptional machinery for expression. Thus, new properties can emerge when devices that had been characterized in isolation are expressed concurrently. We show in this report that, similar to physical systems, the Escherichia coli (E. coli) transcriptional system can exhibit linear behavior under "small" perturbation conditions. This, in turn, allows devices to be treated as independent modules.We developed a framework and model system consisting of three devices to investigate linear system behavior in E. coli. Our framework employed the transfer curve concept to determine the amount of nonlinearity elicited by the E. coli transcriptional system in response to the devices. To this effect, the model system was quantitatively characterized using real-time quantitative PCR to produce device transfer curves (DTCs). Two of the devices encoded the bacterial neomycin phosphotransferase II (nptII) and chloramphenicol acetyl transferase (cat), while the third encoded the jellyfish-originating green fluorescent protein (gfp). The gfp device was the most nonlinear in our system, with nptII and cat devices eliciting linear responses. Superposition experiments verified these findings, with independence among the three devices having been lost when gfp was present at copy numbers above the lowest one used.We show that linear system behavior is possible in E. coli. Elucidation of the mechanism underlying the nonlinearity observed in gfp may lead to design rules that ensure linear system behavior, enabling the accurate prediction of the quantitative behavior of a system assembled from individually characterized devices. Our work suggests that biological systems follow principles similar to physical ones, and that concepts borrowed from the latter (such as DTCs) may be of use in the characterization and design of biological systems.

The increasing cost of energy and concerns about the environment have emphasized the need to find new sources of fuel, with the microbial production of high-energy fuels a promising approach. Here, Escherichia coli is engineered to produce more complex biofuels — fatty esters (biodiesel), fatty alcohols and waxes — directly from simple sugars. Some cells are further engineered to express hemicellulases, a step towards producing these compounds directly from hemicellulose.

A large number of diterpenes have been isolated from Euphorbiaceae plants, many of which are of interest due to toxicity or potential therapeutic activity. Specific Euphorbiaceae diterpenes of medical interest include the latent HIV-1 activator prostratin (and related 12-deoxyphorbol esters), the analgesic resiniferatoxin, and the anticancer drug candidate ingenol 3-angelate. In spite of the large number of diterpenes isolated from these plants and the similarity of their core structures, there is little known about their biosynthetic pathways. Other than the enzymes involved in gibberellin biosynthesis, the only diterpene synthase isolated to date from the Euphorbiaceae has been casbene synthase, responsible for biosynthesis of a macrocyclic diterpene in the castor bean (Ricinus communis). Here, we have selected five Euphorbiaceae species in which to investigate terpene biosynthesis and report on the distribution of diterpene synthases within this family. We have discovered genes encoding putative casbene synthases in all of our selected Euphorbiaceae species and have demonstrated high-level casbene production through expression of four of these genes in a metabolically engineered strain of Saccharomyces cerevisiae. The only other diterpene synthase found among the five plants was a neocembrene synthase from R. communis (this being the first report of a neocembrene synthase gene). Based on the prevalence of casbene synthases, the lack of other candidates, and the structure of the casbene skeleton, we consider it likely that casbene is the precursor to a large number of Euphorbiaceae diterpenes. Casbene production levels of 31 mg/L were achieved in S. cerevisiae and we discuss strategies to further increase production by maximizing flux through the mevalonate pathway.Casbene synthase is commonly found in Euphorbiaceae species and is likely to be the precursor to the majority of complex diterpenes found in this family.

Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L−1 in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.

Isotopomer-assisted metabolite analysis was used to investigate the central metabolism of Mycobacterium smegmatis and its transition from normal growth to a non-replicating state under a hypoxic environment. Tween 80 significantly promoted aerobic growth by improving O2 transfer, while only small amount was degraded and metabolized via the TCA cycle for biomass synthesis. As the bacillus encountered hypoxic stress, isotopomer analysis suggested: (1) isocitrate lyase activity increased, which further induced glyoxylate pathway and glycine dehydrogenase for replenishing NAD+; (2) the relative amount of acetyl-CoA entering the TCA cycle was doubled, whereas little entered the glycolytic and pentose phosphate pathways.

Covering: up to the end of 2007

Nature has balanced most metabolic pathways such that no one enzyme in the pathway controls the flux through that pathway. However, unnatural or nonnative, constructed metabolic pathways may have limited product flux due to unfavorable in vivo properties of one or more enzymes in the pathway. One such example is the mevalonate-based isoprenoid biosynthetic pathway that we previously reconstructed in Escherichia coli. We have used a probable mechanism of adaptive evolution to engineer the in vivo properties of two enzymes (3-hydroxy-3-methylglutaryl-CoA reductase [tHMGR] and many terpene synthases) in this pathway and thereby eliminate or minimize the bottleneck created by these inefficient or nonfunctional enzymes. Here, we demonstrate how we significantly improved the productivity (by ∼1000 fold) of this reconstructed biosynthetic pathway using this strategy. We anticipate that this strategy will find broad applicability in the functional construction (or reconstruction) of biological pathways in heterologous hosts.

The richness and versatility of biological systems make them ideally suited to solve some of the world’s most significant challenges, such as converting cheap, renewable resources into energy-rich molecules; producing high-quality, inexpensive drugs to fight disease; and remediating polluted sites. Over the years, significant strides have been made in engineering microorganisms to produce fuels, bulk chemicals, and valuable drugs from inexpensive starting materials; to detect and degrade nerve agents as well as less toxic organic pollutants; and to accumulate metals and reduce radionuclides. The components needed to engineer the chemistry inside a microbial cell are significantly different from those commonly used to overproduce pharmaceutical proteins. Synthetic biology has had and will continue to have a significant impact on the development of these components to engineer cellular metabolism and microbial chassis to host the chemistry. The ready availability of more well-characterized gene expression components and hosts for chemical synthesis, standards for the connection of these components to make larger functioning devices, computer-aided design software, and debugging tools for biological designs will decrease both the time and the support needed to construct these designs. Some of the most important tools for engineering bacterial metabolism and their use for production of the antimalarial drug artemisinin are reviewed.Keywords: All-or-none induction or expression: The induction of an inducible promoter to full strength in only a fraction of the cells of a population, the fraction dependent on the concentration of the inducer in the medium.; Artemisinin: A sesquiterpene lactone endoperoxide extracted from Artemisia annua L that is highly effective against multi-drug-resistant Plasmodium spp.; Metabolic burden: The consumption of macromolecule precursors and energy due to the expression of foreign genes in a cell.; Metabolic engineering: The manipulation of intracellular metabolic reactions for the production or degradation of a target chemical.; Synthetic biology: The design and construction of new biological components, such as enzymes, genetic circuits, and cells, or the redesign of existing biological systems.Keywords: CAD: Computer-aided design; Internal ribosome entry sequence (IRES): A sequence between two coding regions on a multicistronic mRNA that allows a eukaryotic ribosome to initiate translation of the 3′ coding region.; Operon: A group of genes under the control of a single promoter or multiple promoters.; Tunable intergenic region (TIGR): Untranslated region between two coding regions of a multicistronic mRNA, which contains sequences that control mRNA processing and translation of one or both of the surrounding coding regions.

Protein polymers (long-chain proteins in which a specific amino acid sequence “monomer” is repeated through the molecule) are found widely in nature, and these materials exhibit a diverse array of physical properties. One class of self-assembling proteins is hydrophobic-polar (HP) protein polymers capable of self-assembly under the appropriate solution conditions. We generated a chimeric protein consisting of an HP protein polymer monomer unit, EAK1 (sequence n-AEAEAKAKAEAEAKAK-c), and a silaffin peptide, R5 (sequence: n-SSKKSGSYSGSKGSKRRIL-c). First identified in diatoms, silaffins represent a class of proteins and peptides capable of directing silica precipitation in vitro at neutral pH and ambient temperatures. The EAK1-R5 chimera demonstrated self-assembly into hydrogels and the ability to direct silica precipitation in vitro. This chimera is capable of generating silica morphologies and feature sizes significantly different from those achievable with the R5 peptide alone, indicating that fusions of silaffins with self-assembling proteins may be a route to controlling the morphology of artificially produced silica matrices.

Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages.

Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required.Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL-1 in shake-flask cultures and 1 g L-1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast.The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.

A gene encoding a plant terpene cyclase, Artemisia annua amorpha-4,11-diene synthase (ADS), was expressed in Aspergillus nidulans under control of a strong constitutive promoter, (p)gpdA. The transformants produced only small amounts of amorphadiene, but much larger amounts of similar sesquiterpenes normally produced as minor by-products in planta. In contrast, expression of ADS in Escherichia coli produced almost exclusively amorpha-4,11-diene. These results indicate that the host environment can greatly impact the terpenes produced from terpene synthases.

Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol.Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

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.

Magnesium is an important divalent ion for organisms. There have been a number of studies in vitro suggesting that magnesium affects enzyme activity. Surprisingly, there have been few studies to determine the cellular mechanism for magnesium regulation. We wished to determine if magnesium levels could be regulated in vivo. It is known that Saccharomyces cerevisiae has two magnesium transporters (ALR1 and ALR2) across the plasma membrane. We created S. cerevisiae strains with deletion of one (alr1 or alr2) or both (alr1 alr2) transporters. The deletion of ALR1 resulted in a decrease in intracellular magnesium levels. An increase from 5 to 100 mM in the exogenous magnesium level increased the intracellular levels of magnesium in the alr1 and alr1 alr2 strains, whereas the expression of magnesium transporters from S. cerevisiae or Arabidopsis thaliana led to a change of the intracellular levels of magnesium in those strains. The deletion of magnesium transporters in A. cerevisiae and overexpression of magnesium transporters from A. thaliana also affected the intracellular concentrations of a range of metal ions, which suggests that cells use non-specific transporters to help regulate metal homeostasis.

Isoprenoid secondary metabolites are a rich source of commercial products that have not been fully explored. At present, there are isoprenoid products used in cancer therapy, the treatment of infectious diseases, and crop protection. All isoprenoids share universal prenyl diphosphate precursors synthesized via two distinct pathways. From these universal precursors, the biosynthetic pathways to specific isoprenoids diverge resulting in a staggering array of products. Taking advantage of this diversity has been the focus of much effort in metabolic engineering heterologous hosts. In addition, the engineering of the mevalonate pathway has increased levels of the universal precursors available for heterologous production. Finally, we will describe the efforts to produce to commercial terpenoids, paclitaxel and artemisinin.

The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-δ-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-δ-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-δ-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-δ-cadinene synthase suggested that the G helix plays a very important role in (+)-δ-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

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.Graphical abstractDownload high-res image (151KB)Download full-size imageHighlights► E. coli provides key elements of an ideal host for polyketide production. ► Modular type I PKSs offer engineering opportunities in a programmed manner. ► Modular type I PKSs, a platform of synthetic biology.

Amorphadiene, a sesquiterpene precursor to the anti-malarial drug artemisinin, is synthesized by the cyclization of farnesyl pyrophosphate (FPP). Saccharomyces cerevisiae produces FPP through the mevalonate pathway using acetyl-CoA as a starting compound. In order to enhance the supply of acetyl-CoA to the mevalonate pathway and achieve high-level production of amorphadiene, we engineered the pyruvate dehydrogenase bypass in S. cerevisiae. Overproduction of acetaldehyde dehydrogenase and introduction of a Salmonella enterica acetyl-CoA synthetase variant increased the carbon flux into the mevalonate pathway resulting in increased amorphadiene production. This work will be generally applicable to the production of a broad range of isoprenoids in yeast.

Engineering biosynthetic pathways in microbes for the production of complex chemicals and pharmaceuticals is an attractive alternative to chemical synthesis. However, in transferring large pathways to alternate hosts and manipulating expression levels, the native regulation of carbon flux through the pathway may be lost leading to imbalances in the pathways. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway [Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., Keasling, J.D., 2003. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat. Biotechnol. 21, 796–802]. The strain produces high levels of isoprenoids, but upon further investigation we discovered that the accumulation of pathway intermediates limited flux and that high-level expression of the mevalonate pathway enzymes inhibited cell growth. Gene titration studies and metabolite profiling using liquid chromatography–mass spectrometry linked the growth inhibition phenotype with the accumulation of the pathway intermediate 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA). Such an accumulation implies that the activity of HMG-CoA reductase was insufficient to balance flux in the engineered pathway. By modulating HMG-CoA reductase production, we eliminated the pathway bottleneck and increased mevalonate production. These results demonstrate that balancing carbon flux through the heterologous pathway is a key determinant in optimizing isoprenoid biosynthesis in microbial hosts.

Microorganisms have been rich sources for natural products, some of which have found use as fuels, commodity chemicals, specialty chemicals, polymers, and drugs, to name a few. The recent interest in production of transportation fuels from renewable resources has catalyzed numerous research endeavors that focus on developing microbial systems for production of such natural products. Eliminating bottlenecks in microbial metabolic pathways and alleviating the stresses due to production of these chemicals are crucial in the generation of robust and efficient production hosts. The use of systems-level studies makes it possible to comprehensively understand the impact of pathway engineering within the context of the entire host metabolism, to diagnose stresses due to product synthesis, and provides the rationale to cost-effectively engineer optimal industrial microorganisms.

Many metabolic pathways in microbial hosts have been created, modified and engineered to produce useful molecules. The titer and yield of a final compound is often limited by the inefficient use of cellular resources and imbalanced metabolism. Engineering sensory-regulation devices that regulate pathway gene expression in response to the environment and metabolic status of the cell have great potential to solve these problems, and enhance product titers and yields. This review will focus on recent developments in biosensor design, and their applications for controlling microbial behavior.

We describe a novel biosensor strain for detection and quantification of a small molecule, mevalonate. The biosensor strain is an Escherichia coli mevalonate auxotroph that expresses the green fluorescent protein and reports on the mevalonate concentration in the growth medium through a change in growth rate. A model describing the growth rate dependence on mevalonate was developed in order to use the biosensor strain for high-throughput screening (HTS) and quantitative measurement of mevalonate in the extracellular environment. In general, this method should be applicable to the quantification of any small molecule for which an auxotroph can be developed and will be useful for HTS of evolved metabolic pathways for which there is no readily available screen or selection.

•ORF27 from Streptomyces aizunensis catalyzes formation of 2-pyrrolidone from γ-aminobutyrate.•Recombinant Escherichia coli with GadB and ORF27 produces 2-pyrrolidone from glutamate.•Engineered strain capable of producing 1.1 g/L of 2-pyrrolidone from 9 g/L of glutamate within 31 h.2-Pyrrolidone is a valuable bulk chemical with myriad applications as a solvent, polymer precursor and active pharmaceutical intermediate. A novel 2-pyrrolidone synthase, ORF27, from Streptomyces aizunensis was identified to catalyze the ring closing dehydration of γ-aminobutyrate. ORF27's tendency to aggregate was resolved by expression at low temperature and fusion to the maltose binding protein (MBP). Recombinant Escherichia coli was metabolically engineered for the production of 2-pyrrolidone from glutamate by expressing both the genes encoding GadB, a glutamate decarboxylase, and ORF27. Incorporation of a GadB mutant lacking H465 and T466, GadB_ΔHT, improved the efficiency of one-pot 2-pyrrolidone biosynthesis in vivo. When the recombinant E. coli strain expressing the E. coli GadB_ΔHT mutant and the ORF27-MBP fusion was cultured in ZYM-5052 medium containing 9 g/L of l-glutamate, 7.7 g/L of l-glutamate was converted to 1.1 g/L of 2-pyrrolidone within 31 h, achieving 25% molar yield from the consumed substrate.Download high-res image (76KB)Download full-size image

•Modular polyketide synthases have the potential to produce many existing and novel compounds.•Accessible compounds include several commodity chemicals with large markets.•The potential of polyketide synthases greatly surpasses our current ability to engineer them.•We suggest adopting a design–build–test–learn paradigm for engineering polyketide synthases.•We suggest several strategies to improve the design–build–test–learn cycle.Engineering modular polyketide synthases (PKSs) has the potential to be an effective methodology to produce existing and novel chemicals. However, this potential has only just begun to be realized. We propose the adoption of an iterative design–build–test–learn paradigm to improve PKS engineering. We suggest methods to improve engineered PKS design by learning from laboratory-based selection; adoption of DNA design software and automation to build constructs and libraries more easily; tools for the expression of engineered proteins in a variety of heterologous hosts; and mass spectrometry-based high-throughput screening methods. Finally, lessons learned during iterations of the design–build–test–learn cycle can serve as a knowledge base for the development of a single retrosynthesis algorithm usable by both PKS experts and non-experts alike.Download high-res image (86KB)Download full-size image

Production of biofuels from renewable resources such as cellulosic biomass provides a source of liquid transportation fuel to replace petroleum-based fuels. This endeavor requires the conversion of cellulosic biomass into simple sugars, and the conversion of simple sugars into biofuels. Recently, microorganisms have been engineered to convert simple sugars into several types of biofuels, such as alcohols, fatty acid alkyl esters, alkanes, and terpenes, with high titers and yields. Here, we review recently engineered biosynthetic pathways from the well-characterized microorganisms Escherichia coli and Saccharomyces cerevisiae for the production of several advanced biofuels.Highlights► Several biosynthetic pathways have been recently engineered in E. coli or S. cerevisiae to produce advanced biofuels. ► The advanced biofuels include alcohols, terpenes, fatty acid alkyl esters, alkanes, and alkenes. ► Many of the advanced biofuels were converted from simple sugars with high titers and yields. ► It is advantageous to use native high flux pathways to provide key precursors for fuel molecule biosynthesis. ► Limiting the use of precursors by competing pathways can increase flux toward product. ► Creating irreversible steps in the engineered pathway may be beneficial for biofuel production.

The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost-effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.

An immense array of naturally occurring biological systems have evolved that convert simple substrates into the products that cells need for growth and persistence. Through the careful application of metabolic engineering and synthetic biology, this biotransformation potential can be harnessed to produce chemicals that address unmet clinical and industrial needs. Developing the capacity to utilize biology to perform chemistry is a matter of increasing control over both the function of synthetic biological systems and the engineering of those systems. Recent efforts have improved general techniques and yielded successes in the use of synthetic biology for the production of drugs, bulk chemicals, and fuels in microbial platform hosts. Synthetic promoter systems and novel RNA-based, or riboregulator, mechanisms give more control over gene expression. Improved methods for isolating, engineering, and evolving enzymes give more control over substrate and product specificity and better catalysis inside the cell. New computational tools and methods for high-throughput system assembly and analysis may lead to more rapid forward engineering. We highlight research that reduces reliance upon natural biological components and point to future work that may enable more rational design and assembly of synthetic biological systems for synthetic chemistry.