Biosynthetic Engineering Research Articles

Strategies for the Discovery and Development of New Antibiotics from Natural Products: Three Case Studies.

Natural products continue to be a predominant source for new anti-infective agents. Research at the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) and the Helmholtz Centre for Infection Research (HZI) is dedicated to the development of new lead structures against infectious diseases and, in particular, new antibiotics against hard-to-treat and multidrug-resistant bacterial pathogens. In this chapter, we introduce some of the concepts currently being employed in the field of antibiotic discovery. In particular, we will exemplarily illustrate three approaches: (1) Current sources for novel compounds are mainly soil-dwelling bacteria. In the course of our antimicrobial discovery program, a biodiverse collection of myxobacterial strains has been established and screened for antibiotic activities. Based on this effort, one successful example is presented in this chapter: Antibacterial cystobactamids were discovered and their molecular target, the DNA gyrase, was identified soon after the analysis of myxobacterial self-resistance making use of the information found in the respective biosynthesis gene cluster. (2) Besides our focus on novel natural products, we also apply strategies to further develop either neglected drugs or widely used antibiotics for which development of resistance in the clinical setting is an issue: Antimycobacterial griselimycins were first described in the 1960s but their development and use in tuberculosis therapy was not further pursued. We show how a griselimycin derivative with improved pharmacokinetic properties and enhanced potency against Mycobacterium tuberculosis revealed and validated a novel target for antibacterial therapy, the DNA sliding clamp. (3) In a third approach, biosynthetic engineering was used to modify and optimize natural products regarding their pharmaceutical properties and their production scale: The atypical tetracycline chelocardin is a natural product scaffold that was modified to yield a more potent derivative exhibiting activity against multidrug-resistant pathogens. This was achieved by genetic engineering of the producer strain and the resulting compound is now subject to further optimization by medicinal chemistry approaches.

Structural and functional analysis of two di-domain aromatase/cyclases from type II polyketide synthases

Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-β-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7-C12 and C9-C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7-C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: "nonreducing" ARO/CYCs, which act on nonreduced poly-β-ketones, and "reducing" ARO/CYCs, which act on cyclized C9 reduced poly-β-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono- and di-domain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.

Biosynthetic engineering and fermentation media development leads to gram-scale production of spliceostatin natural products in Burkholderia sp.

A key challenge in natural products drug discovery is compound supply. Hundreds of grams of purified material are needed to advance a natural product lead through preclinical development. Spliceostatins are polyketide-nonribosomal peptide natural products that bind to the spliceosome, an emerging target in cancer therapy. The wild-type bacterium Burkholderia sp. FERM BP-3421 produces a suite of spliceostatin congeners with varying biological activities and physiological stabilities. Hemiketal compounds such as FR901464 were the first to be described. Due to its improved properties, we were particularly interested in a carboxylic acid precursor analog that was first reported from Burkholderia sp. MSMB 43 and termed thailanstatin A. Inactivation of the iron/α-ketoglutarate-dependent dioxygenase gene fr9P had been shown to block hemiketal biosynthesis. However, a 4-deoxy congener of thailanstatin A was the main product seen in the dioxygenase mutant. We show here that expression of the cytochrome P450 gene fr9R is a metabolic bottle neck, as use of an l-arabinose inducible system led to nearly complete conversion of the 4-deoxy analog to the target molecule. By integrating fermentation media development approaches with biosynthetic engineering, we were able to improve production titers of the target compound >40-fold, going from the starting ~60mg/L to 2.5g/L, and to achieve what is predominantly a single component production profile. These improvements were instrumental in enabling preclinical development of spliceostatin analogs as chemotherapy.

Molecular variation of the nonribosomal peptide-polyketide siderophore yersiniabactin through biosynthetic and metabolic engineering.

The production of the mixed nonribosomal peptide-polyketide natural product yersiniabactin (Ybt) has been established using E. coli as a heterologous host. In this study, precursor-directed biosynthesis was used to generate five new analogs of Ybt, demonstrating the flexibility of the heterologous system and the biosynthetic process in allowing compound diversity. A combination of biosynthetic and cellular engineering was then used to influence the production metrics of the resulting analogs. First, the cellular levels and activity of FadL, a hydrocarbon transport protein, were tested for subsequent influence upon exogenous precursor uptake and Ybt analog production with a positive correlation observed between FadL over-production and analog formation. Next, a Ybt biosynthetic editing enzyme was removed from the heterologous system which decreased native compound production but increased analog formation. A final series of experiments enhanced endogenous anthranilate towards complete pathway formation of the associated analog which showed a selective ability to bind gold.

Rapid identification of atypical tetracyclines using tandem mass spectrometric fragmentation patterns

When applying biosynthetic engineering approaches at the early stages of drug discovery, e.g. aiming to develop novel tetracycline analogues, target compounds are generally produced by engineered microorganisms in low yields. Rapid and reliable identification of metabolites with desired structural modification directly from bacterial cultures is therefore of great importance. Structural elucidation of atypical tetracyclines was carried out by fragmentation applying electrospray ionisation tandem mass spectrometry (ESI-MS/MS) (triple quadrupole - linear ion trap; Applied Biosystems 4000 QTRAP) and a high-resolution mass spectrometer (Agilent Technologies 6224 TOF). Fragmentation patterns were obtained either with direct injection or by applying separation of target compounds with high-performance liquid chromatography (HPLC) prior to mass spectrometry. In-source and CID fragmentation were compared. Theoretical calculations of target structures using the Gaussian programme suite were carried out with the aim of strengthening experimental structural elucidation. Recombinant strains of Amycolatopsis sulphurea producing atypical tetracyclines chelocardin, modified chelocardin analogues (9-demethylchelocardin and 2-carboxyamido-2-deacetyl-chelocardin (CDCHD), and anhydrotetracycline (ATC) were analysed by collision-induced dissociation (CID) fragmentation with higher collision energies to yield structurally important fragments which were identified. We have demonstrated that ATC is more prone to fragmentation compared to its epimer, which was further supported by comparison of both structures calculated with ab initio calculations. We have demonstrated that fragmentation patterns of atypical tetracyclines in CID-MS spectra enable rapid structural elucidation of target metabolites produced by cultures of genetically engineered bacteria. This method is of significant importance for early stages of drug development considering that isolation of target metabolites produced at low concentration is challenging. Copyright © 2015 John Wiley & Sons, Ltd.

Engineered biosynthesis of enduracidin lipoglycopeptide antibiotics using the ramoplanin mannosyltransferase Ram29

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.

Biosynthetic Studies of Telomycin Reveal New Lipopeptides with Enhanced Activity.

Telomycin (TEM) is a cyclic depsipeptide antibiotic active against Gram-positive bacteria. In this study, five new natural telomycin analogues produced by Streptomyces canus ATCC 12646 were identified. To understand the biosynthetic machinery of telomycin and to generate more analogues by pathway engineering, the TEM biosynthesis gene cluster has been characterized from S. canus ATCC 12646: it spans approximately 80.5 kb and consists of 34 genes encoding fatty acid ligase, nonribosomal peptide synthetases (NRPSs), regulators, transporters, and tailoring enzymes. The gene cluster was heterologously expressed in Streptomyces albus J1074 setting the stage for convenient biosynthetic engineering, mutasynthesis, and production optimization. Moreover, in-frame deletions of one hydroxylase and two P450 monooxygenase genes resulted in the production of novel telomycin derivatives, revealing these genes to be responsible for the specific modification by hydroxylation of three amino acids found in the TEM backbone. Surprisingly, natural lipopeptide telomycin precursors were identified when characterizing an unusual precursor deacylation mechanism during telomycin maturation. By in vivo gene inactivation and in vitro biochemical characterization of the recombinant enzyme Tem25, the maturation process was shown to involve the cleavage of previously unknown telomycin precursor-lipopeptides, to yield 6-methylheptanoic acid and telomycins. These lipopeptides were isolated from an inactivation mutant of tem25 encoding a (de)acylase, structurally elucidated, and then shown to be deacylated by recombinant Tem25. The TEM precursor and several semisynthetic lipopeptide TEM derivatives showed rapid bactericidal killing and were active against several multidrug-resistant (MDR) Gram-positive pathogens, opening the path to future chemical optimization of telomycin for pharmaceutical application.

A Subdomain Swap Strategy for Reengineering Nonribosomal Peptides

Nonribosomal peptide synthetases (NRPSs) protect microorganisms from environmental threats by producing diverse siderophores, antibiotics, and other peptide natural products. Their modular molecular structure is also attractive from the standpoint of biosynthetic engineering. Here we evaluate a methodology for swapping module specificities of these mega-enzymes that takes advantage of flavodoxin-like subdomains involved in substrate recognition. Nine subdomains encoding diverse specificities were transplanted into the Phe-specific GrsA initiation module of gramicidin S synthetase. All chimeras could be purified as soluble protein. One construct based on a Val-specific subdomain showed sizable adenylation activity and functioned as a Val-Pro diketopiperazine synthetase upon addition of the proline-specific GrsB1 module. These results suggest that subdomain swapping could be a viable alternative to previous NRPS design approaches targeting binding pockets, domains, or entire modules. The short length of the swapped sequence stretch may facilitate straightforward exploitation of the wealth of existing NRPS modules for combinatorial biosynthesis.

Interspecies Transfer and Regulation of Pseudomonas stutzeri A1501 Nitrogen Fixation Island in Escherichia coli.

Until now, considerable effort has been made to engineer novel nitrogen-fixing organisms through the transfer of nif genes from various diazotrophs to non-nitrogen fixers; however, regulatory coupling of the heterologous nif genes with the regulatory system of the new host is still not well understood. In this work, a 49 kb nitrogen fixation island from P. stutzeri A1501 was transferred into E. coli using a novel and efficient transformation strategy, and a series of recombinant nitrogen-fixing E. coli strains were obtained. We found that the nitrogenase activity of the recombinant E. coli strain EN-01, similar to the parent strain P. stutzeri A1501, was dependent on external ammonia concentration, oxygen tension, and temperature. We further found that there existed a regulatory coupling between the E. coli general nitrogen regulatory system and the heterologous P. stutzeri nif island in the recombinant E. coli strain. We also provided evidence that the E. coli general nitrogen regulator GlnG protein was involved in the activation of the nif-specific regulator NifA via a direct interaction with the NifA promoter. To the best of our knowledge, this work plays a groundbreaking role in increasing understanding of the regulatory coupling of the heterologous nitrogen fixation system with the regulatory system of the recipient host. Furthermore, it will shed light on the structure and functional integrity of the nif island and will be useful for the construction of novel and more robust nitrogen-fixing organisms through biosynthetic engineering.

Uncovering the formation and selection of benzylmalonyl-CoA from the biosynthesis of splenocin and enterocin reveals a versatile way to introduce amino acids into polyketide carbon scaffolds.

Selective modification of carbon scaffolds via biosynthetic engineering is important for polyketide structural diversification. Yet, this scope is currently restricted to simple aliphatic groups due to (1) limited variety of CoA-linked extender units, which lack aromatic structures and chemical reactivity, and (2) narrow acyltransferase (AT) specificity, which is limited to aliphatic CoA-linked extender units. In this report, we uncovered and characterized the first aromatic CoA-linked extender unit benzylmalonyl-CoA from the biosynthetic pathways of splenocin and enterocin in Streptomyces sp. CNQ431. Its synthesis employs a deamination/reductive carboxylation strategy to convert phenylalanine into benzylmalonyl-CoA, providing a link between amino acid and CoA-linked extender unit synthesis. By characterization of its selection, we further validated that AT domains of splenocin, and antimycin polyketide synthases are able to select this extender unit to introduce the phenyl group into their dilactone scaffolds. The biosynthetic machinery involved in the formation of this extender unit is highly versatile and can be potentially tailored for tyrosine, histidine and aspartic acid. The disclosed aromatic extender unit, amino acid-oriented synthetic pathway, and aromatic-selective AT domains provides a systematic breakthrough toward current knowledge of polyketide extender unit formation and selection, and also opens a route for further engineering of polyketide carbon scaffolds using amino acids.

Construction of a New Class of Tetracycline Lead Structures with Potent Antibacterial Activity through Biosynthetic Engineering

AbstractAntimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad‐spectrum antibiotic lead was generated with significantly improved activity, including against all Gram‐negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering.

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering.

Insights into methyltransferase specificity and bioactivity of derivatives of the antibiotic plantazolicin.

Peptide antibiotics represent a class of conformationally constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide a rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).

AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.

The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation.

Saturation mutagenesis of TsrA Ala4 unveils a highly mutable residue of thiostrepton A.

Thiopeptides are post-translationally processed macrocyclic peptide metabolites, characterized by extensive backbone and side chain modifications that include a six-membered nitrogeneous ring, thiazol(in)e/oxazol(in)e rings, and dehydrated amino acid residues. Thiostrepton A, one of the more structurally complex and well-studied thiopeptides, contains a second macrocycle bearing a quinaldic acid moiety. Antibacterial, antimalarial, and anticancer properties have been described for thiostrepton A and other thiopeptides, although the molecular details for binding the cellular target in each case are not fully elaborated. We previously demonstrated that a mutation of the TsrA core peptide, Ala4Gly, supported the successful production of the corresponding thiostrepton variant. To more thoroughly probe the thiostrepton biosynthetic machinery's tolerance toward structural variation at the fourth position of the TsrA core peptide, we report here the saturation mutagenesis of this residue using a fosmid-dependent biosynthetic engineering method and the isolation of 16 thiostrepton analogues. Several types of side chain substitutions at the fourth position of TsrA, including those that introduce polar or branched hydrophobic residues are accepted, albeit with varied preferences. In contrast, proline and amino acid residues inherently charged at physiological pH are not well-tolerated at the queried site by the thiostrepton biosynthetic system. These newly generated thiostrepton analogues were assessed for their antibacterial activities and abilities to inhibit the proteolytic functions of the eukaryotic 20S proteasome. We demonstrate that the identity of the fourth amino acid residue in the thiostrepton scaffold is not critical for either ribosome or proteasome inhibition.

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.

Inside Cover: Site‐Specific Modification of the Anticancer and Antituberculosis Polyether Salinomycin by Biosynthetic Engineering (ChemBioChem 14/2014)

Inside Cover: Site‐Specific Modification of the Anticancer and Antituberculosis Polyether Salinomycin by Biosynthetic Engineering (ChemBioChem 14/2014)

Site-specific modification of the anticancer and antituberculosis polyether salinomycin by biosynthetic engineering.

The complex bis-spiroacetal polyether ionophore salinomycin has been identified as a uniquely selective agent against cancer stem cells and is also strikingly effective in an animal model of latent tuberculosis. The basis for these important activities is unknown. We show here that deletion of the salE gene abolishes salinomycin production and yields two new analogues, in both of which the C18=C19 cis double bond is replaced by a hydroxy group stereospecifically located at C19, but which differ from each other in the configuration of the bis-spiroacetal. These results identify SalE as a novel dehydratase and demonstrate that biosynthetic engineering can be used to redirect the reaction cascade of oxidative cyclization to yield new salinomycin analogues for use in mechanism-of-action studies.

Assembly of highly standardized gene fragments for high-level production of porphyrins in E. coli.

Standardization of molecular cloning greatly facilitates advanced DNA engineering, parts sharing, and collaborative efforts such as the iGEM competition. All of these attributes facilitate exploitation of the wealth of genetic information made available by genome and RNA sequencing. Standardization also comes at the cost of reduced flexibility. We addressed this paradox by formulating a set of design principles aimed at maximizing standardization while maintaining high flexibility in choice of cloning technique and minimizing the impact of standard sequences. The design principles were applied to formulate a molecular cloning pipeline and iteratively assemble and optimize a six-gene pathway for protoporphyrin IX synthesis in Escherichia coli. State of the art production levels were achieved through two simple cycles of engineering and screening. The principles defined here are generally applicable and simplifies the experimental design of projects aimed at biosynthetic pathway construction or engineering.

Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002

The plant terpenoids limonene (C10H16) and α-bisabolene (C15H24) are hydrocarbon precursors to a range of industrially relevant chemicals. High-titer microbial synthesis of limonene and α-bisabolene could pave the way for advances in in vivo engineering of tailor-made hydrocarbons, and production at commercial scale. We have engineered the fast-growing unicellular euryhaline cyanobacterium Synechococcus sp. PCC 7002 to produce yields of 4 mg L−1 limonene and 0.6 mg L−1 α-bisabolene through heterologous expression of the Mentha spicata l-limonene synthase or the Abies grandis (E)-α-bisabolene synthase genes, respectively. Titers were significantly higher when a dodecane overlay was applied during culturing, suggesting either that dodecane traps large quantities of volatile limonene or α-bisabolene that would otherwise be lost to evaporation, and/or that continuous product removal in dodecane alleviates product feedback inhibition to promote higher rates of synthesis. We also investigate limonene and bisabolene production in the ΔglgC genetic background, where carbon partitioning is redirected at the expense of glycogen biosynthesis. The Synechococcus sp. PCC 7002 ΔglgC mutant excreted a suite of overflow metabolites (α-ketoisocaproate, pyruvate, α-ketoglutarate, succinate, and acetate) during nitrogen-deprivation, and also at the onset of stationary growth in nutrient-replete media. None of the excreted metabolites, however, appeared to be effectively utilized for terpenoid metabolism. Interestingly, we observed a 1.6- to 2.5-fold increase in the extracellular concentration of most excreted organic acids when the ΔglgC mutant was conferred with the ability to produce limonene. Overall, Synechococcus sp. PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.