Natural Products: Discourse, Diversity, and Design

By Rebecca Goss

Natural Products: Discourse, Diversity and Design provides an informative and accessible overview of discoveries in the area of natural products in the genomic era, bringing together advances across the kingdoms.  As genomics data makes it increasingly clear that the genomes of microbes and plants contain far more genes for natural product synthesis than had been predicted from the numbers of previously identified metabolites, the potential of these organisms to synthesize diverse natural products is likely to be far greater than previously envisaged.  Natural Products addresses not only the philosophical questions of the natural role of these metabolites, but also the evolution of single and multiple pathways, and how these pathways and products may be harnessed to aid discovery of new bioactives and modes of action.

 

Edited by recognized leaders in the fields of plant and microbial biology, bioorganic chemistry and natural products chemistry, and with contributions from researchers at top labs around the world, Natural Products is unprecedented in its combination of disciplines and the breadth of its coverage. Natural Produces: Discourse, Diversity and Design  will appeal to advanced students and experienced researchers, from academia to industry, in diverse areas including ecology, industrial biotechnology, drug discovery, medicinal chemistry, agronomy, crop improvement, and natural product chemistry.

• Language: English
• Publisher: Wiley
• Release date: Apr 2, 2014
• ISBN: 9781118794609

Book preview

Section I Natural Products in the Natural World

Part 1 Role and Reason

1 The Role of Phytochemicals in Relationships of Plants with Other Organisms

Paweł Bednarek

2 Designer Microbial Ecosystems—Toward Biosynthesis with Engineered Microbial Consortia

David M. Babson, Mark Held, and Claudia Schmidt-Dannert

3 Marine Natural Products – Chemical Defense/Chemical Communication in Sponges and Corals

Elodie Quévrain, Isabelle Domart-Coulon, and Marie-Lise Bourguet-Kondracki

Part 2 Self-Protection – Avoiding Autotoxicity

4 How Plants Avoid the Toxicity of Self-Produced Defense Bioactive Compounds

Supaart Sirikantaramas, Mami Yamazaki, and Kazuki Saito

Part 3 Fishing and Pharming

5 Marine Bioprospecting

Amanda M. Fenner and William H. Gerwick

6 Myxobacteria: Chemical Diversity and Screening Strategies

Alberto Plaza and Rolf Müller

7 Fungal Endophytes of Grasses and Morning Glories, and Their Bioprotective Alkaloids

Christopher L. Schardl, Li Chen, and Carolyn A. Young

8 Fungal-Actinomycete Interactions—Wakening of Silent Fungal Secondary Metabolism Gene Clusters via Interorganismic Interactions

Volker Schroeckh, Hans-Wilhelm Nützmann, and Axel A. Brakhage

9 Secondary Metabolites Produced by Plant Pathogens

Barbara J. Howlett

Part 1

Role and Reason

1

The Role of Phytochemicals in Relationships of Plants with Other Organisms

Paweł Bednarek

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland

1.1 Introduction

Plants produce a large number of low-molecular-weight compounds classified as secondary metabolites. According to the original definition, these metabolites are synthesized by living cells, but are not necessary for new cell production.¹,² They can be derived from various primary metabolite precursors, and collectively display substantial chemical diversity, with specific compound classes restricted to particular plant phylogenetic clades.³,⁴ In addition, plant secondary metabolites show variability in their biosynthetic modes. Some phytochemicals are produced and stored, typically in specialized cell types, as a part of the developmental program independent of external factors.⁵,⁶ Other compounds are synthesized de novo in response to environmental cues by transcriptional activation of specific genes.⁷

Despite early attempts to demonstrate secondary metabolite function in plants, for many years these compounds were thought to lack any physiological significance.¹,⁸ The last 50 years of experimental research, however, has strongly supported multiple functions of phytochemicals in plant interactions with other organisms and adaptations to varied environmental conditions.¹,⁹ These include the attraction of beneficial organisms, that is, pollinators, seed dispersers, symbionts, and predators of plant enemies and defense against herbivores, pathogens, and competing plant species. Current secondary metabolite studies employ modern molecular methodologies and genetic tools. These include model organisms, as well as loss-of-function mutants and transgenic lines with alternations in plant biosynthetic pathways, or in the case of interacting organisms, mechanisms critical for adaptation to specific phytochemicals.

This chapter serves to provide a brief overview of small molecule function in interactions of plants with other organisms. Because of the substantial structural diversity and multiple roles of plant secondary metabolites, however, only select structurally and functionally characterized phytochemicals are addressed.

1.2 Glucosinolates

One of the most thoroughly studied plant metabolite groups with complex impacts on the surrounding environment are glucosinolates, amino acid–derived β-thioglucoside-N-hydroxysulfates (Figure 1.1). Glucosinolates are produced and accumulated constitutively by many plants belonging to the order Brassicales (=Capparales).⁵,¹⁰ The model plant Arabidopsis thaliana (Brassicaceae) accumulates two major groups of these compounds, methionine-derived aliphatic (AG), and tryptophan-derived indolic glucosinolates (IG). The wealth of genetic tools available for A. thaliana enabled detailed studies of glucosinolate biosynthesis and their functions in interactions with the environment. Glucosinolates are not biologically active; however, in response to different environmental stimuli, they can be metabolized to a range of different products, including molecules with high chemical reactivity.¹¹–¹³ This process can be initiated through the action of β-thioglucoside glucohydrolases (TGGs, known as myrosinases) that are usually compartmentalized separately from glucosinolates at the cellular or subcellular level in intact plant tissue¹⁴,¹⁵. Tissue damage, caused for instance by feeding herbivores, brings glucosinolates together with myrosinases and initiates glucosinolate hydrolysis, releasing chemically unstable aglycones (thiohydroximate-O-sulfonates) that can decompose into different types of end products (Figure 1.1).¹¹,¹³

Figure 1.1 (a) Simplified scheme of glucosinolate hydrolysis and further decomposition of the resulting aglycone (thiohydroximate-O-sulfonate). Only some of the possible end products are indicated. Note, that only indole glucosinolate–derived unstable isothiocyanates can be metabolized to Brassicaceae phytoalexins and indole-3-carbinol derivatives. TGG = β-thioglucoside glucohydrolases. (b) Structures of selected glucosinolates and their metabolism products.

1.2.1 Glucosinolates Affect Insect–Plant Relationship

The two-component glucosinolate–myrosinase defense system, known as the mustard oil bomb, was initially studied as a mechanism that generated molecules, which effectively deterred insects.¹⁶ Mustard oil bomb contribution to interactions of glucosinolate-producing plants with chewing insects was confirmed by enhanced feeding of several Lepidopteran species larvae on A. thaliana mutants defective in glucosinolate biosynthesis or hydrolysis. These included the myb28 myb29 line, which lacks two MYB-transcription factors regulating AG biosynthesis and does not produce any methionine-derived glucosinolates; the cyp79B2 cyp79B3 double knockout line deficient in several tryptophan derived metabolite production, including IGs; and the tgg1 tgg2 double knockout, depleted in leaf myrosinase activity.¹⁵,¹⁷,¹⁸ Furthermore, these studies revealed that AGs affected a broader range of Lepitopteran species than IGs.¹⁸ The AG deterrent function in these interactions was attributed to isothiocyanates, which possess the highest chemical reactivity and biological activity among all types of reported glucosinolate hydrolysis products.¹² Glucosinolates, however, can also decompose to compounds other than isothiocyanates (Figure 1.1). Generation of such products is usually supported by the presence of so-called specifier proteins.¹³ For example, the epithiospecifier protein promotes the formation of nitriles as major glucosinolate hydrolysis products in some A. thaliana accessions. In accordance with the presumed isothiocyanate function in insect deterrence, the cabbage looper (Trichoplusia ni) larvae fed more readily on nitrile-producing than isothiocyanate-producing A. thaliana lines.¹⁹ Shifting the profile of glucosinolate hydrolysis products from isothiocyanates to nitriles by overexpression of epithiospecifier protein, however, leads to elevated attraction of Cotesia rubecula, a Hymenopterid parasitoid specialized on Pieris rapae, during the feeding of its host larvae on the respective A. thaliana lines.²⁰ This suggested that glucosinolate hydrolysis products, depending on their chemical nature, might contribute to plant defense by direct toxicity, or indirectly by attracting parasites of infesting insects.

Despite its proven function in interactions with members of the Lepidoptera, cell destruction–based glucosinolate activation is not effective against all herbivorous insect groups. For example, Hemiptera aphids use slender stylets inserted intercellularly to feed from phloem sieve elements to prevent plant tissue damage; however, experimental evidence has indicated that glucosinolates can deter aphids, even in the absence of tissue damage or plant TGG activity. Myzus persicae (green peach aphid) absorbed glucosinolates with phloem sap while feeding on A. thaliana. AGs passed intact through the gut, during which time less stable IGs decomposed to a series of different indole-3-carbinol derivatives (Figure 1.1).²¹,²² The importance of IG-derived molecules in plant defense was supported when cyp79B2 cyp79B3 double knockout plants succumbed to M. persicae more rapidly than did the wild-type plants, and aphid reproduction on atr1D mutants, characterized by elevated IG levels, significantly decreased.²² Further studies demonstrated that these defense functions were particularly associated with products derived during gut metabolism of IGs substituted at the position 4 of the indole core, which are generated by the CYP81F2 monooxygenase.²²,²³

Glucosinolate-based defense mechanisms are typically effective against generalist insects (e.g., as demonstrated in chewing and phloem-feeding insects), whereas studies indicate that specialist insects evolved different biochemical mechanisms to avoid toxic glucosinolate breakdown products.²⁴ The diamondback moth (Plutella xylostella) larvae produce sulfatase that converted glucosinolates to desulfo-glucosinolates, which are not accepted by myrosinases as substrates.²⁵ The cabbage white butterfly (Pieris rapae) larvae redirected the decomposition of glucosinolate aglycones from isothiocyanates to less toxic nitriles using a species-specific nitrile-specifier protein.²⁶ The cabbage aphid, Brevicoryne brassicae, represents a very interesting example of protection against glucosinolates. Experimental evidence has suggested that this insect sequesters plant glucosinolates and uses them along with its own myrosinase as a defense mechanism against its natural enemies.²⁷ Remarkably, many insects specialized on Brassicaceae (including representatives of Coleoptera, Lepidoptera, and Diptera) use glucosinolates or their metabolic products as an obligatory oviposition or feeding cue.²⁴,²⁸,²⁹ This shows that metabolites produced for plant defense may become not only useless but also disadvantageous in interactions with adapted pests.

1.2.2 Glucosinolates in Plant Immunity

The chemical reactivity and in vitro antimicrobial activity of glucosinolate-derived isothiocyanates suggested that similar to functions in plant–insect interactions, these compounds might also contribute to the restriction of microbial pathogens.¹²,³⁰ The putative AG-derived isothiocyanate function in immunity was recently demonstrated in A. thaliana nonhost resistance to the bacterial pathogen Pseudomonas syringae. The virulence of P. syringae strains adapted on A. thaliana was shown to be supported with the sax (survival in Arabidopsis extracts) operon, which encodes proteins enabling detoxification and extrusion of AG-derived isothiocyanates. P. syringae strains lacking sax genes were less virulent on young leaves of wild type A. thaliana compared with the AG-deficient myb28 myb29 double knockout line.³¹ Furthermore, myb28 myb29 plants were also more susceptible to the pathogenic ascomycete Sclerotonia sclerotiorum, suggesting that AG function in plant immunity is not restricted to bacterial pathogens.³²

Like aphids, however, many microbial pathogens can colonize plants without remarkable tissue or cell damage. For example, biotrophic or hemibiotrophic fungal and oomycete pathogens are able to keep penetrated epidermal cells alive.³³ It seems rather unlikely that in such cases, tissue damage–based glucosinolate activation contributes significantly to plant immunity. It is notable that in Brassicaceae, pathogen challenge or recognition of microbe-associated molecular patterns (MAMPs) elicited cell damage–independent metabolism of IGs.³⁴–³⁷ This process was dependent on an atypical myrosinase PENETRATION2, which was redirected to the cell periphery at fungal penetration sites and mediated formation of end products different from those reported to contribute to plant–insect interactions.³⁴,³⁸ Results in A. thaliana demonstrated this metabolic pathway was critical for preinvasive defense responses against a broad range of fungal and oomycete pathogens with varied hosts and feeding strategies.³⁸–⁴⁰ As in plant–aphid interactions, IG function in plant immunity was shown to be mediated primarily by their structural variants substituted by the CYP81F2 monooxygenase at the position 4 of the indole ring.²³,³⁴,³⁷,⁴⁰ This suggests IG activation during A. thaliana interactions with pathogens and phloem-feeding, but not chewing, insects may share some common biochemical mechanisms.

1.3 Benzoxazinone Glucosides

Glucosinolates represent only one of many classes of glycosylated plant secondary metabolites, which are produced constitutively and may serve as a source of biologically active molecules in response to environmental stimuli⁴¹. Another group of such phytochemicals are benzoxazinone glucosides (Figure 1.2), present in monocot and dicot species. Like glucosinolates, however, benzoxazinone glucosides are not ubiquitous throughout the plant kingdom.⁶ This class of compounds is proposed to accumulate in vacuoles, and β-glucosidase, which is required for their hydrolysis was detected in plastids. Benzoxazinone glucoside activation is hypothesized to occur following pest attack, and subsequent cell disintegration.⁶,⁴¹ In maize (Zea mays), biosynthesis of these phytochemicals was severely reduced in a benzoxazineless1 (bx1) mutant line.⁴²,⁴³ Notably, bx1 plants were extremely susceptible to Setosphaeria turcica, a causal agent of Northern corn leaf blight.⁴⁴ Increased susceptibility corresponded with elevated S. turcica entry rates on the bx1 line compared with wild-type maize.⁴⁵ These results suggested benzoxazinone glucosides were activated in intact epidermal cells, and respective products restricted fungal growth at the preinvasive stage, similar to A. thaliana IG-derived metabolites.³⁴ An analogous role of IGs and benzoxazinone glucosides in preinvasive fungal pathogen resistance is not the only similarity between the functions of these different classes of molecules in plant interactions with other organisms. Benzoxazinone glucosides have been shown to be integral in plant resistance to aphids, as indicated by enhanced proliferation of the cereal aphid Rhopalosiphum padi on the bx1 line.⁴⁵ Furthermore, in response to MAMPs, bx1 maize mutants and A. thaliana lines defective in IG-biosynthesis or metabolism were also defective in callose (β-1,3-glucan) deposition, which is an immune response observed in all the plant species tested so far.³⁷,⁴⁵,⁴⁶ These results suggest that conserved across the plant kingdom mechanisms of interaction with other organisms can utilize as signals or executors phytochemicals that are unique to particular plant phylogenetic clades.

Figure 1.2 Benzoxazinone glucosides and their hydrolysis products. DIMBOA = 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, MBOA = 6-methoxy-2-benzoxazolinone.

Despite the beneficial features of benzoxazinone glucosides, these compounds, similar to other phytochemicals, exhibit adverse effects on plants. For example, the nutritious crown roots of maize showed relatively high 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (Figure 1.2) accumulation levels that deterred the generalist banded cucumber beetle Diabrotica balteata, but not the specialist western corn rootworm Diabrotica virgifera, which preferred and grew best on these tissues.⁴⁷ Choice experiments with the bx1 mutant line and purified bezoxazinones suggested that D. virgifera uses these compounds as foraging cues.⁴⁷

1.4 Strigolactones

Another striking example of the multiple and complex roles of phytochemicals are strigolactones (Figure 1.3), carotenoid-derived terpenoid lactones that are relatively conserved across the plant kingdom.⁴⁸ These metabolites were originally identified owing to their capacity to stimulate seed germination in the achlorophyllous root parasites Orobanche and Striga.⁴⁹ Production of disadvantageous compounds was rather unlikely to remain conserved across many plant genera and species; therefore, these compounds were supposed to have another core function. Research in mycorrhizal fungi has identified strigolactones as critical signals for the establishment of arbuscular mycorrhizal symbioses.⁵⁰,⁵¹ Arbuscular mycorrhizal fungi form mutualistic symbiotic relationships with roots of more than 80% of terrestrial plants, which serve to improve acquisition of nutrients, such as phosphate and nitrogen, by the host.⁵² Evidence strongly supports that this form of symbiosis played a key role in land colonization by the first primitive plants. Strigolactones have been identified in the charophyte green algae representing order Charales, which implies that strigolactone production predates arbuscular mycorrhizal symbiosis, and suggests that these compounds made an integral contribution to the current shape of life on Earth.⁵³

Figure 1.3 Selected strigolactone structures.

Evidence from other studies demonstrates that strigolactone significance in plants is much more complex. These compounds function as plant growth hormones that suppress subapical shoot outgrowth.⁵⁴,⁵⁵ Following this finding, other functions of this new class of hormones have been reported, including regulation of root architecture and root hair elongation.⁵⁶ Strigolactones also exhibit hormonelike functions in more primitive plant species, including stimulating growth of protonema in Physcomitrella patens,⁵⁷ and rhizoid elongation in the charophytic green algae Chara coralina.⁵³ The latter finding has suggested that strigolactones evolved primarily as plant hormones, which during land plant evolution became specific signals enabling mycorrhizal fungi localization of a suitable symbiotic partner.⁵³,⁵⁸ Because of their double function, that is, serving as plant hormones and integral signals to establish advantageous symbiotic associations, strigolactones became almost indispensable for most land plants. This in turn made these phytochemicals effective cues informing parasitic plant regarding potential host vicinity and activating their seed germination.

1.5 Phytoalexins – Inducible Defense Metabolites

Exclusive of constitutive phytochemicals, plants also produce secondary metabolites in response to environmental cues. Phytoalexins are a well-studied group of such compounds. This term was originally proposed for a hypothetical antimicrobial substance induced in potato tuber tissue by an incompatible strain of the oomycete Phytophthora infestans.⁵⁹ Currently the term phytoalexin refers to low-weight antimicrobial metabolites that are synthesized de novo and accumulate in plants following pathogen challenge.⁷ Consistent with constitutive metabolites, inducible defense phytochemicals are characterized by substantial structural diversity, where unique classes of compounds are restricted to specific phylogenetic clades. However, all plant species tested so far were found to be capable of synthesizing de novo a set of phytoalexins upon microbial invasion, which suggests that this capacity is evolutionarily ancient and indispensable.

1.5.1 Phenyalanine-derived Phytoalexins

One of the interesting phenylalanine-derived phytoalexins is resveratrol (Figure 1.4), induced following infection in several phylogenetically unrelated species, including grapevine (Vitis vinifera)⁶⁰ and peanut (Arachis hypogaea).⁶¹ Biosynthesis of this particular compound requires the presence of only one unique enzyme, stilbene synthase (STS).⁶² The precursor molecules, p-coumaroyl-CoA and malonyl-CoA, are constitutively synthesized by plants, consequently resveratrol a viable candidate molecule for metabolic engineering.⁶³ Heterologous expression of the grapevine STS gene VST1 under control of its native promoter in tobacco (Nicotiana tabacum) lead to pathogen-inducible accumulation of VST1 mRNA and resveratrol, which correlated with enhanced disease resistance to the necrotrophic fungal pathogen Botritis cinerea⁶⁴. This early successful plant metabolite engineering demonstrated the significance of resveratrol in plant immunity.

Figure 1.4 Biosynthesis of phenylalanine-derived phytoalexins and related compounds. STS = stilbene synthase, CHS = chalcone synthase, CHR = chalcone reductase, CHI = chalcone isomerase, IFS = isoflavone synthase.

Particular types of isoflavonoids constitute another group of phenylalanine-derived phytoalexins and are primarily produced by members of the Fabaceae. These include, among many others, glyceollins (Figure 1.4) synthesized by soybean (Glycine max)⁶⁵ and pisatin (Figure 1.4) from pea (Pisum sativum).⁶⁶ Despite the well-documented in vitro antimicrobial activity of these metabolites, direct in vivo evidence for isoflavonoid function in plant immunity had not been generated until recently. Strains of the soybean-adapted pathogenic bacterium P. syringae, were shown to produce and deliver the HopZ1 effector into plant cells.⁶⁷ HopZ1 physically interacted with the 2-hydroxyisoflavanone dehydratase, an enzyme required for production of dadzein (Figure 1.4), a precursor of glyceollins. The interaction led to reduced 2-hydroxyisoflavanone dehydratase levels, most likely owing to degradation by HopZ1, and correspondingly reduced isoflavonoid leveles, which promoted bacterial multiplication in soybean.⁶⁸

Some plants produce flavonoid-type phytoalexins, which are biosynthetically closely related to isoflavonoids. A well-studied example are 3-deoxyanthocyanidins (Figure 1.4) that were reported to accumulate after pathogen challenge in sorghum (Sorghum bicolor).⁶⁹ Interestingly, many Fabaceae species use flavonoids (e.g., naringenin and luteolin; Figure 1.4) to communicate with their symbiotic partners nitrogen fixing rhizobia bacteria.⁷⁰ Perception of flavonoid signals by rhizobia activates NodD (NodulationD) transcription factor, which controls expression of Nod genes required for the synthesis of bacterial lipochitooligosaccharide Nod factors.⁷¹ These oligosaccharides are perceived by the host plants and induce development of root nodules, specialized organs, which accommodate rhizobia.⁷² This indicates that different sub-branches of the same metabolic pathway may deliver compounds with different functions in interactions of plants with the surrounding organisms. The significance of flavonoids and isoflavonoids is not restricted to symbiotic signals and phytoalexin functions. Studies with white clover (Trifolium repens) revealed that flavonoids and isoflavonoids accumulated specifically in cells undergoing early nodule organogenesis.⁷³ The importance of this result was initially not clear, however, experimental evidence suggested involvement of these compounds in auxin transport regulation, not only in Fabaceae, but also in other plant species.⁷⁴ Changes in auxin transport and accumulation appeared, in turn, to be critical for functional nodule formation.⁷⁵,⁷⁶ Despite clear significance of flavonoid and isoflavonoid accumulation in host plant root nodules, disadvantages have been demonstrated. The clover root weevil, Sitona lepidus, preferentially feeds on nitrogen-fixing root nodules because of high amino acid concentrations in the organs. Experimental evidence has suggested that this pest identifies nodules on the basis of increased isoflavonoid formononetin (Figure 1.4) concentrations in root nodules relative to the rest of the root.⁷⁷

1.5.2 Phytoalexins in Brassicaceae

Brassicaceae plant species produce sulfur-containing tryptophan-derived alkaloid phytoalexins in response to infection.⁷⁸ The most thoroughly studied is camalexin (Figure 1.5),⁷⁹ which was found in A. thaliana and other closely related species representing the Camelinae tribe.³⁶,⁸⁰ Similar to that for glucosinolates, the genetic tools available for A. thaliana supported studies of camalexin biosynthesis and in vivo function in defense responses.⁷,⁷⁹ For example, isolated in a genetic screen the phytoalexin-deficient3 (pad3) line carrying mutation in a gene encoding a P450 monooxygenase catalyzing the final step in camalexin biosynthesis⁸¹,⁸² displayed enhanced susceptibility to several fungal and oomycete pathogens providing evidence for camalexin function in A. thaliana immunity.⁴⁰,⁸³,⁸⁴ Furthermore, experimental evidence has suggested that camalexin possesses complementary to IGs immune function, which restricts pathogen growth following successful penetration into plant epidermal cells.³⁴,⁴⁰

Figure 1.5 Representative structures of Brassicaceae phytoalexins.

Further studies in A. thaliana have demonstrated that the PAD3 gene expression was up-regulated, not only following pathogen inoculation but also after attacks by several phloem-feeding insects, including M. persicae⁸⁵,⁸⁶ and B. brassicae.⁸⁷ In addition, pad3-1 mutant plants were more sensitive to B. brassicae;⁸⁷ however, the number of M. persicae offspring in this line was unaffected compared with wild-type plants.⁸⁸ These findings have indicated that phytoalexin function is likely not only restricted to antimicrobial activity, but these compounds may affect some insects.

Deciphering Brassicaceae phytoalexin biosynthesis also revealed that respective biosynthetic pathways are strongly interconnected with glucosinolate biosynthesis. For example, camalexin biosynthesis shares at least two enzymes with the glucosinolate pathway necessary to incorporate reduced sulfur into the core structure,⁸⁹ whereas other representatives of these phytochemicals, including brassinin, cyclobrassinin, and wasalexins (Figure 1.5), were shown to be synthesized in planta directly from IGs.⁹⁰ This immediately raises questions regarding metabolic networks between constitutive and inducible defense molecules in other plant species.

1.6 Conclusions

Recent years have brought enormous progress in our understanding of the function of phytochemicals in plant interactions with the environment; however, assignment of selected secondary metabolites to particular functions in host–plant interactions with other organisms remains a major challenge. This results from the complexity of plant metabolic pathways and the complexity of interorganismal associations. Additionally, phytochemical roles are often obscured by an evolutionary arms race between plants and their enemies, which continuously evolve new protective strategies against plant bioactive metabolites. For example, herbivores and pathogens may achieve this through phytochemical excretion and metabolism or by behavioral adaptations. Consequently, specific plant defense metabolites are functional only against a subgroup of potential target organisms. Plant enemies are even capable of learning to use selected phytochemicals for their own benefit, for example, to localize their favorite plant host. Consequently, it is often difficult to unequivocally quantify the contribution of a particular compound or metabolite group to plant fitness. Typically, the influence of a certain compound is a balance between benefits and trade-offs, which varies for the same plant species among different ecotypes and ecological niches. Some of these issues should be addressed in the near future from a more ecological perspective by shifting research from bi- to multiorganismal interactions.

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2

Designer Microbial Ecosystems – Toward Biosynthesis with Engineered Microbial Consortia

David M. Babson, Mark Held, and Claudia Schmidt-Dannert

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, St. Paul, MN, USA

2.1 Introduction

Large-scale bioproduction of bioactive compounds, fine chemicals, and commodity chemicals is typically carried out using pure cultures of genetically engineered synthetic strains. In contrast, processes such as waste water treatment, aqueous nitrogen removal, anaerobic digestion, and targeted bioremediation employ complex microbial communities to facilitate the desired biological conversions.¹–³ The interactions among these microbial communities are natural, accomplish tasks that individual members could not perform independently, and respond well to external inputs that control the microbial environment and community structure. Before the development of recombinant DNA technologies, mixed cultures were also extensively investigated as production platforms for major biotechnological commodities such as organic acids and amino acids.⁴

Biochemical synthesis of many desirable natural products proceeds through numerous metabolic steps creating a number of confounding problems for metabolic pathway engineers. Without utilizing microbial-mediated synthesis, production of many of these compounds would be economically impractical, as it would require complex reactor configurations and generate low yields. From an economic perspective, bioprocesses that employ pure cultures of metabolically engineered cells are inherently simplistic and less expensive compared with more complex, multireactor processes.

Although synthetic biology can confer new functionality to genetically tractable microorganisms, complex multistep pathways are difficult to establish in a single, non-native host. As the complexity of the engineered biosynthetic reaction cascade increases, the heterologous production process becomes less robust and efficient due to the increased metabolic burden placed on the microbial host. Thus, the logical extension would be to harness the tools developed by synthetic biologists and metabolic engineers to divide metabolic functions across different, engineered microorganisms and create synthetic microbial consortia that perform highly specialized bioconversions by working collectively to generate desirable products (Figure 2.1).

Figure 2.1 Bioprocessing using pure or mixed cultures. Current bioprocesses typically use homogenous cultures of genetically modified microorganisms that convert one or more substrate(s) into a desired product using single or multiple reactor processes (a). In the future, synthetic microbial consortia may be engineered that collectively convert in a single reactor substrate(s) into desired product(s) via shared intermediates. A synthetic population control system would automatically adjust cell densities of consortia members to the specific bioconversion requirements (b).

By compartmentalizing metabolic functions into separate microbial populations it becomes possible to optimize the metabolic network of each strain for distinct metabolic tasks that together result in the production of a desired compound. Such division of labor (i) reduces the metabolic burden imposed on each strain, (ii) allows separate engineering or optimization of metabolic pathways that normally would not efficiently operate simultaneously, (iii) allows recombinant assembly of long multistep pathways divided over two or more engineered strains, and (iv) allows the unique metabolic features and cellular properties present in different strains to be exploited. The different microbial strains cocultured in a bioprocess can thus be considered individual metabolic modules that may be exchanged or augmented with additional modules.

For example, the use of mixed cultures specifically selected for bioprocessing has recently been rediscovered for biofuel production. Here, consolidated bioprocessing (CBP)—the combination of multiple processing steps into a single reactor process—aims to combine hydrolysis of recalcitrant lignocellulose and subsequent conversion of the released sugars into biofuels.

Although engineering microbial consortia has been recognized as a new frontier in biotechnology that could significantly impact future biotechnological production processes, the design of artificial microbial consortia has mostly been limited to model systems.⁵–¹⁵ The concept of establishing consolidated bioprocesses using synthetically engineered consortia is straightforward, but it functionally presents three specific challenges. First, the specialized consortia members need to be developed using genetic and metabolic engineering techniques to perform highly specialized tasks efficiently in a synthetic environment. Second, the synthetic environment needs to be engineered to meet the essential needs of the various microbial members, as well as the overall biosynthetic pathway to be carried out. Finally, some form of cell-to-cell communication is required to modulate relative population densities on the basis of process reaction kinetics and metabolic rates. Each of these points represents a large, multivariable problem and demands significant research-based efforts for optimization, but it is the development of communication circuits that will be crucial for the design of robust, synthetic microbial bioprocessing consortia. The engineering of biological networks with new emergent properties employed in the field of synthetic biology¹⁶ has provided tools and strategies with which to design such circuits.

This chapter explores engineering options for the design of synthetic microbial consortia in the context of multi-step, CBP in the context of biofuel and natural product synthesis. It highlights the application of synthetic cell-to-cell communication circuits, based on bacterial quorum-sensing (QS) systems, as a means of not only controlling product synthesis, but also reactor homeostasis.

2.2 Bacterial Cell-to-Cell Communication via Quorum-Sensing Systems

Forty years of research into bacterial cell-to-cell communication has provided an increasing understanding about the mechanism by which bacteria communicate, coordinate, and affect each other's behaviors. (Summarized in Stevens).¹⁷ Regulatory processes by which bacteria coordinate population behavior in response to cell densities in their environment—referred to as quorum sensing (QS)—are best understood and can be engineered to control gene expression in heterologous bacterial systems.¹⁸–²⁰ Microorganisms capable of QS produce a signal compound that is secreted from the cell into the environment. As the numbers of similar cells proliferate in the environment, the signal compound accumulates. At a threshold concentration, cells recognize the presence of the signal compound, and by extension, the cell population of their immediate environment. The signal compound can then elicit specific cellular functions that allow the entire population to coordinate metabolic functions (Figure 2.2). Several different types of QS signals have been identified. Some of them are recognized by many species, allowing interspecies communication, while others are species-specific. In addition, many microorganisms employ more than one QS system as part of complex signal transduction networks²¹.

Figure 2.2 Gram-negative and Gram-positive bacteria use different quorum sensing systems to coordinate population behavior in response to cell densities. Many Gram-negative bacteria produce AHLs (3-oxo-C6 homo serine lactone of the Vibrio. fisheri LuxR/I system shown) as QS signals that are produced by an autoinducer synthase (I) and diffuse out of the cell. As the microbe proliferates in the environment the signal compound accumulates in the environment. At some threshold concentration the receiver module consisting of a response regulator R senses the signal chemical, and initiates downstream gene expression via binding to cognate promoter elements of target (T) genes. Gram-positive produce auto-inducing peptides (shown is the mature AIP from S. aureus) as QS signal. AIPs are translated as pro-peptides that are processed and secreted by a dedicated set or membrane-bound proteins (A, B). Signal recognition involves binding to a transmembrane histidine kinase (HK) that triggers a phosphorelay resulting in the activation of a cognate response regulator (RR) and subsequent expression of downstream target (T) genes.

This form of bacterial communication is not thought to be very diverse mechanistically,²² with only three general classes of chemical compounds commonly associated with cell-to-cell signaling in bacteria: acyl homoserine lactones (AHLs), oligopeptides, and the LuxS/autoinducer-2 (AI-2) type compounds. In general, the QS machinery is divided among sender module components that are involved in the production of diffusible signal compounds and receiver module components that confer the capability of signal compound recognition. In all of these systems, secreted QS signals are recognized by an intracellular transcription factor, either through direct interaction, or via phosphorylation initiated by a trans-membrane histidine kinase (HK).

Many Gram-negative QS systems employ an autoinducer synthase that produces small molecule AHL signals that can diffuse in and out of cells. AHLs share a homoserine lactone basal group with a variable acyl or aryl side chain.¹⁸,²³–²⁵ Specificity is afforded by differences in the length, saturation, and/or composition of R groups affixed to these side chains.²⁶ Conversely, the receiver module consists of intracellular transcription factors that are homologs of the Vibrio fischeri LuxR protein. At certain concentrations, AHLs bind to LuxR-type transcription factors, causing their activation and subsequent translocation to cognate promoters. This results in the transcriptional regulation of downstream signaling networks.

Many Gram-positive bacteria on the other hand, utilize a much more complex two-component HK response regulator system that recognizes extracellular oligopeptides (autoinducing peptides, AIPs). The sender module consists of genes encoding the AIP precursor and dedicated transport machinery that facilitates its maturation and secretion. During this process, post-translational modifications such as formation of a cyclic thiolactone (AgrD, Staphylococcus aureus) or geranylation of certain amino acid residues (ComX, Bacillus subtilis) may be added to the AIP.¹⁸,²⁰,²⁷ Once the quorum threshold is achieved, the interaction of extracellular AIPs with a transmembrane HK triggers a phosphorelay that activates a downstream, cognate response regulator (RR). Subsequently, the activated RR then binds to responsive promoters and regulates transcription of QS-controlled genes. In contrast to the fairly promiscuous AHL-based systems, the AIP-based systems are extremely species- and even strain-specific.

The receiver and sender units of natural QS systems lend themselves as modules for the design of genetic control circuits to establish cell-to-cell communication that directs the population behavior of heterologous cultures as discussed next in this chapter.

2.3 Engineering Population Control into Designer Bioproduction Consortia

In natural microbial consortia, homeostasis of subpopulations is maintained by the exchange of metabolites and signaling molecules that control growth. A number of studies have investigated mechanisms that maintain cooperative behavior in this manner.⁹,¹¹,²⁸–³⁰ These studies all suggest that the employment of multiple levels of homeostatic controls such as signaling molecules, multifaceted interactions, metabolic codependence, as well as spatial organization of cell populations contribute to the homeostasis of a consortium.

In a synthetic consortium, it is most efficient to distribute the metabolic burden among several organisms where each member has been selected and optimized for a specific role with the goal of cooperatively converting a substrate pool into a desired product. One member of such a consortium could function to produce specific precursor chemicals and another to produce catalytic enzymes and yet another to carry out final product generation. Consequently, in such a highly synthetic system, natural growth selection systems do not exist and need to be artificially incorporated to control population growth rates in response to cell densities and metabolite concentrations in order to achieve maximum process efficiency.

Growth control can be obtained by coupling signal exchange and recognition to the expression of a growth selection marker, which may be an antibiotic-resistant gene or essential metabolic gene. This strategy enhances the stability of a synthetic consortium to perform a bioprocess by selecting strongly against cheaters (microorganisms benefiting from the synthetic ecological environment without contributing the desired bioprocess). Components from naturally occurring bacterial QS systems discussed previously can be transferred into consortia members to facilitate growth control when interfaced with the expression of a specific growth selecting trait. In fact, the less complex AHL-type QS of Gram-negative bacteria have been widely used to engineer such genetic circuits into Escherichia coli.⁵,⁶,⁸,³¹–³⁷ Apart from being engineerable, AHL-type QS systems pose a smaller metabolic burden for a cell as AHL-signal production requires less energy (between 0 and 8 ATP) compared with the production of the Gram-positive oligopeptide QS signals (184 ATP).¹⁹ In addition, many homologs of the acyl-HSL machinery have been characterized and engineered with specific response capabilities providing a toolbox with which to fine-tune growth control.³⁸–⁴¹

Many different circuits can be constructed, and tailored by the tasks required of the synthetic ecological system. These circuits are inherently modular, with sender and receiver components representing independent units that can be isolated, cloned, and reassembled into a desired host. The manner in which these circuits are reconstructed in the new host organisms controls the type of communication achieved. For example, an intact QS system, where the sender and receiver modules are associated by the signal compound (as in natural systems) could be used to establish functional controls in a pure culture. This would allow specific gene expression to result only at certain cell densities.

More complex engineered circuits could be created simply by separating the sender and receiver modules to create various types of directional communication systems. For example, a unidirectional communication system can be produced from a single QS system by separating the sender and receiver modules and constructing them in two independent organisms (Figure 2.3a). ³²,³⁵ In this scenario, a sender cell and receiver cell would be produced. Gene expression in the receiver cells could be controlled as a function of the sender cells' population density, but there would not be the possibility of reciprocal control. By using two QS systems, bidirectional communication circuits can be established.⁶,⁸,³⁴ In this more complex scenario, sender and receiver modules that are not associated by the same signal compound are transplanted into the same cell. When coupled with opposing sender and receiver modules in a second cell type, this scenario results in cells that would be a sender for, and receiver to the other cell type (Figure 2.3b).

Figure 2.3 Engineering cell-to-cell communication circuits in microbial consortia. (a) Uni-directional communication circuit can be established by expressing an autoinducer synthase I in one cell (sender cell) and a cognate response regulator (R) in a second cell (receiver cell). Signal recognition by the receiver cell triggers expression of target genes by binding of the response regulator to cognate promoters. (b) Bidirectional communication circuit. Autoinducer synthases (I1, I2) in each engineered cell-type (receiver/sender cells) make different homo serine lactone (HSL) signals that diffuse out of the cell. Response regulators (R1, R2) in each cell recognize their cognate HSL signals and activate expression of target genes (i.e., resistance marker, reporter, etc.) by binding to promoters (PR1, PR2).

Still more complex communication scenarios can be envisioned by increasing the number of QS systems employed and/or configuring the directional communication in different manners as illustrated in Figure 2.4. For example in a complex biosynthesis using three micro-organisms, a master controller could be used to produce a signal compound received by multiple other types of cells in the consortium; however, once a control circuit has been designed and established, it must be tuned to control and maintain the desired population densities that best support efficient production of the final product, which will be discussed later.

Figure 2.4 The master controller scenario. A single engineered microbe can act as a master controller within a consortia and direct population dynamics of multiple cell types. (a) In a unidirectional sense,