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Abstract Background Phenylpropanoids are the precursors to a range of important plant metabolites such as the cell wall constituent lignin and the secondary metabolites belonging to the flavonoid/stilbene class of compounds. The latter class of plant natural products has been shown to function in a wide range of biological activities. During the last few years an increasing number of health benefits have been associated with these compounds. In particular, they demonstrate potent antioxidant activity and the ability to selectively inhibit certain tyrosine kinases. Biosynthesis of many medicinally important plant secondary metabolites, including stilbenes, is frequently not very well understood and under tight spatial and temporal control, limiting their availability from plant sources. As an alternative, we sought to develop an approach for the biosynthesis of diverse stilbenes by engineered recombinant microbial cells. Results A pathway for stilbene biosynthesis was constructed in Escherichia coli with 4-coumaroyl CoA ligase 1 4CL1) from Arabidopsis thaliana and stilbene synthase (STS) cloned from Arachis hypogaea. E. coli cultures expressing these enzymes together converted the phenylpropionic acid precursor 4-coumaric acid, added to the growth medium, to the stilbene resveratrol (>100 mg/L). Caffeic acid, added in the same way, resulted in the production of the expected dihydroxylated stilbene, piceatannol (>10 mg/L). Ferulic acid, however, was not converted to the expected stilbene product, isorhapontigenin. Substitution of 4CL1 with a homologous enzyme, 4CL4, with a preference for ferulic acid over 4-coumaric acid, had no effect on the conversion of ferulic acid. Accumulation of tri- and tetraketide lactones from ferulic acid, regardless of the CoA-ligase expressed in E. coli, suggests that STS cannot properly accommodate and fold the tetraketide intermediate to the corresponding stilbene structure. Conclusion Phenylpropionic acids, such as 4-coumaric acid and caffeic acid, can be efficiently converted to stilbene compounds by recombinant E. coli cells expressing plant biosynthetic genes. Optimization of precursor conversion and cyclization of the bulky ferulic acid precursor by host metabolic engineering and protein engineering may afford the synthesis of even more structurally diverse stilbene compounds.

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs., (© 2021. The Author(s).)

We herein describe a bioinspired solid-phase assembly of a multienzyme system scaffolded on an artificial cellulosome. An alcohol dehydrogenase and an ω-transaminase were fused to cohesin and dockerin domains to drive their sequential and ordered coimmobilization on agarose porous microbeads. The resulting immobilized scaffolded enzymatic cellulosome was characterized through quartz crystal microbalance with dissipation and confocal laser scanning microscopy to demonstrate that both enzymes interact with each other and physically colocalize within the microbeads. Finally, the assembled multifunctional heterogeneous biocatalyst was tested for the one-pot conversion of alcohols into amines. By using the physically colocalized enzymatic system confined into porous microbeads, the yield of the corresponding amine was 1.3 and 10 times higher than the spatially segregated immobilized system and the free enzymes, respectively. This work establishes the basis of a new concept to organize multienzyme systems at the nanoscale within solid and porous immobilization carriers.

Two decades of structural and functional studies have revealed functions, structures and diversity of bacterial microcompartments. The protein-based organelles encapsulate diverse metabolic pathways in semipermeable, icosahedral or pseudo-icosahedral shells. One of the first discovered and characterized microcompartments are those involved in ethanolamine degradation. This review will summarize their function and assembly along with shared and unique characteristics with other microcompartment types. The modularity and self-assembling properties of their shell proteins make them valuable targets for bioengineering. Advances and prospects for shell protein engineering in vivo and in vitro for synthetic biology and biotechnology applications will be discussed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The presence of endophytes promotes the biosynthesis of secondary plant metabolites. In this study, endophytic fungi were isolated from Schinus terebinthifolius to investigate their diversity and antimicrobial activity. A total of 272 endophytic fungi was obtained. These belonged to nine different genera: Alternaria, Colletotrichum, Diaporthe, Epicoccum, Fusarium, Pestalotiopsis, Phyllosticta, Xylaria, and Cryptococcus. Notably, Diaporthe foliorum was introduced as a new species, with accompanying morphological descriptions, illustrations, and a multigene phylogenetic analysis (using ITS, TEF1, TUB, HIS, and CAL). Among the 26 fungal morphotypes evaluated for antimicrobial activity, five strains had inhibitory effects against pathogenic microorganisms. Xylaria allantoidea CMRP1424 extracts showed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Diaporthe terebinthifolii CMRP1430 and CMRP1436 showed antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Meanwhile, D. foliorum CMRP1321 and D. malorum CMRP1438 extracts inhibited C. albicans alone. Three classes of chemical compounds were identified in D. foliorum CMRP1438 extracts: ferric chloride, potassium hydroxide, and vanillin-sulfuric acid. In conclusion, the endophytic isolates were able to produce bioactive agents with pharmaceutical potential as antibacterial and antifungal agents. As such, they may provide fresh leads in the search for new, biological sources of drug therapies., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multicatalytic enzymes contain several glycoside hydrolase domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd 1381 from the actinobacterium Jonesia denitrificans , that uniquely combines two different polysaccharide-degrading activities. We found that Jd 1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans , degraded both α- and β-chitin. Both the chitinase and the LPMO activities of Jd 1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd 1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd 1381 was comparable with or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens Importantly, comparison of the chitinolytic efficiency of Jd 1381 with the efficiencies of combinations of truncated variants- Jd LPMO10 and Jd CBM5-Chi18 or Jd LPMO10-CBM5 and Jd Chi18-indicated that optimal Jd 1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Mekasha et al.)

Edible mushrooms are an important source of nutraceuticals and for the discovery of bioactive metabolites as pharmaceuticals. In this work, the OSMAC (One Strain, Many Active Compounds) approach was used to isolate two new compounds ( 1 and 2 ) along with seven known compounds ( 3 - 9 ) from a mycelial culture of a unique North American edible mushroom Hericium sp. The fruiting body was collected in Marine on St. Croix, Minnesota (USA), and mycelial cultures were grown on four different solid and liquid media. Extracts from the mycelial cultures were screened for antimicrobial activity and only the extract from the Cheerios substrate culture exhibited antifungal activity. Bioassay guided fractionation and HPLC analysis were used to isolate nine pure compounds and the structures of the known compounds were established by analysis of the NMR and mass spectrometry data and comparison to published reports. Compound 1 is a new erinacerin alkaloid and 2 is an aldehyde derivative of 4-hydroxy chroman. Four chlorinated orcinol derivatives ( 3 - 6 ), a pyran ( 7 ), erinaceolactone ( 8 ), and erinacine ( 9 ) were identified. Compound 4 showed antifungal activity against C. albicans and C. neoformans (MIC = 31.3-62.5 μg/mL, respectively). Compound 4 also inhibited biofilm formation of C. albicans and C. neoformans at 7.8 μg/mL. These results suggest that mycelial cultures of edible fungi may provide useful, bioactive compounds.

Immobilization of enzymes is required for most biocatalytic processes, but chemistries used in enzyme immobilization are limited and can be challenging. Genetically encoded protein-based biomaterials could provide easy-to-use immobilization platforms for biocatalysts. We recently developed a self-assembling protein scaffold that covalently immobilized SpyTagged enzymes by engineering the bacterial microcompartment protein EutM from Salmonella enterica with a SpyCatcher domain. We also identified a range of EutM homologues as robust protein nanostructures with diverse architectures and electrostatic surface properties. In this work, we created a modular immobilization platform with tunable surface properties by developing a toolbox of self-assembling, robust EutM-SpyCatcher scaffolds. Using an alcohol dehydrogenase as model biocatalyst, we show that the scaffolds improve enzyme activity and stability. This work provides a modular, easy-to-use immobilization system that can be tailored for the optimal function of biocatalysts of interest.

Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae , which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining. IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products., (Copyright © 2019 American Society for Microbiology.)

The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5 N225D S229T allele. This allele restores non-trichothecene synthesis but not trichothecene synthesis. The tri5 N225D S229T allele also restores toxisome structure which is lacking in the Δtri5 deletion strain. Our results indicate that the Tri5 protein, but not its enzymatic activity, is also required for the synthesis of non-trichothecene related sesquiterpenes and the formation of toxisomes. Toxisomes thus not only may be important for DON synthesis, but also for the synthesis of other sesquiterpene mycotoxins such as culmorin by F. graminearum., (Published by Elsevier Inc.)

A novel inducible gene expression system using p-isopropyl benzoate (cumate) as an inducer was developed for the industrial production hosts, Bacillus subtilis and Bacillus megaterium. Cumate is non-toxic to the host, inexpensive, and carbon source-independent inducer which provides an economical option for large-scale production of valuable proteins and chemicals from Bacillus strains. The synthetic cumate-inducible system was constructed by combining the strong constitutive Bacillus promoter P veg with regulatory elements of the Pseudomonas putida, CymR repressor, and its operator sequence CuO. The designed expression cassette containing a sfGFP reporter under the cumate-inducible promoter was assembled into a Bacillus-E. coli shuttle and gene expression investigated in the two Bacillus strains. Characterization of gene expression levels, expression kinetics, and dose-response to cumate inducer concentration confirmed high-level, but tightly controlled GFP reporter expression in tunable, cumate concentration-dependent manner. Unexpectedly, this expression system works equally well for Escherichia coli, resulting in a platform that can be used both in gram-positive and gram-negative expression host. Its tight regulation and controllable expression makes this system useful for metabolic engineering, synthetic biology studies as well industrial protein production.

Biocatalysis is emerging as an alternative approach to chemical synthesis of industrially relevant complex molecules. To obtain suitable yields of compounds in a cost-effective manner, biocatalytic reaction cascades must be efficient, robust, and self-sufficient. One approach is to immobilize biocatalysts on a solid support, stabilizing the enzymes and providing optimal microenvironments for reaction sequences. Protein-based scaffolds can be designed as immobilization platforms for biocatalysts, enabling the genetically encoded spatial organization of single enzymes and multistep enzyme cascades. Additionally, protein scaffolds are versatile, are easily adapted, and remain robust under different reaction conditions. In this chapter, we describe methods for the design and production of a self-assembling protein scaffold system for in vitro coimmobilization of biocatalytic cascade enzymes. We provide detailed methods for the characterization of the protein scaffolds, as well as approaches to load biocatalytic cargo enzymes and test activity of immobilized cascades. In addition, we also discuss methods for the development of a scaffold building block toolbox with different surface properties, which could be adapted for a diversity of biocatalysts requiring alternative microenvironments for function., (© 2019 Elsevier Inc. All rights reserved.)

Biological materials that are genetically encoded and can self-assemble offer great potential as immobilization platforms in industrial biocatalysis. Protein-based scaffolds can be used for the spatial organization of enzymes, to stabilize the catalysts and provide optimal microenvironments for reaction sequences. In our previous work, we created a protein scaffold for enzyme localization by engineering the bacterial microcompartment shell protein EutM from Salmonella enterica. Here, we sought to expand this work by developing a toolbox of EutM proteins with different properties, with the potential to be used for future immobilization of enzymes. We describe the bioinformatic identification of hundreds of homologs of EutM from diverse microorganisms. We specifically select 13 EutM homologs from extremophiles for characterization, based on phylogenetic analyses. We synthesize genes encoding the novel proteins, clone and express them in E. coli, and purify the proteins. In vitro characterization shows that the proteins self-assemble into robust nano- and micron-scale architectures including protein nanotubes, filaments, and scaffolds. We explore the self-assembly characteristics from a sequence-based approach and create a synthetic biology platform for the coexpression of different EutM homologs as hybrid scaffolds with integrated enzyme attachment points. This work represents a step towards our goal of generating a modular toolbox for the rapid production of self-assembling protein-based materials for enzyme immobilization.

The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters. IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide access to this chemodiversity for the discovery and synthesis of molecules with new bioactivities. The identification and successful cloning of the previously elusive hirsutene synthase from the S. hirsutum provide important insights and strategies for biosynthetic gene discovery in Basidiomycota. The finding of a terpene synthase-HMGS fusion, the discovery of other sesquiterpenoid biosynthetic gene clusters with dedicated HMGS genes, and HMGS gene duplications in fungal genomes give new importance to the role of HMGS as a key regulatory enzyme in isoprenoid and sterol biosynthesis that should be exploited for metabolic engineering., (Copyright © 2018 American Society for Microbiology.)

Throughout the past decade, the field of synthetic biology has grown rapidly. By using assembly platforms such as BioBricks™, scientists can quickly and easily build gene circuits or multi-step pathways. One limitation, however, is that most of these parts were designed and characterized with Escherichia coli as the target chassis. As a consequence, there exists a lack of standardized and well characterized or BioBrick™ compatible plasmid backbones that replicate in other potential non-model chassis organisms. The Gram-positive bacteria of the genus Rhodococcus represent an interesting chassis for biotechnological applications due to their tremendous metabolic capabilities. In this report we describe our progress toward developing a BioBrick™ compatible plasmid system for Rhodococcus. We demonstrate its utility for heterologous protein expression through flow cytometric analysis of the lac promoter in the oleaginous strain Rhodococcus opacus PD630., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Spatial organization via encapsulation of enzymes within recombinant nanocompartments may increase efficiency in multienzyme cascades. Previously, we reported the encapsulation of single cargo proteins within nanocompartments in the heterologous host Escherichia coli. This was achieved by coexpression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK, with a signal sequence EutC1-19 cargo protein fusion. Optimization of this system, leading to the targeting of more than one cargo protein, requires an understanding of the encapsulation mechanism. In this work, we report that the signal sequence EutC1-19 targets cargo to the interior of nanocompartments via a hydrophobic interaction with a helix on shell protein EutS. We confirm that EutC1-19 does not interact with other Eut BMC shell proteins, EutMNLK. Furthermore, we show that a second signal sequence EutE1-21 interacts specifically with the same helix on EutS. Both signal sequences appear to compete for the same EutS helix to simultaneously colocalize two cargo proteins to the interior of recombinant nanocompartments. This work offers the first insights into signal sequence-shell protein interactions required for cargo sequestration within Eut BMCs. It also provides a basis for the future engineering of Eut nanocompartments as a platform for the potential colocalization of multienzyme cascades for synthetic biology applications.

Advances in biological engineering and systems biology have provided new approaches and tools for the industrialization of biology. In the next decade, advanced biocatalytic systems will increasingly be used for the production of chemicals that cannot be made by current processes and/or where the use of enzyme catalysts is more resource efficient with a much reduced environmental impact. We expect that in the future, manufacture of chemicals and materials will utilize both biocatalytic and chemical synthesis synergistically. The realization of such advanced biomanufacturing processes currently faces a number of major challenges. Ready-to-deploy portfolios of biocatalysts for design to production must be created from biological diverse sources and through protein engineering. Robust and efficient multi-step enzymatic reaction cascades must be developed that can operate simultaneously in one-pot. For this to happen, bio-orthogonal strategies for spatial and temporal control of biocatalyst activities must be developed. Promising approaches and technologies are emerging that will eventually lead to the design of in vitro biocatalytic systems that mimic the metabolic pathways and networks of cellular systems which will be discussed in this roadmap., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products., (Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.

Basidiomycota fungi have received little attention for applications in biocatalysis and biotechnology and remain greatly understudied despite their importance for carbon recycling, ecosystem functioning and medicinal properties. The steady influx of genome data has facilitated detailed studies aimed at understanding the evolution and function of fungal lignocellulose degradation. These studies and recent explorations into the secondary metabolomes have uncovered large portfolios of enzymes useful for biocatalysis and biosynthesis. This review will provide an overview of the biocatalytic repertoires of Basidiomycota characterized to date with the hope of motivation more research into the chemical toolkits of this diverse group of fungi., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) boost enzymatic depolymerization of recalcitrant polysaccharides, such as chitin and cellulose. We have studied a chitin-active LPMO domain (JdLPMO10A) that is considerably smaller (15.5 kDa) than all structurally characterized LPMOs so far and that is part of a modular protein containing a GH18 chitinase. The 1.55 Å resolution structure revealed deletions of interacting loops that protrude from the core β-sandwich scaffold in larger LPMO10s. Despite these deletions, the enzyme is active on alpha- and beta-chitin, and the chitin-binding surface previously described for larger LPMOs is fully conserved. JdLPMO10A may represent a minimal scaffold needed to catalyse the powerful LPMO reaction., (© 2015 Federation of European Biochemical Societies.)

Fungal 1,11 cyclizing sesquiterpene synthases are product specific under typical reaction conditions. However, in vivo expression of certain Δ(6)-protoilludene synthases results in dual 1,11 and 1,10 cyclization. To determine the factors regulating this mechanistic variation, in-depth in vitro characterization of Δ(6)-protoilludene synthases was conducted. Divalent metal ions determine cyclization specificity and this product variability. Promiscuity in metal binding is mediated by secondary metal-binding sites away from the conserved D(D/E)XX(D/E) motif in sesquiterpene synthases. Phylogenetic analysis revealed a divergent evolution of Basidiomycota trans-humulyl cation producing sesquiterpene synthases, results that indicate a wider diversity in function than previously predicted. This study provides key insights into the function and evolution of 1,11 cyclizing fungal sesquiterpene synthases., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tens of thousands of terpenoid natural products have been isolated from plants and microbial sources. Higher fungi (Ascomycota and Basidiomycota) are known to produce an array of well-known terpenoid natural products, including mycotoxins, antibiotics, antitumor compounds, and phytohormones. Except for a few well-studied fungal biosynthetic pathways, the majority of genes and biosynthetic pathways responsible for the biosynthesis of a small number of these secondary metabolites have only been discovered and characterized in the past 5-10 years. This chapter provides a comprehensive overview of the current knowledge on fungal terpenoid biosynthesis from biochemical, genetic, and genomic viewpoints. Enzymes involved in synthesizing, transferring, and cyclizing the prenyl chains that form the hydrocarbon scaffolds of fungal terpenoid natural products are systematically discussed. Genomic information and functional evidence suggest differences between the terpenome of the two major fungal phyla--the Ascomycota and Basidiomycota--which will be illustrated for each group of terpenoid natural products.

Hydroxycinnamic acid esters (HCEs) are widely-distributed phenylpropanoid-derived plant natural products. Rosmarinic acid (RA), the most well-known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, microbial production of HCEs could be a sustainable, controlled means of production. Through the overexpression of a six-enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof-of-concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non-natural HCEs., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)