Narrow Substrate Range Research Articles

Bottom-up approach to deciphering the targets of the ubiquitin-proteasome system in porcine sperm capacitation

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

Cultivation and genomic characterization of novel and ubiquitous marine nitrite-oxidizing bacteria from the Nitrospirales

Nitrospirales, including the genus Nitrospira, are environmentally widespread chemolithoautotrophic nitrite-oxidizing bacteria. These mostly uncultured microorganisms gain energy through nitrite oxidation, fix CO2, and thus play vital roles in nitrogen and carbon cycling. Over the last decade, our understanding of their physiology has advanced through several new discoveries, such as alternative energy metabolisms and complete ammonia oxidizers (comammox Nitrospira). These findings mainly resulted from studies of terrestrial species, whereas less attention has been given to marine Nitrospirales. In this study, we cultured three new marine Nitrospirales enrichments and one isolate. Three of these four NOB represent new Nitrospira species while the fourth represents a novel genus. This fourth organism, tentatively named “Ca. Nitronereus thalassa”, represents the first cultured member of a Nitrospirales lineage that encompasses both free-living and sponge-associated nitrite oxidizers, is highly abundant in the environment, and shows distinct habitat distribution patterns compared to the marine Nitrospira species. Partially explaining this, “Ca. Nitronereus thalassa” harbors a unique combination of genes involved in carbon fixation and respiration, suggesting differential adaptations to fluctuating oxygen concentrations. Furthermore, “Ca. Nitronereus thalassa” appears to have a more narrow substrate range compared to many other marine nitrite oxidizers, as it lacks the genomic potential to utilize formate, cyanate, and urea. Lastly, we show that the presumed marine Nitrospirales lineages are not restricted to oceanic and saline environments, as previously assumed.

Protein engineering for natural product biosynthesis: expanding diversity for therapeutic applications

In this dispensation of the fourth industrial revolution, protein engineering has become a popular approach for increasing enzymatic activity, stability, and titer in the biosynthesis of natural products. This is attributed to its numerous advantages (over direct isolation from plants or via chemical synthesis), including decreasing or eliminating reaction byproducts, high precision, moderate handling of intricate and chemically unstable chemicals, overall reusability, and cost efficiency. Recently, protein engineering tools have advanced to redesign and enhance natural product biosynthesis. These methods include direct evolution, substrate engineering, medium engineering, enzyme engineering and immobilization, structure-assisted protein engineering, and advanced computational. Recent successes in implementing these emerging protein engineering technologies were critically discussed in this article. Also, the advantages, limitations, and applications in industrial and medical biotechnology were discussed. Last, future research directions and potential were also highlighted.

Overexpression of Leishmania major protein arginine methyltransferase 6 reduces parasite infectivity in vivo

Arginine methylation is catalysed by Protein Arginine Methyltransferases (PRMTs) and can affect how a target protein functions and how it interacts with other macromolecules, which in turn impacts on cell metabolism and gene expression control. Leishmania parasites express five different PRMTs, and although the presence of each individual PRMT is not essential per se, the imbalanced activity of these PRMTs can impact the virulence of Leishmania parasites in vitro and in vivo. Here we created a Leishmania major cell line overexpressing PRMT6 and show that similar to what was observed for the T. brucei homologous enzyme, L. major PRMT6 probably has a narrow substrate range. However, its overexpression notably impairs the infection in mice, with a mild reduction in the number of viable parasites in the lymph nodes. Our results indicate that arginine methylation by LmjPRMT6 plays a significant role in the adaptation of the parasite to the environment found in the mammalian host.

Fe-Catalyzed Direct Synthesis of Nitriles from Carboxylic Acids with Electron-Deficient N-Cyano-N-aryl-arylsulfonamide.

Established carboxylic acids to nitriles conversion methods suffer from expensive catalysts, tedious steps, high temperatures (>200 °C), high pressure, or a narrow substrate range. Herein, we demonstrate a concise and efficient access to diverse nitrile compounds from ubiquitous carboxylic acids with electron-deficient N-cyano-N-aryl-arylsulfonamide (NCAS) in moderate to excellent yields. This strategy is promoted by an inexpensive iron catalyst and is generally compatible with primary, secondary, tertiary, and aryl carboxylic acids, as well as a variety of functional groups.

Ecotin: A versatile protease inhibitor of bacteria and eukaryotes.

Serine protease inhibitors are a large family of proteins involved in important pathways and processes, such as inflammatory responses and blood clotting. Most are characterized by a precise mode of action, thereby targeting a narrow range of protease substrates. However, the serine-protease inhibitor ecotin is able to inhibit a broad range of serine proteases that display a wide range of specificities. This specificity is driven by special structural features which allow unique flexibility upon binding to targets. Although frequently observed in many human/animal-associated bacteria, ecotin homologs may also be found in plant-associated taxa and environmental species. The purpose of this review is to provide an update on the biological importance, role in host-microbe interactions, and evolutionary relationship between ecotin orthologs isolated from Eukaryotic and Prokaryotic species across the Tree of Life.

Engineering Extracellular Electron Transfer in Escherichia coli for Microbial Electrochemical Devices

Microbes hold great potential for green electrochemical synthesis as well as bio-electricity generation from organic waste. The key to the successful development of microbial electrochemical devices lies with the ability of the microbes to transport electrons across their outer membranes. These devices thus often rely on the use of microbes known as exoelectrogens, which include bacteria that have evolved metabolic pathways that enable extracellular electron transfer (EET) to solid substrates for cellular respiration. While exoelectrogens demonstrate facilitated electron transfer with the electrodes of these devices, natural exoelectrogens are often restricted to utilizing a narrow range of substrates that limits their versatility for processing different organic wastes and electrosynthesizing a rich diversity of high-value chemicals. By contrast, non-exoelectrogenic microbes such as Escherichia coli that lack efficient EET pathways are host to rich metabolic reaction networks that can be more readily tailored for various applications using synthetic biology.In this work, we biologically engineer E. coli for enhanced EET. To this end, we expressed different combinations of proteins from the natural exoelectrogen S. oneidensis MR-1, as well as E. coli native proteins to optimize EET of the bioengineered strain. The bioengineered strains show significant improvements in electron transfer rates, as confirmed through colorimetric reduction assays of soluble electron acceptors and electrochemical characterizations following electrode reduction. The improved EET demonstrated in this work paves the way for increasing the efficiency of existing E. c oli - based electrochemical devices while opening the doors to new applications that benefit from the broad chemical repertoire of these microbes.

Biochemical characterization and synthetic application of aromatic L-amino acid decarboxylase from Bacillus atrophaeus.

Aromatic L-amino acid decarboxylases (AADCs) are ubiquitously found in higher organisms owing to their physiological role in the synthesis of neurotransmitters and alkaloids. However, bacterial AADC has not attracted much attention because of its rather limited availability and narrow substrate range. Here, we examined the biochemical properties of AADC from Bacillus atrophaeus (AADC-BA) and assessed the synthetic feasibility of the enzyme for the preparation of monoamine neurotransmitters. AADC-BA was expressed in Escherichia coli BL21(DE3) and the purified enzyme showed a specific activity of 2.6 ± 0.4 U/mg for 10 mM L-phenylalanine (L-Phe) at 37 °C. AADC-BA showed optimal pH and temperature ranges at 7-8 and 37-45 °C, respectively. The KM and kcat values for L-Phe were 7.2 mM and 7.4 s-1, respectively, at pH 7.0 and 37 °C. Comparison of the kinetic constants at different temperatures revealed that the temperature dependency of the enzyme was mainly determined by catalytic turnover rather than substrate binding. AADC-BA showed a broad substrate scope for various aromatic amino acids, including L-Phe, L-tryptophan (610% relative to L-Phe), L-tyrosine (12%), 3,4-dihydroxyphenyl-L-alanine (24%), 5-hydroxy-L-tryptophan (L-HTP, 71%), 4-chloro-L-phenylalanine (520%), and 4-nitro-L-phenylalanine (450%). Homology modeling and docking simulations were carried out and were consistent with the observed substrate specificity. To demonstrate the synthetic potential of AADC-BA, we carried out the production of serotonin by decarboxylation of L-HTP. The reaction yield of serotonin reached 98% after 1 h at the reaction conditions of 50 mM L-HTP and 4 U/mL AADC-BA. Moreover, we carried out preparative-scale decarboxylation of L-Phe (100 mM in 40-mL reaction mixture) and isolated the resulting 2-phenylethylamine (51% recovery yield). We expect that the broad substrate specificity of AADC-BA can be exploited to produce various aromatic biogenic amines. KEY POINTS: • AADC-BA showed broad substrate specificity for various aromatic amino acids. • The substrate specificity was elucidated by in silico structural modeling. • The synthetic potential of AADC-BA was demonstrated for the production of biogenic amines.

Current perspective on improved fermentative production and purification of fungal cellulases for successful biorefinery applications: a brief review

Cellulolytic enzymes play the most indispensable and centralized role in the hydrolysis of cellulosic material. Effective enzymatic hydrolysis is one of the major prerequisites in the development of a successful lignocellulosic biorefinery. However, the high cost of cellulase production yet remains to be the major bottleneck for large-scale commercial realization of lignocellusic bio-refinery. In addition, the lack of specificity and inconsistent bioconversion efficiencies of enzymes have restricted their applications towards bioconversion of narrow range of substrates. In view of addressing such bottlenecks associated with cellulase applications and further to improve the applicability of cellulase enzyme in various industrial processes, the current review essentially captures up-to-date information of various strategies adopted for the prospective production and purification of cellulase enzymes. Some holistic strategies deliberated in this review, which evolved over time and have brought about notable improvements in the final titer, yield, specificity, and productivity of the cellulolytic enzymes. It includes utilization of low-cost industrial or agricultural wastes for onsite production, application of enzyme, strain improvement through mutagenic, metabolic engineering and genetic engineering approaches, application of suitable bioprocess optimization techniques, development of ideal bioreactor configuration based on mode of fermentation process, and adaptation of the most intensive purification schemes. Additionally, to address the present existing challenges, more emphasizes have been concentrated towards economically sustainable and comprehensive production strategies of the enzyme.

Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation.

Shewanella oneidensis MR-1, amodel strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.

Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes.

Ammonia monooxygenase (AMO) is a key nitrogen-transforming enzyme belonging to the same copper-dependent membrane monooxygenase family (CuMMO) as the particulate methane monooxygenase (pMMO). The AMO from ammonia-oxidizing archaea (AOA) is very divergent from both the AMO of ammonia-oxidizing bacteria (AOB) and the pMMO from methanotrophs, and little is known about the structure or substrate range of the archaeal AMO. This study compares inhibition by C2 to C8 linear 1-alkynes of AMO from two phylogenetically distinct strains of AOA, "Candidatus Nitrosocosmicus franklandus" C13 and "Candidatus Nitrosotalea sinensis" Nd2, with AMO from Nitrosomonas europaea and pMMO from Methylococcus capsulatus (Bath). An increased sensitivity of the archaeal AMO to short-chain-length alkynes (≤C5) appeared to be conserved across AOA lineages. Similarities in C2 to C8 alkyne inhibition profiles between AMO from AOA and pMMO from M. capsulatus suggested that the archaeal AMO has a narrower substrate range than N. europaea AMO. Inhibition of AMO from "Ca Nitrosocosmicus franklandus" and N. europaea by the aromatic alkyne phenylacetylene was also investigated. Kinetic data revealed that the mechanisms by which phenylacetylene inhibits "Ca Nitrosocosmicus franklandus" and N. europaea are different, indicating differences in the AMO active site between AOA and AOB. Phenylacetylene was found to be a specific and irreversible inhibitor of AMO from "Ca Nitrosocosmicus franklandus," and it does not compete with NH3 for binding at the active site.IMPORTANCE Archaeal and bacterial ammonia oxidizers (AOA and AOB, respectively) initiate nitrification by oxidizing ammonia to hydroxylamine, a reaction catalyzed by ammonia monooxygenase (AMO). AMO enzyme is difficult to purify in its active form, and its structure and biochemistry remain largely unexplored. The bacterial AMO and the closely related particulate methane monooxygenase (pMMO) have a broad range of hydrocarbon cooxidation substrates. This study provides insights into the AMO of previously unstudied archaeal genera, by comparing the response of the archaeal AMO, a bacterial AMO, and pMMO to inhibition by linear 1-alkynes and the aromatic alkyne, phenylacetylene. Reduced sensitivity to inhibition by larger alkynes suggests that the archaeal AMO has a narrower hydrocarbon substrate range than the bacterial AMO, as previously reported for other genera of AOA. Phenylacetylene inhibited the archaeal and bacterial AMOs at different thresholds and by different mechanisms of inhibition, highlighting structural differences between the two forms of monooxygenase.

Engineering transketolase to accept both unnatural donor and acceptor substrates and produce α-hydroxyketones.

A narrow substrate range is a major limitation in exploiting enzymes more widely as catalysts in synthetic organic chemistry. For enzymes using two substrates, the simultaneous optimisation of both substrate specificities is also required for the rapid expansion of accepted substrates. Transketolase (TK) catalyses the reversible transfer of a C2 -ketol unit from a donor substrate to an aldehyde acceptor and suffers the limitation of narrow substrate scope for industrial applications. Herein, TK from Escherichiacoli was engineered to accept both pyruvate, as a novel donor substrate, and unnatural acceptor aldehydes, including propanal, pentanal, hexanal and 3-formylbenzoic acid (FBA). Twenty single-mutant variants were first designed and characterised experimentally. Beneficial mutations were then recombined to construct a small library. Screening of this library identified the best variant with a 9.2-fold improvement in the yield towards pyruvate and propionaldehyde, relative to wild-type (WT). Pentanal and hexanal were used as acceptors to determine stereoselectivities of the reactions, which were found to be higher than 98% enantiomeric excess (ee) for the S configuration. Three variants were identified to be active for the reaction between pyruvate and 3-FBA. The best variant was able to convert 47% of substrate into product within 24h, whereas no conversion was observed for WT. Docking experiments suggested a cooperation between the mutations responsible for donor and acceptor recognition, which would promote the activity towards both the acceptor and donor. The variants obtained have the potential to be used for developing catalytic pathways to a diverse range of high-value products.

UDP-Glucuronic Acid Transport Is Required for Virulence of Cryptococcus neoformans.

ABSTRACTGlycans play diverse biological roles, ranging from structural and regulatory functions to mediating cellular interactions. For pathogens, they are also often required for virulence and survival in the host. In Cryptococcus neoformans, an opportunistic pathogen of humans, the acidic monosaccharide glucuronic acid (GlcA) is a critical component of multiple essential glycoconjugates. One of these glycoconjugates is the polysaccharide capsule, a major virulence factor that enables this yeast to modulate the host immune response and resist antimicrobial defenses. This allows cryptococci to colonize the lung and brain, leading to hundreds of thousands of deaths each year worldwide. Synthesis of most glycans, including capsule polysaccharides, occurs in the secretory pathway. However, the activated precursors for this process, nucleotide sugars, are made primarily in the cytosol. This topological problem is resolved by the action of nucleotide sugar transporters (NSTs). We discovered that Uut1 is the sole UDP-GlcA transporter in C. neoformans and is unique among NSTs for its narrow substrate range and high affinity for UDP-GlcA. Mutant cells with UUT1 deleted lack capsule polysaccharides and are highly sensitive to environmental stress. As a result, the deletion mutant is internalized and cleared by phagocytes more readily than wild-type cells are and is completely avirulent in mice. These findings expand our understanding of the requirements for capsule synthesis and cryptococcal virulence and elucidate a critical protein family.

Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi.

We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3–4 M KCl. However, specific activity values were two-fold higher in the presence of 3–4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.

Pushing the bounds of biocatalysis

Enzymes often elicit envy from organic chemists. Highly evolved by nature, these biocatalysts can carry out reactions with a selectivity that surpasses most synthetic catalysts. Yet they tend to act on such a narrow range of substrates that chemists haven’t been able to exploit their abilities in a general way. In an effort to broaden enzymes’ reach, researchers led by Alison Narayan at the University of Michigan explored how three known enzymes—called TropB, AzaH, and SorbC—carry out the same reaction (Nat. Chem. 2017, DOI:10.1038/nchem.2879). In the reaction, known as an oxidative dearomatization, a catalyst breaks the aromaticity of a planar ring system to install an oxygen species, forming a valuable chiral building block. Narayan and her team determined which of the three enzymes worked best with which substrates from a group, allowing them to maximize the reaction’s enantioselectivity and site selectivity and enabling the chemists to target specific positions and

Functional differentiation of 3-ketosteroid \u03941-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

BackgroundThe Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications.ResultsThe three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters.ConclusionsAt least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated substrates. KstDs differences in their catalytic properties was somehow related to structural differences revealed by a preliminary structural modelling. Transcription of R. ruber kstD genes is driven from specific promoters. The three genes are constitutively transcribed, although an additional induction is observed in kstD1 and kstD3. These enzymes have a wide versatility and allow a fine tuning-up of the KstD cellular activity.

Protein engineering and de novo designing of a biocatalyst.

Proteins as a biomolecule have been recognized as a "molecule with manifold biological functions". The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by-products and their non-hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf-life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter-relationships. Copyright © 2016 John Wiley & Sons, Ltd.

FudC, a protein primarily responsible for furfural detoxification in Corynebacterium glutamicum.

Lignocellulosic hydrolysates contain compounds that inhibit microbial growth and fermentation, thereby decreasing the productivity of biofuel and biochemical production. In particular, the heterocyclic aldehyde furfural is one of the most toxic compounds found in these hydrolysates. We previously demonstrated that Corynebacterium glutamicum converts furfural into the less toxic compounds furfuryl alcohol and 2-furoic acid. To date, however, the genes involved in these oxidation and reduction reactions have not been identified in the C. glutamicum genome. Here, we show that Cgl0331 (designated FudC) is mainly responsible for the reduction of furfural into furfuryl alcohol in C. glutamicum. Deletion of the gene encoding FudC markedly diminished the in vivo reduction of furfural to furfuryl alcohol. Purified His-tagged FudC protein from Escherichia coli was also shown to convert furfural into furfuryl alcohol in an in vitro reaction utilizing NADPH, but not NADH, as a cofactor. Kinetic measurements demonstrated that FudC has a high affinity for furfural but has a narrow substrate range for other aldehydes compared to the protein responsible for furfural reduction in E. coli.

Secretive expression of heterologous β-glucosidase in Zymomonas mobilis

Background: Zymomonas mobilis is an efficient ethanol fermentation strain, but its narrow substrate range limits its fermentation in lignocellulose hydrolysate. As a potential consolidated bioprocessing (CBP) stain for bioethanol production, the ability of cellulose utilization was necessary. In this study, extracellular expression of β-glucosidase on Z. mobilis was studied as the first step for construction of a practical CBP strain to reduce the use of β-glucosidase in the cellulase components. Results: The heterologous β-glucosidase from Bacillus polymyxa was expressed in the ethanologenic strain Z. mobilis (ZM4) and secreted extracellularly by an endogenous signal peptide and a fusion protein. The signal peptide SP1086 of the endoglucanase gene ZMO1086 from Z. mobilis was identified and facilitated 12 % of the endoglucanase encoded by ZMO1086 from Z. mobilis ZM4 and 16 % of the β-glucosidase encoded by bglB gene secreted out of the membrane of Z. mobilis ZM4. Another method for enhancement of the β-glucosidase secretion is to fuse the β-glucosidase encoded by bglB with the levansucrase encoded by sacB from Z. mobilis ZM4 to achieve the secretive expression. Its expression level was enhanced two times but only showed a 2 % secretion ratio in this situation. Conclusions: The SP1086 signal peptide showed an obviously secreting capacity of the β-glucosidase protein. The fusion protein with SacB also showed the secretion effect, but it was less efficient.

Functional characterization of two acyltransferases from Populus trichocarpa capable of synthesizing benzyl benzoate and salicyl benzoate, potential intermediates in salicinoid phenolic glycoside biosynthesis

Salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Although their biosynthetic pathway is not known, recent work has suggested that benzyl benzoate may be a potential biosynthetic intermediate. Two candidate genes, named PtACT47 and PtACT49, encoding BAHD-type acyl transferases were identified and are predicted to produce such benzylated secondary metabolites. Herein described are the cDNA cloning, heterologous expression and in vitro functional characterization of these two BAHD acyltransferases. Recombinant PtACT47 exhibited low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8nM−1s−1) and lowest Km values (45.1μM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA: salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, including benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8nM−1s−1) and lowest Km (55.3μM) were observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA: benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters, such as hexenyl benzoate, at trace levels. Although the activities demonstrated are consistent with roles in salicinoid biosynthesis, direct tests of this hypothesis using transgenic poplar must still be performed.