Alternative splicing analysis of lignocellulose-degrading enzyme genes and enzyme variants in Aspergillus niger.

Alternative splicing (AS) greatly expands the protein diversity in eukaryotes. Although AS variants have been frequently reported existing in filamentous fungi, it remains unclear whether lignocellulose-degrading enzyme genes in industrially important fungi undergo AS events. In this work, AS events of lignocellulose-degrading enzymes genes in Aspergillus niger under two carbon sources (glucose and wheat straw) were investigated by RNA-Seq. The results showed that a total of 23 out of the 56 lignocellulose-degrading enzyme genes had AS events and intron retention was the main type of these AS events. The AS variant enzymes from the annotated endo-ß-1,4-xylanase F1 gene (xynF1) and the endo-ß-1,4-glucanase D gene (eglD), noted as XYNF1-AS and EGLD-AS, were characterized compared to their normal splicing products XYNF1 and EGLD, respectively. The AS variant XYNF1-AS displayed xylanase activity whereas XYNF1 did not. As for EGLD-AS and EGLD, neither of them showed annotated endo-ß-1,4-glucanase activity. Instead, both showed lytic polysaccharide monooxygenase (LPMO) activity with some differences in catalytic properties. Our work demonstrated that the AS variants in A. niger were good sources for discovering novel lignocellulose-degrading enzymes. KEY POINTS: • AS events were identified in the lignocellulose-degrading enzyme genes of A. niger. • New ß-1,4-xylanase and LPMO derived from AS events were characterized.

Broadening the Substrate Specificity of Cellobiose Phosphorylase from Clostridium thermocellum for Improved Transformation of Cellodextrin to Starch.

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182-7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin's phosphorolysis.

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Response of Black Soldier Fly (Hermetia illucens) Larvae Intestinal Microbes and Reduction Mechanisms to High Concentrations of Tetracycline.

Black soldier fly (Hermetia illucens L) larvae (BSFL) possess remarkable antibiotic degradation abilities due to their robust intestinal microbiota. However, the response mechanism of BSFL intestinal microbes to the high concentration of antibiotic stress remains unclear. In this study, we investigated the shift in BSFL gut microbiome and the functional genes that respond to 1250 mg/kg of tetracycline via metagenomic and metatranscriptomic analysis, respectively. The bio-physiological phenotypes showed that the survival rate of BSFL was not affected by tetracycline, while the biomass and substrate consumption of BSFL was slightly reduced. Natural BSFL achieved a 20% higher tetracycline degradation rate than the germ-free BSFL after 8 days of rearing. Metagenomic and metatranscriptomic sequencing results revealed the differences between the entire and active microbiome. Metatranscriptomic analysis indicated that Enterococcus, Vagococcus, Providencia, and Paenalcaligenes were the active genera that responded to tetracycline. Furthermore, based on the active functional genes that responded to tetracycline pressure, the response mechanisms of BSFL intestinal microbes were speculated as follows: the Tet family that mediates the expression of efflux pumps expel tetracycline out of the microbes, while tetM and tetW release it from the ribosome. Eventually, tetracycline was degraded by deacetylases and novel enzymes. Overall, this study provides novel insights about the active intestinal microbes and their functional genes in insects responding to the high concentration of antibiotics.

Biochar Can Improve Absorption of Nitrogen in Chicken Manure by Black Soldier Fly.

(1) Background: There is growing interest in using insects to treat nutrient-rich organic wastes, such as the black soldier fly (BSF), one of the most efficient organic waste recyclers for upcycling nutrients into the food system. Although biochar (BC) was shown to enhance nutrient retention and the final product quality during the composting of livestock and poultry manure in many previous studies, little information is available on the effect of BC on livestock manure bioconversion by black soldier fly larvae (BSFL). (2) Methods: This study investigated the effect of adding a small amount of BC to chicken manure (CM) on the bioconversion system of the black soldier fly (including N2O and NH3 emissions and the final distribution of nitrogen during the treatment process). (3) Results: The lowest N2O and NH3 emission and highest residual nitrogen in the substrate were observed in the 15% BC treatment. The highest bioconversion rate of CM (8.31%) and the peak of larval biomass was obtained in the 5% BC treatment. (4) Conclusions: The results demonstrate the feasibility of adding 5% BC to reduce pollution and achieve a satisfactory BSFL-based CM bioconversion efficiency.

Biosynthesis of artificial starch and microbial protein from agricultural residue.

Growing populations and climate change pose great challenges to food security. Humankind is confronting a serious question: how will we feed the world in the near future? This study presents an out-of-the-box solution involving the highly efficient biosynthesis of artificial starch and microbial proteins from available and abundant agricultural residue as new feed and food sources. A one-pot biotransformation using an in vitro coenzyme-free synthetic enzymatic pathway and baker's yeast can simultaneously convert dilute sulfuric acid-pretreated corn stover to artificial starch and microbial protein under aerobic conditions. The ß-glucosidase-free commercial cellulase mixture plus an ex vivo two-enzyme complex containing cellobiose phosphorylase and potato α-glucan phosphorylase displayed on the surface of Saccharomyces cerevisiae, showed better cellulose hydrolysis rates than a commercial ß-glucosidase-rich cellulase mixture. This is because the channeling of the hydrolytic product from the solid cellulosic feedstock to the yeast mitigated the inhibition of the cellulase cocktail. Animal tests have shown that the digestion of artificial amylose results in slow and relatively small changes in blood sugar levels, suggesting that it could be a new health food component that prevents obesity and diabetes. A combination of the utilization of available agricultural residue and the biosynthesis of starch and microbial protein from non-food biomass could address the looming food crisis in the food-energy-water nexus.

Bacillus velezensis EEAM 10B Strengthens Nutrient Metabolic Process in Black Soldier Fly Larvae (Hermetia illucens) via Changing Gut Microbiome and Metabolic Pathways.

Insects are a potential alternative protein source to solve the food shortage crisis. Previous studies have illustrated that probiotics can improve the substrate conversion efficiency of insects and increase insect protein content. However, the effects of probiotics on insect physiology and nutrient metabolism are still not well understood. Here, the black soldier fly larvae (BSFL), Hermetia illucens (Diptera: Stratiomyidae), was used as a study subject to deeply investigate the specific interaction among a novel probiotic, Bacillus velezensis EEAM 10B (10B), intestinal microbiota, and the host. In this study, the effects of 10B on the survival and physiology of BSFL were first analyzed. It shows that 10B significantly elevated the substrate conversion rate, average dry weight, and protein content of BSFL by 5%, 0.13 g/pc, and 8%, respectively. Then, we assessed the effect of 10B on the microbial community composition in the gut and frass of BSFL using Illumina Miseq sequencing. It shows that 10B significantly altered the microbial composition of the gut, but not that of the frass. Pearson's correlation analysis further showed that the Bacillus, unclassified_of_Caloramatoraceae, and Gracilibacillus were positively correlated with the survival rate, crude protein content, and substrate conversion rate of BSFL. To further investigate the effect of 10B on host metabolism, metabolic analyses on germ-free BSFL, monobacterial intestinal BSFL, and natural BSFL were also performed. The results proved that 10B (i) played a vital role in the survival of BSFL; and (ii) regulated the amino acid synthetic and metabolic process of BSFL, thus leading to the rise of the protein content of BSFL. In addition, vitamin backfill assays verified that the BSFL survival rate was significantly improved by supplying the germ-free BSFL with riboflavin, which further suggests that 10B determines the survival of BSFL via delivering riboflavin. Overall, this study provides a reference for understanding the comprehensive contribution of a specific probiotic to its host.

Enzymatic Enantioselective anti-Markovnikov Hydration of Aryl Alkenes.

The addition of water to alkenes is an important method for the synthesis of alcohols, but the regioselectivity of acid-catalyzed hydration of terminal alkenes yields secondary alcohols according to Markovnikov's rule, making it difficult to obtain primary alcohols. Here we report a styrene monooxygenase that catalyzes the anti-Markovnikov hydration of the terminal aryl alkenes under anaerobic conditions. This hydration provides primary alcohols in good yields (up to 100 %), excellent anti-Markovnikov regioselectivity (>99 : 1), and good enantiomeric purity (60-83 % ee). Residues Asn46, Asp100, and Asn309 are essential for catalysis suggesting an acid-base mechanism with a carbanion-like intermediate that could account for the anti-Markovnikov regioselectivity. Our work reveals a new enzymatic tool with unusual regioselectivity based on the promiscuous catalytic activity of a monooxygenase.

Enantioselectivity and key residue of Herbaspirillum huttiense monooxygenase in asymmetric epoxidation of styrenes.

Styrene monooxygenases (SMOs) are powerful enzymes for the synthesis of enantiopure epoxides, but these SMOs have narrow substrate spectra, and the residues in controlling enantioselectivity of SMOs remains unclear. A monooxygenase from Herbaspirillum huttiense (HhMO) was found to have excellent enantioselectivities and diastereoselectivities in the epoxidation of unconjugated terminal alkenes. Here we found that HhMO could also transfer styrene into styrene epoxide with 75% ee, and it could also catalyze the epoxidation of styrene derivatives into the corresponding epoxides with enantioselectivities up to 99% ee. Meanwhile, site 199 in the substrate access channel of HhMO was found to play an important role in the controlling enantioselectivity of the epoxidation. The E199L variant catalyzed the epoxidation of styrene with > 99% ee. The identification of critical residue that affects the enantioselectivity of SMOs would thus be valuable for creating efficient monooxygenases for the preparation of essential enantiopure epoxides. KEY POINTS: • Bioexpoxidation of both conjugated and unconjugated alkenes by HhMO with excellent enantioselectivities. • Gating residue 199 played an essential role in controlling the enantioselectivity of SMO. • HhMO E199L catalyzed the epoxidation of styrenes with up to > 99% ee.

Fusion of cellobiose phosphorylase and potato alpha-glucan phosphorylase facilitates substrate channeling for enzymatic conversion of cellobiose to starch.

We previously reported an in vitro enzymatic pathway for conversion of nonfood cellulose to starch (PNAS,110 (18): 7182-7187, 2013), in which the two sequential enzymes cellobiose phosphorylase (CBP) from Clostridium thermocellum and potato alpha-glucan phosphorylase (PGP) from Solanum tuberosum were the two key enzymes responsible for the whole conversion rate. In this work CBP and PGP were fused to form a large enzyme and it turned out that the fusion protein could exhibit a good bifunctionality when PGP moiety was put at the N-terminus and CBP moiety at the C-terminus (designated as PGP-CBP). Although the coupled reaction rate of PGP-CBP was decreased by 23.0% compared with the free enzymes, substrate channeling between the two active sites in PGP-CBP was formed, demonstrated by the introduction of the competing enzyme of PGP to the reaction system. The potential of PGP-CBP fusion enzyme being applied to the conversion of cellulose to amylose was discussed.

Strategies for Controlling the Sporulation in Fusarium spp.

Fusarium species are the most destructive phytopathogenic and toxin-producing fungi, causing serious diseases in almost all economically important plants. Sporulation is an essential part of the life cycle of Fusarium. Fusarium most frequently produces three different types of asexual spores, i.e., macroconidia, chlamydospores, and microconidia. It also produces meiotic spores, but fewer than 20% of Fusaria have a known sexual cycle. Therefore, the asexual spores of the Fusarium species play an important role in their propagation and infection. This review places special emphasis on current developments in artificial anti-sporulation techniques as well as features of Fusarium's asexual sporulation regulation, such as temperature, light, pH, host tissue, and nutrients. This description of sporulation regulation aspects and artificial anti-sporulation strategies will help to shed light on the ways to effectively control Fusarium diseases by inhibiting the production of spores, which eventually improves the production of food plants.

Enzymatic regeneration and conservation of ATP: challenges and opportunities.

Adenosine triphosphate (ATP), the universal energy currency of life, has a central role in numerous biochemical reactions with potential for the synthesis of numerous high-value products. ATP can be regenerated by three types of mechanisms: substrate level phosphorylation, oxidative phosphorylation, and photophosphorylation. Current ATP regeneration methods are mainly based on substrate level phosphorylation catalyzed by one enzyme, several cascade enzymes, or in vitro synthetic enzymatic pathways. Among them, polyphosphate kinases and acetate kinase, along with their respective phosphate donors, are the most popular approaches for in vitro ATP regeneration. For in vitro artificial pathways, either ATP-free or ATP-balancing strategies can be implemented via smart pathway design by choosing ATP-independent enzymes. Also, we discuss some remaining challenges and suggest perspectives, especially for industrial biomanufacturing. Development of ATP regeneration systems featuring low cost, high volumetric productivity, long lifetime, flexible compatibility, and great robustness could be one of the bottom-up strategies for cascade biocatalysis and in vitro synthetic biology.

Identification of an intracellular ß-glucosidase in Aspergillus niger with transglycosylation activity.

Aspergillus niger is featured with its copious amount of extracellular ß-glucosidase which is generally used to balance the cellulolytic enzyme cocktails for lignocellulose saccharification. However, whether or not A. niger produces any intracellular ß-glucosidase remains obscure. In this study, we analyzed a total of fifteen putative ß-glucosidase genes (bgls) in A. niger CBS 513.88 genome and the five of them were predicted as intracellular bgls due to the lack of signal peptide of extracellular proteins. After further characterization of these five genes through a Saccharomyces cerevisiae in vivo system, only An03g03740 (designated bgl1B) was confirmed to be a ß-glucosidase gene. Western blot and mass spectrometry analysis confirmed BGL1B protein localization inside the cell. BGL1B exhibited the maximal activity at 40 °C and pH 5.6. The Km for p-nitrophenyl-ß-D-glucopyranoside and Ki for glucose were 0.233 ± 0.058 mM and 119.8 ± 4.35 mM, respectively. BGL1B showed a strong transglycosylation activity while hydrolyzing cellodextrins with sophorose, laminaribiose, and cellotriose formed from cellobiose, and sophorose and laminaribiose formed from cellotriose. The confirmation of the intracellular ß-glucosidase BGL1B in A. niger further extends our understanding of how A. niger utilizes lignocellulose. KEY POINTS: • Identification of putative genes revealed a novel ß-glucosidase in Aspergillus niger. • Newly identified ß-glucosidase BGL1B was an intracellular enzyme of A. niger. • BGL1B exhibited a strong transglycosylation activity.

Identification and Characterization of a Cellodextrin Transporter in Aspergillus niger.

Aspergillus niger produces a wide spectrum of extracellular polysaccharide hydrolases that hydrolyze cellulose into soluble glucose and cellodextrins. Transporters are essential for sugar uptake, yet it is not clear whether cellodextrin transporters exist in A. niger. Here, one cellulose inducible cellodextrin transporter CtA was identified in A. niger B2. It was found that CtA not only could transport cellobiose, but also cellotriose, cellotetraose, and cellopentaose. The yeast strain YPßG-CtA, expressing cellodextrin transporter CtA and an intracellular ß-glucosidase, grew on cellobiose with the cell growth rate of 0.0830 ± 0.0113 h-1 under aerobic condition. Furthermore, the engineered yeast could produce 1.1 g/L ethanol anaerobically on cellobiose in 2 days. The identification of CtA provides evidence that the cellodextrin uptake is a complementary strategy of cellulose utilization in A. niger, and the CtA could be a strong transporter candidate for constructing engineered cellodextrin-utilizing microorganisms.

Effects of intron retention on properties of ß-glucosidase in Aspergillus niger.

Intron retention, one of the major types of alternative splicing in plants and animals, has also been reported existing in filamentous fungi's glycoside hydrolases. In this study, an intron-retained ß-glucosidase gene transcript (bgl1B) from A. niger B2 strain was obtained. Compared with the normally spliced transcript bgl1A, bgl1B had an extra 51bp insertion, which was confirmed to be the sixth (the last) intron of this ß-glucosidase gene. The bgl1A and bgl1B were expressed in Pichia pastoris and the purified enzymes were used to compare their catalytic properties. The results showed that the intron retention didn't impair the catalytic function. Instead, the intron-retained enzyme BGL1B had a better thermostability with a higher optimal temperature and a longer half-life under 50 °C. Also it exhibited a little higher kcat for 4-nitrophenyl-ß-d-glucopyranoside (PNPG) and a noticeable higher hydrolysis efficiency towards geniposide. This work suggested that the ß-glucosidase gene in A. niger most likely underwent an alternative splicing presented as intron retention type, and intron retention might be a source of enzyme diversity in fungi.

Upgrade of wood sugar d-xylose to a value-added nutraceutical by in vitro metabolic engineering.

The upgrade of D-xylose, the most abundant pentose, to value-added biochemicals is economically important to next-generation biorefineries. myo-Inositol, as vitamin B8, has a six-carbon carbon-carbon ring. Here we designed an in vitro artificial NAD(P)-free 12-enzyme pathway that can effectively convert the five-carbon xylose to inositol involving xylose phosphorylation, carbon-carbon (C-C) rearrangement, C-C bond circulation, and dephosphorylation. The reaction conditions catalyzed by all thermostable enzymes from hyperthermophilic microorganisms Thermus thermophiles, Thermotoga maritima, and Archaeoglobus fulgidus were optimized in reaction temperature, buffer type and concentration, enzyme composition, Mg2+ concentration, and fed-batch addition of ATP. The 11-enzyme cocktail, whereas a fructose 1,6-bisphosphatase from T. maritima has another function of inositol monophosphatase, converted 20 mM xylose to 16.1 mM inositol with a conversion efficiency of 96.6% at 70 °C. Polyphosphate was found to replace ATP for xylulose phosphorylation due to broad substrate promiscuity of the T. maritima xylulokinase. The Tris-HCl buffer effectively mitigated the Maillard reaction at 70 °C or higher temperature. The co-production of value-added biochemicals, such as inositol, from wood sugar could greatly improve economics of new biorefineries, similar to oil refineries that make value-added plastic precursors to subsidize gasoline/diesel production.

Self-adaptive terahertz spectroscopy from atmospheric vapor based on Hilbert-Huang transform.

Absorption lines of atmospheric vapor commonly appear in terahertz (THz) spectra measured in a humid air environment. However, these effects are generally undesirable because they may mask critical spectroscopic information. Here, a self-adaptive method is demonstrated for effectively identifying and eliminating atmospheric vapor noise from THz spectra of an all-fiber THz system with the Hilbert-Huang transform. The THz signal was decomposed into eight components in different time scales called the intrinsic mode functions and the interference of atmospheric vapor was accurately isolated. A series of experiments confirmed the effectiveness and strong self-adaptiveness of the proposed system in vapor noise elimination.

Structural Characteristics of the Novel Polysaccharide FVPA1 from Winter Culinary-Medicinal Mushroom, Flammulina velutipes (Agaricomycetes), Capable of Enhancing Natural Killer Cell Activity against K562 Tumor Cells.

FVPA1, a novel polysaccharide, has been isolated from fruiting bodies of the culinary-medicinal mushroom Flammulina velutipes, a historically popular, widely cultivated and consumed functional food with an attractive taste, beneficial nutraceutical properties such as antitumor and immunomodulatory effects, and a number of essential biological activities. The average molecular weight was estimated to be ~1.8 × 104 Da based on high-performance size exclusion chromatography. Sugar analyses, methylation analyses, and 1H, 13C, and 2-dimensional nuclear magnetic resonance spectroscopy revealed the following structure of the repeating units of the FVPA1 polysaccharide Identification of this structure would conceivably lead to better understanding of the nutraceutical functions of this very important edible fungus. Bioactivity tests in vitro indicated that FVPA1 could significantly enhance natural killer cell activity against K562 tumor cells.

[Progress in metabolic engineering of ß-carotene synthesis].

ß-carotene is an important natural plant pigment and has various physiological functions in organisms. With the proposition of systematic biology and progress in carotenoids biosynthesis since the 1960s, metabolic engineering has played a significant role in enhancing carotenoid production. In this review, we present ß-carotene's traditional production methods and metabolic engineering strategies for constructing ß-carotene-producing strains. Meanwhile, main problems and corresponding solutions to improve ß-carotene yield of engineered strains were further analyzed, for further efficient microbial production of ß-carotene.

Biochemical properties of GH94 cellodextrin phosphorylase THA_1941 from a thermophilic eubacterium Thermosipho africanus TCF52B with cellobiose phosphorylase activity.

A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.

Molecular Cloning and Characterizations of Xylanase Inhibitor Protein from Wheat (Triticum Aestivum).

Xylanase inhibitor proteins (XIPs) were regarded to inhibit the activity of xylanases during baking and gluten-starch separation processes. To avoid the inhibition to xylanases, it is necessary to define the conditions under which the inhibition takes place. In this study, we cloned the XIP gene from 2 different variety of Triticum aestivum, that is, Zhengmai 9023 and Zhengmai 366, and investigated the properties of XIP protein expressed by Pichia pastoris. The results showed that the 2 XIP genes (xip-9023 and xip-366) were highly homologous with only 3 nucleotide differences. XIP-9023 showed the optimal inhibition pH and temperature were 7 °C and 40 °C, respectively. Inhibition of xylanase by XIP-9023 reached the maximum in 40 min. At 50% inhibition of xylanase, the molar ratio of inhibitor: xylanase was 26:1. XIP-9023 was active to various fungal xylanases tested as well as to a bacterial xylanase produced by Paenibacillus sp. isolated from cow rumen.