Microbial Platform Research Articles

Isolation, characterization and application of theophylline-degrading Aspergillus fungi

BackgroundCaffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion.ResultsSeven tea-derived fungi were collected and identified by ITS, β-tubulin and calmodulin gene sequences, Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p < 0.01). 1,3-dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively by HPLC in A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively.ConclusionsFor the first time, we confirmed that isolated A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.

Structure-Guided Engineering of a Scoulerine 9-O-Methyltransferase Enables the Biosynthesis of Tetrahydropalmatrubine and Tetrahydropalmatine in Yeast

Benzylisoquinoline alkaloids (BIAs) are an important class of plant natural products with diverse pharmacological properties. Microbial platforms can produce BIAs through heterologous biosynthesis ...

Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.

Draft Genome Assemblies of Ionic Liquid-Resistant Yarrowia lipolytica PO1f and Its Superior Evolved Strain, YlCW001

Adaptive laboratory evolution of Yarrowia lipolytica PO1f in the benchmark ionic liquid (IL; 1-ethyl-3-methylimidazolium acetate) produced a superior IL-tolerant microorganism, strain YlCW001. Here, we report the genome sequences of PO1f and YlCW001 to study the robustness of Y. lipolytica and its potential use as a microbial platform for producing fuels and chemicals.

Thermonuclease test accuracy is preserved in methicillin-resistant Staphylococcus aureus isolates.

Introduction The nuc gene encodes a thermonuclease which is present in Staphylococcus aureus but not in coagulase-negative staphylococci (CoNS) and is the target of the rapid phenotypic thermonuclease test. The effect of nuc gene variation in methicillin-resistant S. aureus (MRSA) on the performance of PCR testing has been noted, although there are no reports about the effect of MRSA on the activity of the thermonuclease enzyme.Aim Our goals were to examine the sensitivity and specificity of the thermonuclease test used to distinguish S. aureus from CoNS cultured from blood. In addition, we aimed to assess differences in the sensitivity, specificity and accuracy of the thermonuclease test between methicillin-sensitive S. aureus (MSSA) and MRSA isolates.Methodology We performed a retrospective analysis of 1404 isolates. Each isolate from a positive blood culture was identified as a Gram-positive coccus by microscopy then analysed with the thermonuclease test (Southern Group Laboratory) prior to confirmatory identification using VITEK microbial identification platforms (bioMérieux) and cefoxitin disc diffusion testing.Results Of 1331 samples included in the final analysis, 189 were thermonuclease-positive, of which 176 were identified as S. aureus . Of the 1142 thermonuclease-negative samples, 13 were finally identified as S. aureus , giving a sensitivity of 93.1 % (95 % confidence interval [CI] 88.5–96.3) and specificity of 98.9 % (95 % CI 98.1–99.4). Of the nine proven MRSA samples, eight were thermonuclease-positive, giving a sensitivity of 88.9 % (95 % CI 51.8–99.7). Thermonuclease test accuracy for MSSA and MRSA isolates was 98.1 % (95 % CI 97.2–98.8) versus 98.8 % (95 % CI 98.0–99.3), respectively.Conclusions In the era of increasing use of molecular-based microbiology assays, the thermonuclease test remains a simple, inexpensive and robust test for the presumptive identification of S. aureus cultured from blood, irrespective of methicillin sensitivity.

Characterization of Guaiene Synthases from Stellera chamaejasme L. Flowers and Their Application in De novo Production of (-)-Rotundone in Yeast.

Four terpene synthases for the biosynthesis of volatile terpenoids were identified from the transcriptome of Stellera chamaejasme L. flowers, including SchTPS1, SchTPS2, SchTPS3, and SchTPS4. Their functions were characterized by synthetic biology approaches in Escherichia coli and in vitro enzymatic assays. SchTPS1, SchTPS2, and SchTPS3 are guaiene synthases, while SchTPS4 is an (E,E)-geranyl linalool synthase. Next, SchTPS1 and α-guaiene 2-oxidase VvSTO2 were co-expressed in Saccharomyces cerevisiae to reconstruct the biosynthetic pathway of (-)-rotundone, which is a unique aroma compound in fruits, vegetables, and wines. This is the first report for the construction of a (-)-rotundone-producing microbial platform.

In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate.

Formaldehyde is a highly reactive compound that participates in multiple spontaneous reactions, but these are mostly deleterious and damage cellular components. In contrast, the spontaneous condensation of formaldehyde with tetrahydrofolate (THF) has been proposed to contribute to the assimilation of this intermediate during growth on C1 carbon sources such as methanol. However, the in vivo rate of this condensation reaction is unknown and its possible contribution to growth remains elusive. Here, we used microbial platforms to assess the rate of this condensation in the cellular environment. We constructed Escherichia coli strains lacking the enzymes that naturally produce 5,10-methylene-THF. These strains were able to grow on minimal medium only when equipped with a sarcosine (N-methyl-glycine) oxidation pathway that sustained a high cellular concentration of formaldehyde, which spontaneously reacts with THF to produce 5,10-methylene-THF. We used flux balance analysis to derive the rate of the spontaneous condensation from the observed growth rate. According to this, we calculated that a microorganism obtaining its entire biomass via the spontaneous condensation of formaldehyde with THF would have a doubling time of more than three weeks. Hence, this spontaneous reaction is unlikely to serve as an effective route for formaldehyde assimilation.

Advanced Strategies for Production of Natural Products in Yeast.

Natural products account for more than 50% of all small-molecule pharmaceutical agents currently in clinical use. However, low availability often becomes problematic when a bioactive natural product is promising to become a pharmaceutical or leading compound. Advances in synthetic biology and metabolic engineering provide a feasible solution for sustainable supply of these compounds. In this review, we have summarized current progress in engineering yeast cell factories for production of natural products, including terpenoids, alkaloids, and phenylpropanoids. We then discuss advanced strategies in metabolic engineering at three different dimensions, including point, line, and plane (corresponding to the individual enzymes and cofactors, metabolic pathways, and the global cellular network). In particular, we comprehensively discuss how to engineer cofactor biosynthesis for enhancing the biosynthesis efficiency, other than the enzyme activity. Finally, current challenges and perspective are also discussed for future engineering direction.

Biosynthesis of zinc oxide nanoparticles with antimicrobial, anticancer, antioxidant and photocatalytic activities by the endophytic Alternaria tenuissima.

Zinc oxide nanoparticles (ZnONPs) were successfully synthesized using the culture filtrate of the endophytic fungus Alternaria tenuissima as a rapid, eco-friendly and cost-effective method. The rapid synthesis of ZnONPs was completed after 20min as confirmed by UV-Vis spectroscopy. The synthesized ZnONPs showed a single-phase crystalline structure. Dynamic light scattering analysis showed that the synthesized ZnONPs were monodispersed and the recorded polydispersity index value was 0·311. Zeta potential value of -23·92mV indicated the high stability of ZnONPs. Transmission electron microscope revealed the spherical shape and the mean particle size was 15.45nm. Functional groups present in the prepared samples of ZnONPs were confirmed by Fourier transform infrared spectroscopy. Additionally, the biological activities of in vitro antimicrobial, anticancer, antioxidant as well as the photocatalytic activities were evaluated. ZnONPs showed broad spectrum of antimicrobial potential against all the tested plant and human pathogens. Based on the MTT assay, ZnONPs inhibited the proliferation of normal human melanocytes, human breast and liver cancer cell lines with IC50 concentrations of 55·76, 18·02 and 16·87µgml-1 . ZnONPs exhibited promising antioxidant potential with 50% inhibitory concentration of 102·13µgml-1 . Moreover, ZnONPs showed efficient degradation of methylene blue dye. The synthesized ZnONPs showed promising activities that can be better explored in the near future for many medical, agricultural and industrial applications. This study suggests a new and alternate approach with the excellent biotechnological potentiality for the production of ZnONPs which could open up the way for the industrial manufacture of nanoparticles using microbial platforms.

Agro-industrial wastes for production of paclitaxel by irradiated Aspergillus fumigatus under solid-state fermentation.

Paclitaxel is the most profitable drug ever developed in cancer chemotherapy; however, the yield of paclitaxel from microbial platforms is still far from the commercial purpose. Thus, this study was conducted to explore the possibility of solid-state fermentation (SSF) for production of paclitaxel by fungal fermentation. Different agro-industrial wastes were screened as solid substrates for production of paclitaxel by the endophytic Aspergillus fumigatus TXD105 under SSF. Sugarcane bagasse followed by wheat bran, and rice bran were the most suitable substrates for maximum production of paclitaxel. In the effort to increase the paclitaxel production, selection of the most proper moistening agent that supports the production of paclitaxel by the fungal strain was investigated. The effect of varying inoculum concentrations on the production of paclitaxel was also studied. Moreover, optimization of SSF conditions (moisture level, substrate concentrations and nutrients concentration) was adopted using response surface methodology. SSF carried out under the optimum conditions of 20g sugarcane bagasse, twofold nutrients concentration of the MM1D broth, 80% moisture level and inoculum concentration of 107 spores per ml intensified the paclitaxel concentration to 145·61mgkg-1 which represents a 10-fold increase. The production of paclitaxel by the fungal strain was further improved via exposure to UV and gamma radiation at specific doses. The paclitaxel concentrations were intensified following UV and gamma radiation to 209·91 and 351·82mgkg-1 . This is the first report on the production of paclitaxel using agro-industrial wastes as cheap source that may contribute in lowering the cost of producing paclitaxel. These findings offer new and alternate sources with excellent biotechnological potential for paclitaxel production by fungal fermentation.

Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast.

Sandalwood oil has been widely used in perfumery industries and aromatherapy. Santalols are its major components. Herein, we attempted to construct santalol-producing yeasts. To alter flux from predominant triterpenoid/steroid biosynthesis to sesquiterpenoid production, expression of ERG9 (encoding yeast squalene synthase) was depressed by replacing its innate promotor with PHXT1 and fermenting the resulting strains in galactose-rich media. And the genes related to santalol biosynthesis were overexpressed under control of GAL promotors, which linked santalol biosynthesis to GAL regulatory system. GAL4 (a transcriptional activator of GAL promotors) and PGM2 (a yeast phosphoglucomutase) were overexpressed to overall promote this artificial santalol biosynthetic pathway and enhance galactose uptake. 1.3 g/L santalols and 1.2 g/L Z-α-santalol were achieved in the strain WL17 expressing SaSS (α-santalene synthase from Santalum album) and WL19 expressing SanSyn (α-santalene synthase from Clausena lansium) by fed-batch fermentation, respectively. This study constructed the microbial santalol-producing platform for the first time.

Engineering of Saccharomyces cerevisiae for efficient fermentation of cellulose.

Conversion of lignocellulosic biomass to biofuels using microbial fermentation is an attractive option to substitute petroleum-based production economically and sustainably. The substantial efforts to design yeast strains for biomass hydrolysis have led to industrially applicable biological routes. Saccharomyces cerevisiae is a robust microbial platform widely used in biofuel production, based on its amenability to systems and synthetic biology tools. The critical challenges for the efficient microbial conversion of lignocellulosic biomass by engineered S. cerevisiae include heterologous expression of cellulolytic enzymes, co-fermentation of hexose and pentose sugars, and robustness against various stresses. Scientists developed many engineering strategies for cellulolytic S. cerevisiae strains, bringing the application of consolidated bioprocess at an industrial scale. Recent advances in the development and implementation of engineered yeast strains capable of assimilating lignocellulose will be reviewed.

Investigating Nutrient Limitation Role on Improvement of Growth and Poly(3-Hydroxybutyrate) Accumulation by Burkholderia sacchari LMG 19450 From Xylose as the Sole Carbon Source.

Burkholderia sacchari LMG19450, a non-model organism and a promising microbial platform, was studied to determine nutrient limitation impact on poly(3-hydroxybutyrate) [P(3HB)] production and bacterial growth from xylose, a major hemicellulosic residue. Nitrogen and phosphorus limitations have been studied in a number of cases to enhance PHA accumulation, but not combining xylose and B. sacchari. Within this strategy, it was sought to understand how to control PHA production and even modulate monomer composition. Nitrogen-limited and phosphorus-limited fed-batch experiments in bioreactors were performed to evaluate each one's influence on cell growth and poly(3-hydroxybutyrate) production. The mineral medium composition was defined based on yields calculated from typical results so that nitrogen was available during phosphorus limitation and residual phosphorus was available when limiting nitrogen. Sets of experiments were performed so as to promote cell growth in the first stage (supplied with initial xylose 15 g/L), followed by an accumulation phase, where N or P was the limiting nutrient when xylose was fed in pulses to avoid concentrations lower than 5 g/L. N-limited fed-batch specific cell growth (around 0.19 1/h) and substrate consumption (around 0.24 1/h) rates were higher when compared to phosphorus-limited ones. Xylose to PHA yield was similar in both conditions [0.37 gP(3HB)/gxyl]. We also described pst gene cluster in B. sacchari, responsible for high-affinity phosphate uptake. Obtained phosphorus to biomass yields might evidence polyphosphate accumulation. Results were compared with studies with B. sacchari and other PHA-producing microorganisms. Since it is the first report of the mentioned kinetic parameters for LMG 19450 growing on xylose solely, our results open exciting perspectives to develop an efficient bioprocess strategy with increased P(3HB) production from xylose or xylose-rich substrates.

Generation of ionic liquid tolerant Pseudomonas putida KT2440 strains via adaptive laboratory evolution

Pseudomonas putida KT2440, a promising microbial platform for industrial biotechnology was tolerized to low-cost biomass decomposing ionic liquids via the adaptive laboratory evolution.

Clostridium thermocellum: A microbial platform for high-value chemical production from lignocellulose.

Second generation biorefining, namely fermentation processes based on lignocellulosic feedstocks, has attracted tremendous interest (owing to the large availability and low cost of this biomass) as a strategy to produce biofuels and commodity chemicals that is an alternative to oil refining. However, the innate recalcitrance of lignocellulose has slowed progress toward economically viable processes. Consolidated bioprocessing (CBP), i.e., single-step fermentation of lignocellulose may dramatically reduce the current costs of 2nd generation biorefining. Metabolic engineering has been used as a tool to develop improved microbial strains supporting CBP. Clostridium thermocellum is among the most efficient cellulose degraders isolated so far and one of the most promising host organisms for application of CBP. The development of efficient and reliable genetic tools has allowed significant progress in metabolic engineering of this strain aimed at expanding the panel of growth substrates and improving the production of a number of commodity chemicals of industrial interest such as ethanol, butanol, isobutanol, isobutyl acetate and lactic acid. The present review aims to summarize recent developments in metabolic engineering of this organism which currently represents a reference model for the development of biocatalysts for 2nd generation biorefining.

Microbial Secretion Platform for 3-Hydroxybutyrate Oligomer and Its End-Capped Forms Using Chain Transfer Reaction-Mediated Polyhydroxyalkanoate Synthases.

The biodegradable polyester 3-hydroxybutyrate (3HB) polymer [P(3HB)] is intracellularly synthesized and accumulated in recombinant Escherichia coli. In this study, native polyhydroxyalkanoate (PHA) synthases are used to attempt to microbially secrete 3HB homo-oligomers (3HBOs), which are widely distributed in nature as physiologically active substances. High secretory production is observed, especially for the two PHA synthases from Aeromonas caviae and Bacillus cereus YB4. Surprisingly, an ethyl ester at the carboxy terminus (ethyl ester form) of 3HBOs is identified for most of the PHA synthases tested. Next, 3HBOs with a functional carboxyl group (carboxyl form of 3HBO) are obtained by using the alcohol dehydrogenase gene (adhE)-deficient mutant strain, suggesting that the endogenous ethanol produced in E. coli acts as a chain transfer (CT) agent in the generation of 3HBOs. Furthermore, an in vitro polymerization assay reveals that CT agents such as ethanol and free 3HB are involved in the generation of ethyl ester and carboxyl form of 3HBO, respectively. The microbial platform established herein allows the secretion of 3HBOs with desirable end structures by supplementation with various CT agents. The obtained 3HBOs and their end-capped forms may be used as physiologically active substances and building blocks for polymeric materials.

Synchronized changes in shallow water carbonate production during the Carnian Pluvial Episode (Late Triassic) throughout Tethys

Abstract Quantitative petrologic analysis on carbonates was carried out on stratigraphic sections encompassing the Carnian Pluvial Episode from northwestern Sichuan Basin, South China and eastern Southern Alps. The Carnian Pluvial Episode, or CPE, is a period of climate change that occurred between the early Carnian and the beginning of the late Carnian (Late Triassic) and coincides with multiple, sharp negative excursions in the record of δ13C that are thought to be evidence of perturbations of the global carbon cycle. During the CPE, relevant environmental modifications and biological turnovers occurred in the marine realm. In particular, microbial carbonate platforms, that were dominant in northwestern Sichuan Basin and throughout Tethys, underwent widespread demise and the microbial component in shelf carbonate sediments sharply decreased and was replaced by ooid- and skeletal grains. Our results show a Tethys-wide coincidence between this change in the carbonate factory. Most notably, both in eastern and western Tethys microbial carbonate production recovered in the late Tuvalian, and the timing of recovery was not influenced by differences of basin evolution and geodynamic setting that characterize such distant domains.

Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

AbstractLignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.

Photosynthetic conversion of CO2 to hyaluronic acid by engineered strains of the cyanobacterium Synechococcus sp. PCC 7002

Hyaluronic acid (HA), consisting of alternating N-acetylglucosamine and glucuronic acid units, is a natural polymer with diverse cosmetic and medical applications. Currently, HA is produced by overexpressing HA synthases from gram-negative Pasteurella multocida (encoded by pmHAS) or gram-positive Streptococcus equisimilis (encoded by seHasA) in various heterotrophic microbial production platforms. Here we introduced these two different types of HA synthase into the fast-growing cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) to explore the capacity for producing HA in a photosynthetic system. Our results show that both HA synthases enable Syn7002 to produce HA photoautotrophically, but that overexpression of the soluble HA synthase (PmHAS) is less deleterious to cell growth and results in higher production. Genetic disruption of the competing cellulose biosynthetic pathway increased the HA titer by over 5-fold (from 14mg/L to 80mg/L) and the relative proportion of HA with molecular mass greater than 2MDa. Introduction of glmS and glmU, coding for enzymes involved in the biosynthesis of the precursor UDP-N-acetylglucosamine, in combination with partial glycogen depletion, allowed photosynthetic production of 112mg/L of HA in 5 days, an 8-fold increase in comparison to the initial PmHAS expressing strain. Addition of tuaD and gtaB (coding for genes involved in UDP-glucuronic acid biosynthesis) also improved the HA yield, albeit to a lesser extent. Overall our results have shown that cyanobacteria hold promise for the sustainable production of pharmaceutically important polysaccharides from sunlight and CO2.

Single mutation at a highly conserved region of chloramphenicol acetyltransferase enables isobutyl acetate production directly from cellulose by Clostridium thermocellum at elevated temperatures

BackgroundEsters are versatile chemicals and potential drop-in biofuels. To develop a sustainable production platform, microbial ester biosynthesis using alcohol acetyltransferases (AATs) has been studied for decades. Volatility of esters endows high-temperature fermentation with advantageous downstream product separation. However, due to the limited thermostability of AATs known, the ester biosynthesis has largely relied on use of mesophilic microbes. Therefore, developing thermostable AATs is important for ester production directly from lignocellulosic biomass by the thermophilic consolidated bioprocessing (CBP) microbes, e.g., Clostridium thermocellum.ResultsIn this study, we engineered a thermostable chloramphenicol acetyltransferase from Staphylococcus aureus (CATSa) for enhanced isobutyl acetate production at elevated temperatures. We first analyzed the broad alcohol substrate range of CATSa. Then, we targeted a highly conserved region in the binding pocket of CATSa for mutagenesis. The mutagenesis revealed that F97W significantly increased conversion of isobutanol to isobutyl acetate. Using CATSa F97W, we demonstrated direct conversion of cellulose into isobutyl acetate by an engineered C. thermocellum at elevated temperatures.ConclusionsThis study highlights that CAT is a potential thermostable AAT that can be harnessed to develop the thermophilic CBP microbial platform for biosynthesis of designer bioesters directly from lignocellulosic biomass.