The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei.

BACKGROUND: Lignocellulose is the most abundant renewable resource in the world and has attracted widespread attention. It can be hydrolyzed into sugars with the help of cellulases and hemicellulases that are secreted by filamentous fungi. Several studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes, including synthesis of metabolites, sporulation, and cell growth and differentiation. However, it remains unknown how and to what extent Ras small GTPases participate in cellulase production. RESULTS: In this study, we found that the putative Ras small GTPase RSR1 negatively regulated the expression of cellulases and xylanases. Deletion of rsr1 (∆rsr1) significantly increased cellulase production and decreased the expression levels of ACY1-cAMP-protein kinase A (PKA) signaling pathway genes and the concentration of intracellular cyclic adenosine monophosphate (cAMP). Loss of acy1 based on ∆rsr1 (∆rsr1∆acy1) could further increase cellulase production and the expression levels of cellulase genes, while overexpression of acy1 based on ∆rsr1 (∆rsr1-OEacy1) significantly reduced cellulase production and transcriptional levels of cellulase genes. In addition, our results revealed that RSR1 negatively controlled cellulase production via the ACY1-cAMP-PKA pathway. Transcriptome analysis revealed significantly increased expression of three G-protein coupled receptors (GPCRs; tre62462, tre58767, and tre53238) and approximately two-fold higher expression of ACE3 and XYR1, which transcriptionally activated cellulases with the loss of rsr1. ∆rsr1∆ tre62462 exhibited a decrease in cellulase activity compared to ∆rsr1, while that of ∆rsr1∆tre58767 and ∆rsr1∆tre53238 showed a remarkable improvement compared to ∆rsr1. These findings revealed that GPCRs on the membrane may sense extracellular signals and transmit them to rsr1 and then to ACY1-cAMP-PKA, thereby negatively controlling the expression of the cellulase activators ACE3 and XYR1. These data indicate the crucial role of Ras small GTPases in regulating cellulase gene expression. CONCLUSIONS: Here, we demonstrate that some GPCRs and Ras small GTPases play key roles in the regulation of cellulase genes in Trichoderma reesei. Understanding the roles of these components in the regulation of cellulase gene transcription and the signaling processes in T. reesei can lay the groundwork for understanding and transforming other filamentous fungi.

Mechanism of Zn2+ regulation of cellulase production in Trichoderma reesei Rut-C30.

BACKGROUND: Trichoderma reesei Rut-C30 is a hypercellulolytic mutant strain that degrades abundant sources of lignocellulosic plant biomass, yielding renewable biofuels. Although Zn2+ is an activator of enzymes in almost all organisms, its effects on cellulase activity in T. reesei have yet to be reported. RESULTS: Although high concentrations of Zn2+ severely suppressed the extension of T. reesei mycelia, the application of 1-4 mM Zn2+ enhanced cellulase and xylanase production in the high-yielding cellulase-producing Rut-C30 strain of T. reesei. Expression of the major cellulase, xylanase, and two essential transcription activator genes (xyr1 and ace3) increased in response to Zn2+ stimulation. Transcriptome analysis revealed that the mRNA levels of plc-e encoding phospholipase C, which is involved in the calcium signaling pathway, were enhanced by Zn2+ application. The disruption of plc-e abolished the cellulase-positive influence of Zn2+ in the early phase of induction, indicating that plc-e is involved in Zn2+-induced cellulase production. Furthermore, treatment with LaCl3 (a plasma membrane Ca2+ channel blocker) and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) indicated that calcium signaling is partially involved in this process. Moreover, we identified the zinc-responsive transcription factor zafA, the transcriptional levels of which declined in response to Zn2+ stress. Deletion of zafA indicates that this factor plays a prominent role in mediating the Zn2+-induced excessive production of cellulase. CONCLUSIONS: For the first time, we have demonstrated that Zn2+ is toxic to T. reesei, although promotes a marked increase in cellulase production. This positive influence of Zn2+ is facilitated by the plc-e gene and zafA transcription factor. These findings provide insights into the role of Zn2+ in T. reesei and the mechanisms underlying signal transduction in cellulase synthesis.

Rational identification of a high catalytic efficiency leucine dehydrogenase and process development for efficient synthesis of l-phenylglycine.

Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid (BFA) for producing l-phenylglycine (l-Phg), was directly identified from 4695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4253.8 U mg-1 . Through reaction process optimization, a high-concentration substrate (0.7 m) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 m BFA could be aminated to l-Phg with conversion of >99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large-scale application of l-Phg.

Anti-Hyperuricemic, Nephroprotective, and Gut Microbiota Regulative Effects of Separated Hydrolysate of α-Lactalbumin on Potassium Oxonate- and Hypoxanthine-Induced Hyperuricemic Mice.

SCOPE: This study aims to investigate the anti-hyperuricemic and nephroprotective effects and the potential mechanisms of the separated gastrointestinal hydrolysates of α-lactalbumin on hyperuricemic mice. METHODS AND RESULTS: The gastrointestinal hydrolysate of α-lactalbumin, the hydrolysate fraction with molecular weight (MW) < 3 kDa (LH-3k), and the fragments with smallest MW among LH-3K harvested through dextran gel chromatography (F5) are used. Hyperuricemia mice are induced via daily oral gavage of potassium oxonate and hypoxanthine. F5 displays the highest in vitro xanthine oxidase (XO) inhibition among all the fractions separated from LH-3k. Oral administration of F5 significantly reduces the levels of serum uric acid (UA), creatinine, and urea nitrogen. F5 treatment could ameliorate kidney injury through alleviating oxidative stress and inflammation. F5 alleviates hyperuricemia in mice by inhibiting hepatic XO activity and regulating the expression of renal urate transporters. Gut microbiota analysis illustrates that F5 administration increases the abundance of some SCFAs producers, and inhibits the growth of hyperuricemia and inflammation associated genera. LH-3k exhibits similar effects but does not show significance as those of the F5 fraction. CONCLUSION: The anti-hyperuricemia and nephroprotective functions of F5 are mediated by inhibiting hepatic XO activity, ameliorating oxidative stress and inflammation, regulating renal urate transporters, and modulating the gut microbiota in hyperuricemic mice.

Roles of PKAc1 and CRE1 in cellulose degradation, conidiation, and yellow pigment synthesis in Trichoderma reesei QM6a.

PURPOSE: This study aimed to reveal the roles of the protein kinase A catalytic subunit 1 (pkac1) and carbon catabolite repressor cre1 genes in cellulase production by Trichoderma reesei wild-type strain QM6a. Our strategy might be useful to construct a high-yielding cellulase strain for its wide application. METHODS: This paper describes cellulase activity, plate conidiation, and yellow pigment synthesis assays of QM6a with the disruption of pkac1 and cre1. RESULTS: Deletion of pkac1 (Δpkac1) had no effect on cellulase production or transcript levels of major cellulase genes in the presence of cellulose. Disruption of cre1 (Δcre1) resulted in a remarkable increase in cellulase production and expression of the four major cellulase genes. Double disruption of pkac1 and cre1 significantly improved enzyme activity and protein production. The double disruption also resulted in a significant reduction in yellow pigment production and abrogated conidial production. CONCLUSION: Double deletion of pkac1 and cre1 led to increased hydrolytic enzyme production in T. reesei using cellulose as a carbon source.

Mn2+ modulates the production of mycophenolic acid in Penicillium brevicompactum NRRL864 via reactive oxygen species signaling and the investigation of pb-pho.

Although Penicillium brevicompactum is a widely used commercial strain for the manufacture of mycophenolic acid, there are few findings of Ca2+, Mn2+, and reactive oxygen species (ROS) interaction. Metal ions play a crucial role in physiological metabolism. Calcium, as the important second messenger, influences fungus growth, virulence, and stress responses. The concentration of cytosolic Ca2+ was influenced by the Mn2+, which demonstrated the crosstalk between calcium and manganese. In the previous study, the crosstalk between calcium and ROS has been discovered and verified, which modified the secondary metabolism and enhanced the yield of MPA (Mycophenolic Acid). A higher concentration of Mn2+ in the fermentation broth causes an increase in cytoplasmic Ca2+ and ROS (Reactive Oxygen Species), enhancing the yield of MPA by about 20 % higher and disclosing the cascade regulation with the Mn2+, Ca2+, and ROS. To be more specific, the intracellular concentration of ROS at 6 mM Mn2+ is about 1.5 times higher than that at 0.6 mM. Furthermore, we identify an Mn2+ transport protein, designated as Pb-PHO, which shows 71.2 % identity to the inorganic phosphate transporter PHO84 (Q0CBJ6) from Aspergillus terreus. At the same time, the △Pb-pho exhibits damage to the cell wall integrity, while the OE-pho displays a more normal phenotype at high osmotic stress. The high-affinity Ca2+ channel, Pb-CCH, is examined via knockdown to demonstrate the crosstalk between Mn2+ and Ca2+. The results show that the addition of Mn2+ remits the negative influence of pb-cch knockdown and the addition of Ca2+ remits the negative influence of pb-pho knockdown, demonstrating the relationship between cytoplasmic Mn2+ and Ca2+. Taken together, our results demonstrate the mechanism of a manganese-induced cascade of manganese-calcium-ROS and reveal a signal pathway-relative method to illustrate the manganese-induced increase of MPA production in Penicillium brevicompactum. Furthermore, we discover and identify an Mn2+ transport protein, Pb-PHO, which is subcellular localized at the plasma membrane and proved to affect the cell wall integrity.

The effects of angiotensin I-converting enzyme inhibitory peptide VGINYW and the hydrolysate of α-lactalbumin on blood pressure, oxidative stress and gut microbiota of spontaneously hypertensive rats.

VGINYW is a highly active angiotensin I-converting enzyme (ACE) inhibitory peptide discovered from α-lactalbumin by an in vitro-in silico high throughput screening strategy. The aim of this study was to evaluate the antihypertensive effect of the peptide and the α-lactalbumin hydrolysates under 3 kDa (LH-3k), and illustrate the possible mechanism in spontaneously hypertensive rats (SHRs). SHRs were administered with VGINYW and LH-3k at doses of 5 mg per kg BW and 100 mg per kg BW, respectively. VGINYW and LH-3k could markedly decrease the systolic blood pressure (SBP) of the SHRs, and the maximal drops of 21 mmHg (2 h after administration) and 17 mmHg (4 h after administration) were achieved during the 8 hour test, respectively. When the agents were given once per day for 4 weeks, they caused a long-term decrease of 16 mmHg of SBP. VGINYW and LH-3k control the blood pressure through regulating the renin-angiotensin system by inhibiting the ACE activity and diminishing the angiotensin II level, and further upregulating the expression levels of the angiotensin-converting enzyme 2 and angiotensin type 2 receptor, and downregulating the expression of the angiotensin type 1 receptor. VGINYW and LH-3k could notably ameliorate the oxidative stress in the SHR as well. It is more important that the gavage of VGINYW and LH-3k could alleviate hypertension-associated intestinal microbiota dysbiosis by recovering the diversity of the gut microbiota and altering the key floras which are short chain fatty acid producers. In conclusion, VGINYW and LH-3k are effective functional ingredients for blood pressure control.

Synergistic Regulation of Metabolism by Ca2+/Reactive Oxygen Species in Penicillium brevicompactum Improves Production of Mycophenolic Acid and Investigation of the Ca2+ Channel.

Although Penicillium brevicompactum is a very important industrial strain for mycophenolic acid production, there are no reports on Ca2+/reactive oxygen species (ROS) synergistic regulation and calcium channels, Cch-pb. This study initially intensified the concentration of the intracellular Ca2+ in the high yielding mycophenolic acid producing strain NRRL864 to explore the physiological role of intracellular redox state in metabolic regulation by Penicillium brevicompactum. The addition of Ca2+ in the media caused an increase of intracellular Ca2+, which was accompanied by a strong increase, 1.5 times, in the higher intracellular ROS concentration. In addition, the more intensive ROS sparked the production of an unreported pigment and increase in mycophenolic acid production. Furthermore, the Ca2+ channel, the homologous gene of Cch1, Cch-pb, was investigated to verify the relationship between Ca2+ and the intracellular ROS. The Vitreoscilla hemoglobin was overexpressed, which was bacterial hemoglobin from Vitreoscilla, reducing the intracellular ROS concentration to verify the relationship between the redox state and the yield of mycophenolic acid. The strain pb-VGB expressed the Vitreoscilla hemoglobin exhibited a lower intracellular ROS concentration, 30% lower, and decreased the yield of mycophenolic acid as 10% lower at the same time. Subsequently, with the NRRL864 fermented under 1.7 and 28 mM Ca2+, the [NADH]/[NAD+] ratios were detected and the higher [NADH]/[NAD+] ratios (4 times higher with 28 mM) meant a more robust primary metabolism which provided more precursors to produce the pigment and the mycophenolic acid. Finally, the 10 times higher calcium addition in the media resulted in 25% enhanced mycophenolic acid production to 6.7 g/L and induced pigment synthesis in NRRL864.

Induction of cellulase production by Sr2+ in Trichoderma reesei via calcium signaling transduction.

Trichoderma reesei RUT-C30 is a well-known high-yielding cellulase-producing fungal strain that converts lignocellulose into cellulosic sugar for resource regeneration. Calcium is a ubiquitous secondary messenger that regulates growth and cellulase production in T. reesei. We serendipitously found that adding Sr2+ to the medium significantly increased cellulase activity in the T. reesei RUT-C30 strain and upregulated the expression of cellulase-related genes. Further studies showed that Sr2+ supplementation increased the cytosolic calcium concentration and activated the calcium-responsive signal transduction pathway of Ca2+-calcineurin-responsive zinc finger transcription factor 1 (CRZ1). Using the plasma membrane Ca2+ channel blocker, LaCl3, we demonstrated that Sr2+ induces cellulase production via the calcium signaling pathway. Supplementation with the corresponding concentrations of Sr2+ also inhibited colony growth. Sr2+ supplementation led to an increase in intracellular reactive oxygen species (ROS) and upregulated the transcriptional levels of intracellular superoxide dismutase (sod1) and catalase (cat1). We further demonstrated that ROS content was detrimental to cellulase production, which was alleviated by the ROS scavenger N-acetyl cysteine (NAC). This study demonstrated for the first time that Sr2+ supplementation stimulates cellulase production and upregulates cellulase genes via the calcium signaling transduction pathway. Sr2+ leads to an increase in intracellular ROS, which is detrimental to cellulase production and can be alleviated by the ROS scavenger NAC. Our results provide insights into the mechanistic study of cellulase synthesis and the discovery of novel inducers of cellulase.

Efficient expression and purification of soluble HarpinEa protein by translation initiation region codon optimization.

HarpinEa protein can stimulate plants to produce defense responses to resist the attack of pathogens, improve plant immune resistance, and promote plant growth. This has extremely high application value in agriculture. To efficiently express soluble HarpinEa protein, in this study, we expressed HarpinEa protein with a 6× His-tag in Escherichia coli BL21 (DE3). Because of the low level of expression of HarpinEa protein in E. coli, three rounds of synonymous codon optimization were performed on the +53 bp of the translation initiation region (TIR) of HarpinEa. Soluble HarpinEa protein after optimization accounted for 50.3% of the total soluble cellular protein expressed. After purification using a Ni Bestarose Fast Flow column, the purity of HarpinEa protein exceeded 95%, and the yield reached 227.5 mg/L of culture medium. The purified HarpinEa protein was sensitive to proteases and exhibited thermal stability. It triggered visible hypersensitive responses after being injected into tobacco leaves for 48 h. Plants treated with HarpinEa showed obvious growth-promoting and resistance-improving performance. Thus, the use of TIR synonymous codon optimization successfully achieved the economical, efficient, and soluble production of HarpinEa protein.

Developing a medium combination to attain similar glycosylation profile to originator by DoE and cluster analysis method.

Glycosylation is critical for monoclonal antibody production because of its impact on pharmacokinetics and pharmacodynamics. Modulation of glycan profile is frequently needed in biosimilar development. However, glycosylation profile is not a single value like that of cell culture titer, hence making it challenging for the Design of Experiment (DoE) methodology to be directly applied. In this study, a Her2-binding antibody was developed as a biosimilar to Herceptin. Cluster analysis was introduced to demonstrate the similarity of glycan profiles between the samples and the reference with specific value-distance. The glycosylation was subsequently optimized with the DoE method. Basal medium and feed medium were found to be the significant factors to the glycosylation pattern. Moreover, a combination of medium and feed strategy was developed to attain the most similar glycoprotein molecule to that of the originator biologic drug. This study may provide an additional option to evaluate multivariable factors and assess biosimilarity and/or comparability in monoclonal antibody production.

cAMP activates calcium signalling via phospholipase C to regulate cellulase production in the filamentous fungus Trichoderma reesei.

BACKGROUND: The filamentous fungus Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression. RESULTS: We found that Mn2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E. CONCLUSIONS: The findings of this study reveal the presence of a signal transduction pathway in which Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP-PLC-calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.

Characterization and Function of a Novel Welan Gum Lyase From Marine Sphingomonas sp. WG.

Welan gum, a kind of microbial exopolysaccharides, produced by the genus Sphingomonas, have great potential for application in many fields, such as the food industry, cement production, and enhanced oil recovery. But there are still challenges to reduce the cost, enhance the production and the quality. Herein, the bioinformatics analysis of WelR gene was preformed, and the characterization and function of WelR, welan gum lyase, from Sphingomonas sp. WG were investigated for the first time. The results indicated that 382nd (Asn), 383rd (Met), 494th (Asn), and 568th (Glu) were the key amino acid residues, and C-terminal amino acids were essential to keeping the stability of WelR. The optimal temperature and pH of the enzymatic activity were found to be 25°C and 7.4, respectively. And WelR was good low temperature resistance and alkali resistant. K+, Mg2+, Ca2+, Mn2+, and EDTA increased WelR activities, in contrast to Zn2+. Coupled with the change in glucose concentration and growth profile, the qRT-PCR results indicated that WelR may degrade welan gum existing in the culture to maintain bacterial metabolism when glucose was depleted. This work will lay a theoretical foundation to establish new strategies for the regulation of welan gum biosynthesis.

High-level expression of a ß-mannanase (manB) in Pichia pastoris GS115 for mannose production with Penicillium brevicompactum fermentation pretreatment of soybean meal.

An endo-1,4-ß-mannanase gene (manB) from a Bacillus pumilus Nsic-2 grown in a stinky tofu emulsion was cloned and expressed in Pichia pastoris GS115. After characterized, the endo-1,4-ß-mannanase (manB) show maximum activity at pH 6.0 and 50 °C with LBG as substrate and perform high stability at a range of pH 6-8. After applying for a shake flask fermentation, the specific activity of manB reached 3462 U/mg. To produce mannose, the soybean meal (SBM) was pretreated by biological fermentation for 11 days with Penicillium brevicompactum, and then hydrolyzed by manB. As a result, mannose yield reached 3.58 g per 1 kg SBM which indicated that 0.358% SBM was converted into mannose after hydrolyzation, and mean a total 20% mannan of SBM converting into mannose, while the control group demonstrated only 1.78% conversion. An effective ß-mannanase for the bioconversion of mannan-rich biomasses and an efficient method to produce mannose with soybean meal were introduced.

A Multifunctional Electrochemical Sensor for the Simultaneous Detection of Ascorbic Acid, Dopamine, Uric Acid, and Nitrite.

BACKGROUND: Ascorbic acid (AA), dopamine (DA), uric acid (UA), and nitrite (NO2-) are essential biomarkers for human metabolism and can be used to indicate some chronic diseases and metabolic disorders, including scurvy, Parkinson's disease, hyperuricemia, and kidney disease. OBJECTIVE: A multifunctional electrochemical sensor that can integrate the detection of these species was constructed using nanoporous gold (NPG) as a recognition element to modify glassy carbon electrode (GCE). METHODS: The electrochemical performance of the multifunctional electrochemical sensor was investigated toward AA, DA, UA, and NO2- in citrate buffer solution (CBS, 100 mM, pH 4.0) and human serum using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) methods. RESULTS: In the quaternary mixture detection, the resulting NPG/GCE electrode displayed four independent oxidation peaks with wide peak separations. Further, the NPG/GCE electrode showed good linear responses with the sensitivities of 32, 1103, 71, and 147 µA/mM/cm2 and the detection limits of 1.58, 0.17, 0.37, and 0.36 µM for AA, DA UA, and NO2-, respectively. Additionally, the NPG/GCE electrode exhibited great anti-interference and was successfully applied in human serum samples. CONCLUSIONS: These results indicate that the NPG/GCE electrode can simultaneously and selectively detect AA, DA, UA, and NO2-, which has the potential for application and diagnosis in the screening and diagnosis of chronic diseases and metabolic disorders. HIGHLIGHTS: A multianalyte electrochemical sensor was fabricated for human metabolites detection. The sensor displayed good performance in the simultaneous detection of AA, DA, UA, and NO2- and applied to human serum samples.

Similarity assessment by multivariate statistics method based on the distance between biosimilar and originator.

The development of biosimilar products or follow-on biologics has been flourishing in recent years because of their lower price than the originators. In this study, a multivariate data analysis method based on JMP software was proposed to assess the glycosylation pattern similarity of antibody candidates from different conditions in optimization experiments with a reference. A specific distance was generated by this method and indicated the glycoform similarity between the biosimilar and the reference. This method can be applied to analyze the similarity of other physicochemical and functional characteristics between follow-on biologics and originators. Then, the design of experimental methods can be realized to optimize the conditions of cell culture to attain similar antibody candidates. A higher concentration of GlcNAc added to the basal media made the glycan of the antibody more similar to the glycan of the reference in this study.

Functional expression of a novel methanol-stable esterase from Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system.

BACKGROUND: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. RESULTS: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of pNP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate pNP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). CONCLUSIONS: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG.

To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 °C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 °C. The optimal pH and temperature of UGPase were 9.0 and 45 °C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 °C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.

N,N-dimethylformamide induces cellulase production in the filamentous fungus Trichoderma reesei.

BACKGROUND: The filamentous fungus Trichoderma reesei produces cellulase enzymes that are widely studied for lignocellulose bioconversion to biofuel. N,N-dimethylformamide (DMF) is a versatile organic solvent used in large quantities in industries. RESULTS: In this study, we serendipitously found that biologically relevant concentrations of extracellular DMF-induced cellulase production in the T. reesei hyper-cellulolytic mutant Rut-C30 and wild-type strain QM6a. Next, by transcriptome analysis, we determined that plc-e encoding phospholipase C was activated by DMF and revealed that cytosolic Ca2+ plays a vital role in the response of T. reesei to DMF. Using EGTA (a putative extracellular Ca2+ chelator) and LaCl3 (a plasma membrane Ca2+ channel blocker), we demonstrated that DMF induced a cytosolic Ca2+ burst via extracellular Ca2+ and Ca2+ channels in T. reesei, and that the cytosolic Ca2+ burst induced by DMF-mediated overexpression of cellulase through calcium signaling. Deletion of crz1 confirmed that calcium signaling plays a dominant role in DMF-induced cellulase production. Additionally, 0.5-2% DMF increases the permeability of T. reesei mycelia for cellulase release. Simultaneous supplementation with 1% DMF and 10 mM Mn2+ to T. reesei Rut-C30 increased cellulase activity approximately fourfold compared to that without treatment and was also more than that observed in response to either treatment alone. CONCLUSIONS: Our results reveal that DMF-induced cellulase production via calcium signaling and permeabilization. Our results also provide insight into the role of calcium signaling in enzyme production for enhanced cellulase production and the development of novel inducers of cellulase.