Production Of Soluble Protein Research Articles

Fine Structural Analysis of Degummed Fibroin Fibers Reveals Its Superior Mechanical Capabilities.

Bombyx mori silk fibroin fibers constitute a class of protein building blocks capable of functionalization and reprocessing into various material formats. The properties of these fibers are typically affected by the intense thermal treatments needed to remove the sericin gum coating layer. Additionally, their mechanical characteristics are often misinterpreted by assuming the asymmetrical cross-sectional area (CSA) as a perfect circle. The thermal treatments impact not only the mechanics of the degummed fibroin fibers, but also the structural configuration of the resolubilized protein, thereby limiting the performance of the resulting silk-based materials. To mitigate these limitations, we explored varying alkali conditions at low temperatures for surface treatment, effectively removing the sericin gum layer while preserving the molecular structure of the fibroin protein, thus, maintaining the hierarchical integrity of the exposed fibroin microfiber core. The precise determination of the initial CSA of the asymmetrical silk fibers led to a comprehensive analysis of their mechanical properties. Our findings indicate that the alkali surface treatment raised the Young's modulus and tensile strength, by increasing the extent of the fibers' crystallinity, by approximately 40 % and 50 %, respectively, without compromising their strain. Furthermore, we have shown that this treatment facilitated further production of high-purity soluble silk protein with rheological and self-assembly characteristics comparable to those of native silk feedstock, initially stored in the animal's silk gland. The developed approaches benefits both the development of silk-based materials with tailored properties and the proper mechanical characterization of asymmetrical fibrous biological materials made of natural building blocks.

Feasibility assessment and underlying mechanisms of metabisulfite pretreatment for enhanced volatile fatty acids production from anaerobic sludge fermentation

Employing chemical pretreatment for waste activated sludge (WAS) fermentation is crucial to achieving sustainable sludge management. This study investigated the feasibility of metabisulfite (MS) pretreatment for enhancing volatile fatty acids (VFAs) production from WAS. The results show that after 24-h MS pretreatment, the content of soluble organic matter and loosely bound extracellular polymeric substances (LB-EPS), especially proteins, increased significantly. During the fermentation, MS pretreatment under alkaline conditions was more efficient, with VFA peaking on the fifth day, showing a 140 % increase compared to the alkaline control group. Correlation analysis suggests that the dosage of MS, rather than pH, is closely related to the levels of soluble protein, polysaccharides, LB-EPS, and subsequential VFAs production, while alkaline conditions facilitate the dissolution of total organic carbon. Furthermore, sulfite radicals (SO3•−) are attributed to cell inactivation and lysis, while alkaline conditions initially reduce the size of the flocs, further promoting MS for attacking flocs, thereby improving the performance of fermentation. The study also found that MS pretreatment reduced microbial community diversity, enriched hydrolytic and fermentation bacteria (Actinobacteriota and Firmicutes), and suppressed methanogens (Methanobacteriaceae and Methanosaetaceae), making it a safe, viable, and cost-effective chemical agent for sustainable sludge management.

Transcriptomic and Metabolomic Analyses of Soybean Protein Isolate on Monascus Pigments and Monacolin K Production.

Monascus pigments (MPs) and monacolin K (MK) are important secondary metabolites produced by Monascus spp. This study aimed to investigate the effect of soybean protein isolate (SPI) on the biosynthesis of MPs and MK based on the analysis of physiological indicators, transcriptomes, and metabolomes. The results indicated that the growth, yellow MPs, and MK production of Monascus pilosus MS-1 were significantly enhanced by SPI, which were 8.20, 8.01, and 1.91 times higher than that of the control, respectively. The utilization of a nitrogen source, protease activity, the production and utilization of soluble protein, polypeptides, and free amino acids were also promoted by SPI. The transcriptomic analysis revealed that the genes mokA, mokB, mokC, mokD, mokE, mokI, and mokH which are involved in MK biosynthesis were significantly up-regulated by SPI. Moreover, the glycolysis/gluconeogenesis, pyruvate metabolism, fatty acid degradation, tricarboxylic acid (TCA) cycle, and amino acid metabolism were effectively up-regulated by SPI. The metabolomic analysis indicated that metabolisms of amino acid, lipid, pyruvate, TCA cycle, glycolysis/gluconeogenesis, starch and sucrose, and pentose phosphate pathway were significantly disturbed by SPI. Thus, MPs and MK production promoted by SPI were mainly attributed to the increased biomass, up-regulated gene expression level, and more precursors and energies.

Streamlining heterologous expression of top carbonic anhydrases in Escherichia coli: bioinformatic and experimental approaches

BackgroundCarbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications.ResultsIn this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity.ConclusionsOverall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.

From growth inhibition to ultrastructural changes: Toxicological assessment of lambda cyhalothrin and fosetyl aluminium against Bacillus subtilis and Pseudomonas aeruginosa

In modern agricultural practices, agrochemicals and pesticides play an important role in protecting the crops from pests and elevating agricultural productivity. This strategic utilization is essential to meet global food demand due to the relentless growth of the world's population. However, the indiscriminate application of these substances may result in environmental hazards and directly affect the soil microorganisms and crop production. Considering this, an in vitro study was carried out to evaluate the pesticides' effects i.e. lambda cyhalothrin (insecticide) and fosetyl aluminum (fungicide) at lower, recommended, and higher doses on growth behavior, enzymatic profile, total soluble protein production, and lipid peroxidation of bacterial specimens (Pseudomonas aeruginosa and Bacillus subtilis). The experimental findings demonstrated a concentration-dependent decrease in growth of both tested bacteria, when exposed to fosetyl aluminium concentrations exceeding the recommended dose. This decline was statistically significant (p < 0.000). However, lambda cyhalothrin at three times of recommended dose induces 10% increase in growth of Pseudomonas aeruginosa (P. aeruginosa) and 76.8% decrease in growth of Bacillus subtilis (B. subtilis) respectively as compared to control. These results showed the stimulatory effect of lambda cyhalothrin on P. aeruginosa and inhibitory effect on B. subtilis. Pesticides induced notable alterations in biomarker enzymatic assays and other parameters related to oxidative stress among bacterial strains, resulting in increased oxidative stress and membrane permeability. Generally, the maximum toxicity of both (P. aeruginosa and B. subtilis) was shown by fosetyl aluminium, at three times of recommended dose. Fosetyl aluminium induced morphological changes like cellular cracking, reduced viability, aberrant margins and more damage in both bacterial strains as compared to lambda cyhalothrin when observed under scanning electron microscope (SEM). Conclusively the, present study provide an insights into a mechanistic approach of pyrethroid insecticide and phosphonite fungicide induced cellular toxicity towards bacteria.

Modifications of the 5' region of the CASPONTM tag's mRNA further enhance soluble recombinant protein production in Escherichia coli.

Escherichia coli is one of the most commonly used host organisms for the production of biopharmaceuticals, as it allows for cost-efficient and fast recombinant protein expression. However, challenging proteins are often produced with low titres or as inclusion bodies, and the manufacturing process needs to be developed individually for each protein. Recently, we developed the CASPONTM technology, a generic fusion tag-based platform process for high-titer soluble expression including a standardized downstream processing and highly specific enzymatic cleavage of the fusion tag. To assess potential strategies for further improvement of the N-terminally fused CASPONTM tag, we modified the 5'UTR and 5' region of the tag-coding mRNA to optimize the ribosome-mRNA interactions. In the present work, we found that by modifying the 5'UTR sequence of a pET30acer plasmid-based system, expression of the fusion protein CASPONTM-tumour necrosis factor α was altered in laboratory-scale carbon-limited fed-batch cultivations, but no significant increase in expression titre was achieved. Translation efficiency was highest for a construct carrying an expression enhancer element and additionally possessing a very favourable interaction energy between ribosome and mRNA (∆Gtotal). However, a construct with comparatively low transcriptional efficiency, which lacked the expression enhancer sequence and carried the most favourable ∆Gtotal tested, led to the highest recombinant protein formation alongside the reference pET30a construct. Furthermore, we found, that by introducing synonymous mutations within the nucleotide sequence of the T7AC element of the CASPONTM tag, utilizing a combination of rare and non-rare codons, the free folding energy of the nucleotides at the 5' end (-4 to + 37) of the transcript encoding the CASPONTM tag increased by 6kcal/mol. Surprisingly, this new T7ACrare variant led to improved recombinant protein titres by 1.3-fold up to 5.3-fold, shown with three industry-relevant proteins in lab-scale carbon limited fed-batch fermentations under industrially relevant conditions. This study reveals some of the complex interdependencies between the ribosome and mRNA that govern recombinant protein expression. By modifying the 5'UTR to obtain an optimized interaction energy between the mRNA and the ribosome (ΔGtotal), transcript levels were changed, highlighting the different translation efficiencies of individual transcripts. It was shown that the highest recombinant titre was not obtained by the construct with the most efficient translation but by a construct with a generally high transcript amount coupled with a favourable ΔGtotal. Furthermore, an unexpectedly high potential to enhance expression by introducing silent mutations including multiple rare codons into the 5'end of the CAPONTM tag's mRNA was identified. Although the titres of the fusion proteins were dramatically increased, no formation of inclusion bodies or negative impact on cell growth was observed. We hypothesize that the drastic increase in titre is most likely caused by better ribosomal binding site accessibility. Our study, which demonstrates the influence of changes in ribosome-mRNA interactions on protein expression under industrially relevant production conditions, opens the door to the applicability of the new T7ACrare tag in biopharmaceutical industry using the CASPONTM platform process.

Using Vibrio natriegens for High-Yield Production of Challenging Expression Targets and for Protein Perdeuteration.

Production of soluble proteins is essential for structure/function studies; however, this usually requires milligram amounts of protein, which can be difficult to obtain with traditional expression systems. Recently, the Gram-negative bacterium Vibrio natriegens emerged as a novel and alternative host platform for production of proteins in high yields. Here, we used a commercial strain derived from V. natriegens (Vmax X2) to produce soluble bacterial and fungal proteins in milligram scale, which we struggled to achieve in Escherichia coli. These proteins include the cholera toxin (CT) and N-acetyl glucosamine-binding protein A (GbpA) from Vibrio cholerae, the heat-labile enterotoxin (LT) from E. coli and the fungal nematotoxin CCTX2 from Coprinopsis cinerea. CT, GbpA, and LT are secreted by the Type II secretion system in their natural hosts. When these three proteins were produced in Vmax, they were also secreted and could be recovered from the growth media. This simplified the downstream purification procedure and resulted in considerably higher protein yields compared to production in E. coli (6- to 26-fold increase). We also tested Vmax for protein perdeuteration using deuterated minimal media with deuterium oxide as solvent and achieved a 3-fold increase in yield compared to the equivalent protocol in E. coli. This is good news, since isotopic labeling is expensive and often ineffective but represents a necessary prerequisite for some structural biology techniques. Thus, Vmax represents a promising host for production of challenging expression targets and for protein perdeuteration in amounts suitable for structural biology studies.

Production of recombinant HPV11/16 E6/E7-MBP-His6 fusion proteins and their potential to induce cytokine secretion by immune cells in peripheral blood

Human papillomavirus (HPV) infection poses a significant threat to public health worldwide. Targeting the function of HPV E6 and E7 proteins and activating the host immune response against these proteins represent promising therapeutic strategies for combating HPV-related diseases. Consequently, the efficient production of soluble, high-purity E6 and E7 proteins is crucial for function and host immune response studies. In this context, we selected the pMCSG19 protein expression vector for Escherichia coli to produce soluble MBP-His6 tagged HPV11/16 E6/E7 proteins, achieving relatively high purity and yield. Notably, these proteins exhibited low toxicity to peripheral blood mononuclear cells (PBMCs) and did not compromise their viability. Additionally, the recombinant proteins were capable of inducing the secretion of multiple cytokines by immune cells in peripheral blood, indicating their potential to elicit immune responses. In conclusion, our study offers a novel approach for the production of HPV11/16 E6/E7 fusion proteins with relatively high purity and yield. The fusing HPV11/16 E6/E7 proteins to MBP-His6 tag may serve as a valuable method for large-scale protein production in future research endeavors.

Valorization of chicken feather waste using recombinant bacillus subtilis cells by solid-state fermentation for soluble proteins and serine alkaline protease production

Feather waste, a by-product of the poultry industry, is rich in proteins, peptides, and amino acids. Improper disposal of feathers can cause environmental pollution. Solid-state fermentation (SSF) is a viable alternative to submerged fermentation due to its simplicity, productivity, and lower cost. The study goal is a biorefinery of chicken feather waste supplemented with wheat bran using a recombinant Bacillus subtilis strain to produce soluble proteins and a serine alkaline protease. Plackett-Burman Design and Central Composite Design were utilized in a statistical-mathematical model to optimize the process. Multi-factorial design optimization resulted in 80 % substrate degradation efficiency, an alkaline protease with dual activities (1423 proteolytic units and 190 keratinolytic units), 214 mg soluble proteins/g substrate, and 87 % model validation. Scaling up the SSF process to 50 g of substrate significantly enhanced the end products of feather biodegradation to 1616 proteolytic units, 2844 keratinolytic units, and 127 mg soluble proteins/g substrate. Aim and scope of the manuscriptThe aim of the present study is to utilize chicken feather waste (alone or supplemented with other materials) through recombinant Bacillus subtilis cells using solid state fermentation (SSF) at a laboratory scale. The plan study provides a promising waste management in the environmental field concerning biodegradation of such recalcitrant keratinous wastes supplemented with agricultural residues via recombinant microorganism. On semi-pilot scale, high production and quality of soluble protein, protease, and keratinase activity were produced according to the statistically optimised first stage fermentation in the laboratory scale. The bioconversion process took place as a major goal to obtain valuable products, with low utilities and energy requirements. Therefore, this will consider as an economically feasible and environmentally friendly alternative. Moreover, this study is considered as first step fermentation for feather waste to pave the road for directing it to a second step fermentation for biogas production and bioenergy generation through bio-electrochemical systems (Manuscript under publication).

The Functional Characteristics and Soluble Expression of Saffron CsCCD2.

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on β-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also β-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.

Aspergillus nomiae and fumigatus Ameliorating the Hypoxic Stress Induced by Waterlogging through Ethylene Metabolism in Zea mays L.

Transient and prolonged waterlogging stress (WS) stimulates ethylene (ET) generation in plants, but their reprogramming is critical in determining the plants' fate under WS, which can be combated by the application of symbiotically associated beneficial microbes that induce resistance to WS. The present research was rationalized to explore the potential of the newly isolated 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing fungal endophytic consortium of Aspergillus nomiae (MA1) and Aspergillus fumigatus (MA4) on maize growth promotion under WS. MA1 and MA4 were isolated from the seeds of Moringa oleifera L., which ably produced a sufficient amount of IAA, proline, phenols, and flavonoids. MA1 and MA4 proficiently colonized the root zone of maize (Zea mays L.). The symbiotic association of MA1 and MA4 promoted the growth response of maize compared with the non-inoculated plants under WS stress. Moreover, MA1- and MA4-inoculated maize plants enhanced the production of total soluble protein, sugar, lipids, phenolics, and flavonoids, with a reduction in proline content and H2O2 production. MA1- and MA4-inoculated maize plants showed an increase in the DPPH activity and antioxidant enzyme activities of CAT and POD, along with an increased level of hormonal content (GA3 and IAA) and decreased ABA and ACC contents. Optimal stomatal activity in leaf tissue and adventitious root formation at the root/stem junction was increased in MA1- and MA4-inoculated maize plants, with reduced lysigenous aerenchyma formation, ratio of cortex-to-stele, water-filled cells, and cell gaps within roots; increased tight and round cells; and intact cortical cells without damage. MA1 and MA4 induced a reduction in deformed mesophyll cells, and deteriorated epidermal and vascular bundle cells, as well as swollen metaxylem, phloem, pith, and cortical area, in maize plants under WS compared with control. Moreover, the transcript abundance of ethylene-responsive gene ZmEREB180, responsible for the induction of the WS tolerance in maize, showed optimally reduced expression sufficient for induction in WS tolerance, in MA1- and MA4-inoculated maize plants under WS compared with the non-inoculated control. The existing research supported the use of MA1 and MA4 isolates for establishing the bipartite mutualistic symbiosis in maize to assuage the adverse effects of WS by optimizing ethylene production.

Mechanistic insights into hormesis induced by erythromycin in the marine alga Thalassiosira weissflogii

Erythromycin (ERY) is a typical macrolide antibiotic with large production and extensive use on a global scale. Detection of ERY in both freshwaters and coaster seawaters, as well as relatively high ecotoxicity of ERY have been documented. Notably, hormesis has been reported on several freshwater algae under ERY stress, where growth was promoted at relatively lower exposures but inhibited at higher treatment levels. On the contrary, there is limited information of ERY toxicity in marine algae, hampering the risk assessment on ERY in the coaster waters. The presence of hormesis may challenge the current concept of dose-response adopted in chemical risk assessment. Whether and how exposure to ERY can induce dose-dependent toxicity in marine algae remain virtually unknown, especially at environmentally relevant concentrations. The present study used a model marine diatom Thalassiosira weissflogii (T. weissflogii) to reveal its toxicological responses to ERY at different biological levels and decipher the underlying mechanisms. Assessment of multiple apical endpoints shows an evident growth promotion following ERY exposure at an environmentally relevant concentration (1 µg/L), associated with increased contents reactive oxygen species (ROS) and chlorophyll-a (Chl-a), activated signaling pathways related to ribosome biosynthesis and translation, and production of total soluble protein. By contrast, growth inhibition in the 750 and 2500 µg/L treatments was attributed to reduced viability, increased ROS formation, reduced content of total soluble protein, inhibited photosynthesis, and perturbed signaling pathways involved in xenobiotic metabolism, ribosome, metabolism of amino acid, and nitrogen metabolism. Measurements of multiple apical endpoints coupled with de novo transcriptomics analysis applied in the present study, a systems biology approach, can generate detailed mechanistic information of chemical toxicity including dose-response and species sensitivity difference used in environmental risk assessment.

Effects of Selenium on Growth and Biochemical Characters of Tissue Culture Seedlings of Sophora tonkinensis

Background Selenium (Se) plays a key role in controlling several processes in plant development, such as root growth and regulation of enzyme activity. However, research on the effects of Se in Sophora tonkinensis, a medicinal plant, has not been reported. To elucidate the effect of Se on the accumulation of secondary metabolites in tissue-cultured seedlings of S. tonkinensis. Materials and Methods Tissue culture seedlings of S. tonkinensis was treated with different concentrations of Se in the rooting medium. Agronomic traits of roots and concentrations of hormones and some secondary metabolites were measured from seedlings. Results The results show that (1) root growth of tissue culture seedlings were stimulated by Se; (2) a specific amount of Se enhanced the activities of peroxidase (POD) and superoxide dismutase (SOD), as well as the production and storage of soluble proteins and chlorophyll; and (3) Se treatment lowered brassinolide (BR) content, and 15 mg/L Se minimized abscisic acid (ABA) content, while 60 mg/L Se enhanced ABA content. The gibberellin (GA3) content in seedlings increased significantly compared with the control under the treatment of 60 mg/L Se. Conclusion Se concentration of 30 mg/L facilitated matrine and oxymatrine production and accumulation in stem/leaf tissues.

A preliminary study to explain how Streptomyces pactum (Act12) works on phytoextraction: soil heavy metal extraction, seed germination, and plant growth.

Streptomyces pactum (Act12) can both promote plant growth and strengthen heavy metal mobilization. Nevertheless, the mechanisms of how Act12 works during the phytoextraction process are still unknown. The present work investigated whether the metabolites produced by Act12 could influence the seed germination and the growth of potherb mustard and explored its mobilizing effect on soil cadmium (Cd) and zinc (Zn). The results showed that the germination potential and rate of potherb mustard seed treated with Act12 fermentation broth were 1.0- and 0.32-folds higher than those of control, probably due to the interruption of seed dormant stage. We also found that Act12 inoculation not only promoted the dry biomass (6.82%) of potherb mustard, but also increased the leaf chlorophyll (11.8%) and soluble protein (0.35%) production. The boosted seed germination rate under Act12 treatment (up to 63.3%) indicated that Act12 enhanced the resistance of potherb mustard seeds to Cd and Zn and alleviated their physiological toxicity. The generated metabolites during the Act12 fermentation posed positive impact on the availability of soil Cd and Zn. These findings bring new insight into the Act12-assisted phytoextraction of Cd and Zn from contaminated soils.

Bioaccessibility of Maillard Reaction Products from Biscuits Formulated from Buckwheat Flours Fermented by Selected Lactic Acid Bacteria.

The in vitro bioaccessibility of the soluble protein and Maillard reaction products (MRPs) such as furosine (an early indicator of the MR), free FIC (fluorescent intermediate compounds), and FAST index (fluorescence of advanced MRPs and tryptophan), and the level of melanoidins defined by the browning index were analyzed in biscuits formulated from raw and roasted common buckwheat flours fermented by select lactic acid bacteria (LAB). The content of soluble proteins in fermented buckwheat flour and biscuits before and after digestion in vitro was significantly dependent on the LAB applied and the type of flour used and was the highest in the digested biscuits, indicating increased bioaccessibility. Generally, in all analyzed biscuits a lower furosine content was observed as compared to control samples, and its high bioaccessibility was noted after digestion. The free FIC in biscuits was strain-dependent, resulting in low bioaccessibility with the exception of biscuits obtained from both types of flours fermented by Streptococcus thermophilus MK-10. Compared to control biscuits obtained from raw buckwheat flour, the almost twice-increased FAST index was found for samples fermented by L. plantarum IB or Streptococcus thermophilus MK-10. After digestion, at least a fivefold higher value of the browning index was noted in control and tested biscuits, indicating the high bioaccessibility of melanoidins. This study indicates that fermentation of buckwheat flours by selected lactic acid bacteria seems to be a good way to obtain a product with high bioaccessibility of MRPs. However, further research on their functional properties is needed.

Exploring Potent Fungal Isolates from Sanitary Landfill Soil for In Vitro Degradation of Dibutyl Phthalate.

Di-n-butyl phthalate (DBP) is one of the most extensively used plasticizers for providing elasticity to plastics. Being potentially harmful to humans, investigating eco-benign options for its rapid degradation is imperative. Microbe-mediated DBP mineralization is well-recorded, but studies on the pollutant's fungal catabolism remain scarce. Thus, the present investigation was undertaken to exploit the fungal strains from toxic sanitary landfill soil for the degradation of DBP. The most efficient isolate, SDBP4, identified on a molecular basis as Aspergillus flavus, was able to mineralize 99.34% dibutyl phthalate (100 mg L-1) within 15 days of incubation. It was found that the high production of esterases by the fungal strain was responsible for the degradation. The strain also exhibited the highest biomass (1615.33 mg L-1) and total soluble protein (261.73 µg mL-1) production amongst other isolates. The DBP degradation pathway scheme was elucidated with the help of GC-MS-based characterizations that revealed the formation of intermediate metabolites such as benzyl-butyl phthalate (BBP), dimethyl-phthalate (DMP), di-iso-butyl-phthalate (DIBP) and phthalic acid (PA). This is the first report of DBP mineralization assisted with A. flavus, using it as a sole carbon source. SDBP4 will be further formulated to develop an eco-benign product for the bioremediation of DBP-contaminated toxic sanitary landfill soils.

Omics-guided bacterial engineering of Escherichia coli ER2566 for recombinant protein expression.

The goal of bacterial engineering is to rewire metabolic pathways to generate high-value molecules for various applications. However, the production of recombinant proteins is constrained by the complexity of the connections between cellular physiology and recombinant protein synthesis. Here, we used a rational and highly efficient approach to improve bacterial engineering. Based on the complete genome and annotation information of the Escherichia coli ER2566 strain, we compared the transcriptomic profiles of the strain under leaky expression and low temperature-induced stress. Combining the gene ontology (GO) enrichment terms and differentially expressed genes (DEGs) with higher expression, we selected and knocked out 36 genes to determine the potential impact of these genes on protein production. Deletion of bluF, cydA, mngR, and udp led to a significant decrease in soluble recombinant protein production. Moreover, at low-temperature induction, 4 DEGs (gntK, flgH, flgK, flgL) were associated with enhanced expression of the recombinant protein. Knocking out several motility-related DEGs (ER2666-ΔflgH-ΔflgL-ΔflgK) simultaneously improved the protein yield by 1.5-fold at 24°C induction, and the recombinant strain had the potential to be applied in the expression studies of different exogenous proteins, aiming to improve the yields of soluble form to varying degrees in comparison to the ER2566 strain. Totally, this study focused on the anabolic and stress-responsive hub genes of the adaptation of E. coli to recombinant protein overexpression on the transcriptome level and constructs a series of engineering strains increasing the soluble protein yield of recombinant proteins which lays a solid foundation for the engineering of bacterial strains for recombinant technological advances. KEY POINTS: • Comparative transcriptome analysis shows host responses with altered induction stress. • Deletion of bluF, cydA, mngR, and udp genes was identified to significantly decrease the soluble recombinant protein productions. • Synchronal knockout of flagellar genes in E. coli can enhance recombinant protein yield up to ~ 1.5-fold at 24°C induction. • Non-model bacterial strains can be re-engineered for recombinant protein expression.

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

Heavy Metals Exacerbate the Effect of Temperature on the Growth of Chlorella sp.: Implications on Algal Blooms and Management

With the accelerated urbanization and rapid development of the industrial and agricultural sectors, concern about the pollution of water environments is becoming more widespread. Algal blooms of varying sizes are becoming increasingly frequent in lakes and reservoirs; temperatures, nutrients, heavy metals, and dissolved oxygen are the factors that influence algal bloom occurrence. However, knowledge of the combined effect of heavy metals and temperature on algal growth remains limited. Thus, this study investigated how specific concentrations of heavy metals affect algal growth at different temperatures; to this end, two heavy metals were used (0.01 mg/L Pb2+ and 0.05 mg/L Cr6+) at three incubation temperatures (15, 25, and 30 °C) with the alga Chlorella sp. A higher incubation temperature contributed to a rise in soluble proteins, which promoted algal growth. The density of algal cells increased with temperature, and catalase (CAT) decreased with increasing temperature. Chlorella sp. growth and catalase activity were optimal at 30 °C (algal cell density: 1.46 × 107 cell/L; CAT activity: 29.98 gprot/L). Pb2+ and Cr6+ significantly promoted Chlorella sp. growth during incubation at 25 and 30 °C, respectively. At specific temperatures, 0.01 mg/L Pb2+ and 0.05 mg/L Cr6+ promoted the production of soluble proteins and, hence, the growth of Chlorella sp. The results provide a useful background for the mitigation and prevention of algal blooms.

Boosting VFAs production during the anaerobic acidification of lignocellulose waste pulp and paper mill excess sludge: Ultrasonic pretreatment and inoculating rumen microorganisms

Ultrasonic pretreatment combined rumen microorganisms (RM) anaerobic hydrolysis was used as a novel method to boost volatile fatty acids (VFAs) production from lignocellulose waste pulp and paper mill excess sludge (PPES). To investigate the effect of ultrasonic on PPES degradation, various ultrasonic power levels and treatment time were used to treat PPES. The findings showed that ultrasonic pretreatment breaks the structure of PPES distinctly and significantly contributes to promoting PPES degradation. Under ultrasonic power level of 540 W and treatment time of 50 min conditions, the production of soluble chemical oxygen demand (SCOD), ammonia nitrogen (NH4+-N), reducing sugar, and soluble protein was 36.9, 16.9, 29.2, and 26.8 times of control-1, respectively. In the following RM anaerobic acidification experiment, VFAs production of PPES after ultrasonic treatment increased by 92 % compared to that of PPES without ultrasonic treatment and were much higher than that of the control-2 group without inoculating RM. During the fermentation stage, the degradation yield of cellulose, hemicellulose and lignin were 40.35 %, 58.35 % and 16.49 %, respectively. These results demonstrated that ultrasonic-RM treatment could improve the efficiency of the anaerobic acidification process and VFAs production and RM had a strong effect on degradation of hemicellulose and cellulose. In addition, Illumina MiSeq sequencing showed that the number of microbial species decreased, and the dominant flora changed.