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Background The GRAS and oleaginous yeast Yarrowia lipolytica ( Y. lipolytica ) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. Results In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 ( CCD1 ) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of β-ionone of 3.5 mg/L. To further improve the β-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of β-ionone in Y. lipolytica . The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a β-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. Conclusions An efficient β-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest β-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica .

Migraine is a very common, multifactorial, and recurrent central nervous system disorder that causes throbbing headache, photophobia, phonophobia, nausea, and disability. Migraine occurs more often in females, and its complex physiopathology is not yet fully understood. An increasing number of gastrointestinal disorders have been linked to the occurrence of migraine suggesting that gut microbiota might play a pivotal role in migraine through the gut-brain axis. In the present work, we performed a metagenome-wide association study (MWAS) to determine the relationship between gut microbiota and migraine by analyzing 108 shotgun-sequenced fecal samples obtained from elderly women who suffer from migraine and matched healthy controls. Notably, the alpha diversity was significantly decreased in the migraine group at species, genus, and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologous levels. Firmicutes, especially the \"unfriendly\" spp., were significantly enriched in the migraine group. Conversely, the healthy controls held more beneficial microorganisms, such as , and . For functional modules, the migraine group was enriched in gut-brain modules (GBMs) including kynurenine degradation and γ-aminobutyric acid (GABA) synthesis. However, the healthy controls held higher gut metabolic modules (GMMs) including glycolysis, homoacetogenesis, and GBMs including quinolinic acid degradation and -adenosyl methionine (SAM) synthesis. The differences in gut microbiota composition and function between the migraine and healthy groups provided new information as well as novel therapeutic targets and strategies for migraine treatment, which could help to improve the early diagnosis of the disease, as well as the long-term prognosis and the life quality of patients suffering from migraine.

The sophisticated antibiotic resistance mechanism of Pseudomonas aeruginosa has urged the development of alternative antibacterial strategies. Phage therapy has been proven successful for the treatment of multidrug-resistant infections. In this study, we reported two virulent P. aeruginosa phages, vB_PaeM_SCUT-S1 (S1) and vB_PaeM_SCUT-S2 (S2), which were characterized at morphological, genomic, and proteomic levels. Phages S1 and S2 were assigned to the Myoviridae family. The genome sequencing showed that the genome size of Phage S1 was 66,046 bp and that of Phage S2 was 94,434 bp. The phylogenetic tree indicated that the two phages were distantly related to each other and were classified in the genera Pbunavirus and Pakpunavirus respectively. Thirty-one proteins were identified for each phage by mass spectrometry and were used to substantiate the function of the predicted coding genes. The two phages inhibited the growth of P. aeruginosa strain PAO1 at low multiplicity of infection levels and had good performance both on preventing biofilm formation and eradicating preformed biofilms. They were also stable over a wide range of temperature and pH values, supporting their potential use in the treatment of P. aeruginosa infections.

Background Compound–protein interaction site and binding affinity predictions are crucial for drug discovery and drug design. In recent years, many deep learning-based methods have been proposed for predications related to compound–protein interaction. For protein inputs, how to make use of protein primary sequence and tertiary structure information has impact on prediction results. Results In this study, we propose a deep learning model based on a multi-objective neural network, which involves a multi-objective neural network for compound–protein interaction site and binding affinity prediction. We used several kinds of self-supervised protein embeddings to enrich our protein inputs and used convolutional neural networks to extract features from them. Our results demonstrate that our model had improvements in terms of interaction site prediction and affinity prediction compared to previous models. In a case study, our model could better predict binding sites, which also showed its effectiveness. Conclusion These results suggest that our model could be a helpful tool for compound–protein related predictions.

Background Microbial organisms hold significant potential for converting renewable substrates into valuable chemicals. Low pH fermentation in industrial settings offers key advantages, including reduced neutralizer usage and decreased wastewater generation, particularly in the production of amino acids and organic acids. Engineering acid-tolerant strains represents a viable strategy to enhance productivity in acidic environments. Synthetic biology provides dynamic regulatory tools, such as gene circuits, facilitating precise expression of acid resistance (AR) modules in a just-in-time and just-enough manner. Results In this study, we aimed to enhance the robustness and productivity of Escherichia coli , a workhorse for amino acid and organic acid production, in industrial fermentation under mild acidic conditions. We employed an Esa-type quorum sensing circuit to dynamically regulate the expression of an AR module (DsrA-Hfq) in a just-in-time and just-enough manner. Through careful engineering of the critical promoter P esaS and stepwise evaluation, we developed an optimal Esa-P BD (L) circuit that conferred upon an industrial E. coli strain SCEcL3 comparable lysine productivity and enhanced yield at pH 5.5 compared to the parent strain at pH 6.8. Conclusions This study exemplifies the practical application of gene circuits in industrial environments, which present challenges far beyond those of well-controlled laboratory conditions.

The cleavable self-aggregating tag 2.0 (cSAT2.0) incorporates the propeptide (PEP) of subtilisin from Deinococcus gobiensis, enabling protein purification with over 98% purity in a single, column-free step.Purified protein yields ranged from 24 mg/l to 89 mg/l for one model peptide and five model proteins in shake-flask experiments.A yield of 1.4 g/l of purified caplacizumab with over 99% purity was achieved in a 5-l fermenter.The cSAT2.0 method efficiently produces the model peptide and proteins in a high-throughput 96-well plate format. Protein purification remains a formidable and costly technical obstacle in biotechnology. Here, we present a new column-free method, utilizing the cleavable self-aggregating tag 2.0 (cSAT2.0) scheme, to streamline protein production in Escherichia coli, yielding high quantities with exceptional purity. In shake-flask experiments using lysogeny broth (LB) medium, the cSAT2.0 scheme successfully produced one peptide and five proteins, with yields ranging from 24 mg/l to 89 mg/l, and purity levels exceeding 98%. The cSAT2.0 scheme also enabled high-throughput protein preparation on microplates. Furthermore, we scaled up the fermentation process for caplacizumab, achieving 1.4 g/l of highly purified protein in a 5-l fermenter. Our results demonstrate that the cSAT2.0 scheme can serve as an economical and robust platform for protein production from microplate to fermenter scales. [Display omitted] We present the cleavable self-aggregating tag 2.0 (cSAT2.0) scheme, a single-step, column-free scheme for protein purification in Escherichia coli, achieving over 98% purity across a range of peptides and proteins. This versatile approach is scalable from microplates to fermenters, offering significant potential for applications in biological and pharmaceutical research and industry. The cleavable self-aggregating tag 2.0 (cSAT2.0) system has been validated across a diverse set of targets, including one peptide and five proteins (one therapeutic protein, two nanobodies, and two enzymes), primarily at the laboratory scale. When applied to caplacizumab production in a 5-l fermenter, it yielded an impressive 1.4 g/l with a purity >99%. More recently, we obtained preliminary data demonstrating the production of human growth hormone (hGH) at 2.5 g/l and >98% purity in a 5-l fermenter. These results underscore the effectiveness and robustness of this method for potential industrial applications. Based on this progress, we propose that the cSAT2.0 system has reached NASA’s Technology Readiness Level (TRL) 6, indicating its readiness for real-world implementation.

Doc number: 51 Abstract Background: Furfural and 5-hydroxymethylfurfural (HMF) are the degradation products of lignocellulose during pretreatment operations and significantly inhibit the consequent enzymatic hydrolysis and fermentation processes. The biodetoxification fungus Amorphotheca resinae ZN1 had demonstrated its excellent capacity on degrading lignocellulose derived inhibitors and helped the fermentation processes to achieve high yield of ethanol and biochemicals. Analysis of the biological degradation performance of furfural and HMF by A. resinae ZN1 will provide essential information for their fast and complete removal from the pretreated lignocellulose materials and facilitate the consequent ethanol fermentation. Results: The degradation performance of furfural and HMF by A. resinae ZN1 was investigated by capturing intermediate metabolic products at various culture conditions. A. resinae ZN1 converts furfural/HMF into furfuryl/HMF alcohols and furoic/HMF acids simultaneously at aerobic condition, and only the corresponding furfuryl/HMF alcohols are obtained at anaerobic condition. The existence of glucose accelerates the degradation rate of furfural and HMF by A. resinae ZN1 and the cell mass growth rate aerobically. Remarkably, glucose is not consumed before furfural or HMF is degraded to a low threshold concentration. The finding suggests that furfural or HMF has a substrate priority of utilization by A. resinae ZN1 than glucose. This property may help the detoxification of furfural and HMF to be operated without consuming glucose. Conclusions: The biological degradation performance of furfural and HMF by A. resinae ZN1 was investigated experimentally. Oxygen supply is important on the complete biodegradation of furfural and HMF by A. resinae ZN1. Furfural or HMF has the priority of substrate utilization than glucose by A. resinae ZN1. This study provided important information for detoxification enhancement and strain modification.

Background Protein purification remains a critical need for biosciences and biotechnology. It frequently requires multiple rounds of chromatographic steps that are expensive and time-consuming. Our lab previously reported a cleavable self-aggregating tag (cSAT) scheme for streamlined protein expression and purification. The tag consists of a self-assembling peptide (SAP) and a controllable self-cleaving intein. The SAP drives the target protein into an active aggregate, then by intein-mediated cleavage, the target protein is released. Here we report a novel cSAT scheme in which the self-assembling peptide is replaced with a salt inducible self-assembling peptide. This allows a target protein to be expressed first in the soluble form, and the addition of salt then drives the target protein into the aggregated form, followed by cleavage and release. Results In this study, we used MpA (MKQLEDKIEELLSKAAMKQLEDKIEELLSK) as a second class of self-assembling peptide in the cSAT scheme. This scheme utilizes low salt concentration to keep the fusion protein soluble, while eliminating insoluble cellular matters by centrifugation. Salt then triggers MpA-mediated self-aggregation of the fusion, removing soluble background host cell proteins. Finally, intein-mediated cleavage releases the target protein into solution. As a proof-of-concept, we successfully purified four proteins and peptides (human growth hormone, 22.1 kDa; LCB3, 7.7 kDa; SpyCatcherΔN-ELP-SpyCatcherΔN, 26.2 kDa; and xylanase, 45.3 kDa) with yields ranging from 12 to 87 mg/L. This was comparable to the classical His-tag method both in yield and purity (72–97%), but without the His-tag. By using a further two-step column purification process that included ion-exchange chromatography and size-exclusion chromatography, the purity was increased to over 99%. Conclusion Our results demonstrate that a salt-inducible self-assembling peptide can serve as a controllable aggregating tag, which might be advantageous in applications where soluble expression of the target protein is preferred. This work also demonstrates the potential and advantages of utilizing salt inducible self-assembling peptides for protein separation.

Background During fermentation, industrial microorganisms encounter multiple stresses that inhibit cell growth and decrease fermentation yields, in particular acid stress, which is due to the accumulation of acidic metabolites in the fermentation medium. Although the addition of a base to the medium can counteract the effect of acid accumulation, the engineering of acid-tolerant strains is considered a more intelligent and cost-effective solution. While synthetic biology theoretically provides a novel approach for devising such tolerance modules, in practice it is difficult to assemble stress-tolerance modules from hundreds of stress-related genes. Results In this study, we designed a set of synthetic acid-tolerance modules for fine-tuning the expression of multi-component gene blocks comprising a member of the proton-consuming acid resistance system ( gadE ), a periplasmic chaperone ( hdeB ), and reactive oxygen species (ROS) scavengers ( sodB and katE ). Directed evolution was used to construct an acid-responsive asr promoter library, from which four variants were selected and used in the synthetic modules. The module variants were screened in a stepwise manner under mild acidic conditions (pH 5–6), first by cell growth using the laboratory Escherichia coli strain MG1655 cultured in microplates, and then by lysine production performance using the industrial lysine-producing E. coli strain MG1655 SCEcL3 cultured first in multiple 10-mL micro-bioreactors, and then in 1.3-L parallel bioreactors. The procedure resulted in the identification of a best strain with lysine titer and yield at pH 6.0 comparable to the parent strain at pH 6.8. Conclusion Our results demonstrate a promising synthetic-biology strategy to enhance the growth robustness and productivity of E. coli upon the mildly acidic conditions, in both a general lab strain MG1655 and an industrial lysine-producing strain SCEcL3, by using the stress-responsive synthetic acid-tolerance modules comprising a limited number of genes. This study provides a reliable and efficient method for achieving synthetic modules of interest, particularly in improving the robustness and productivity of industrial strains.

Background In recent years, it has been gradually realized that bacterial inclusion bodies (IBs) could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D -amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further. Results In this paper, we report that an ionic self-assembling peptide ELK16 (LELELKLK) 2 was able to effectively induce the formation of cytoplasmic inclusion bodies in Escherichia coli ( E. coli ) when attached to the carboxyl termini of four model proteins including lipase A, amadoriase II, β-xylosidase, and green fluorescent protein. These aggregates had a general appearance similar to the usually reported cytoplasmic inclusion bodies (IBs) under transmission electron microscopy or fluorescence confocal microscopy. Except for lipase A-ELK16 fusion, the three other fusion protein aggregates retained comparable specific activities with the native counterparts. Conformational analyses by Fourier transform infrared spectroscopy revealed the existence of newly formed antiparallel beta-sheet structures in these ELK16 peptide-induced inclusion bodies, which is consistent with the reported assembly of the ELK16 peptide. Conclusions This has been the first report where a terminally attached self-assembling β peptide ELK16 can promote the formation of active inclusion bodies or active protein aggregates in E. coli . It has the potential to render E. coli and other recombinant hosts more efficient as microbial cell factories for protein production. Our observation might also provide hints for protein aggregation-related diseases.