Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (PsADH2). The library of 30 selected promoters showing 0.4- to 5.5-fold of the PsADH2 activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type PsADH2. Their hypoxia-inducible properties were confirmed with two additional reporters: β-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.

Yeastolate is often used as a media supplement in industrial mammalian cell culture or as a major media component for microbial fermentations. Yeastolate variability can significantly affect process performance, but analysis is technically challenging because of compositional complexity. However, what may be adequate for manufacturing purposes is a fast, inexpensive screening method to identify molecular variance and provide sufficient information for quality control purposes, without characterizing all the molecular components. Here we used Size Exclusion Chromatography (SEC) and chemometrics as a relatively fast screening method for identifying lot-to-lot variance (with Principal Component Analysis, PCA) and investigated if Partial Least Squares, PLS, predictive models which correlated SEC data with process titer could be obtained. SEC provided a relatively fast measure of gross molecular size hydrolysate variability with minimal sample preparation and relatively simple data analysis. The sample set comprised of 18 samples from 12 unique source lots of an ultra-filtered yeastolate (10kDa molecular weight cut-off) used in a mammalian cell culture process. SEC showed significant lot-to-lot variation, at 214 and 280nm detection, with the most significant variation, that correlated with process performance, occurring at a retention time of ~6minutes. PCA and PLS regression correlation models provided fast identification of yeastolate variance and its process impact. The primary drawback is the limited column lifetime (<300 injections) caused by the complex nature of yeastolate and the presence of zinc. This limited long term reproducibility because these age-related, non-linear changes in chromatogram peak positions and shapes were very significant.

In microbial biotechnology, there is a constant demand for functional peptides to give new functionality to engineered proteins to address problems such as direct delivery of functional proteins into bacterial cells, enhanced protein solubility during the expression of recombinant proteins, and efficient protein secretion from bacteria. To tackle these critical issues, we selected three types of functional small peptides: cell-penetrating peptides (CPPs) enable the delivery of diverse cargoes into bacterial cytoplasm for a variety of purposes, protein-solubilizing peptide tags demonstrate remarkable efficiency in solubilizing recombinant proteins without folding interference, and signal peptides play a key role in enabling the secretion of recombinant proteins from bacterial cells. In this review, we introduced these three functional small peptides that offer effective solutions to address emerging problems in microbial biotechnology. Additionally, we summarized various engineering efforts aimed at enhancing the activity and performance of these peptides, thereby providing valuable insights into their potential for further applications.

Reduced levels of the Sortilin-related receptor with A-type repeats (SORLA) in different brain regions as well as in the cerebrospinal fluid have been associated with Alzheimer's disease. Methods and reagents to develop reliable detection assays to quantify SORLA and its specific isoforms are therefore much needed. Nanobodies (Nbs) are unique biomolecules derived from the blood of camelids that display advantageous physicochemical and antigen affinity properties, making them attractive tools with great relevance to both diagnostic and therapeutic applications. Here, we purified and characterized eight Nbs that were isolated from the blood of an alpaca immunized with the recombinant extracellular domain of SORLA. The selected Nbs showed high affinity to SORLA in the low nanomolar range as observed by surface plasmon resonance. For mapping of the Nbs' epitopes within the antigen, we transiently transfected HEK293 cells with a panel of SORLA deletion constructs, and developed a protocol of immunostaining by applying fluorescent dye conjugated Nbs. With this method, we showed that the selected Nbs specifically recognize a part of SORLA containing Fibronectin-type III domains, representing promising tools not only for disease clarifying research, but also for translational medicine as candidates for clinical diagnostic purposes.

β-Amyrin is a pentacyclic triterpenoid and has anti-viral, anti-bacterial and anti-inflammatory activities. The synthetic pathway of β-amyrin has been analyzed and its heterogeneous synthesis has been achieved in Saccharomyces cerevisiae. Squalene epoxidase (SQE) catalyzes the oxygenation of squalene to form 2,3-oxidosqualene and is rate-limiting in the synthetic pathways of β-amyrin. The endogenous SQE in S. cerevisiae is insufficient for high production of β-amyrin. Herein, eight squalene epoxidases derived from different plants were selected and characterized in S. cerevisiae for improved biosynthesis of β-amyrin. Among them, the squalene epoxidase from Oryza sativa (OsSQE52) showed the best performance compared to other plant-derived sources. Through protein remodeling, the mutant OsSQE52L256R, obtained based on modeling analysis, increased the titer of β-amyrin by 2.43-fold compared to that in the control strain with ERG1 overexpressed under the same conditions. Moreover, the expression of OsSQE52L256R was optimized with the improvement of precursor supply to further increase the production of β-amyrin. Finally, the constructed strains produced 66.97 mg/L β-amyrin in the shake flask, which was 6.45-fold higher than the original strain. Our study provides alternative SQEs for efficient production of β-amyrin as well as other triterpenoids derived from 2,3-oxidosqualene.

Effective strategies to optimize algal growth and lipid productivity are critical for the sustainable production of biomass for various applications. Light management has emerged as a promising approach, but the intricate relationship between light intensity, spectral quality, and algal responses remains poorly understood. This study investigated the effects of different light qualities (blue, red-orange, and white-yellow) and intensities (45-305 μmol/m2·s) on Chlamydomonas reinhardtii. Red-orange light exhibited the highest promotion of biomass growth and lipid productivity, with specific growth rates of 1.968 (d-1) and biomass productivity of 0.284 (g/L/d) at 155 μmol/m2·s and 205 μmol/m2·s, respectively. Within the intensity range of 205 μmol/m2·s to 305 μmol/m2·s, lipid mass fractions ranged from 10.5% w/w to 11.0% w/w, accompanied by lipid concentrations ranging from 68.6 mg/L to 74.9 mg/L. Red-orange light positively influenced carbohydrate accumulation, while blue light promoted protein synthesis. These findings highlight the importance of optimizing light quality and intensity to enhance algal biomass productivity and manipulate biochemical composition. Understanding the complex relationship between light parameters and algal physiology will contribute to sustainable algal cultivation practices and the use of microalgae as a valuable bioresource.

Increasing the screening efficiency and maintaining the N-terminal cleavage pattern are key factors in the development of an in vitro synthetic signal peptide screening system for high therapeutic protein production in Chinese hamster ovary (CHO) cells. This study improved the in vitro screening system of synthetic signal peptides in CHO cells for therapeutic protein production by modifying the expression vector. Incorporating a leaky stop codon with IgG transmembrane and cytoplasmic domains into the expression vector improved the proportion of high producers in establishing stable CHO cell pools. The selected signal peptides from stable CHO cell pools that were generated using degenerate codon-based oligonucleotides with a conserved polar carboxy-terminal domain in the native signal peptide showed similar N-terminal cleavage patterns to the native one. In addition, replacing native signal peptide with selected synthetic signal peptides did not influence the sialylated N-linked glycan formation and biological activity of therapeutic Fc-fusion glycoprotein in CHO cells. Thus, an in vitro synthetic signal peptide screening system can be used for therapeutic Fc-fusion glycoprotein production in CHO cells with an enhanced specific protein productivity while maintaining the N-terminal cleavage pattern similar to the native one.

Carotenoids are a vast group of natural pigments that come in a variety of colors ranging from red to orange. Apocarotenoids are derived from these carotenoids, which are hormones, pigments, retinoids, and volatiles employed in the textiles, cosmetics, pharmaceutical, and food industries. Due to the high commercial value and poor natural host abundance, they are significantly undersupplied. Microbes like Saccharomyces cerevisiae and Escherichia coli act as heterologous hosts for apocarotenoid production. This article briefly reviews categories of apocarotenoids, their biosynthetic pathway commencing from the MVA and MEP, its significance, the tool enzymes for apocarotenoid biosynthesis like CCDs, their biotechnological production in microbial factories, and future perspectives.

As the world grapples with an imminent energy crisis brought on by the depletion of nonrenewable resources, such as petroleum, the necessity for alternative and eco-friendly power sources becomes increasingly apparent. In this regard harnessing knowledge gained from natural microorganisms to produce electricity using economical substrates is a promising solution through microbial fuel cells (MFCs). Microbial fuel cells leverage microbes' catabolic abilities to break down organic matter and release electrons that are subsequently transported across an external circuit for electricity generation. This article delves into the fundamental components involved in MFC construction and explores crucial factors that impact their performance including substrate oxidation, electron transfer, and internal resistance. Additionally, it offers a comprehensive analysis of existing microbial fuel cell designs while highlighting their respective strengths and weaknesses. Finally, the article showcases cost-effective MFC models based on thorough studies conducted worldwide while illuminating potential practical applications of this renewable energy technology.

Improved understanding of expression of recombinant immunoglobulin (IgG)-based therapies can decrease manufacturing process costs and bring down costs to patients. Deletion of C-terminal Lysine (C-Lys) from IgG molecules has been shown to greatly impact yield. This study set out to characterise structural components of IgG C-terminal variants which modulate protein expression by examination of the consequences of mutations at the C-terminal of IgG on expression and by the use of fluorescent C-terminal fragment fusion proteins. Cell-based and cell-free experiments were also implemented to characterise how the C-terminal differentially engages with cellular pathways to modulate expression. IgG variants engineered by removal of the C-terminal Lys were expressed at significantly lower rates than control variants by CHO (and HEK) cells. Engineered constructs of mCherry fused with short regions of the C-terminal regions of IgG mimicked the ordering of expressability observed for IgG variants. These fluorescent C-terminal fragment fusions offered the potential to profile how sequences (and point mutations) modified expression. Via combinations of cell and cell-free systems, screening across a range of variants of IgG and mCherry reporter constructs has shown that interactions between specific C-terminal amino acid sequences and the ribosome can regulate the rate and extent of expression. This study highlights the importance of amino acid sequence regulatory events determining the efficiency of production of desirable recombinant proteins, showing that wildtype C-terminal lysine is a necessary capping molecule for IgG1 expression. From a wider perspective, these data are especially significant towards the design of novel entities. The approach has also provided information about novel short C-terminal tags which may be used to provide selective synthesis of specific subunits in the production of multisubunit products. Alternative strategies for removing C-terminal amino acid heterogeneity whilst maintaining efficient rates of expression have been provided.

Heat-shock proteins (Hsps) are a family of proteins essential in preserving the vitality and functionality of proteins under stress conditions. Cucumber (Cucumis sativus) is a widely grown plant with high nutritional value and is used as a model organism in many studies. This study employed a genomics, transcriptomics, and metabolomics approach to investigate cucumbers' Hsps against abiotic stress conditions. Bioinformatics methods were used to identify six Hsp families in the cucumber genome and to characterize family members. Transcriptomics data from the Sequence Read Archive (SRA) database was also conducted to select CsHsp genes for further study. Real-time PCR was used to evaluate gene expression levels under different stress conditions, revealing that CssHsp-08 was a vital gene for resistance to stress conditions; including drought, salinity, cold, heat stresses, and ABA application. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of plant extracts revealed that amino acids accumulate in leaves under high temperatures and roots under drought, while sucrose accumulates in both tissues under applied most stress factors. The study provides valuable insights into the structure, organization, evolution, and expression profiles of the Hsp family and contributes to a better understanding of plant stress mechanisms. These findings have important implications for developing crops that can withstand environmental stress conditions better.

The fermentation process of milk to yoghurt using Lactobacillus delbrueckii subsp. bulgaricus in co-culture with Streptococcus thermophilus is hallmarked by the breakdown of lactose to organic acids such as lactate. This leads to a substantial decrease in pH - both in the medium, as well as cytosolic. The latter impairs metabolic activities due to the pH-dependence of enzymes, which compromises microbial growth. To quantitatively elucidate the impact of the acidification on metabolism of L. bulgaricus in an integrated way, we have developed a proton-dependent computational model of lactose metabolism and casein degradation based on experimental data. The model accounts for the influence of pH on enzyme activities as well as cellular growth and proliferation of the bacterial population. We used a machine learning approach to quantify the cell volume throughout fermentation. Simulation results show a decrease in metabolic flux with acidification of the cytosol. Additionally, the validated model predicts a similar metabolic behaviour within a wide range of non-limiting substrate concentrations. This computational model provides a deeper understanding of the intricate relationships between metabolic activity and acidification and paves the way for further optimization of yoghurt production under industrial settings.

DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2 µM for HPLC-UV and 0.02 µM for HPLC-MS of methylated cytosine.

Production of crops is increasing day by day in agriculture sectors. The insecurity of food is a main reason of plant disease and is a main global issue that humans face these days. With the design of contemporary environmental agriculture, more focus is devised for yielding the crop and elevating its quality. The occurrence of crops has elevated in years and the kind of disease has become more and more complex. The disease in plants and the pernicious insects are the major risks in agriculture field. Thus, earlier discovery and treatment of this disease are imperative. The major design of Deep Learning (DL) model helped in detecting the plant disease and grants a dynamic tool with accurate results. This paper presents DL-assisted technique for detecting and classifying the tomato disease and used deep batch-normalized eLu Alex Net (DbneAlexnet) for classifying the tomato plant leaves. Initially, tomato plant leaf images are taken as an input from specific dataset represented and it is subjected to preprocessing phase to eliminate unwanted distortions using anisotropic filtering. Then, the segmentation is carried out using U-net, which is trained by Gradient-Golden search optimization (Gradient-GSO) Algorithm and it is incorporation of both Golden search optimization (GSO) and Gradient concept. Thereafter the segmented image is given to image augmentation process, where position augmentation and color augmentation are considered. Finally, the multiclass plant leaf disease is classified using DbneAlexnet and is trained using proposed Gradient Jaya- Golden search optimization (GJ-GSO). Here, the GJ-GSO is devised with the integration of Gradient concept, Jaya algorithm, and GSO algorithm. The proposed GJ-GSO-based DbneAlexnet outperformed highest accuracy of 92.4%, True positive rate (TPR) of 91.9%, True negative rate (TNR) of 92.2% and smallest False Positive Rate (FPR) of 0.078. Hence, the technique with unified segmentation and classification is effectual for identifying the plant disease and the empirical research verifies the benefits of the developed model.

Although the presence of silica in many living organisms offers advanced properties including cell protection, the different in vitro attempts to build living materials in pure silica never favoured the cells viability. Thus, little attention has been paid to host-guest interactions to modify the expected biologic response. Here we report the physiological changes undergone by Escherichia coli K-12 in silica from colloidal solution to gel confinement. We show that the physiological alterations in growing cultures are not triggered by the initial oxidative Reactive Oxygen Species (ROS) response. Silica promotes the induction of alternative metabolic pathways along with an increase of growth suggesting the existence of rpoS polymorphisms. Since the functionality of hybrid materials depends on the specific biologic responses of their guests, such cell physiological adaptation opens perspectives in the design of bioactive devices attracting for a large field of sciences.

In this study, an anaerobic pneumatic mechanical digester (PMD) was designed for the first time to investigate the impact of pneumatic agitator on increasing the bioethanol production and compared with a mechanical digester (MD). Fermentation was performed during an optimized pretreatment and hydrolysis process by RSM (Response Surface Method). Ultrasound optimized points (the time values, the acid concentration, and the biomass load) were 30 min, 1.95% v/v, and 6%, and hydrolysis was done within 45 min at the acid concentration of 2.04% v/v and temperature of 148.4 °C. The hydrolysis solutions were poured and the fermentation process took place within 20 days in the PMD and MD. The sampling sequence was every 5 days. According to the results, the PMD could produce bioethanol more than the MD by 27.94%. Besides, CO, H2S and O2 were measured through fermentation. In PMD, the amount of H2S and O2 was lower than the MD, but then the production of CO in the PMD was meaningfully higher. Finally, by the application of the PMD, the amount of harmful mixtures produced throughout the process can be controlled. It can be said that with the new method designed in this study, it is possible to take an important step in the biorefinery and use the biomass produced in nature in an economical and environmentally friendly way.

Avian Influenza, the most studied virus, is of high concern due to its zoonotic pandemic potential. In recent years, several influenza vaccines have been used with the broad goal of managing and in certain cases, eliminating the disease. The matrix 2 extracellular domain (M2e), is one of the key targets of the universal influenza vaccine, a liner peptide that is conserved throughout all influenza A subtypes virus. Many recombinant influenza proteins have been expressed in yeast and plants for vaccine development. A remarkable development has been made in the field of biotechnology to explore the potential of microalga as an expression host. In this study, we designed a fusion gene code for M2e peptide and CTB protein as M2e's natural form has a low level of immunogenicity. The fusion gene was cloned in the Chloroplast transformation vector pSRSapI and expressed in the TN72 mutant strain of Chlamydomonas reinhardii. The expression of the targeted protein was confirmed by ECL western blot analysis. A GM1-ELISA was carried out to detect the affinity of fusion protein for GM1 monosialoganglioside and the significant P-value is lower than 0.05. Immunogenicity assay on chicken detected the anti-M2e bodies in chicken serum. This study gives evidence of therapeutic protein production through algae chloroplast and a stable, selection free and low cost oral delivery for universal vaccine against influenza A virus.

As a renewable carbon source produced from organic wastes by acidogenic fermentation, volatile fatty acids (VFAs) are important intermediates in chemical and biological fields and beneficial to resource recovery and carbon neutrality. Maximizing VFA production by some strategies without additional chemicals is critical to increasing economic and environmental benefits. In this study, the effects of initial organic load (OL) on the performance of VFA production, variations of intermediate metabolites, and the thermogravimetric properties of potato peel waste (PPW) during batch acidogenic fermentation were studied. The results showed that the concentration of VFAs increased with the increase of initial OL, while the VFA yield decreased with the increase of initial OL. When the initial OL was in the range of 28.4 g VS/L-91.3 g VS/L, the fermentation type of PPW was butyric acid fermentation. The highest butyric acid proportion of 61.3% was achieved with the initial OL of 71.5 g VS/L. With the increase of initial OL, the proportion of acetic acid and the utilization rate of protein in the PPW decreased. VFAs were produced from proteins and carbohydrates in the early stage and mainly produced from carbohydrates in the later stage. The production efficiency of VFA was relatively high with the initial OL of 71.5 g VS/L, because more easily-biodegradable compounds were solubilized. The results showed that suitably increased initial OL could accelerate acidogenesis, reduce hydrolysis time, and increase the proportion of butyric acid. The findings in this work suggest that PPW is a promising feedstock for butyric acid biosynthesis and appropriate initial OL is beneficial to VFA production.

In this study, the Fenton oxidized lignin was prepared to investigate the effect of Fenton oxidation modification on the activity of lignin immobilized β-glucosidase (β-GL). The results demonstrated that Fenton oxidation could significantly improve the activity and stability of immobilized β-GL. This is because the Fenton oxidation increased the electrostatic, hydrogen bonding, and hydrophobic forces between lignin and β-GL, resulting in increased lignin adsorption onto β-GL. The Fenton oxidation also changed the chemical structure of lignin, altering the lignin-β-GL binding site and reducing the negative effect of lignin on the β-GL catalytic domain. This research will improve understanding of the effect of Fenton lignin oxidation on immobilized β-GL activity and expand the use of lignin in enzyme immobilization.

The Asian citrus psyllid (ACP) Diaphorina citri Kuwayama (Hemiptera: Liviidae) is the vector of Candidatus Liberibacter spp., the bacteria associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. HLB management has heavily relied on insecticide applications to control the ACP, although there are efforts towards more sustainable alternatives. In previous work, our group assessed the potential bioactivity of different strains of Bacillus thuringiensis (Eubacteriales: Bacillaceae) (Bt) containing cry/cyt genes as feasible tools to control ACP nymphs. Here, we report an attempt to use the cry11A gene from Bt to produce transgenic sweet orange plants using two promoters. For the genetic transformation, 'Hamlin' and 'Valencia' sweet orange seedlings were used as sources of explants. Transgenic plants were detected by polymerase chain reaction (PCR) with specific primers, and the transgene copy number was confirmed by Southern blot analyses. Transcript expression levels were determined by qPCR. Mortality assays of D. citri nymphs were carried out in a greenhouse, and the effect of the events tested ranged from 22% to 43% at the end of the five-day exposure period. To our knowledge, this is the first manuscript reporting the production of citrus plants expressing the Bt cry11A gene for the management of D. citri nymphs.