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The growing preference for incorporating microbial aspartic proteases in industries is due to their high catalytic function and high degree of substrate selectivity. These properties, however, are attributable to molecular alterations in their structure and a variety of other characteristics. Molecular tools, functional genomics, and genome editing technologies coupled with other biotechnological approaches have aided in improving the potential of industrially important microbial proteases by addressing some of their major limitations, such as: low catalytic efficiency, low conversion rates, low thermostability, and less enzyme yield. However, the native folding within their full domain is dependent on a surrounding structure which challenges their functionality in substrate conversion, mainly due to their mutual interactions in the context of complex systems. Hence, manipulating their structure and controlling their expression systems could potentially produce enzymes with high selectivity and catalytic functions. The proteins produced by microbial aspartic proteases are industrially capable and far-reaching in regulating certain harmful distinctive industrial processes and the benefits of being eco-friendly. This review provides: an update on current trends and gaps in microbial protease biotechnology, exploring the relevant recombinant strategies and molecular technologies widely used in expression platforms for engineering microbial aspartic proteases, as well as their potential industrial and biotechnological applications. [ABSTRACT FROM AUTHOR]

Candida tropicalis is distinguished from Saccharomyces cerevisiae by its great xylose utilization ability, maximizing its capability to ferment both hexoses and xylose to produce high-value products, and its ability to overcome the problem of end-product inhibition derived from lignocellulosic hydrolysis. However, little or no information about the molecular evolution mechanism of C. tropicalis to inhibitory compounds (acetic acid, phenol, and furfural) derived from lignocellulosic hydrolysis is known. In this study, the adaptation of a C. tropicalis strain SHC-03 to corn stover hydrolysate after mutagenesis through stepwise evolution resulted in multi-generation evolved strains after 60 rounds of evolution using the C. tropicalis strain SHC-03 as the parental strain. The evolved strain, generation B-60, (GB-60) showed the highest tolerance (15.81 g/L) and resistance as well as the lowest subcellular damage to inhibitory compounds derived from corn stover hydrolysis. Comparative transcriptome and genomic analyses between the evolved strain GB-60 and the control strain SHC-03 revealed enhanced functional and mutated genes including CTRG_03726, CTRG_01923, CTRG_04945, CTRG_05115, CTRG_03102, CTRG_05127, CTRG_00952 and CTRG_03034 involved in increased cell wall biosynthesis and remodeling, pentose phosphate pathway, metabolic and carbon pathways, detoxification of inhibitory compounds, and the multidrug/multixenobiotic resistance (MDR/MXR) transporters. These genes also played active roles in oxidative stress response, mitochondrial stress response, ER stress response, vacuole stress response and autophagy, DNA repair, and chromatin protection resulting in less ROS accumulation, as well as lower damage to some subcellular organelles in the evolved strain. The information from this study will provide guidelines for the development of engineering strains with high tolerance towards inhibitors derived from corn stover hydrolysis. [Display omitted] • Acquisition of improved evolved strain of C. tropicalis SHC-03, GB-60. • Increased resistance of evolved strain to inhibitors derived from corn stover hydrolysis. • Discovery of genes and pathways involved in increased resistance to and degradation of inhibitors. [ABSTRACT FROM AUTHOR]

The formulation in which biochemical enzymes are administered in polymer science plays a key role in retaining their catalytic activity. The one-step synthesis of polymers with highly sequence-controlled enzymes is a strategy employed to provide enzymes with higher catalytic activity and thermostability in material sustainability. Enzyme-catalyzed chain growth polymerization reactions using activated monomers, protein–polymer complexation techniques, covalent and non-covalent interaction, and electrostatic interactions can provide means to develop formulations that maintain the stability of the enzyme during complex material processes. Multifarious applications of catalytic enzymes are usually attributed to their efficiency, pH, and temperature, thus, progressing with a critical structure-controlled synthesis of polymer materials. Due to the obvious economics of manufacturing and environmental sustainability, the green synthesis of enzyme-catalyzed materials has attracted significant interest. Several enzymes from microorganisms and plants via enzyme-mediated material synthesis have provided a viable alternative for the appropriate synthesis of polymers, effectively utilizing the one-step approach. This review analyzes more and deeper strategies and material technologies widely used in multi-enzyme cascade platforms for engineering polymer materials, as well as their potential industrial applications, to provide an update on current trends and gaps in the one-step synthesis of materials using catalytic enzymes. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae able to tolerate lignocellulose-derived inhibitors like furfural. Yeast strain performance tolerance has been measured by the length of the lag phase for cell growth in response to the furfural inhibitor challenge. The aims of this work were to obtain RDS1 yeast tolerant strain against furfural through overexpression using a method of in vivo homologous recombination. Here, we report that the overexpressing RDS1 recovered more rapidly and displayed a lag phase at about 12 h than its parental strain. Overexpressing RDS 1 strain encodes a novel aldehyde reductase with catalytic function for reduction of furfural with NAD(P)H as the co-factor. It displayed the highest specific activity (24.8 U/mg) for furfural reduction using NADH as a cofactor. Fluorescence microscopy revealed improved accumulation of reactive oxygen species resistance to the damaging effects of inhibitor in contrast to the parental. Comparative transcriptomics revealed key genes potentially associated with stress responses to the furfural inhibitor, including specific and multiple functions involving defensive reduction–oxidation reaction process and cell wall response. A significant change in expression level of log2 (fold change >1) was displayed for RDS1 gene in the recombinant strain, which demonstrated that the introduction of RDS1 overexpression promoted the expression level. Such signature expressions differentiated tolerance phenotypes of RDS 1 from the innate stress response of its parental strain. Overexpression of the RDS1 gene involving diversified functional categories is accountable for stress tolerance in yeast S. cerevisiae to survive and adapt the furfural during the lag phase. [ABSTRACT FROM AUTHOR]

The pupae is one of the most valuable components of the silkworm, a by-product of silk reeling. Employing them as a raw material to create valued products will help to recycle and reuse silkworm pupae, as well as contribute to the food industry's long-term development. Silkworm pupae protein (SPP) and pupae oil (SPO) are high in nutritional and bioactive components that have a variety of applications in the food industry as well as health-promoting characteristics. In this review, we first elaborate on the modification, nutritional, and functional properties of silkworm pupae in terms of requirements for protein and lipids processing as well as current extraction technology. In the subsequent sections, we summarize the modification techniques including the science and technology involved in attaining fatty acid components of SPO. Finally, we focus on recent studies related to the utilization of SPP and SPO subject to their inclusion in foods for their health benefits. The key to silkworm pupae product design may be to fully exploit and utilize the inherent characteristics of the pupae to produce functional peptides and structural lipids with high nutritional content, which may necessitate further improvements in the bioactive and functional properties. Designing more ecologically friendly, low-cost, and efficient processes for producing protein and oil from silkworm pupae is also a trend in technology. This may further provide new insights into the mechanisms behind modifying silkworm pupae as food and its health advantages. [Display omitted] • Modifications and functions of silkworm pupae protein (SPP) and oil (SPO) were reviewed. • Modification of SPP and SPO reveal valuable nutritional properties. • Inclusion of SPP and SPO in food and feed applications were summarized. • Peptide composition in silkworm pupae aid in valuable biological activities. • Further research to compare SPP and SPO with standard proteins and oils is recommended. [ABSTRACT FROM AUTHOR]

Aldehydes are the main inhibitors generated during the pretreatment of lignocellulosic biomass, which can inhibit cell growth and disturb subsequent fermentation. Saccharomyces cerevisiae has the intrinsic ability to in situ detoxify aldehydes to their less toxic or nontoxic alcohols by numerous aldehyde dehydrogenases/reductases during the lag phase. Herein, we report that an uncharacterized open reading frame YMR152W from S. cerevisiae encodes a novel aldehyde reductase with catalytic functions for reduction of at least six aldehydes, including two furan aldehydes (furfural and 5-hydroxymethylfurfural), three aliphatic aldehydes (acetaldehyde, glycolaldehyde, and 3-methylbutanal), and an aromatic aldehyde (benzaldehyde) with NADH or NADPH as the co-factor. Particularly, Ymr152wp displayed the highest specific activity (190.86 U/mg), and the best catalytic rate constant (K cat), catalytic efficiency (K cat /K m), and affinity (K m) when acetaldehyde was used as the substrate with NADH as the co-factor. The optimum pH of Ymr152wp is acidic (pH 5.0–6.0), but this enzyme is more stable in alkaline conditions (pH 8.0). Metal ions, chemical protective additives, salts, and substrates could stimulate or inhibit enzyme activities of Ymr152wp in varying degrees. Ymr152wp was classified into the quinone oxidoreductase (QOR) subfamily of the medium-chain dehydrogenase/reductase (MDR) family based on the results of amino acid sequence analysis and phylogenetic analysis. Although Ymr152wp was grouped into the QOR family, no quinone reductase activity was observed using typical quinones (9,10-phenanthrenequinone, 1,2-naphthoquinone, and p -benzoquinone) as the substrates. This study provides guidelines for exploring more uncharacterized aldehyde reductases in S. cerevisiae for in situ detoxification of aldehyde inhibitors derived from lignocellulosic hydrolysis. • YMR152W from Saccharomyces cerevisiae encodes an aldehyde reductase. • Ymr152wp showed much better specific activity for reduction of acetaldehyde. • Overexpression of YMR152W conferred resistance to aldehyde inhibitors. • Ymr152wp is classified into the QOR subfamily of the MDR family. [ABSTRACT FROM AUTHOR]

At least 24 aldehyde reductases from Saccharomyces cerevisiae have been characterized and most function in in situ detoxification of lignocellulosic aldehyde inhibitors, but none is classified into the polyol dehydrogenase (PDH) subfamily of the medium-chain dehydrogenase/reductase (MDR) superfamily. This study confirmed that two (2R,3R)-2,3-butanediol dehydrogenases (BDHs) from industrial (denoted Y)/laboratory (denoted B) strains of S. cerevisiae, Bdh1p(Y)/Bdh1p(B) and Bdh2p(Y)/Bdh2p(B), were members of the PDH subfamily with an NAD(P)H binding domain and a catalytic zinc binding domain, and exhibited reductive activities towards lignocellulosic aldehyde inhibitors, such as acetaldehyde, glycolaldehyde, and furfural. Especially, the highest enzyme activity towards acetaldehyde by Bdh2p(Y) was 117.95 U/mg with cofactor nicotinamide adenine dinucleotide reduced (NADH). Based on the comparative kinetic property analysis, Bdh2p(Y)/Bdh2p(B) possessed higher specific activity, substrate affinity, and catalytic efficiency towards glycolaldehyde than Bdh1p(Y)/Bdh1p(B). This was speculated to be related to their 49% sequence differences and five nonsynonymous substitutions (Ser41Thr, Glu173Gln, Ile270Leu, Ile316Met, and Gly317Cys) occurred in their conserved NAD(P)H binding domains. Compared with BDHs from a laboratory strain, Bdh1p(Y) and Bdh2p(Y) from an industrial strain displayed five nonsynonymous mutations (Thr12, Asn61, Glu168, Val222, and Ala235) and three nonsynonymous mutations (Ala34, Ile96, and Ala369), respectively. From a first analysis with selected aldehydes, their reductase activities were different from BDHs of laboratory strain, and their catalytic efficiency was higher towards glycolaldehyde and lower towards acetaldehyde. Comparative investigation of kinetic properties of BDHs from S. cerevisiae as aldehyde reductases provides a guideline for their practical applications in in situ detoxification of aldehyde inhibitors during lignocellulose bioconversion. Key Points • Two yeast BDHs have enzyme activities for reduction of aldehydes. • Overexpression of BDHs slightly improves yeast tolerance to acetaldehyde and glycolaldehyde. • Bdh1p and Bdh2p differ in enzyme kinetic properties. • BDHs from strains with different genetic backgrounds differ in enzyme kinetic properties. [ABSTRACT FROM AUTHOR]

In industrial production, the excessive discharge of furfural can pose harm to soil microorganisms, aquatic animals and plants, as well as humans. Therefore, it is crucial to develop efficient and cost-effective methods for degrading furfural in the environment. Currently, the use of Saccharomyces cerevisiae for furfural degradation in water has shown effectiveness, but there is a need to explore improved efficiency and tolerance in S. cerevisiae for this purpose. In this study, we isolated and evolved highly efficient furfural degradation strains, namely YBA_08 and F60C. These strains exhibited remarkable capabilities, degrading 59% and 99% furfural in the YPD medium after 72 h of incubation, significantly higher than the 31% achieved by the model strain S288C. Through analysis of the efficient degradation mechanism in the evolutionary strain F60C, we discovered a 326% increase in the total amount of NADH and NADPH. This increase likely promotes faster furfural degradation through intracellular aldehyde reductases. Moreover, the decrease in NADPH content led to a 406% increase in glutathione content at the background level, which protects cells from damage caused by reactive oxygen species. Mutations and differential expression related to cell cycle and cell wall synthesis were observed, enabling cell survival in the presence of furfural and facilitating rapid furfural degradation and growth recovery. Based on these findings, it is speculated that strains YBA_08 and F60C have the potential to contribute to furfural degradation in water and the production of furfuryl alcohol, ethanol, and FDCA in biorefinery processes. [Display omitted] • Furfural induced damage of biological macromolecules and growth inhibition. • A 326% increase of NAD(P)H promoted the rapid degradation of furfural. • A 406% increase of glutathione in background level reduced oxidative stress. • DNA protection and extended lifespan ensured cell survival. [ABSTRACT FROM AUTHOR]

The aldehyde reductases from the short-chain dehydrogenase/reductase (SDR) family were identified as a series of critical enzymes for the improved tolerance of Saccharomyces cerevisiae to the aldehydes by catalyzing the detoxification reactions of aldehydes. Herein, we report that a novel aldehyde reductase Ykl107wp deduced from YKL107W from S. cerevisiae belongs to the classical SDR group and can catalyze the reduction reactions of acetaldehyde (AA), glycolaldehyde (GA), furfural (FF), formaldehyde (FA), and propionaldehyde (PA) but cannot reduce the six representative ketones. Ykl107wp displayed the best maximum velocity (Vmax), catalytic rate constant (Kcat), catalytic efficiency (Kcat/Km), and highest affinity (Km) to acetaldehyde. The optimum pH of Ykl107wp was 6.0 for the reduction of AA and 7.0 for the reduction of GA and FF, and the optimum temperatures were 40, 35, and 30 °C for the reduction of AA, GA, and FF, respectively. Ykl107wp for the reduction of AA was greatly affected by metal ions, chemical additives, and salts and showed poor thermal and pH stability, but its stability was slightly affected by a substrate. Ykl107wp was localized in endoplasmic reticulum and prevented the yeast cells from damage caused by furfural via the detoxification of furfural to furfural alcohol. This research provides guidelines for the study of uncharacterized classical SDR aldehyde reductases and exploration of their protective mechanisms on the corresponding organelles. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) disease outbreaks have resulted in many fatalities, yet no licensed vaccines are available to prevent infection. Recombinant glycoprotein (GP) production may contribute to finding a cure for Ebola virus disease, which is the key candidate protein for vaccine preparation. To explore GP1,2 expression in BmN cells, EBOV-GP1,2 with its native signal peptide or the GP64 signal peptide was cloned and transferred into a normal or gp64 null Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid via transposition. The infectivity of the recombinant bacmids was investigated after transfection, expression and localization of EBOV-GP were investigated, and cell morphological changes were analyzed by TEM. The GP64 signal peptide, but not the GP1,2 native signal peptide, caused GP1,2 localization to the cell membrane, and the differentially localized GP1,2 proteins were cleaved into GP1 and GP2 fragments in BmN cells. GP1,2 expression resulted in dramatic morphological changes in BmN cells in the early stage of infection. However, GP1,2 expression did not rescue GP64 deficiency in BmNPV infection. This study provides a better understanding of GP expression and processing in BmN cells, which may lay a foundation for EBOV-GP expression using the BmNPV baculovirus expression system. [ABSTRACT FROM AUTHOR]