Applied Microbiology and Biotechnology最新文献

Leloir glycosyltransferases are instrumental in the synthesis of glycoconjugates. Nucleotide sugars as their donor substrates are still considered expensive making preparative enzymatic syntheses economically unattractive. The review highlights the development and advancements of in situ regeneration cycles that utilize nucleotides as byproducts from glycosyltransferase reactions to synthesize respective nucleotide sugars. This approach reduces costs and avoids inhibition of Leloir glycosyltransferases. Regeneration cycles for ten nucleotide sugars are explored emphasizing enzyme cascades from salvage pathways and nucleotide biosynthesis. Additionally, the review highlights advancements involving sucrose synthase for the in situ regeneration of nucleotide sugars from sucrose. Sucrose synthase as the first example of a reversible glycosyltransferase reaction paved the way to establish economic syntheses of glycosylated natural products. Important aspects like enzyme immobilization and protein fusion to optimize processes are discussed. Overall, the review underscores the significance of advanced in situ regeneration cycles for nucleotide sugars for cost-effective access to high-value glycoconjugates.

• Enzyme cascades for in situ regeneration of nucleotide sugars

• Effective cycles for large-scale synthesis of glycoconjugates

• Regeneration of nucleotide sugars from sucrose by sucrose synthase

Cancer is a predominant contributor to global morbidity and mortality, affecting populations worldwide. Marine Micromonospora species have been identified as significant sources of anticancer compounds. This work aimed to perform a polyphasic approach of isolated strain and conduct comparative metabolomic and genomic analyses to identify compounds with anticancer activity. The study utilized a polyphasic approach on isolated strains for anticancer compound identification. Taxonomic analysis of strain 2MTK254 revealed unique pigment and fatty acid patterns, designating it as a novel Micromonospora carbonacea subsp. caeruleus. Its crude extract displayed significant anti-colorectal activity (66.03% inhibition). Molecular network analysis classified metabolites into eight classes, highlighting a polycyclic tetramate macrolactams (PTMs) compound (P1, C₂₉H₃₈N₂O₄) with 99.31% inhibitory activity against HCT-116 cell lines (IC₅₀ at 0.125 µM). Genome analysis identified 32 biosynthetic gene clusters (BGCs), including unique PTMs BGCs (83% similarity) linked to the P1 compound. Thus, M. carbonacea subsp. caeruleus 2MTK254 holds promise as a source of novel PTMs with anti-colorectal cancer potential.

• A novel strain of Micromonospora carbonacea subsp. caeruleus 2MTK254 was isolated in Thailand

• A new polycyclic tetramate macrolactam (PTM) with anticancer activity was identified in 2MTK254

• The genome of 2MTK254 has unique secondary metabolite gene clusters

β-1,2-Glucans are physiologically important polymers for interactions such as symbiosis and pathogenesis between organisms and adaptation to environmental changes. However, rarity of β-1,2-glucans in nature limits exploration of related enzymes. Recently, many β-1,2-glucan-degrading enzymes have been found after identification of a novel phosphorylase acting on β-1,2-glucooligosaccharides. The expansion of the repertoire has reached revelation of the cyclization mechanism of cyclic β-1,2-glucan synthase and led to finding of new enzymes catalyzing cyclization of β-1,2-glucans in a manner different from cyclic β-1,2-glucan synthase. In this review, we mainly focus on newly found enzymes that catalyze cyclization of β-1,2-glucans along with existence of β-1,2-glucan-associated carbohydrates in nature and introduction of the repertoire of β-1,2-glucan-degrading enzymes.

• Newly found domain which cyclizes β-1,2-glucan created a new glycoside hydrolase family.

• Cyclization is performed with a unique mechanism.

• α-1,6-Cyclized β-1,2-glucan is produced by an enzyme in another newly found family.

The control of biodeteriogenic microorganisms is essential for the management of heritage sites. Many conventional biocides are no longer available because they have lost their efficacy or have been withdrawn from the market due to their danger to humans and the environment. Therefore, new effective and sustainable biocides are needed, such as plant extracts that could be a good alternative. In this study, essential oils (EOs) of Ocimum basilicum L., Cinnamomum verum Presl, Lavandula angustifolia Mill., Origanum vulgare L., Thymus vulgaris L. and Melaleuca alternifolia Maiden & Betche were tested as green biocides against microorganisms collected from biofilms in the hypogeum of the Colosseum (Rome, Italy). Biocidal screening was first carried out on phototrophic microorganisms grown on BG11 agar culture medium. The efficacy was assessed by measuring photosynthetic activity with a mini-PAM portable fluorometer, and by determining morphological changes or the absence of autofluorescence using light microscopy and confocal laser scanning microscopy. The most effective EOs against phototrophs were further tested to inhibit the growth of heterotrophic fungi and bacteria in order to identify those with a broad-spectrum action. The EOs of cinnamon, oregano and thyme at 5% concentration (v/v) were the most effective against the microorganisms isolated from the biofilms in the Colosseum. These EOs represent a green alternative to traditional chemical biocides due to their activity against a wide range of microorganisms and their complex composition which suggests the potential to reduce the risk of microbial resistance.

The biological production of butanol via ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum has a storied history of over 100 years, initially driven by the demand for synthetic rubber during World War I and later for industrial applications. Despite its decline due to the rise of petrochemical alternatives, renewed interest has emerged due to the global shift towards sustainable energy sources and rising oil prices. This review highlights the challenges in the cultivation process of C. acetobutylicum, such as strain degeneration, solvent toxicity, and substrate costs, and presents recent advancements aimed at overcoming these issues. Detailed documentation of the entire cultivation process including cell conservation, pre-culture, and main culture is seen as a fundamental step to facilitate further progress in research. Key strategies to improve production efficiency were identified as controlling pH to facilitate the metabolic shift from acidogenesis to solventogenesis, employing in situ product removal techniques, and advancing metabolic engineering for improved solvent tolerance of C. acetobutylicum. Furthermore, the use of renewable resources, particularly lignocellulosic biomass, positions ABE fermentation as a viable solution for sustainable solvent production. By focusing on innovative research avenues, including co-cultivation and bioelectrochemical systems, the potential for C. acetobutylicum to contribute significantly to a bio-based economy can be realized.

• Historical significance and revival of ABE fermentation with Clostridium acetobutylicum

• Current challenges and innovative solutions in cultivating C. acetobutylicum

• New avenues for enhancing productivity and sustainability

In this study, we investigated MalS, a periplasmic α-enzyme from Escherichia coli K12, known for its unique biochemical properties related to polysaccharide utilization. Evolutionarily, MalS has inherited the glycosyl hydrolase catalytic domain from the glycoside hydrolase family 13, with the protein sequences highly conserved across Enterobacteria, including Salmonella and Shigella. MalS exhibited optimal activity at 65 °C, significantly higher than other E. coli enzymes. Although its reaction pattern resembled that of typical α-amylases, its catalytic efficiency on polysaccharides was notably lower. Intriguingly, MalS demonstrated a strong binding affinity for various glucose polymers, including β-cyclodextrin and glycogen, which significantly enhanced its thermostability. Despite full-length MalS binding strongly to glycogen, neither its N-terminal domain, predicted by AlphaFold2 to belong to the Carbohydrate-Binding Module family 69, nor the remaining parts of the enzyme showed binding affinity toward polysaccharides. Kinetic studies revealed that MalS had a 2.5-fold lower K_m and 1.4-fold higher catalytic efficiency toward glycogen compared to amylopectin, which contrasts starkly with pancreatic α-amylases. However, over prolonged reactions, glycogen hydrolysis by MalS was slower than that of amylopectin. In the early initial stage, MalS predominantly degraded glycogen to maltopentaose (G5) rather than maltohexaose (G6) as usual. Taken together, these findings suggest MalS may play a role in recognizing glycogen-type polysaccharides in the bacterial periplasm during adaptation to new environments. Given the crucial role of glycogen in the survival and infection processes of pathogenic bacteria, understanding MalS’s interaction with glycogen-type polysaccharides could offer valuable insights into bacterial survival mechanisms and their ability to infect hosts.

• MalS has unique structure and properties but conserved among many enterobacteria

• Binding of MalS with polysaccharides significantly enhanced its thermostability

• Unlike other amylases, MalS showed 2.5-fold lower K_m on glycogen than amylopectin

A comparative genomic analysis of feline coronavirus (FCoV) and feline panleukopenia virus (FPLV) was performed. Based on the conserved regions of the two viruses, specific probes and real-time PCR (qPCR) primers were designed, and a duplex TaqMan qPCR-based assay was established for detecting FCoV and FPLV. The results showed high analytical specificity, and no cross-response with other feline viruses was observed. This method is highly versatile and can be used to detect all FCoV strains stored in laboratories and recombinant plasmids constructed according to the sequences of blank FCoV strains in laboratories. The analytical sensitivity of this method in detecting FCoV and FPLV was as low as 50 copies/μL, which is approximately 20-fold greater than that of conventional PCR. The coefficients of variation (CVs) for the intra- and interbatch coefficients of variation were less than 2%. After 75 clinical samples were tested, the percentage of FCoV- and FPLV-positive samples was 5.34% greater than that of conventional PCR methods, a finding robustly supported by sequencing identification. As validated by clinical samples, the method was sensitive, specific, general, and reproducible and holds great potential for the rapid identification and diagnosis of FCoV and FPLV infections, as well as for epidemiological investigations.

• One-step duplex TaqMan real-time PCR detection method can detect FCoV and FPLV in clinical samples simultaneously and steadily.

• Almost all the currently known FCoV and FPLV strains can be detected.

• This method has high sensitivity, specificity and generality.

Despite the lack of implementation of consolidated bioprocessing (CBP) at an industrial scale, this bioconversion strategy still holds significant potential as an economically viable solution for converting lignocellulosic biomass (LCB) into biofuels and green chemicals, provided an appropriate organism can be isolated or engineered. The use of Saccharomyces cerevisiae for this purpose requires, among other things, the development of a cellulase expression system within the yeast. Over the past three decades, numerous studies have reported the expression of cellulase-encoding genes, both individually and in combination, in S. cerevisiae. Various strategies have emerged to produce a core set of cellulases, with differing degrees of success. While one-step conversion of cellulosic substrates to ethanol has been reported, the resulting titers and productivities fall well below industrial requirements. In this review, we examine the strategies employed for cellulase expression in yeast, highlighting the successes in developing basic cellulolytic CBP-enabled yeasts. We also summarize recent advancements in rational strain design and engineering, exploring how these approaches can be further enhanced through modern synthetic biology tools to optimize CBP-enabled yeast strains for potential industrial applications.

• S. cerevisiae’s lack of cellulolytic ability warrants its engineering for industry.

• Advancements in the expression of core sets of cellulases have been reported.

• Rational engineering is needed to enhance cellulase secretion and strain robustness.

• Insights gained from omics strategies will direct the future development of CBP strains.

The E2 subunit vaccine has been considered a promising alternative to an attenuated classical swine fever (CSF) vaccine. However, it fails to induce a good cellular immune response. Given that immunogenic adjuvants can regulate the cellular immunity to achieve a maximum efficacy against antigens, immunostimulatory effects of porcine IL-28B on the CSF virus (CSFV) E2 subunit vaccine were evaluated in the present study. We expressed recombinant proteins E2-IL28B, E2, and IL-28B using CHO-S mammalian cells as an antigen expression platform, and three types of CSFV E2 subunit vaccines based on antigens E2-IL28B, E2 + IL-28B, and E2 were prepared, respectively. We found that both E2-IL28B and E2 + IL-28B antigens exhibited superior immunogenicity with dramatically induced antibody titers and neutralizing antibody levels than the E2 alone. Moreover, E2-IL28B or E2 + IL-28B, instead of E2, boosted cellular immune responses via obviously increasing the percentages of CD3⁺CD4⁺ T lymphocytes, promoting the lymphocyte proliferations, and enhancing the release of Th1-type cytokines. All results revealed that the inclusion of IL-28B, whether fused or mixed with E2, significantly elevated E2-induced immune potencies, suggesting that IL-28B could be used as a molecular adjuvant to optimize the design of E2 subunit vaccine for more effective controls of the CSF disease.

• New CSF E2 subunit vaccine candidates were developed in which IL-28B was an immunoadjuvant

• IL-28B significantly elevated the E2-induced immune potency whether it was fused or mixed with E2

• This study provided novel insights into the immunoregulatory properties of IL-28B used for the optimized subunit vaccine design

The high mortality rate associated with single-use CRISPR-Cas9 in Escherichia coli limits its application. Recently, new CRISPR-based techniques for E.coli gene editing have emerged. Research aims to develop a system for rapid, marker-free, multi-site, and multi-copy genome editing in E.coli to advance synthetic biology. ATP, essential for energy in living organisms, plays a crucial role in various metabolic processes. To reduce the cost of ATP-requiring reactions, it is crucial to identify and efficiently express genes in ATP synthesis pathway. This study identified a single ppk gene (No.8) capable of completing the cyclic reaction. Using MUCICAT technology, the ppk gene (No.8) was inserted into various positions and copy numbers in the E.coli genome, resulting in different activity levels. The findings suggest that the difficulty of inserting the ppk gene (No.8) into the genome follows this order: IS186 < 8array < IS186 + 8array < IS1. A single genome insertion can mimic plasmid expression level. This study explores promoter competition and offers solutions, inspiring researchers in constructing the AMP-ATP cycle system in E.coli.

• The single ppk gene (No.8) can regenerate the AMP-ATP cycle, crucial for ATP-dependent reactions.

• Inserting the ppk gene (No.8) into the cr5 site of the E.coli genome achieves expression levels comparable to the pET29a plasmid.

• The expression level of the ppk gene (No.8) is not significantly affected by its copy number in the E.coli genome.