Applied Microbiology and Biotechnology最新文献

Fucoxanthin, a bioactive carotenoid derived from algae, has attracted considerable attention for its applications in health, cosmetics, and nutrition. Advances in metabolic engineering, such as the overexpression of pathway-specific enzymes and enhancement of precursor availability, have shown promising results in improving production efficiency. However, despite its high value, the biosynthetic pathway of fucoxanthin remains only partially elucidated, posing significant challenges for metabolic engineering efforts. Recent studies have identified previously unknown enzymes and regulatory elements within the pathway, providing opportunities for further productivity enhancements through targeted metabolic modifications. Additionally, adaptive evolution, mutagenesis-driven strain development, and optimized cultivation conditions have demonstrated significant potential to boost fucoxanthin yields. This review consolidates the latest insights into the biosynthetic pathway of fucoxanthin and highlights metabolic engineering strategies aimed at enhancing the production of fucoxanthin and related carotenoids, offering approaches to design high-yielding strains. Furthermore, recent advancements in random mutagenesis and cultivation technology are discussed. By integrating these developments, more economically viable and environmentally sustainable fucoxanthin production systems can be achieved.

• Insights into fucoxanthin biosynthesis enable targeted metabolic engineering.

• ALE and cultivation strategies complement metabolic engineering efforts.

• Balanced push–pull-block strategies improve fucoxanthin production efficiency.

Snake venom has long-term physiological effects on survivor’s life. An electrochemical immunosensor based on samarium-cobalt-doped antimony tungstate (Sb₂WO₄@Sm-Co) is developed via a solvothermal method to detect snake venom antigens (SVA). The fabricated nanospheres are functionalized with carboxyl groups to enhance the linkage of the 3-mercaptopropionic acid linker (3-MPA). This modification increases the conjugation of antivenom polyvalent antibody with the nanomaterial on a glassy carbon electrode (Sb₂WO₄@Sm-Co-COOH-MPA-Ab/GCE). The modified nanospheres are characterized by UV–VIS spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS). The electrochemical performance of formulated immunosensor for antigen sensing is tested by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), linear sweep voltammetry (LSV), and chronoamperometry. This developed immunosensor has a wide linear range of 5–30 ng/mL with LODs of 0.08 ng/mL and 0.1 ng/mL from DPV and LSV, respectively. The amperometric immunosensor increases the tested antibody's loading capacity and accelerates the electron transfer rate. The analytical parameters reveal that this immunosensor is ultrasensitive, stable, reproducible, and selective for measuring SVA and can have potential applications in diagnostic clinics.

• The hierarchical Sb₂WO₄@Sm-Co-COOH NPs were synthesized through a one-step solvothermal method

• Monitoring the effect of doping Sm and Co on the characteristics of Sb₂WO₄

• MPA-linked IgG antibodys-based immunosensor was synthesized with good dispersity and high surface functional groups for capturing SVAs

Tick-borne severe fever with thrombocytopenia syndrome (SFTS) is an emerging zoonotic disease caused by the SFTS virus (SFTSV). Serological assays based on the nucleocapsid protein and partial glycoprotein of this virus have been used for detecting SFTSV infections in humans and animals. However, whether the complete SFTSV glycoprotein (Gn/Gc) can induce the assembly of virus-like particles (VLPs) which can be used for serological surveillance and vaccine production remains unclear. In this study, we successfully expressed and secreted SFTSV Gn/Gc antigens by using a single plasmid encoding the complete glycoprotein sequence of the dominant genotype B virus. HEK293T and COS-1 cells were transfected with the aforementioned plasmid; cultivating these cells at 32 °C, instead of 37 °C, led to 4.0- and 3.3-fold higher antigen recovery, respectively. The secreted Gn/Gc antigens at 32 °C retained epitopes resembling those of the virion; these epitopes were recognized by a SFTS human–derived monoclonal antibody. Sucrose density gradient centrifugation, followed by transmission electron microscopy, confirmed the formation of VLPs with a diameter of approximately 100 nm. Overall, our findings highlight the potential of SFTSV VLPs for serological surveillance and vaccine development.

• Cultivating transfected cells at 32 °C boosts SFTSV glycoprotein production.

• Complete SFTSV glycoprotein expression facilitates virus-like particle assembly.

• The assembly does not require any other viral proteins or RNA.

Xylose is the most abundant pentose in nature. However, it is usually obtained in mixtures with glucose, leading to carbon catabolite repression in many microorganisms. Among E. coli lineages, significant metabolic and regulatory differences exist, requiring distinct metabolic engineering strategies to develop a xylose-selective phenotype in the strains W, K-12, and C. In this study, strain ES02 was engineered from Escherichia coli BL21 (DE3) as a xylose-selective strain by deleting the glk, ptsG, and manZ genes. However, when grown in a mixture of xylose and glucose, this strain’s specific growth rate and xylose consumption rate decreased by about 50% compared to cultures with only xylose. A modified version of the xylose-responsive transcriptional activator XylR^Q31K was utilized to overcome this issue. The resulting strain ES04 (BL21 (DE3) Δglk, ΔmanZ, ΔptsG, xylR::Km^r, lacZ::xylR^C91A-Gm^r) efficiently used xylose as carbon source either alone or in a mixture with glucose, with a specific xylose consumption rate 75% higher than that of the wild-type strain BL21(DE3). Unexpectedly, strain ES04 partially recovers the ability to grow and consume glucose at a low rate, preferentially consuming xylose over glucose in sugar mixtures, revealing an altered carbon catabolite repression phenotype. Transcriptomics analysis suggested that glucose assimilation in this strain was related to the overexpression of the galactitol operon gatDCBAZY. Further inactivation of this operon confirmed its participation in glucose assimilation.

• XylR^Q31K alleviates carbon catabolite repression in the xylose-selective strain ES04.

• Galactitol operon overexpression in ES04 links to partial glucose utilization.

• ES04 strain preferentially uses xylose over glucose, revealing altered CCR.

Clostridium butyricum inhabits various anoxic environments, including soil and the human gut. Here, this common bacterium comes into contact with abundant plant-derived flavonoids. Metabolization of these bioactive polyphenols has been studied in recent years, particularly focusing on gut bacteria due to the proposed health-promoting properties of these dietary constituents. Based on an initial report in 1997 on eriodictyol degradation (Miyake et al. 1997, J Agric Food Chem, 45:3738–3742), the present study systematically investigated C. butyricum for its ability to convert a set of structurally diverse flavonoids. Incubation experiments revealed that C. butyricum deglycosylated flavonoid O-glucosides but only when glucose was absent. Moreover, aglycone members of flavone, flavanone, dihydrochalcone, and flavanonol subclasses were degraded. The C-ring cleavage of the flavanones, naringenin and eriodictyol, was stereospecific and finally resulted in formation of the corresponding hydroxyphenylpropionic acids. Stereospecific C-ring cleavage of the flavanonol taxifolin led to taxifolin dihydrochalcone. C. butyricum did neither cleave flavonols and isoflavones nor catalyze de-rhamnosylation, demethylation, or dehydroxylation of flavonoids. Genes encoding potential flavonoid-metabolizing enzymes were detected in the C. butyricum genome. Overall, these findings indicate that C. butyricum utilizes flavonoids as alternative substrates and, as observed for the dihydrochalcone phloretin, can eliminate growth-inhibiting flavonoids through degradation.

• Clostridium butyricum deglycosylated flavonoid O-glucosides.

• Clostridium butyricum converted members of several flavonoid subclasses.

• Potential flavonoid-metabolizing enzymes are encoded in the C. butyricum genome.

Recently developed plasmid-based reverse genetics systems for rotavirus A (RVA) enable rapid engineering of reassortants carrying human RVA antigens. However, complete genome segment sequences are required for successful generation of such reassortants, and sequencing of the untranslated regions (UTRs) of field strains is often not accomplished. To address this problem, we established a system that permits the generation of reassortants using only the open reading frame (ORF) nucleotide sequence information. Plasmids containing the VP7-ORF nucleotide sequence of six human RVA field strains (genotypes G2, G5, G8, G9, G12 and G29) derived from GenBank and flanked by the UTR sequences of simian RVA strain SA11 were constructed. Using these plasmids, four VP7 (G2, G5, G9 and G12) reassortants in an SA11 backbone were successfully generated. In contrast, the G8 and G29 reassortants were not viable. BLASTp search of the G8 and G29 sequences revealed an unusual amino acid substitution in each sequence, which was not present in related field strains. Site-directed reversion of the corresponding C656T mutation in G8 led to effective rescue of reassortant virus. However, reverting the G84C mutation in G29 did not result in replicating virus. The results suggest that most human RVA VP7 UTRs can be substituted with simian RVA UTRs. However, generation of reassortants might be impeded by potential sequencing errors or intrinsic reassortment limitations. The established system could help to broaden the antigenic repertoire for rapid engineering of potential novel RVA vaccine strains.

• Generation of diverse rotavirus vaccine strains is impeded by missing UTR sequences.

• UTRs from SA11 can be used instead of missing UTR sequences from field strains.

• Human RVA reassortants of genotypes G2, G5, G8, G9, G12 were successfully rescued.

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.