Engineering Microbiology最新文献

This research identified four amino acid residues (Leu174, Asn297, Tyr301, and Gln291) that contribute to substrate recognition by the high-affinity glucose transporter Xltr1p from Trichoderma reesei. Potential hotspots affecting substrate specificity were selected through homology modeling, evolutionary conservation analyses, and substrate-docking modeling of Xltr1p. Variants carrying mutations at these hotspots were subsequently obtained via in silico screening. Replacement of Leu174 or Asn297 in Xltr1p with alanine resulted in loss of hexose transport activity, indicating that Leu174 and Asn297 play essential roles in hexose transport. The Y301W variant exhibited accelerated mannose transport, but lost galactose transport capacity, and mutation of Gln291 to alanine greatly accelerated mannose transport. These results suggest that amino acids located in transmembrane α-helix 7 (Asn297, Tyr301, and Gln291) play critical roles in substrate recognition by the hexose transporter Xltr1p. Our results will help expand the potential applications of this transporter and provide insights into the mechanisms underlying its function and specificity.

African swine fever virus (ASFV) infection poses enormous threats and challenges to the global pig industry; however, no effective vaccine is available against ASFV, attributing to the huge viral genome (approximately189 kb) and numerous encoding products (>150 genes) due to the limited understanding on the molecular mechanisms of viral pathogenesis. Elucidating the host-factor/viral-protein interaction network will reveal new targets for developing novel antiviral therapies. Using proteomic analysis, we identified 255 cellular proteins that interact with the ASFV-encoded pE301R protein when transiently expressed in HEK293T cells. Gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment, and protein-protein interaction (PPI) network analyses revealed that pE301R-interacting host proteins are potentially involved in various biological processes, including protein translation and folding, response to stimulation, and mitochondrial transmembrane transport. The interactions of two putative cellular proteins (apoptosis inducing factor mitochondria associated 1 (AIFM1) and vimentin (VIM)) with pE301R-apoptosis inducing factor have been verified by co-immunoprecipitation. Our study revealed the inhibitory role of pE301R in interferon (IFN) induction that involves VIM sequestration by pE301R, identified interactions between ASFV pE301R and cellular proteins, and predicted the potential function of pE301R and its associated biological processes, providing valuable information to enhance our understanding of viral protein function, pathogenesis, and potential candidates for the prevention and control of ASFV infection.

Carbazomycins (1–8) are a subgroup of carbazole derivatives that contain oxygen at the C3 and C4 positions and an unusual asymmetric substitution pattern. Several of these compounds exhibit antifungal and antioxidant activities. To date, no systematic biosynthetic studies have been conducted on carbazomycins. In this study, carbazomycins A and B (1 and 2) were isolated from Streptomyces luteosporeus NRRL 2401 using a one-strain-many-compound (OSMAC)-guided natural product mining screen. A biosynthetic gene cluster (BGC) was identified, and possible biosynthetic pathways for 1 and 2 were proposed. The in vivo genetic manipulation of the O-methyltransferase-encoding gene cbzMT proved indispensable for 1 and 2 biosynthesis. Size exclusion chromatography indicated that CbzMT was active as a dimer. In vitro biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4, producing monomethylated 2 and dimethylated 1. Monomethylated carbazomycin B (2) is not easily methylated; however, CbzMT seemingly prefers the dimethylation of the dihydroxyl substrate (12) to 1, even with a low conversion efficiency. These findings not only improve the understanding of carbazomycin biosynthesis but also expand the inventory of OMT-catalyzing iterative methylations on different acceptor sites, paving the way for engineering biocatalysts to synthesize new active carbazomycin derivatives.

Microbial fuel cells (MFCs) employing Pseudomonas putida B6-2 (ATCC BAA-2545) as an exoelectrogen have been developed to harness energy from various conventional substrates, such as acetate, lactate, glucose, and fructose. Owing to its metabolic versatility, P. putida B6-2 demonstrates adaptable growth rates on diverse, cost-effective carbon sources within MFCs, exhibiting distinct energy production characteristics. Notably, the anode chamber's pH rises with carboxylates' (acetate and lactate) consumption and decreases with carbohydrates' (glucose and fructose) utilization. The MFC utilizing fructose as a substrate achieved the highest power density at 411 mW m⁻². Initial analysis revealed that P. putida B6-2 forms biofilms covered with nanowires, contributing to bioelectricity generation. These microbial nanowires are likely key players in direct extracellular electron transport through physical contact. This study established a robust foundation for producing valuable compounds and bioenergy from common substrates in bioelectrochemical systems (BESs) utilizing P. putida as an exoelectrogen.

The human intestinal microbiota that comprise over 1,000 species thrive in dark and anaerobic environments. They are recognized for the production of diverse low-molecular-weight metabolites crucial to human health and diseases. Carotenoids, low-molecular-weight pigments known for their antioxidative activity, are delivered to humans through oral intake. However, it remains unclear whether human intestinal bacteria biosynthesize carotenoids as part of the in-situ microbiota. In this study, we investigated carotenoid synthesis genes in various human gut and probiotic bacteria. As a result, novel candidates, the crtM and crtN genes, were identified in the carbon monoxide-utilizing gut anaerobe Eubacterium limosum and the lactic acid bacterium Leuconostoc mesenteroides subsp. mesenteroides. These gene candidates were isolated, introduced into Escherichia coli, which synthesized a carotenoid substrate, and cultured aerobically. Structural analysis of the resulting carotenoids revealed that the crtM and crtN gene candidates of E. limosum and L. mesenteroides mediate the production of 4,4′-diaponeurosporene through 15-cis-4,4′-diapophytoene. Evaluation of the crtE-homologous genes in these bacteria indicated their non-functionality for C₄₀-carotenoid production. E. limosum and L. mesenteroides, along with the known carotenogenic lactic acid bacterium Lactiplantibacillus plantarum, were observed to produce no carotenoids under strictly anaerobic conditions. The two lactic acid bacteria synthesized detectable levels of 4,4′-diaponeurosporene under semi-aerobic conditions. The findings highlight that the obligate anaerobe E. limosum retains aerobically functional C₃₀-carotenoid biosynthesis genes, potentially with no immediate self-utility, suggesting an evolutionary direction in carotenoid biosynthesis. (229 words)

The consumption of non-renewable fossil fuels has directly contributed to a dramatic rise in global carbon dioxide (CO₂) emissions, posing an ongoing threat to the ecological security of the Earth. Microbial electrosynthesis (MES) is an innovative energy regeneration strategy that offers a gentle and efficient approach to converting CO₂ into high-value products. The cathode chamber is a vital component of an MES system and its internal factors play crucial roles in improving the performance of the MES system. Therefore, this review aimed to provide a detailed analysis of the key factors related to the cathode chamber in the MES system. The topics covered include inward extracellular electron transfer pathways, cathode materials, applied cathode potentials, catholyte pH, and reactor configuration. In addition, this review analyzes and discusses the challenges and promising avenues for improving the conversion of CO₂ into high-value products via MES.

Adenoviruses typically cause mild illnesses, but severe diseases may occur primarily in immunodeficient individuals, particularly children. Recently, adenoviruses have garnered significant interest as a versatile tool in gene therapy, tumor treatment, and vaccine vector development. Over the past two decades, the advent of recombineering, a method based on homologous recombination, has notably enhanced the utility of adenoviral vectors in therapeutic applications. This review summarizes recent advancements in the use of human adenoviral vectors in medicine and discusses the pivotal role of recombineering in the development of these vectors. Additionally, it highlights the current achievements and potential future impact of therapeutic adenoviral vectors.

Lignocellulosic biomass is an abundant and renewable bioresource for the production of biofuels and biochemical products. The classical biorefinery process for lignocellulosic degradation and conversion comprises three stages, i.e., pretreatment, enzymatic saccharification, and fermentation. However, the complicated pretreatment process, high cost of cellulase production, and insufficient production performance of fermentation strains have restricted the industrialization of biorefinery. Consolidated bioprocessing (CBP) technology combines the process of enzyme production, enzymatic saccharification, and fermentation in a single bioreactor using a specific microorganism or a consortium of microbes and represents another approach worth exploring for the production of chemicals from lignocellulosic biomass. The present review summarizes the progress made in research of CBP technology for lignocellulosic biomass conversion. In this review, different CBP strategies in lignocellulose biorefinery are reviewed, including CBP with natural lignocellulose-degrading microorganisms as the chassis, CBP with biosynthetic microorganisms as the chassis, and CBP with microbial co-culturing systems. This review provides new perspectives and insights on the utilization of low-cost feedstock lignocellulosic biomass for production of biochemicals.

The association between cigarette smoking and the gut microbiota remains unclear, and there is no agreement on how smoking affects the composition of gut microorganisms. In this study, the relationship between smoking status and gut microbial composition was investigated by performing 16S rRNA gene amplicon sequencing analysis of stool samples from 80 healthy Chinese adults. The results showed that smoking did not cause significant changes to the composition and microbial functional pathways of the gut microbiota. However, smoking altered the relative abundance of several specific taxa, where Phascolarctobacterium and Fusobacterium increased and Dialister decreased. Notably, our analysis revealed that smoking introduced more microbial interactions to the interaction network and decreased its modularity. Overall, this study provides new insights into the association between smoking status and the gut microbiota.

Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals. Marine microbial natural products exhibit diverse chemical structures and bioactivities with substantial potential for the development of novel pharmaceuticals. However, discovering compounds with new skeletons from marine microbes remains challenging. In recent decades, multiple approaches have been developed to discover novel marine microbial natural products, among which heterologous expression has proven to be an effective method. Facilitated by large DNA cloning and comparative metabolomic technologies, a few novel bioactive natural products from marine microorganisms have been identified by the expression of their biosynthetic gene clusters (BGCs) in heterologous hosts. Heterologous expression is advantageous for characterizing gene functions and elucidating the biosynthetic mechanisms of natural products. This review provides an overview of recent progress in heterologous expression-guided discovery, biosynthetic mechanism elucidation, and yield optimization of natural products from marine microorganisms and discusses the future directions of the heterologous expression strategy in facilitating novel natural product exploitation.