Engineering Microbiology最新文献

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the importance of developing novel vaccines. An ideal vaccine should trigger an intense immune reaction without causing significant side effects. In this study we found that substitution of tryptophan located in the cores of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein structures with certain smaller amino acids resulted in variants with melting temperatures of 33–37 °C. An enzyme activity assay indicated that the proteolytic activity of the main proteinase (3CL^pro) decreased sharply when the environmental temperature exceeded the melting temperature, implying that other protein variants may lose most of their functions under the same conditions. This finding suggests that a virus variant containing engineered proteins with melting temperatures of 33–37 °C may only be functional in the upper respiratory tract where the temperature is about 33 °C, but will be unable to invade internal organs, which maintain temperatures above 37 °C, thus making it possible to construct temperature-sensitive attenuated vaccines.

The adaptive survival mechanisms of bacterial pathogens under host-induced stress are crucial for understanding pathogenesis. Recently, Uppalapati et al. revealed a unique dual function of the Gifsy-1 prophage terminase in Salmonella enterica: it acts as a transfer ribonuclease (tRNase) under oxidative stress. The Gifsy-1 prophage terminase targets and fragments tRNA^Leu to halt translation and temporarily impairs bacterial growth when exposed to high levels of ROS generated by the host immune cells. This response not only preserves genomic integrity by facilitating DNA repair but also inhibits prophage mobilization, thereby aiding in bacterial survival within vertebrate hosts. This study highlights a novel intersection between phage biology and bacterial adaptive strategies.

Myxobacteria are well known for multicellular social behaviors and valued for biosynthesis of natural products. Myxobacteria social behaviors such as clumping growth severely hamper strain cultivation and genetic manipulation. Using Myxococcus xanthus DK1622, we engineered Hu04, which is deficient in multicellular behavior and pigmentation. Hu04, while maintaining nutritional growth and a similar metabolic background, exhibits improved dispersed growth, streamlining operational procedures. It achieves high cell densities in culture and is promising for synthetic biology applications.

The recent discovery of the CRISPR-Cas-mediated acquired immunity system highlights the fact that our knowledge of phage/virus defense mechanisms encoded in bacterial and archaeal genomes is far from complete. Indeed, new prokaryotic immune systems are now continually being discovered. A recent report described a novel glycosylase that recognizes α-glycosyl-hydroxymethyl cytosin (α-Glu-hmC), a modified base observed in the T4 phage genome, where it produces an abasic site, thereby inhibiting the phage propagation.

Terpenoids are widely used as medicines, flavors, and biofuels. However, the use of these natural products is largely restricted by their low abundance in native plants. Fortunately, heterologous biosynthesis of terpenoids in microorganisms offers an alternative and sustainable approach for efficient production. Various genome-editing technologies have been developed for microbial strain construction. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) is the most commonly used system owing to its outstanding efficiency and convenience in genome editing. In this review, the basic principles of CRISPR-Cas9 systems are briefly introduced and their applications in engineering bacteria for the production of plant-derived terpenoids are summarized. The aim of this review is to provide an overview of the current developments of CRISPR-Cas9-based genome-editing technologies in bacterial engineering, concluding with perspectives on the challenges and opportunities of these technologies.

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)