Journal of bioscience and bioengineering最新文献

Synthetic biology approaches enable the creation of promising chassis for practical application in various fields, though engineering of microbial metabolism often imposes a metabolic burden, potentially driving adaptive evolution during long-term cultivation. A previously established phosphite (Pt)-dependent metabolic system has proven to be an effective strategy for the containment of genetically engineered microorganisms, although its implementation accompanied a slight growth retardation. Here, we investigated the effect of long-term serial passaging cultivation on the Pt-dependent strain of Synechococcus elongatus PCC 7942, RH714. Compared with the originally constructed RH714, the passaged population of RH714 exhibited improved growth and a higher rate of Pt consumption in culture medium. Sequence analysis revealed point mutations within the introduced htxBCDE transporter genes, which are required for selective incorporation of Pt as a phosphorus nutrition. Introduction of the mutated gene cluster into S. elongatus PCC 7942 reproduced the traits of the passaged RH714 population, suggesting that these genetic changes enhance Pt transport activity and account for the observed phenotypes. Disruption of endogenous phosphate (Pi) transporter genes in the strains expressing the mutated htxBCDE-ptxD cluster abolished growth in Pi-containing medium, suggesting that the mutations in the transporter genes did not alter substrate specificity toward Pi. These results indicated that long-term passage cultivation developed an optimized mutant capable of efficient proliferation under the Pt metabolizing conditions without compromising its biocontainment capability.

Dendritic cells (DCs) are professional antigen-presenting cells that play a central role in initiating and shaping adaptive immune responses. Targeting antigens to DCs has emerged as a promising strategy to enhance vaccine efficacy and tailor desired immune responses. While antibodies are well established as targeting molecules for DCs, the use of peptides remains underexplored despite their favorable tissue penetration and their ability to offer design flexibility. Here, we report the identification of dectin-1-binding peptides using a ribosome display-based in vitro directed evolution system. Dectin-1 is a C-type lectin receptor expressed on murine CD11b⁺ and human CD1c⁺ DCs, which plays a key role in antigen uptake and in directing immune responses toward specific pathways, particularly the Th17 pathway. Using recombinant murine dectin-1 as bait, we performed four rounds of ribosome display selection and obtained peptides with high affinity. Selected peptides fused to enhanced green fluorescent protein showed binding to recombinant dectin-1 and native dectin-1-expressing cells, including bone marrow-derived DCs. These results demonstrated the feasibility of peptide-based molecular targeting toward dectin-1⁺ DCs, which would not only provide a versatile platform for the development of next-generation vaccines and immunotherapies, but also a valuable tool for dissecting the functional roles of dectin-1⁺ DCs in immune regulation.

Single-cell genomics (SCG) complements culture-independent metagenomics for accessing fungal genomes, particularly from lineages that remain uncultured. We contrast metagenomics, which excels when profiling community composition and metabolic potential but often underrepresents low-abundance fungi, with SCG, which first isolates individual cells or nuclei to generate single-amplified genomes (SAGs) and can recover rare or microdiverse taxa. We then organize existing fungal SCG applications into three subgroups: spore-level sequencing from host-enriched or environmental material; single-nucleus genomics for multinucleate fungi; and single-spore sequencing of haploid progeny for diploid linkage and chromosome phasing. Across studies, pooling and co-assembly of cognate cells improves completeness; key hurdles persist in wall lysis, whole-genome amplification bias, and contamination control. Practical advances include shallow sequencing for QC triage, nuclei pooling with normalized co-assembly, and hybrid long- and short-read assembly. SCG adds unique value where strain resolution and genotypic context matter, including host-to-mobile-element linkage, recovery of large biosynthetic gene clusters, and karyotype validation against telomere-to-telomere references. Used alongside metagenomics, SCG enables a strain-resolved view of fungal biodiversity and function, with incremental improvements across the SCG pipeline promising routine access to genomes from early-diverging and other environmentally embedded fungi.

Microcarrier surface topography and fluid shear stress (FSS) critically regulate cellular behavior. An edible zein/fucoidan microcarrier with 200 μm grooves was designed for this study. Computational fluid dynamics (CFD) simulations and experiments were combined to analyze surface microfluidic characteristics during dynamic culture and their cellular effects. The research demonstrates controlled myogenic differentiation through groove topography and FSS modulation for scalable cultured meat production. The results demonstrated that the grooved microcarriers exhibited excellent cell attachment and proliferation capacity in spinner flask dynamic culture, achieving a maximum cell density of 1.16 × 10⁶ cells/mL, comparable to commercial Cultispher-S microcarriers. The groove structure promoted cell alignment through contact guidance, significantly enhancing the gene expression of myogenic differentiation markers (myogenic differentiation 1, MyoD1; α-actinin; myosin heavy chain, MHC) and cell fusion (myomaker, MYMK). CFD simulations revealed that the grooves created a low-shear microenvironment (minimum average FSS: 3.93 × 10^-2 Pa, maximum: 1.18 × 10^-1 Pa), which effectively avoided high FSS-induced damage while maintaining mechanical stimulation. This optimal mechanical microenvironment further activated the expression of key genes involved in early-stage (MyoD1; myogenin, MyoG; myocyte enhancer factor 2C, MEF2C) and late-stage (α-actinin; myosin heavy chain 2, Myh2) myogenic differentiation. Flat and spherical microcarriers showed lower myogenic differentiation efficiency. This study elucidates the synergistic mechanism between groove structures and the low FSS microenvironment within grooves, providing a novel scaffold design rationale that combines biomimetic topology with fluid dynamics compatibility for large-scale cultured meat production.

The natural abundances of stable isotopes were used to determine prey-predator relationships and material flows in a biological treatment reactor for municipal wastewater. The reactor used in this study was a down-flow hanging sponge (DHS), which is an alternative trickling filter that uses sponge as the packing material. The sponge retained sludge containing a wide variety of organisms, including microfauna. Stable isotope analysis revealed spatial, temporal, and biotic variations in the carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) of the retained sludge and microfauna (water mites and fly larvae). In addition, adult flies and spiders were present and analyzed. The δ¹³C and δ¹⁵N in sludge were temporally and spatially similar along the reactor. The isotopic signature was associated with treatment characteristics such as a low nitrification efficiency in the DHS reactor. The δ¹³C and δ¹⁵N of sympatric fly larvae differed from those of water mites, which indicated dietary differences between the taxa. Interestingly, the water mites had higher δ¹³C and δ¹⁵N than the retained sludge, which indicated that they were in a higher trophic position in the food web. In addition, the δ¹³C and δ¹⁵N values of spiders were approximately 1 ‰-3‰ higher than those of adult flies. This strongly suggested that a prey-predator relationship existed between adult flies and spiders.

A high growth rate is essential for increasing protein production efficiency in liquid fermentation of filamentous fungi, such as Aspergillus oryzae. However, the increase in culture viscosity due to fungal growth constrains the overall yield. We have demonstrated that culture viscosity is lower in A. oryzae AGΔ-GAGΔ strains, which are deficient in the cell surface polysaccharides α-1,3-glucan (AG) and galactosaminogalactan (GAG), than in the wild-type (WT) strains. Nevertheless, even in aerated fermentation, an increase in AGΔ-GAGΔ viscosity results in oxygen depletion, which limits fungal growth and enzyme production. In this study, we investigated viscosity dynamics and protein production during high-cell-density fermentation of AGΔ-GAGΔ under pure oxygen aeration. Fed-batch cultivation of the WT and AGΔ-GAGΔ strains, expressing recombinant xylanase (XynF1), was used to compare the effects of air and pure oxygen aeration at the same flow rate. At 60 h, AGΔ-GAGΔ under pure oxygen aeration showed higher cell density (1.2× WT under pure oxygen aeration, 2.1× AGΔ-GAGΔ under air aeration) and XynF1 activity (1.8× WT under pure oxygen aeration, 2.3× AGΔ-GAGΔ under air aeration). Under pure oxygen aeration, AGΔ-GAGΔ showed lower viscosity (0.32×) and mixing time (0.50×) than WT. At 60 h, fine mycelial pellets (micropellets; 200-700 μm) were clearly observed in AGΔ-GAGΔ under pure oxygen but not under air aeration. These findings suggest that oxygen enrichment during AGΔ-GAGΔ cultivation mitigated the increase in viscosity, thereby promoting higher cell density and protein production. The formation of micropellets in AGΔ-GAGΔ likely contributed to a reduction in culture viscosity.

In this study, Pseudomonas sp. NGC7 was engineered to selectively produce vanillate (VA) from aromatic compounds in the sugarcane bagasse alkaline extract. A VA O-demethylase gene (vanA4B4)-disrupted strain derived from NGC7 grew and produced VA from the extract containing no saccharides. The organic acids in the extract promoted the strain to grow. The aromatics in the extract were further concentrated by the solid phase extraction with DIAION HP20 resin, and the organic acids were fractionated into the flow-through fraction. A fed-batch culture of NGC7ΔvanA4B4 strain using this concentrated alkaline extract exhibited increased VA production; however, the accumulation of syringate (SA) and 4-hydroxybenzoate (HBA) was also observed along with VA production, despite the strain possessing the genes responsible for SA and HBA degradation. Analysis of the mutants capable of degrading SA while producing VA revealed that mutations in vanR2, a transcriptional repressor of the genes responsible for SA degradation, enabled SA degradation during VA production. In addition, the expression of praI, an HBA hydroxylase derived from Paenibacillus sp. JJ-1b, was suitable for efficient HBA degradation. Thus, the mutation in vanR2 and the expression of praI represented the key engineering strategies for achieving the selective VA production. As the growth of the engineered strains was promoted by the organic acids present in the extract, VA production from the concentrated extract was evaluated in a flow-through-based medium supplemented with mineral salts and metals. Finally, the engineered VA-producing strain produced 4.30 mM VA selectively at a yield of 77 mol% in the practical medium.

A novel two-enzyme cascade one-pot synthesis of β-alanine from 1,3-diaminopropane (DAP) has been developed. In the first step, DAP was oxidized to 3-aminopropionaldehyde (3-APAL) by diamine oxidase (DAO). In the second step, 3-APAL was oxidized to β-alanine by 3-APAL dehydrogenase (APALDH). Catalase and NADH oxidase were employed to degrade the by-product, H₂O₂, and to regenerate the cofactor, NAD⁺. DAO specific to DAP has not been reported in any prokaryote. Therefore, initial proof of concept was established using commercial eukaryotic DAO_pk (from porcine kidney) and catalase (from bovine liver), along with recombinant APALDH and NOX enzymes sourced from Arthrobacter crystallopoietes and Lactococcus lactis, respectively. β-Alanine was successfully produced via this pathway; 12.2 mM (1.1 g/L) was formed in 41 h with 12 % conversion. To increase the reaction rate, DAO with higher specific activity was identified from Arthrobacter pascens (DAO_Ap). The optimum pH and temperature of DAO_Ap were determined to be 9.0 and 37 °C, respectively. Batch enzymatic biotransformation achieved 6 % conversion, yielding 0.33 mM (29 mg/L) β-alanine in 4 h. The low titre in batch conversion was attributed to substrate inhibition affecting DAO_Ap, NOX, and catalase. Fed-batch enzymatic biotransformation was conducted to overcome substrate inhibition, yielding 47 % conversion, with 2.34 mM (63 mg/L) β-alanine formation in 4 h. Approximately a 7.5-fold increase in conversion was achieved using fed-batch enzymatic biotransformation. This study accomplished a novel two-enzyme cascade biotransformation strategy for one-pot β-alanine synthesis from DAP.

The understanding of white-rot fungi (WRF) and their role in degrading recalcitrant environmental pollutants has significantly advanced due to developments in bioremediation research. Considerable progress has been made in elucidating the degradation capabilities of WRF against lots of environmental pollutants. In this review, research hotspots on the degradation of WRF were identified through bibliometric analysis. Key findings from systematic studies on the degradation of polycyclic aromatic hydrocarbons (PAHs) and bisphenols by WRF are synthesized and discussed. Furthermore, insights into the molecular and genetic basis underlying the enzymatic systems responsible for the degradation of PAHs and bisphenols are highlighted. Advancements and challenges in understanding the degradation capabilities and degradation mechanisms are examined in order to identify opportunities for developing more effective strategies to harness the bioremediation potential of WRF.

Pseudonocardia sp. D17 (D17) is a novel strain capable of aerobically metabolizing cis-1,2-dichloroethene (cDCE), a persistent contaminant in soil and groundwater. This study aimed to investigate the cDCE degradation characteristics of D17 with respect to kinetics, associated degradative enzymes, and degradation pathways. Degradation experiments with cDCE concentrations ranging from 0.267 to 91.3 μM revealed that D17 can efficiently degrade cDCE across this range. The maximum specific degradation rate and half saturation constant for cDCE degradation by D17 were estimated to be 0.418 ± 0.045 nmol/mg-protein/min and 38.5 ± 9.2 μM, respectively. Heterologous expression experiments demonstrated that both group 5 soluble di-iron monooxygenases in D17, namely tetrahydrofuran and propane monooxygenases, can catalyze cDCE degradation with higher catalytic activity observed in the former. This suggests their involvement in cDCE degradation by D17. It was also proposed that D17 completely dechlorinates cDCE through multiple pathways to generate glyoxylic acid, which is either mineralized or incorporated into the glyoxylate cycle, with a minor portion being converted to oxalic acid as a dead-end product. These findings provide novel insights into metabolic aerobic cDCE biodegradation and highlight the potential of D17 as a bioremediation agent.