Journal of bioscience and bioengineering最新文献

Chlamydomonas reinhardtii, which is a unicellular photosynthetic eukaryote, has long served as a model microalga for fundamental biological research and biotechnological applications. Recently, it has attracted attention as a promising biological resource for the sustainable production of bio-oils and high-value biomolecules. To enhance the biotechnological utility of this species, various genetic engineering tools have been developed in recent years. In this study, the tetracycline repressor (TetR)-based transactivation system, which is widely used in mammalian and other eukaryotic cells, was repurposed to establish a synthetic transcriptional activation system for exogenous gene expression in Chlamydomonas. We first constructed a transient expression-based evaluation platform to screen for transcriptional activation domains (TADs) that are functional in Chlamydomonas. Among the analyzed TADs, VP192 (tandem repeat of 12 copies of the core VP16 domain) had the highest transcriptional activity when fused to either TetR or Gal4 DNA-binding domains. Furthermore, fusion proteins comprising VP192 and either TetR or reverse TetR enabled doxycycline-dependent regulation of transgene expression in a dose-dependent manner. Notably, transcriptional inducibility was maintained even when the tetracycline-responsive element (TRE) was fused to the HSP70A promoter. Combining TetR-VP192 and this synthetic chimeric promoter (TRE-P_HSP) yielded transgene expression levels that exceeded those resulting from the strong HSP70A/RbcS2 hybrid promoter by more than 14-fold. These findings suggest that artificial transcription factors and engineered promoters provide a versatile molecular toolkit for regulating exogenous gene expression in Chlamydomonas.

Streptomyces rochei 7434AN4 carries three linear plasmids, pSLA2-L (211 kb), pSLA2-M (113 kb), and pSLA2-S (18 kb). Genome project of this strain revealed that the linear chromosome consists of 8.36 Mb in size and carries more than 35 biosynthetic gene clusters. Three plasmidless mutants (2-39, YN-P7, and YN-P145) showed their chromosomal telomere deletion, concomitantly with a loss of plasmids. To compare the metabolic profile of plasmidless mutants, each strain was cultivated in different mediums [YM and modified oatmeal broth (OMB) medium]. Remarkably, strain YN-P145 showed two distinct UV-active spots when it was grown in OMB medium. These UV-active compounds were purified using Sephadex LH-20 and silica gel chromatographies. The UV-active compound named YN-P145A has a molecular formula of C₁₄H₁₆N₂O₂ and was characterized to be cyclo(Phe-Pro), a diketopiperazine (cyclodipeptide) that is known to have an antifungal activity. Another UV-active compound from strain YN-P145, compound YN-P145B, has a molecular formula of C₈H₁₁NO. Extensive NMR analysis revealed that YN-P145B was identical to a cyclic enamide termed as YM3163-A, 2-(cyclohex-2-en-1-ylidene)acetamide, which has been isolated from the recombinant of SARP-type activator SRO_3163 in S. rochei. Thus, genome-wide deletion occasionally leads to accumulate natural products, whose expression was weak or not at all in its parent strain.

Tobacco fermentation is a crucial process for improving tobacco quality by reducing undesirable substances and enhancing aroma characteristics. In this study, an enzyme-microbe combined fermentation strategy was established and evaluated. Cytobacillus oceanisediminis C4 combined with amylase pretreatment was applied in the fermentation of tobacco powder (TP) to improve the quality of tobacco, yielding an enhanced sensory score of 84.50, compared with 82.00 in the untreated TP. Compared with the control, the reducing sugar content increased significantly, whereas starch and protein contents decreased markedly after combined fermentation. Furthermore, total aromatic components of TP increased by 34.77 %, along with noticeable changes in TP surface structure. Bacterial community structure analysis revealed a noteworthy shift at the genus level, with the relative abundance of Bacillus and Pseudomonas escalating from 0.30 % and 1.15 % to 74.67 % and 4.40 %, respectively. Additionally, fungal community structure analysis revealed that the relative abundance of Monascus surged from 1.69 % to 87.59 %. Metabolomic analysis demonstrated that fermentation reprogrammed the metabolic profile toward flavor-active and aroma-precursor compounds, characterized by increased levels of amino acids and phenolic acids and decreased levels of lipids and flavonoids. These coordinated changes contributed to enhanced aroma, reduced irritation, and overall improvement in tobacco quality. Overall, this study establishes an effective enzyme-microbe combined fermentation strategy, demonstrates its applicability for improving heat-not-burn (HnB) tobacco products, and provides multi-omics insights into the underlying mechanisms of quality enhancement.

This study reports the first known simultaneous production of garviecin Q and garvicin ML and a new bacteriocin (garvieacin FR6) by Lactococcus garvieae FR6. These bacteriocins were isolated from the culture supernatant of the FR6 strain using hydrophobic interaction, cation-exchange, and reverse-phase chromatography. ESI-TOF MS confirmed its predicted molecular masses of 5321.68 Da (garvieacin FR6), 5346.64 Da (garvieacin Q), and 6010.64 Da (garvicin ML), and genome sequencing identified three distinct bacteriocin gene clusters. The genome analysis revealed that garvieacin FR6 belongs to the linear class IId bacteriocin, similar to garvieacin Q, and is characterized by gene clusters that encode essential components for producing these bacteriocins, including immunity proteins and an ATP-binding cassette (ABC) transporter. In addition, garvieacin FR6 has a narrower antimicrobial spectrum compared to garvieacin Q and garvicin ML, particularly with low minimum inhibitory concentrations against Latilactobacillus sakei (0.54 μM) and Lactococcus garvieae strains (0.13 μM). Therefore, the FR6 strain produces three bacteriocins from different subclasses, each with a distinct antimicrobial spectrum. The co-expression of garvieacin Q, garvicin ML, and the novel bacteriocin garvieacin FR6 provides a practical source of bacteriocins for food biopreservation and a platform for the development of targeted antimicrobials. Especially where combined peptides may enhance effectiveness and help prevent resistance by offering complementary inhibition pressure. Future work should focus on performance and synergy in real food matrices, defining the mechanism of action and immunity/resistance determinants, and establishing scalable production, formulation, and safety data to support translational use.

The purpose of this study was to investigate the optimal cultivation strategy for long-term stable ethanol production from sweet potato starch in vertical mass flow bioreactor (VMFB) by co-immobilizing Aspergillus awamori, Rhizopus japonicus and Zymomonas mobilis. In exploring the optimum initial substrate concentration, the maximum ethanol concentration of 45.11 g/L was achieved at the 54th hour at a feed concentration of 12 % (w/v), which resulted in a conversion rate of 66.26 % compared to the theoretical value. In the repeated batch process (multiple cycles), stable ethanol production was achieved from 6 % (w/v, 3cycles) to 12 % (w/v, 5cycles) of sweet potato starch producing total ethanol concentrations of 71.03 g/L (3cycles, 72 h) and 241.54 g/L (5 cycles, 348 h), respectively. In fed-batch fermentation with a total addition of 24 % (w/v) sweet potato starch (four separate additions), the maximum ethanol concentration and yield after 114 h were 102.6 g/L and 0.43, respectively. To evaluate the potential for future industrialization, dried raw starch was used instead of gelatinized starch as a substrate. The results demonstrated that the fed-batch fermentation process had higher ethanol yield and starch conversion rates, with values of 0.32 and 55.96 % at 144 h, respectively.

Bananas (Musa acuminata) are among the world's most popular fruits and are climacteric, continuing to ripen after harvest. In Japan's banana supply chain, unripe green bananas are transported with ripening artificially suppressed. Upon arrival, the bananas are exposed to ethylene gas, which restarts the ripening process. Because these procedures involve artificial treatment and storage, quality control during post-harvest management is critical. However, current quality assessment methods rely on destructive testing, and real-time, non-contact monitoring technologies have not yet been established. This study hypothesized that a non-destructive model for predicting fruit ripeness could be developed using volatile organic compounds (VOCs) generated through biological reactions. VOCs emitted by bananas were collected and analyzed using gas chromatography-mass spectrometry (GC-MS). Orthogonal partial least squares (OPLS) regression analysis was performed, with VOC composition as explanatory variables and ripeness indicators (hue angle and soluble solids content, SSC) as response variables. The resulting model predicted banana ripeness indicators with high accuracy. Esters, which contribute to the characteristic sweet and fruity aroma of bananas, and their precursor alcohols were identified as key contributors to ripeness prediction. To the best of our knowledge, this study represents the first attempt to predict banana ripeness indicators (hue angle and SSC) non-destructively and non-invasively using VOCs emitted by the fruit. The developed model shows promise for application in banana quality control.

Molecular weight of polyhydroxyalkanoate (PHA) is a critical factor governing their physical properties, particularly mechanical strength. It is empirically known that molecular weight of PHA varies depending on factors such as the microbial host and the type of PHA synthase (PhaC). However, methods for controlling the molecular weight of PHA are limited. In this study, we found that the addition of a cyclic polyether, 18-crown-6 ether (18c6), to the culture medium of recombinant Escherichia coli increased molecular weight of poly(3-hydroxybutyrate) [P(3HB)]. Cells expressing an engineered class I PHA synthase, PhaC_AR, and propionyl-coenzyme A transferase from Megasphaera elsdenii were cultivated with the supplementation of 3HB for P(3HB) production. The P(3HB) molecular weight increased by up to 20-fold in an 18c6 concentration-dependent manner. The addition of 18c6 reduced E. coli growth, but it had no significant effect on PhaC expression levels. Furthermore, in vitro assay indicated that 18c6 does not affect the enzymatic activity of PhaC. Therefore, these results suggest that the effect of 18c6 is not attributable to its direct interaction with PhaC. The addition of 18c6 also increased the molecular weight of poly(3-hydroxypropionate), but did not increase that of poly(3-hydroxyhexanoate), indicating that the effect of 18c6 varies depending on the monomer used for the polymer synthesis. The addition of 18c6 could potentially be utilized as a versatile method for producing high-molecular-weight PHAs.

The prolonged use of current anti-inflammatory therapies has significant limitations for chronic inflammation management, requiring safer alternatives. In this study, we investigated the anti-inflammatory effect of methylxanthine derivative, pentoxifylline. MTT assay was used for the determination of the cytotoxicity of pentoxifylline. Lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells were used for the determination of the anti-inflammatory effect of pentoxifylline using Griess assay, qRT-PCR, Western blot, gene reporter assay and cell migration assay. Pentoxifylline exhibited a high level of safe therapeutic window with no cytotoxicity up to 20 μM in the MTT assay on RAW 264.7 cells. Pentoxifylline demonstrated concentration and time-dependent anti-inflammatory effects with significantly (p < 0.05) reduced NO production at 10 and 20 μM concentrations. Gene expression revealed significant downregulation of inflammatory mediator genes: Nos2, Tnfα, and Il1β (p < 0.05). Protein analysis confirmed these effects with reductions in iNOS, TNFα, IL1β, and IL6 (p < 0.05). Pentoxifylline caused a significant reduction (p < 0.05) of transcriptional activity of NF-κB and AP-1. Further, a significant reduction of nuclear translocation of NF-κB by pentoxifylline confirmed the transcriptional inhibition. Additionally, in the scratch assay, pentoxifylline exhibited a significant reduction (p < 0.05) in macrophage migration, supporting its role in inhibiting inflammation progression. These comprehensive multi-target anti-inflammatory effects of pentoxifylline support its promising potential for use as a therapeutic candidate for inflammatory diseases.

White-rot basidiomycete fungi have been the focus of research as edible mushrooms and for industrial utilization, especially due to their unique wood-degrading enzymes. Nevertheless, establishing highly efficient transformation systems for these organisms remains a substantial challenge. In filamentous ascomycetes, plasmids carrying the specific sequence, AMA1, have been widely used to enhance transformation efficiency. However, their functionality in basidiomycetes has not been systematically assessed. In this study, a pAMAHyg plasmid was constructed that harbored AMA1, in addition to a hygromycin B resistance marker gene derived from the pPHT1 plasmid. The pAMAHyg was introduced into the white-rot fungus Pleurotus ostreatus and yielded more hygromycin-resistant colonies than those with the pPHT1, despite its larger size, suggesting a positive impact on transformation efficiency. However, unlike previous reports in ascomycetes, frequent loss of drug resistance was not observed upon subculturing. Plasmid rescue experiments in Escherichia coli suggested that the circular form and original sequence of pPHT1 or pAMAHyg could be maintained in P. ostreatus, with few exceptions. Methylation-sensitive restriction enzyme analysis of the rescued plasmids confirmed the loss of bacterial Dam methylation and the acquisition of CpG methylation, regardless of AMA1 presence, suggesting the replication of these plasmids in P. ostreatus. Southern blot analysis revealed the genomic integration of the introduced plasmid as well. Collectively, these results suggest that the integrated and episomal plasmid sequences may coexist in P. ostreatus transformants. This study provides experimental evidence supporting the extrachromosomal existence of circular plasmids in the fungus, even in the absence of AMA1.

Bark compost serves as an organic fertilizer, originating from the microbial decomposition of tree bark and woody residues. An examination of 84 bacterial isolates from commercially available bark composts identified nine strains capable of degrading N-acylhomoserine lactones (AHLs), which function as signaling molecules in bacterial quorum sensing. Whole-genome sequencing of one AHL-degrading isolate, Ochrobactrum quorumnocens B44, revealed the presence of multiple AHL-degrading gene homologs, with the gene OCHQB44_21820 (aiiO) encoding the principal AHL-degrading enzyme. AiiO efficiently degraded a range of AHL molecules with acyl chain lengths of 6-10, including their 3-oxo derivatives. Contrary to previous reports suggesting that AiiO functions as an AHL acylase, hydrolyzing the amide bond of AHL, biochemical analysis in this study demonstrated that AiiO acts as an AHL lactonase, catalyzing the hydrolytic opening of the lactone ring in AHL. The purified AiiO as a maltose binding protein fusion efficiently degraded AHLs at an optimal temperature of 40 °C, maintaining stability under typical soil conditions. The expression of aiiO in the plant pathogen Pectobacterium carotovorum subsp. carotovorum causes self-degradation of biosynthesized AHL and suppresses potato tissue maceration regulated by AHL-mediated quorum sensing. Co-inoculation of B44 with P. carotovorum subsp. carotovorum attenuated soft rot symptoms in potato slices. These results showed the possibility that the application of bark compost rich in AHL-degrading bacteria enhances soil resistance to bacterial diseases.