Applied Microbiology and Biotechnology最新文献

In this study, we investigated MalS, a periplasmic α-enzyme from Escherichia coli K12, known for its unique biochemical properties related to polysaccharide utilization. Evolutionarily, MalS has inherited the glycosyl hydrolase catalytic domain from the glycoside hydrolase family 13, with the protein sequences highly conserved across Enterobacteria, including Salmonella and Shigella. MalS exhibited optimal activity at 65 °C, significantly higher than other E. coli enzymes. Although its reaction pattern resembled that of typical α-amylases, its catalytic efficiency on polysaccharides was notably lower. Intriguingly, MalS demonstrated a strong binding affinity for various glucose polymers, including β-cyclodextrin and glycogen, which significantly enhanced its thermostability. Despite full-length MalS binding strongly to glycogen, neither its N-terminal domain, predicted by AlphaFold2 to belong to the Carbohydrate-Binding Module family 69, nor the remaining parts of the enzyme showed binding affinity toward polysaccharides. Kinetic studies revealed that MalS had a 2.5-fold lower K_m and 1.4-fold higher catalytic efficiency toward glycogen compared to amylopectin, which contrasts starkly with pancreatic α-amylases. However, over prolonged reactions, glycogen hydrolysis by MalS was slower than that of amylopectin. In the early initial stage, MalS predominantly degraded glycogen to maltopentaose (G5) rather than maltohexaose (G6) as usual. Taken together, these findings suggest MalS may play a role in recognizing glycogen-type polysaccharides in the bacterial periplasm during adaptation to new environments. Given the crucial role of glycogen in the survival and infection processes of pathogenic bacteria, understanding MalS’s interaction with glycogen-type polysaccharides could offer valuable insights into bacterial survival mechanisms and their ability to infect hosts.

• MalS has unique structure and properties but conserved among many enterobacteria

• Binding of MalS with polysaccharides significantly enhanced its thermostability

• Unlike other amylases, MalS showed 2.5-fold lower K_m on glycogen than amylopectin

A comparative genomic analysis of feline coronavirus (FCoV) and feline panleukopenia virus (FPLV) was performed. Based on the conserved regions of the two viruses, specific probes and real-time PCR (qPCR) primers were designed, and a duplex TaqMan qPCR-based assay was established for detecting FCoV and FPLV. The results showed high analytical specificity, and no cross-response with other feline viruses was observed. This method is highly versatile and can be used to detect all FCoV strains stored in laboratories and recombinant plasmids constructed according to the sequences of blank FCoV strains in laboratories. The analytical sensitivity of this method in detecting FCoV and FPLV was as low as 50 copies/μL, which is approximately 20-fold greater than that of conventional PCR. The coefficients of variation (CVs) for the intra- and interbatch coefficients of variation were less than 2%. After 75 clinical samples were tested, the percentage of FCoV- and FPLV-positive samples was 5.34% greater than that of conventional PCR methods, a finding robustly supported by sequencing identification. As validated by clinical samples, the method was sensitive, specific, general, and reproducible and holds great potential for the rapid identification and diagnosis of FCoV and FPLV infections, as well as for epidemiological investigations.

• One-step duplex TaqMan real-time PCR detection method can detect FCoV and FPLV in clinical samples simultaneously and steadily.

• Almost all the currently known FCoV and FPLV strains can be detected.

• This method has high sensitivity, specificity and generality.

Despite the lack of implementation of consolidated bioprocessing (CBP) at an industrial scale, this bioconversion strategy still holds significant potential as an economically viable solution for converting lignocellulosic biomass (LCB) into biofuels and green chemicals, provided an appropriate organism can be isolated or engineered. The use of Saccharomyces cerevisiae for this purpose requires, among other things, the development of a cellulase expression system within the yeast. Over the past three decades, numerous studies have reported the expression of cellulase-encoding genes, both individually and in combination, in S. cerevisiae. Various strategies have emerged to produce a core set of cellulases, with differing degrees of success. While one-step conversion of cellulosic substrates to ethanol has been reported, the resulting titers and productivities fall well below industrial requirements. In this review, we examine the strategies employed for cellulase expression in yeast, highlighting the successes in developing basic cellulolytic CBP-enabled yeasts. We also summarize recent advancements in rational strain design and engineering, exploring how these approaches can be further enhanced through modern synthetic biology tools to optimize CBP-enabled yeast strains for potential industrial applications.

• S. cerevisiae’s lack of cellulolytic ability warrants its engineering for industry.

• Advancements in the expression of core sets of cellulases have been reported.

• Rational engineering is needed to enhance cellulase secretion and strain robustness.

• Insights gained from omics strategies will direct the future development of CBP strains.

The E2 subunit vaccine has been considered a promising alternative to an attenuated classical swine fever (CSF) vaccine. However, it fails to induce a good cellular immune response. Given that immunogenic adjuvants can regulate the cellular immunity to achieve a maximum efficacy against antigens, immunostimulatory effects of porcine IL-28B on the CSF virus (CSFV) E2 subunit vaccine were evaluated in the present study. We expressed recombinant proteins E2-IL28B, E2, and IL-28B using CHO-S mammalian cells as an antigen expression platform, and three types of CSFV E2 subunit vaccines based on antigens E2-IL28B, E2 + IL-28B, and E2 were prepared, respectively. We found that both E2-IL28B and E2 + IL-28B antigens exhibited superior immunogenicity with dramatically induced antibody titers and neutralizing antibody levels than the E2 alone. Moreover, E2-IL28B or E2 + IL-28B, instead of E2, boosted cellular immune responses via obviously increasing the percentages of CD3⁺CD4⁺ T lymphocytes, promoting the lymphocyte proliferations, and enhancing the release of Th1-type cytokines. All results revealed that the inclusion of IL-28B, whether fused or mixed with E2, significantly elevated E2-induced immune potencies, suggesting that IL-28B could be used as a molecular adjuvant to optimize the design of E2 subunit vaccine for more effective controls of the CSF disease.

• New CSF E2 subunit vaccine candidates were developed in which IL-28B was an immunoadjuvant

• IL-28B significantly elevated the E2-induced immune potency whether it was fused or mixed with E2

• This study provided novel insights into the immunoregulatory properties of IL-28B used for the optimized subunit vaccine design

The high mortality rate associated with single-use CRISPR-Cas9 in Escherichia coli limits its application. Recently, new CRISPR-based techniques for E.coli gene editing have emerged. Research aims to develop a system for rapid, marker-free, multi-site, and multi-copy genome editing in E.coli to advance synthetic biology. ATP, essential for energy in living organisms, plays a crucial role in various metabolic processes. To reduce the cost of ATP-requiring reactions, it is crucial to identify and efficiently express genes in ATP synthesis pathway. This study identified a single ppk gene (No.8) capable of completing the cyclic reaction. Using MUCICAT technology, the ppk gene (No.8) was inserted into various positions and copy numbers in the E.coli genome, resulting in different activity levels. The findings suggest that the difficulty of inserting the ppk gene (No.8) into the genome follows this order: IS186 < 8array < IS186 + 8array < IS1. A single genome insertion can mimic plasmid expression level. This study explores promoter competition and offers solutions, inspiring researchers in constructing the AMP-ATP cycle system in E.coli.

• The single ppk gene (No.8) can regenerate the AMP-ATP cycle, crucial for ATP-dependent reactions.

• Inserting the ppk gene (No.8) into the cr5 site of the E.coli genome achieves expression levels comparable to the pET29a plasmid.

• The expression level of the ppk gene (No.8) is not significantly affected by its copy number in the E.coli genome.

Human skin wounds primarily heal through reparative wound healing without pilosebaceous units or other appendages, rather than regenerative wound healing. Hair follicle (HF) regeneration is a significant challenge for skin wound healing. The effects and underlying mechanisms of Isaria cicadae Miquel rice fermentation extract (IMFRE) remain unclear, although it has anti-inflammatory, antioxidant, and reparative effects on oxidative damage in keratinocytes. We assessed the regenerative wound healing ability of IMFRE and its related molecular mechanisms through experimental validation and network pharmacology analysis. Our findings suggest that IMFRE could be an important potential solution for regenerative wound healing of skin hair follicle by utilizing the Hippo pathway regulatory mechanism.

• IMFRE was found to significantly enhance the wound healing rate of mouse skin.

• CK15 and CD34 were significantly increased by high-dose IMFRE intervention.

• IMFRE could inhibit EGFR, GPCR, and Integrin expression.

Filamentous fungi or mycelia are a valuable bioresource to produce several biomolecules and enzymes, especially because of their biodegradation potential and for their key role of enablers of a circular bioeconomy. Filamentous fungi can be grown in submerged cultivation to maximise the volumetric productivity of the bioprocess, instead of using the more established and time-consuming solid-state cultivation. Multicellular mycelia are sensitive to shear stresses induced by mechanical agitation, and this aspect greatly affects their morphology in submerged cultivation (pelletisation) and the connected volumetric productivity. An efficient compromise is the growth of filamentous fungi in airlift bioreactors (ALR) where the volumetric oxygen transfer (K_La) is optimal, but the shear stress is reduced. In this review, we critically analysed the advantages and disadvantages of ALR-based cultivation of filamentous fungi, comparing these bioreactors also with stirred tank reactors and bubble column reactors; we focused on scientific literature that highlights findings for the cultivation of filamentous fungi for both the production of enzymes and the production of myco-biomass in ALR; we included studies for the control of the pelletisation of the fungal biomass in batch and semi-continuous cultivation, highlighting the interlinked hydrodynamics; finally, we included studies regarding the modifications of ALR in order to enhance filamentous fungi production.

• ALR are efficient for batch and prolonged continuous cultivation of filamentous fungi.

• ALR show both optimal gas hold-up and K_La with an airflow that has high superficial velocity and critical bubble diameter (1–6 mm).

• Suspended mycelia aggregates (pellet) maintain a fluidised motion in ALR if their size/density can be controlled.

Immune checkpoint inhibitors (ICIs) have significantly advanced the field of cancer immunotherapy. However, clinical data has shown that many patients have a low response rate or even resistance to immune checkpoint inhibitor alone. The underlying reasons for its poor efficacy include the deficiency of immune infiltration and effective CD28/CD80 costimulatory signal in tumor. Discoidin domain receptor 1 (DDR1) has been reported to be negatively related to immune cell infiltration in tumors. Herein, we constructed a soluble fusion protein using CD80, the natural ligand of CD28, in combination with DDR1 inhibitor. Our results demonstrated that CD80-Fc effectively activated T cells and inhibited tumor growth in vivo, even in tumors with poor efficacy of ICIs. Importantly, CD80-Fc fusion protein had a milder affinity against the targets which suggested a potential higher safety than CD28 agonists. Further, in order to promote tumor immune infiltration, we attempted to combine CD80-Fc fusion protein with DDR1 inhibitor for treatment. Our results indicated that using CD80-Fc fusion protein along with DDR1 inhibitor significantly promoted T cell infiltration in tumor microenvironment and more strongly inhibited tumor growth. Therefore, the combination use of CD80 fusion protein and DDR1 inhibitor could become an effective tumor immunotherapy strategy, potentially benefiting a larger number of patients.

• We successfully constructed, expressed, and purified the recombinant CD80-Fc fusion protein

• We demonstrated that CD80-Fc fusion protein has good safety and anti-tumor activity

• We demonstrated that using CD80-Fc fusion protein along with DDR1 inhibitor can significantly promote immune infiltration of T cells in tumor microenvironment and more strongly inhibit tumor growth

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies, efficient and scalable expansion procedures must be developed. For other adherent human cell types, the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study, a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings, indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion (({N}_{s1u})), thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics, computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner, improved process understanding allowed an expansion factor of ≈ 26 to be achieved, yielding more than 3 × 10⁹ cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability, identity, and differentiation potential throughout cultivation.

• ({N}_{s1u}) can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors

• Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion

• Perfusion is advantageous and supports the cultivation of hiPSCs