[Objective] To obtain a yeast strain efficiently producing the acidic protease PrA for applications in food processing, feed additives, and other related industries. [Methods] We constructed a recombinant strain of Pichia pastoris expressing PrA by fermentation in shake flasks and measured the enzymatic properties of the expressed PrA. Several strategies, such as signal peptide modification, gene dosage optimization, and co-expression with molecular chaperones, were employed to enhance the production of PrA. Additionally, high-density fermentation was employed to further improve the expression level. [Results] The expressed enzyme PrA showcased the specific activity of 3 974.00 U/mg, with the optimum performance at pH 3.0 and 45 ℃. The production of PrA by the parental strain was 738.03 U/mL. The modification of the MF4I signal peptide increased the production of PrA to 1 206.52 U/mL. Moreover, an increase in the copy number of prA further increased the PrA production to 2 406.47 U/mL. Additionally, co-expression with single or combined molecular chaperones increased the PrA production to 4 091.27 U/mL. After undergoing high-density fermentation, the enzyme activity reached 43 088.00 U/mL within 168 h, representing a 58.4-fold increase compared with the initial production. [Conclusion] High-level expression of PrA was achieved in P. pastoris, which laid a foundation for the future industrial applications. The results provide valuable insights into the research and development of PrA for applications in food processing and feed additives.

[Objective] Monensin is a polyether antibiotic produced by Streptomyces cinnamonensis. To enhance the production of monensin by microbial fermentation, we employed metabolic engineering to strengthen the synthesis pathway of the key precursor methylmalonyl-CoA in S. cinnamonensis 2110. [Methods] Firstly, crotonyl-CoA reductase (CCR) was overexpressed to strengthen the acetoacetyl-CoA pathway. Subsequently, methylmalonyl-CoA mutase (MCM) was overexpressed to improve the succinyl-CoA pathway. Finally, an engineered strain with tandem overexpression of CCR and MCM was constructed and evaluated for the fermentation performance. [Results] The overexpression of CCR increased the strain biomass and monensin titer by 10.4% and 19.0%, respectively, after 10 days of shake-flask fermentation. The overexpression of MCM increased the monensin titer by 9.9%, whereas it did not increase the strain biomass after 10 days of shake-flask fermentation. The tandem overexpression of CCR and MCM increased the biomass and monensin titer by 9.4% and 26.8%, respectively, after 10 days of shake-flask fermentation. In a 5 L bioreactor, the engineered strain 2110-CCR-MCM reached the highest biomass of 54.6 g/L and monensin titer of 11.3 kU/mL, which increased by 12.7% and 36.2%, respectively, compared with those of the starting strain 2110. [Conclusion] CCR and MCM mediated the key metabolic pathway of monensin biosynthesis in S. cinnamonensis, and the overexpression of CCR and MCM was highly favorable for monensin synthesis. This study provides technical reference for the engineering of strains with high yields of other polyketides.

[Objective] Outer membrane vesicles (OMVs) are spherical bilayer membrane structures secreted by Gram-negative and some Gram-positive bacteria. OMVs contain abundant surface antigens and are of great research significance in vaccine development. However, the presence of lipopolysaccharides (LPS), which is the primary component of OMVs, arouses safety concern. Therefore, genetically modifying bacterial LPS to produce safe and efficient OMVs is a viable approach to enhance the production and application of OMVs. [Methods] We modified Salmonella minnesota Re595 with O antigen and most core antigen deletions by deleting the acyl chain coding gene msbB and inserting the phosphatase coding gene lpxE from Francisella novicida to reduce acyl chains and phosphate groups on lipid A, thus obtaining less toxic LPS. LPS and OMVs were extracted from the starting strain and modified strain, and their pro-inflammatory activities were compared between the two strains. In addition, inactivated foot-and-mouth disease virus vaccines were prepared with OMVs to assess the immune adjuvant activity of OMVs. [Results] The modification of LPS reduced the endotoxin activity and pro-inflammatory responses while significantly increasing the immune adjuvant activity of OMVs. [Conclusion] This study demonstrates that the modification of LPS can attenuate the toxicity and enhance the immune adjuvant activity of bacterial OMVs. These findings provide a theoretical foundation for utilizing OMVs as immune adjuvants in the future.

Streptococcus pneumoniae causes serious diseases such as pneumoniae and meningitis in humans. Capsular polysaccharides (CPSs) surrounding bacteria are not only key virulence factors but also major antigens. Therefore, CPSs have been prepared into polysaccharide vaccines and polysaccharide conjugate vaccines, which have greatly reduced the infection of pneumococci. CPSs are formed by polymerization of oligosaccharide repeating units which generally have 2−8 monosaccharides. CPSs present complex structures with diverse antigenic epitopes, being the basis of bacterial serotyping. Currently, 107 serotypes of S. pneumoniae have been identified. Each serotype has a unique CPS structure, a stable genetic basis, and specific serological characteristics. The diversity and constant changes of CPS structures explain the difficulty in the eradication of pneumococci. This review summarizes the known chemical structures of 95 CPSs and discusses the genetic basis, biosynthesis mechanism, and purification methods of CPSs. This review aims to enrich the knowledge about CPS diversity and provide a reference for probing into the functions and evolution of CPSs as well as for preparing polysaccharide vaccines.

Isoquercetin is a flavonoid with antioxidant, anti-inflammatory, and immunomodulatory activities. However, the low content in plants poses a challenge to the large-scale production of isoquercetin by the extraction method.[Objective] α-L-rhamnosidase can specifically hydrolyze the terminal L-rhamnose residues of natural glycosides. In this study, we screened the strains capable of efficiently and specifically transforming rutin to produce isoquercetin with rutin as the sole carbon source and applied the α-L-rhamnosidase to the production of isoquercetin, aiming to provide new elements for the large-scale production of isoquercetin. [Methods] The selective culture medium with rutin as the sole carbon source was used to screen and identify the strains that can specifically hydrolyze rutin into isoquercetin. The transcriptome analysis was carried out to obtain highly efficient and specific α-L-rhamnosidase, the domain composition of which was determined by structural simulation. The enzymatic properties and substrate specificity of the α-L-rhamnosidase were studied. Furthermore, the hydrolysis effect of the enzyme heterologously expressed in Pichia pastoris in a 5 L fermenter was determined. [Results] AfRhase had five domains, including one α-domain (domain A) and four β-domains (domains N, E, F, and C). With rutin as the substrate, the recombinant enzyme AfRhase showcased the best performance at 55 ℃ and pH 4.5. AfRhase had a wide range of substrates including rutin, hesperidin, naringin, and epimedin C. In a 5 L fermenter for scaled-up production of isoquercetin, P. pastoris expressing AfRhase generated 61 g isoquercetin by hydrolyzing 120 g crude rutin (purity of 70%), with the molar conversion rate of 95.4% and production efficiency of 2.0 mmol/(L·h). [Conclusion] This study for the first time discovered a highly efficient and specific α-L-rhamnosidase from Aspergillus sp. XT-1 for the production of isoquercetin from rutin and heterologously expressed this enzyme in P. pastoris. The domain composition, enzymatic properties, substrate specificity, and hydrolysis efficiency in a 5 L fermenter of this enzyme were determined. In conclusion, this study broadened the function of a fungus-derived α-L-rhamnosidase for the transformation of rutin and laid a foundation for the industrial production of isoquercetin.

Monocarboxylates such as lactate, pyruvate, and ketone bodies play an important role in the metabolic activities of organisms. As a monocarboxylate transporter, MpMch2 is mainly responsible for the transmembrane transport of monocarboxylates and the maintenance of glucose metabolism balance.[Objective] To analyze the functions of MpMch2 in Monascus purpureus. [Methods] The MpMch2 in M. purpureus Mp-21 was replaced with the hygromycin gene by homologous recombination to construct the deletion strain ∆MpMch2. The colony and cell morphology of Mp-21 and ∆MpMch2 on different media was observed, and the yield of monascus pigment, γ-aminobutyric acid, conidia and ascospores were determined. The expression levels of genes related to conidia and γ-aminobutyric acid were determined by RT-qPCR. [Results] There was no significant difference in the colony morphology between the wild type and ∆MpMch2 on different media. After knockout of MpMch2, the yields of conidia, ascospores, Monascus pigments, and γ-aminobutyric acid decreased, and the expression levels of related genes were down-regulated. [Conclusion] MpMch2 positively regulated the development of conidia and ascospores and the production of Monascus pigments and γ-aminobutyric acid.

Transfer-message RNA (tmRNA) is a ubiquitous and stable non-coding small RNA in bacteria, with characteristic regions similar with both tRNA and mRNA. tmRNA is known to mediate the ribosome rescue mechanism called trans-translation and has effects on the pathogenicity and stress responses of pathogenic bacteria. [Objective] To study the function of tmRNA in Aeromonas veronii causing threats to aquaculture and human public safety, so as to reveal the molecular mechanism for the pathogenicity of A. veronii. [Methods] IntaRNA 2.0 was used to predict the downstream targets binding to tmRNA. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were conducted to predict the biological processes and signaling pathways involving the predicted targets. Real-time qPCR was employed to compare the expression levels of the candidate target genes among the wild type, tmRNA knockout strain, and smpB knockout strain of A. veronii, thus identifying the potential targets regulated by tmRNA in the form of sRNA. [Results] One hundred potential specific downstream targets might bind to tmRNA at the 3′-end tRNA-like domain (tRNA-like domain, TLD), H2 domain, and PK3 and PK4 regions, thus participating in the general metabolic pathways of the pathogen. The results of qPCR indicated that the expression of WP_201994931.1 was regulated by tmRNA in an SmpB-independent manner, while the expression of WP_201954220.1, WP_005335875.1, WP_265062582.1, WP_265061484.1, and WP_265061494.1 was regulated by SmpB. [Conclusion] We preliminarily identified that WP_201994931.1 might constitute a downstream target regulated by tmRNA as an sRNA. This study provides basic information for expanding the functions of tmRNA in the form of sRNA and facilitates the further studies regarding the molecular mechanisms of the pathogenicity and environmental adaptation of A. veronii.

Bacterium-mediated cancer immunotherapy (BCI) presents numerous advantages in cancer treatment, while the immune response mechanism of dexamethasone (DEX) combined with BCI for tumor treatment remains unclear. [Objective] To investigate the therapeutic efficacy and mechanism of dexamethasone in combination with attenuated Salmonella typhimurium St.ΔppGpp-mediated BCI. [Methods] The inhibitory effects of St.ΔppGpp+DEX on cancer were evaluated in a murine model of colorectal cancer. In vivo imaging was utilized to determine the tumor targeting and colonization duration of St.ΔppGpp. Organ toxicity resulted from St.ΔppGpp+DEX treatment was assessed by hematoxylin and eosin (H&E) staining. Macrophage polarization, neutrophil recruitment, and T-cell responses were analyzed by flow cytometry and immunofluorescence assay of sections. The changes in inflammatory cytokines in the tumor microenvironment were examined via qRT-PCR. A mouse model transplanted with human colorectal cancer was employed to confirm the effect of T cell depletion on the therapeutic efficacy of St.ΔppGpp+DEX. [Results] The combined treatment St.ΔppGpp+DEX significantly decreased tumor size and enhanced the survival rate of mice. DEX extended the colonization of St.ΔppGpp in tumor cells. Furthermore, St.ΔppGpp+DEX did not induce damage to vital immune organs, and it facilitated the polarization of macrophages from M2 to M1 phenotype while suppressing neutrophil recruitment. T cell depletion did not influence the efficacy of St.ΔppGpp+DEX. [Conclusion] DEX can enhance the anti-tumor effects of St.ΔppGpp by inhibiting neutrophil recruitment and increasing the proportion of M1 macrophages in the tumor microenvironment.

[Objective] The protein SakA encoded by sakA is a member of the mitogen-activated protein kinase (MAPK) family in Aspergillus niger. However, little is known about the roles of SakA in A. niger. In this study, we constructed the A. niger strains with knockout of sakA to investigate the roles of this gene. [Methods] The Agrobacterium-mediated method was utilized to construct ΔsakA strains from A. niger RAF106 (the wild type, WT). The growth and spore production of ΔsakA and WT were observed on three different media. The sensitivity of ΔsakA and WT to different stress conditions was studied. The intracellular and extracellular levels of amylase, pectinase, and cellulase were compared between ΔsakA and WT. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to determine the relative transcript levels of the genes associated with spore production, amylase, pectinase, cellulase, and hyperosmotic regulation. [Results] Three ΔsakA strains were successfully obtained and verified by PCR and qRT-PCR. The ΔsakA strains had slow growth, delayed spore production, and delayed conidiophore differentiation compared with WT. The ΔsakA strains showcased slower colony growth than WT under the stress conditions of 0.6 mol/L KCl, 0.8 mol/L NaCl, and 1.2 mol/L NaCl. Compared with WT, the knockout of sakA increased the extracellular amylase production by 20.68%–21.43% and decreased the intracellular amylase production by 19.18%–20.26%, decreased the extracellular pectinase production by 36.71%–38.30% and increased the intracellular pectinase production by 35.68%–36.53%, decreased the extracellular cellulase production by 28.04%–33.82% and increased the intracellular cellulase production by 15.28%–18.19%. Compared with WT, the knockout of sakA down-regulated the transcript levels of spore production-related genes (fluG, sfgA, flbA, flbB, flbD, laeA, brlA, abaA, vosA, stuA, and velB) by 8.53%–90.87%. Furthermore, it down-regulated the transcript levels of amylase-related genes (amyC, amyD, amyE, amyF, amyG, and amyH) and the transcription factor (amyR) by 8.87%–87.50%, the pectinase-related genes (aglB, lacA, pexB, pecA, pecC, pecB, endA, endC, and poly) by 23.23%–84.01%, the cellulase-related genes (xlnR, chbA, chbB, and eglB) by 3.75%–81.02%, and the hyperosmotic regulation-related genes (ena1, ena2, sho1, nik1, ypdl, pkA, and hAD) by 5.27%–94.36%. [Conclusion] The sakA gene of A. niger positively regulates spore production and is essential for spore production. The knockout of sakA affects the spore production of A. niger. Furthermore, SakA plays a crucial role in the synthesis and secretion of amylase, pectinase, and cellulase as well as osmotic stress response.

[Objective] To study the morphological and physiological characteristics of hybrids compared with their parents and contribute to research on the mechanisms of speciation and evolution. [Methods] Sonneratia×hainanensis, a natural hybrid of the mangrove plants Sonneratia alba and S. ovata, usually presents hybrid weakness than its parents. In this study, Illumina high-throughput sequencing was employed to compare the rhizosphere microbiomes (including bacteria and fungi) between the hybrid and its parents, on the basis of which the reason for hybrid weakness was explored. [Results] The principal coordinate analysis (PCoA) revealed no significant difference in the rhizosphere bacterial or fungal community structure between the hybrid and its parents. However, the rhizosphere microbiome of the hybrid was different from that of the female parent S. alba with strong survival ability but similar to that of the male parent S. ovata. The rhizosphere bacteria belonged to 388 genera, 320 families of 76 phyla. The dominant phylum Pseudomonadota had the relative abundance above 41.00% in the rhizosphere of the three plant species, reaching 55.33% in the hybrid, which was higher than that in the parents. At the genus level, 18 common genera including Desulfococcus (3.23%) and Rhodoplanes (0.94%) in all the three mangrove plants showed the relative abundance of 15.77%. Among them, 8 salt-tolerant genera such as Mariprofundus showed decreased relative abundance in the hybrid, which may affect the salt tolerance. The rhizosphere fungi were dominated by Ascomycota and Basidiomycota with the relative abundance of 41.89% and 4.53%, respectively, which was significantly lower than that in the parents. Moreover, the predominant fungal genera were different in the three mangrove plants. Functional annotation of prokaryotic taxa (FAPROTAX) predicted that the mangrove prokaryotes were involved in sulfur metabolism and nitrogen metabolism. Although the hybrid had higher Shannon and Simpson indexes of rhizosphere bacteria than S. alba, some dominant taxa such as B-42 (unclassified Trueperaceae), Mariprofundus, and Sulfurimonas participating in the nitrogen cycle were not inherited by the hybrid. The soil total nitrogen (TN) and total phosphorus (TP) of the hybrid was significantly lower than that of S. alba. TN was significantly positively correlated with the relative abundance of Mariprofundus, B-42, Aspergillus, and Rhodotorula, which, however, demonstrated decreased relative abundance in the rhizosphere of the hybrid. [Conclusion] The results help to understand the mechanisms of hybrid weakness in Sonneratia×hainanensis.