Journal of biotechnology最新文献

Promoters are crucial elements for controlling gene expression in cells, yet lactic acid bacteria (LAB) often lack a diverse set of available constitutive promoters with quantitative characterization. To enrich the LAB promoter library, this study focused on the known strong constitutive promoter P₂₃ in LAB. Through error-prone PCR and dNTP analog-induced random mutagenesis, a library of 247 mutants of P₂₃ was generated by using the red fluorescent protein (RFP) fluorescence intensity as a high-throughput screening indicator in Streptococcus thermophilus. The activity of P₂₃ mutants varied from 0.01 to 3.63 times that of P₂₃. Similar trends of promoter strength were observed in Lactobacillus plantarum and Lactococcus lactis, but significant differences in Escherichia coli, indicating the library's specificity to LAB. To assess the application potential of this P₂₃ library, seven promoters with different strengths (0.28-2.58) were selected. The mutant promoters significantly enhanced the enzyme activities of superoxide dismutase (SOD), β-glucuronidase (GusA), and β-galactosidase (β-gal) in S. thermophilus. Notably, the mutant P_23-203 expressing SOD exhibited an enzyme activity of 382.9 U/mg, which was 1.65 times higher than the control (P₂₃). Similarly, the expression of GusA and β-gal were 1.82 and 1.28 times higher than those of P₂₃, respectively. This study provided a set of significantly different P₂₃ constitutive promoter mutant elements for the first time, laying the foundation for metabolic engineering and synthetic biology applications in LAB.

Hairy roots mediated by Agrobacterium rhizogenes can be obtained from the composite plants (plants with hairy roots and untransformed aerial parts) by ex vitro method. Composite plants can produce higher amounts of secondary metabolites by merging hydroponic systems. This provides a stable condition for composite plants, in which various metabolites are produced in different parts. In the present study, composite plants of Glycyrrhiza glabra were produced under ex vitro conditions and transferred into a hydroponic culture medium to produce and extract secondary metabolites. The results showed a 4.8- and 1.8-fold enhancement in the expressions of SQS1 and bAS genes in the roots of the composite plants compared to the control group, respectively. The levels of glycyrrhizin (1.7) and glabridin (3.5) were higher in the leaves of the composite plants compared to the controls. Moreover, higher amounts of glabridin (1.7) were observed in the roots of the composite plants compared to the control group. Investigation of the oxidative enzymes in the composite plants and control group revealed that the plants used more secondary metabolites through Agrobacterium inoculation. The plants needed more antioxidant enzymes to counter the release of oxygen-free radicals in control conditions, but composite plants used secondary metabolites as scavengers. Data revealed that composite plants managed to produce high amounts of various secondary metabolites in a hydroponic system.

This study explores the immobilization of lipase from Candida rugosa (CRL) on hemp tea waste to catalyze the esterification of oleic acid with primary aliphatic C2-C12 alcohols. in a solvent-free system. The immobilization method employed was adsorption, chosen for its simplicity, low cost, and ability to preserve enzyme activity. The esterification of undecanoic acid, lauric acid, and oleic acid with alcohols of varying chain lengths (ethanol, 1-propanol, 1-butanol, 1-octanol, 1-decanol, and 1-dodecanol) was studied. The esterification efficiency was found to be influenced by the type of alcohol, the molar ratio of oleic acid to alcohol, and the water content in the reaction medium. The highest conversions were achieved with ethanol (23% conversion with 18.5% water and a molar ratio of 1:5) and 1-decanol (30% conversion with no added water and a molar ratio of 1:2). Comparatively, the soluble lipase achieved slightly higher conversion of oleic acid using decanol (38%), indicating that the performance may be attributed to origin of lipase, rather than the immobilization procedure. The study demonstrated that the immobilization of lipase on hemp tea waste did not hinder enzyme activity. Additionally, the biocatalyst developed can function in a completely solvent-free system, offering a green solution by repurposing waste materials for industrial ester production.

This study was aimed to develop a highly productive microbial fermentation process for gamma-aminobutyric acid (GABA) production from glucose. For this, an efficient GABA-producing E. coli strain was firstly developed through metabolic engineering with a strategy of increasing the flux of GABA biosynthetic pathway and deleting or repressing the GABA shunt pathways that compete with GABA biosynthesis. According to this strategy, three metabolically engineered E. coli strains of GTB, GTS, and A1S1 were constructed, and through batch cultivation of these strains, E. coli GTS was ultimately selected as the most efficient GABA-producing strain. From flask cultures, E. coli GTS was found to produce 3.96 g/L of GABA, a titer 2.1 times or 17 % higher than that produced by E. coli GTB or E. coli A1S1, respectively. To maximize GABA production from glucose, pH-stat fed-batch culture conditions of the E. coli GTS were optimized in a one-factor-at-a-time manner. Fed-batch cultivation of the E. coli GTS under optimal conditions resulted in the highest GABA production performance with a concentration of 85.9 g/L and a volumetric productivity of 2.37 g/L/h. This result shows that the microbial fermentation process developed in this study has outstanding potential for the mass production of GABA.

Shake flasks are a foundational tool in early process development by allowing high throughput exploration of the design space. However, lack of online data at this scale can hamper rapid decision making. Oxygen transfer rate (OTR) monitoring has been readily applied as an online process characterization tool at the benchtop bioreactor scale. Recent advances in modern sensing technology have allowed OTR monitoring to be available at the shake flask level. It is now possible to multiplex time-of-action (e.g., Induction, temperature shift, pH shift, feeding initiation, point of harvest) characterization studies by relying on careful analysis of OTR profile kinetics. As a result, there is potential to save time and capital expenditures while exploring process intensification studies though accurate and physiologically relevant online data. In this article, we detail the application of OTR monitoring to characterize the impact that recombinant protein production has on an inducible CHO cell line expressing Palivizumab. We then test out time-of-action studies to intensify protein production outcomes. We observe that recombinant protein expression causes a metabolic load that diminishes potential biomass growth. As a result, when compared to a control standard process, delaying induction and temperature shift has the potential to improve viable cell densities (VCD) by 2-fold thus increasing recombinant protein yield by over 30 %. The study also demonstrates that OTR can serve as a useful tool to detect cessation of exponential growth. Consequently, time-of-action points that are characteristic of inducible systems can be formulated accurately and reliably to maximize production performance.

Modern management of chicks hinders the vertical transmission of intestinal microbiota, which is closely related to immunity. Inulin is a substrate that can be utilized by the microbiota. This study aimed to determine whether fecal microbiota transplantation (FMT) combined with inulin played a "1 + 1 > 2" role in enhancing the development and function of immune organs. Chicks were treated with 1 % inulin and/or fecal microbiota suspension on days 1-6. The growth performance, immune organ development, and immune indicators were evaluated on days 7, 14, and 21. Results showed that the combination of FMT and inulin significantly increased the immune organ index on day 7 and promoted the morphological structure and the expression of proliferating cell nuclear antigen (PCNA) in immune organs on days 7, 14, and 21. Each treatment increased the gene expression of interferon-gamma (IFN-γ), interleukin-4 (IL-4), interleukin-2 (IL-2), B cell-activating factor receptor (BAFFR), B cell linker (BLNK), C-X-C Motif Chemokine Ligand 12 (CXCL12), C-X-C Motif Chemokine Receptor 4 (CXCR4), and Biotin (Bu-1) to varying degrees. FMT combined with inulin significantly increased the expression of IgA-positive cells on days 7 and 14. In conclusion, the synergistic effect of FMT and inulin had beneficial impacts on the development and function of immune organs.

Chinese Hamster Ovary (CHO) cells produce monoclonal antibodies and other biotherapeutics at industrial scale. Despite their ubiquitous nature in the biopharmaceutical industry, little is known about the behaviors of individual transfected clonal CHO cells. Most CHO cells are assessed on their stability, their ability to produce the protein of interest over time. But CHO cells have primarily been studied in bulk, instead assuming that these bulk samples are homogenous because of presumed genetic clonality across the sample. This does not address cellular heterogeneity in these ostensibly clonal cells. These variable stability phenotypes may reflect heterogeneity within the clonal samples. In this study, we performed single-cell RNA sequencing on two clonal CHO-K1 cell populations with different stability phenotypes over a 90 day culture period. Our data showed that the instability of one of the clone's gene expression was due in part to the emergence of a low-producing subpopulation in the aged samples. This low-producing subpopulation did not exhibit markers of cellular stress which were expressed in the higher-producing populations. Further multiomic investigation should be performed to better characterize this heterogeneity.

Recombinant adeno-associated viruses (rAAVs) comprise a promising viral vector for therapeutic gene delivery to treat disease. However, the current manufacturing capability of rAAVs must be improved to meet commercial demand. Previously published omics studies indicate that rAAV production through transient transfection triggers antiviral responses and endoplasmic reticulum stress responses in the host cell. Both responses negatively regulate viral production. We demonstrate that the modulation of the antiviral immune response (by blocking interferon signaling pathways) can effectively lower the production of interferon and enhance viral genome production. The use of interferon inhibitors before transfection can significantly increase rAAV production in HEK293 cells, with up to a 2-fold increase in productivity and up to a 6-fold increase in specific productivity. Compared to the untreated groups, the addition of these small molecules generally reduced viable cell density but increased vector productivity. The positive candidates were BX795 (a TBK inhibitor), TPCA-1 (an IKK2 inhibitor), Cyt387 (a JAK1 inhibitor), and ruxolitinib (another JAK1 inhibitor). These candidates were identified using deep well screening, and reproducible titer improvement was achieved in a 30mL shake flask scale. Additionally, genome titer improvement is feasible and scalable in two different media, but the extent of improvement may vary.

11α-Hydroxyandrost-4-ene-3,17-dione (11α-OH AD) is an essential steroid hormone drug intermediate that exhibits low biotransformation efficiency. In this study, a mixed-strain fermentation strategy was established for the efficient production of 11α-OH AD from phytosterols (PS). Initially, strains were screened for efficient transformation of AD to produce 11α-OH AD. Subsequently, a dual-strain mixed-culture fermentation technique was established, with Mycolicibacterium neoaurum CICC 21097 ΔksdD (MNR) showing highly effective results. Ultimately, a one-step conversion process for the production of 11α-OH AD was achieved at a molar yield of 76.5 % under optimal conditions using PS as a substrate, the highest reported yield to date. Additionally, studies revealed synergistic metabolic interactions between MNR and Aspergillus ochraceus in the mixed-culture system. These findings provide valuable insights for the industrial production of high-value products using mixed-strain fermentation.

Chiral azacyclic amine derivatives occupy a vital role of nitrogen-containing compounds, due to serve as foundational motifs in numerous pharmaceuticals and bioactive substances. Novel complementary enantioselective reductive aminases IRED9 and IRED11 were unveiled through comprehensive gene mining from Streptomyces viridochromogenes and Micromonospora echinaurantiaca, respectively, which both demonstrated enantiomeric excess (ee) values and conversion ratios of up to 99 % towards N-Boc-3-pyridinone (NBPO) and cyclopropylamine. IRED9 exhibited the highest activity at pH 8.0 and 45 °C，while IRED11 have optimal conditions at pH 8.0 and 50 °C. A variety of amine donors and ketones could be converted by IRED9 and IRED11 for asymmetric synthesis of piperidinamine and derivatives with complementary enantioselectivity. Through preparative-scale synthesis of (S)- and (R)-3-piperidinamine, IRED9 and IRED11 demonstrate substrate loadings of 120 g·L^-1 and 40 g·L^-1 with 98 % yield and 99 % ee, respectively. The space time yield (STY) reached 142.7 g·L^-1d^-1 and 47.1 g·L^-1d^-1 for the S enantiomer and R enantiomer, respectively. Interaction analysis indicated the substrate orientation and strong charge attraction interaction are vital factors for enantioselectivity of IREDs. This study unveils novel enantioselective reductive aminases for stereodivergent synthesis of piperidinamine and derivatives at high substrate loading.