Biotechnology and Bioprocess Engineering最新文献

Abstract

Effective diagnostics and therapeutics are foundational to modern medical technology. As the drug modality transitions from traditional small-molecule drugs to more intricate protein and nucleic acid-based therapies, there is mounting momentum towards personalized therapeutic approaches. These evolving paradigms challenge conventional biomanufacturing practices, highlighting the need for agile and patient-centric methodologies. Cell-free protein synthesis (CFPS), which facilitates the direct and programmable production of recombinant proteins, emerges as a compelling alternative. It offers rapid, adaptable, and customized protein production capabilities. Furthermore, by leveraging the core molecular mechanisms of gene expression, CFPS is gaining traction in the diagnostics realm, addressing the growing need for sophisticated diagnostic tools. In this review, we survey recent advancements and discuss how CFPS is poised to markedly reshape therapeutic production and diagnostic methods in the foreseeable future.

The importance of a single-use bioreactor (SUB) is continuously increasing in research and industrial fields for biopharmaceutical production. In this study, a newly developed SUB system, the CELBIC^® system, applied with the unique agitation method is introduced, and its physical properties and biological applications are evaluated. The mixing time was in the range of 11.0–129.0 s, and the volumetric mass transfer coefficient, k_La, measured through surface aeration, was in the range of 2.7–15.5 h⁻¹ at a working volume of 1–10 L. Biological evaluations using two Chinese hamster ovary (CHO) cell lines, CHO-DG44 and CHO-S cell, were carried out in the CELBIC^® systems in 1 and 50 L scales, showing similar cell culture performance as that from stirred tank-type bioreactors. These results support the CELBIC^® system as a new SUB system applicable to cell cultures for biopharmaceutical production.

Abstract

Many Halomonas spp. thrive in high-salinity environments, and their resistance to high salt levels allows for their cultivation in non-sterile conditions. Despite their robustness and potential poly (3-hydroxybutyrate) (PHB) production capability, there are relatively few reports on the engineering of various Halomonas species, and there are still some difficulties in genetically engineering novel Halomonas strains. In particular, conjugation as a transformation method has been employed more frequently than electroporation in Halomonas; however, electroporation is necessary for the accelerated engineering of Halomonas and increased time efficiency. To touch this issue, we collected Halomonas strains and evaluated their PHB production and electroporation efficiencies resulting that the Halomonas sp. YK44 showed the highest electroporation efficiency with high PHB production among the various Halomonas strains. A series of electroporation protocol optimization experiments were conducted to identify optimal conditions for Halomonas sp. YK44 such as main culturing for 10 h, utilizing a DNA concentration of 150–200 μg/mL, and performing electroporation at 2.1 kV, followed by a washing step using 10% glycerol and a recovery period of 36 h with pBBR1MCS2. By introducing isobutanol biosynthetic genes using an optimized electroporation protocol, the highest isobutanol production was obtained at 196 mg/L with 63% PHB content simultaneously and the higher PHB production was obtained at 6.6 g/L with 152 mg/L isobutanol. Our approach showed the overall process to identify a suitable Halomonas host by applying general electroporation methods, optimizing electroporation protocols, and demonstrated the first coproduction of PHB and isobutanol in Halomonas.

Cell-free synthesis technology is emerging as a versatile platform for biomanufacturing, particularly for on-site production of essential products. In this study, we address a pivotal challenge encountered when utilizing cell-free protein synthesis (CFPS) for on-site production in resource-limited settings. The efficacy of CFPS critically depends on precise temperature regulation. However, traditional heating methods are impractical for application outside of laboratory environments. To address this challenge, we propose an innovative solution that integrates gold nanorods (GNRs) with photothermal properties into the CFPS reaction mixture. Upon activation by a handheld laser module emitting near-infrared light, these GNRs efficiently convert light energy into heat, enabling rapid and precise temperature control for protein synthesis. Our approach not only achieves optimal synthesis temperatures more rapidly than conventional methods but also significantly enhances protein yields, with an increase of over threefold within a 30-min reaction period. In summary, our findings highlight the potential of photothermal heating as a means to ensure portable and efficient protein synthesis in the field. This advancement brings CFPS closer to achieving real-time, on-site protein production.

Modern chemical processes, vital for diverse product manufacturing, often result in substantial energy consumption and environmental waste. As environmental concerns continue to escalate and industries evolve to meet customized demands, biomanufacturing emerges as a promising alternative due to its efficiency, expandability, and eco-friendliness. Cell-free synthesis systems, which harness cellular extracts for biosynthetic reactions, offer a highly adaptable solution for biomanufacturing, particularly when rapid production is required with limited resources. While conventional cell-free systems encounter challenges related to storage and transportation due to the necessity for ultra-cold temperatures, recent studies have demonstrated that these systems can be lyophilized and rehydrated to enable on-demand biomolecule production. Our study aims to enhance the stability of lyophilized cell-free systems. We have discovered the significant role played by antioxidants, specifically dithiothreitol and sodium nitrite, in preserving translational activity during extended storage. This finding represents a significant step forward for decentralized, on-demand protein production using cell-free methods.

Bacillus subtilis is a natural producer of 2,3-butanediol (2,3-BDO) and has acquired “Generally Regarded as Safe" status. It is reported to produce 2,3-BDO from synthetic sugars as well as complex and economic sugar sources such as molasses. However, the rate-limiting step in the formation of 2,3-BDO is its conversion from acetoin to 2,3-BDO by the enzyme butanediol dehydrogenase (2,3-BDH). Such 2,3-BDHs were screened based on higher affinity (lower K_m) towards acetoin as substrate. The in silico docking studies were conducted for further validation, and they showed a high interaction profile for the PpBDH protein towards acetoin. Heterologous expression of these genes was studied in engineered Bacillus subtilis (BS1A1). In this study, it was seen that 2,3-BDH from Paenibacillus polymyxa ZJ-9 was reported to have higher enzyme activity levels, and in the fermentation studies, it was seen that the ratio of 2,3-BDO to acetoin was increased by 80.25%. The insights encourage further bioprocess optimization for increasing the fermentative production of 2,3-BDO. Our results provide a potential strategy to avoid the back conversion of 2,3-BDO to acetoin in an engineered Bacillus system.

A total of 817 Lactobacillus-like isolates were obtained from different sources, including soil, ruminant gastric juice, and fermented foods. They were screened to select the best isolate for the production of 1,3-propanediol (1,3-PDO). The isolate SY235 was selected and designated as Lactobacillus reuteri based on the 16S rRNA gene sequence. It was then employed in fed-batch fermentation for producing 1,3-PDO. The highest titer of 1,3-PDO (52.6 ± 2.4 g/L) was attained at 60 h, corresponding to a yield of 0.55 ± 0.01 g/g glycerol and a productivity of 0.88 ± 0.04 g/L/h. No further production was observed after 60 h of incubation. These results demonstrate that the newly isolated strain L. reuteri SY235 can efficiently transform glycerol into 1,3-PDO.

Elicitation of antibody and cell-mediated immune responses are crucial for successful vaccine development against tuberculosis (TB). Mycobacterium tuberculosis (Mtb) antigens CFP10 and ESAT6, potent and proven vaccine candidates require appropriate adjuvants to trigger better immune response. Virus-like particles carrying repetitive copies of foreign antigens can induce both T and B cell-mediated immunity required for conferring protection against intracellular pathogens. In this study, we developed hybrid potyvirus-like particles (PVLPs) displaying mycobacterial antigens on their surface by translationally fusing the coat protein (CP) gene derived from Johnson grass mosaic virus with CFP 10 or/and ESAT 6 gene(s). The recombinant plasmids carrying fusion constructs were transformed into Escherichia coli, the fusion proteins, viz. ESAT6-CP, CP-CFP10 and ESAT6-CP-CFP10, were expressed and purified using Ni-NTA²⁺ affinity chromatography under denaturing conditions. The chimeric CP fusion proteins were self-assembled in vitro into PVLPs by the gradual removal of denaturing conditions. The purified hybrid PVLPs carrying Mtb antigens when injected into mice showed enhanced immunogenicity for both ESAT6 and CFP10 antigens compared to the same antigens immunized without any adjuvant. In vitro stimulation of splenocytes derived from mice immunized with chimeric PVLPs upregulates the expression of cytokines involved in TB immune response.

The development of analytical methods for endogenous therapeutic substances is a critical but challenging issue as obtaining a blank matrix without endogenous substance is impossible. To address this issue, we prepared a surrogate biological matrix by removing endogenous bile acids from rat plasma using a charcoal-stripped method and developed an analytical method for ursodeoxycholic acid (UDCA) and its conjugated metabolites, tauroursodeoxycholic (TUDCA) and glycoursodeoxycholic acid (GUDCA), including the use of surrogate matrices and protein precipitation method. In addition, we applied the bioanalytical method to investigate the bioavailability of UDCA-mixed micelle powder formulation (UDCA-MM). The oral bioavailability of UDCA in rats was calculated as 15.2% and increased 3.32-fold following the oral administration of UDCA-MM with the increased production of TUDCA without significant change in GUDCA. The UDCA-MM powder was prepared by thin-layer hydration and subsequent freeze-drying method in a ratio of UDCA/polysorbate 80/poloxamer 407 = 1:1:10 (w/w/w). The UDCA-MM was easily dispersed with a particle size of 16.5 ± 2.2 nm and solubility of 1120 ± 38 μg/mL, which represented a 175.3-fold increase in its solubility of UDCA. In conclusion, we developed and validated a simple and reliable bioanalytical method for UDCA, TUDCA, and GUDCA using the charcoal-stripped plasma as surrogate matrices. Our bioanalytical method successfully supported the assessment of the pharmacokinetics or bioavailability of UDCA, TUDCA, and GUDCA after the intravenous or oral dosing of UDCA and UDCA-MM. The UDCA-MM using poloxamer 407 and polysorbate 80 is a promising technique for increasing the solubility and oral absorption of UDCA.