Engineering in Life Sciences最新文献

The synthesis of enantiopure α-hydroxy ketones, particularly R- and S-phenylacetylcarbinol (PAC), represents an important process in the pharmaceutical industry, serving as a pivotal step in the production of drugs. Recently, two novel enzymes, ephedrine dehydrogenase (EDH) and pseudoephedrine dehydrogenase (PseDH), have been described. These enzymes enable the specific reduction of 1-phenyl-1,2-propanedione (PPD) to R-PAC and S-PAC, respectively. In this study, we transferred these enzymes into Pickering emulsions, which is an attractive reaction set-up for large-scale synthesis. The bioactive w/o Pickering emulsion (bioactive Pickering emulsion [BioPE]), in which methyl tert-butyl ether served as the continuous phase, was stabilized by silica nanoparticles. Formate dehydrogenase from Rhodococcus jostii was utilized for cofactor regeneration. Given the considerable complexity of the BioPE, this reaction system underwent a first-time application of design of experiment (DOE) for systematic engineering. A definitive screening design was employed to identify significant factors affecting space-time yield (STY) and conversion. Response surface methodology was used to optimize the conditions, resulting in the observation of a high STY of 4.2 g L⁻¹ h⁻¹ and a conversion of 83.2% for BioPE with EDH, and an STY of 4.4 g L⁻¹ h⁻¹ and a conversion of 64.5% for BioPE with PseDH.

The demand for lentiviral vectors (LVs) as tools for ex vivo gene therapies is ever-increasing. Despite their promising applications, challenges in LV production remain largely due to the fragile envelope, which challenges the maintenance of vector stability. Thus, downstream processing optimization to enhance efficiency, yield, and product quality is necessary. This study investigated the influence of membrane types and filtration devices during ultrafiltration (UF). Nine different membrane materials consisting of polyethersulfone (PES), regenerated cellulose, or Hydrosart, with distinct molecular weight cutoffs, were evaluated in stirred cells, centrifugal ultrafilters, and crossflow cassettes. The evaluation was based on the ability to retain infectious LV particles and remove impurities. The analysis revealed that a reinforced 100 kDa PES and a 300 kDa Hydrosart membrane had the best overall ability to concentrate infectious LVs and remove DNA, especially when operated in a stirred cell. Challenges were seen in the nonoptimized crossflow cassette process, where infectious LV recovery was generally lower compared to other devices. We demonstrated that membrane material and filtration device have a direct impact on the efficiency of LV UF.

Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L⁻¹, and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.

Analyzing the relationship between cell morphology, rheological characteristics, and production dynamics of cultivations with filamentous microorganisms is a challenging task. The complex interdependencies and the commonly low reproducibility of heterogeneous cultivations hinder the bioprocess development of commercially relevant production systems. The present study aims to characterize process parameters in Actinomadura namibiensis shake flask cultures to gain insights into relationships between culture behavior and rheological characteristics during salt-enhanced labyrinthopeptin A1 production. Plate–plate (PP) and vane–cup rheometer measurements of viscous model fluids and culture broths are compared, revealing a more uniform distribution of broth when measured with the PP system. Additionally, rheological characteristics and culture performance of A. namibiensis cultures are evaluated using online data of the specific power input and the oxygen transfer rate. It is demonstrated that salt-enhancement labyrinthopeptin A1 production by the addition of 50 mM (NH₄)₂SO₄ increases the apparent viscosity of the A. namibiensis culture by four-fold and significantly reduces the reproducibility of the culture resulting in a 46 h difference in lag-phase duration. This approach demonstrates that the culture behavior of complex filamentous cell morphologies is challenging to decipher, but online monitoring of rheology and oxygen transfer can provide valuable insights into the cultivation dynamics of filamentous microbial cultures.

Bioelectrochemical systems (BESs) offer a sustainable method for chemical production, including the enhanced production of succinic acid. By combining fermentation with BES, it could be possible to achieve sustainable succinic acid production and CO₂ fixation using Actinobacillus succinogenes. In literature, the potential application of BES is commonly associated with increased succinate yields, as it is expected to enhance the availability of NADH, thereby influencing the intracellular nicotinamide adenine dinucleotide (NADH/NAD⁺) balance. However, it remains unclear whether BES can improve NADH regeneration and achieve higher NADH/NAD⁺ ratios across all growth phases of A. succinogenes. This study investigates the impact of an applied electrical potential on the intracellular NADH/NAD⁺ ratio during an electrochemical-assisted fermentation process. Using an adapted high-performance liquid chromatography method with a Supelcosil LC-18-T column, it was demonstrated that NADH availability in BES, particularly during the stationary growth phase, improved by up to 1.98-fold compared to the control. This enhancement in reducing power led to a succinate yield of 0.747 ± 0.01 g g⁻¹, representing a 15.65% increase compared to a fermentation without electrochemical assistance. These findings support the expectation that the use of BES could enhance the competitiveness of bio-based succinate production.

Raman spectroscopy, a robust and non-invasive analytical method, has demonstrated significant potential for monitoring biopharmaceutical production processes. Its ability to provide detailed information about molecular vibrations makes it ideal for the detection and quantification of therapeutic proteins and critical control parameters in complex biopharmaceutical mixtures. However, its application in Saccharomyces cerevisiae fermentations has been hindered by the inherent strong fluorescence background from the cells. This fluorescence interferes with Raman signals, compromising spectral data accuracy. In this study, we present an approach that mitigates this issue by deploying Raman spectroscopy on cell-free media samples, combined with advanced chemometric modeling. This method enables accurate prediction of protein concentration and key process parameters, fundamental for the control and optimization of biopharmaceutical fermentation processes. Utilizing variable importance in projection (VIP) further enhances model robustness, leading to lower relative root mean squared error of prediction (RMSEP) values across the six targets studied. Our findings highlight the potential of Raman spectroscopy for real-time, on-line monitoring and control of complex microbial fermentations, thereby significantly enhancing the efficiency and quality of S. cerevisiae-based biopharmaceutical production.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology and biomedical research, but there is still a need to shorten the operation process and reduce the cost of antibodies. This study aims to assess a novel IHC technique that incorporates mechanical microvibration (MMV) to expedite the process, reduce antibody consumption, and enhance staining quality. MMV was generated using coin vibration motors attached to glass slides mounted with consecutive tissue sections. Multiple antibodies targeting various antigens were used to stain cancerous and normal tissues, with and without microvibration. Various parameters were tested, including incubation durations, temperatures, and antibody dilutions. The novel method showed the potential to achieve comparable or superior outcomes in significantly less time, utilizing over 10 times less antibody than controls. MMV improved specific staining quality, yielding stronger, and better-defined positive reactions. This was validated through a multicenter double-blind assessment and quantitative image analysis. The possible mechanisms were also investigated. MMV shortens immunohistochemical staining duration, reduces antibody usage, and enhances staining specificity, likely by accelerating antibody movement and diffusion. These improvements translate to time and cost savings, offering clinical and financial value for diagnostic pathology and biomedical research.