Engineering in Life Sciences最新文献

Industrial biocatalysis, a multibillion dollar industry, relies on the selectivity and efficacy of enzymes for efficient chemical transformations. However, enzymes, evolutionary adapted to mild biological conditions, often struggle in industrial processes that require harsh reaction conditions, resulting in reduced stability and activity. Enzyme immobilization, which addresses challenges such as enzyme reuse and stability, has therefore become a vital strategy for improving enzyme use in industrial applications. Traditional immobilization techniques rely on the confinement or display of enzymes within/on organic or inorganic supports, while recent advances in synthetic biology have led to the development of solely biological in vivo immobilization methods that streamline enzyme production and immobilization. These methods offer added benefits in terms of sustainability and cost efficiency. In addition, the development and use of multifunctional materials, such as magnetic (nano)materials for enzyme immobilization, has enabled improved separation and purification processes. The combination of both “worlds,” opens up new avenues in both (industrial) biocatalysis, fundamental science, and biomedicine. Therefore, in this review, we provide an overview of established and recently emerging methods for the generation of magnetic protein immobilizates, placing a special focus on in vivo immobilization solutions.

The industrially attractive biopolymer poly-γ-glutamic acid (γ-PGA) is commonly produced by species of the genus Bacillus by co-feeding different carbon- and nitrogen-sources. Recent studies have highlighted the pivotal role of co-metabolization of a rapidly degradable carbon source such as glycerol together with citrate for γ-PGA production, independently fueling biomass generation as well as tricarboxylic acid (TCA) cycle precursor supply. With this study, we report that the sole presence of citrate in the production medium greatly influences growth behavior, γ-PGA production, and the viscosity of microbial cultures during biopolymer synthesis. Independent of the citrate concentration in the medium, only minor amounts of citrate were imported by B. subtilis 168 in the presence of glycerol due to carbon catabolite repression. However, a high citrate concentration resulted in a 6-fold increase in γ-PGA titer compared to low exogenous citrate levels. Data suggests that citrate was not used as a precursor in γ-PGA synthesis but rather influenced the fate of imported glutamate. The citrate concentration also affected medium viscosity as depletion resulted in a remarkable spike in culture broth viscosity. Additionally, cellular proteome analysis at different levels of citrate availability revealed significant changes in protein abundance involved in motility and fatty acid degradation.

Practical Application: This research provides critical insights into optimizing γ-PGA production in Bacillus subtilis, particularly by using citrate supplementation to control medium viscosity and improve production yields. The study reveals that citrate not only plays a role in controlling viscosity but also influences intracellular glutamate metabolism, a key factor for γ-PGA synthesis. Citrate interacts with divalent cations such as Mg²⁺ and Ca²⁺, reducing electrostatic interactions and thus decreasing viscosity in the medium. Additionally, while citrate uptake is limited due to carbon catabolite repression (CCR), even the minimal presence of citrate impacts growth and production. The findings suggest that citrate may trigger unexplored regulatory mechanisms affecting glutamate utilization. Their understanding opens new avenues for industrial optimization, which focus on enhancing glutamate synthesis pathways and exploring novel citrate-sensing mechanisms. Overall, this research lays the groundwork for improving the efficiency and consistency of γ-PGA production by fine-tuning media components and understanding their metabolic effects.

This study focuses on applying microbial self-healing cement in repairing cracks in cement-based materials and enhancing its resistance to water penetration performance. Traditional cement is susceptible to environmental influences, leading to the formation of microcracks and a reduction in durability. This research used Bacillus pseudofirmus to prepare microcapsules through sodium alginate gelation technology. We mixed microcapsules into the cement. The results indicate that the microbial self-healing cement, with a 1% self-healing agent added, increased its resistance to water penetration ability by 29.2% after 28 days. This improvement rose to 39.3% after 84 days. Additionally, we used the embedded needle method to make mortar blocks through microcracks, mimicking the cracks found in real cement. The self-healing effect of the microcapsules was especially noticeable for cracks under 0.3 mm in diameter, compared to the commonly used commercial crystallization penetration technology. This is attributed to the crystalline bodies formed by the self-healing agent in the microcapsules blocking the cracks and preventing water penetration. This study provides an environmentally friendly solution for the repair of cracks in cement-based materials using microbial self-healing technology and lays the foundation for improving the repair efficiency and durability and exploring stability and reliability in the future.

Practical Application: This study investigated the application of microbial self-healing cement in repairing cracks in cement-based materials and enhancing its resistance to water penetration properties. Cement, a material widely used in infrastructure, has low tensile strength and often forms microcracks. These microcracks reducing the durability of cement and posing risks to the economy and safety. Adding 1% self-healing agent to microbial self-healing cement significantly increases the resistance to water penetration pressure of the mortar blocks. Compared to the standard specimens, the resistance to water penetration ability increased by 29.2% at 28 days and further increased to 39.3% at 84 days. Microbial self-healing cement could effectively restore the resistance to water penetration performance of the mortar blocks after repairing cracks. The repairing results are significantly better than the methods of mixing or applying cement crystalline materials.

Combined cross-linked enzyme aggregates (combi-CLEAs) represent a promising carrier-free immobilized enzyme technology. This study describes the preparation of combi-CLEAs comprising leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) for the regeneration of cofactor nicotinamide adenine dinucleotide necessary for 2-aminobutyric acid production. Different from traditional methods using ammonium sulfate or organic reagents as precipitant, this work utilized low concentrations of calcium ions to purify and precipitate the histidine-tagged enzymes. We developed a simple and environmentally friendly protocol for combi-CLEAs formation, involving precipitation with 10 mM calcium ions at an enzyme activity ratio of 1:2 for LeuDH and FDH, respectively, followed by cross-linking with 0.15% (w/v) glutaraldehyde at 20°C for 2 h at pH 7.5. The optimal catalytic reaction temperature and pH value for the combi-CLEAs were determined to be a temperature of 37°C and a pH of 7.5. The combi-CLEAs demonstrated enhanced thermal and pH tolerance compared to the free enzyme mixture. Moreover, the combi-CLEAs showed good operational stability, retaining 40% of its initial activity after seven cycles of reuse. These findings suggest that the combi-CLEAs of LeuDH and FDH are an efficient and cost-effective option for 2-aminobutyric acid production.

Retraction: Y.A. Arani, Z. Noormohammadi, B. Rasekh, F. Yazdian, and H. Kazemi, “Evaluation of SDS-Coated Iron Nanostructure on the Gene Expression of Bio Surfactant-Producing Genes by Pseudomonas Aeruginosa,” Engineering in Life Sciences 22, no. 9 (2022): 584–593, https://doi.org/10.1002/elsc.202200002.

The above article, published online on 24 August 2022, in Wiley Online Library (http://onlinelibrary.wiley.com/), has been retracted by agreement between the journal Editors-in-Chief, An-Ping Zeng and Ralf Takors; and Wiley Periodicals LLC. Following an investigation by the publisher, the parties have concluded that this article was accepted solely on the basis of a compromised peer review process. In addition, a third party informed the publisher that Figures 4 and 5 were reproduced from two articles published either previously or in the same year, and were used here in a different scientific context. The publisher has investigated and confirmed this, and found additional image manipulation in Figure 4. Therefore, the article must be retracted. Corresponding author Behnam Rasekh disagrees with this decision.

The enhanced surface cylindrical flask (ESCF) consists of an eight-striped inner arrangement holding 16 standard microscopic slides placed inside a cylindrical vessel. The specially designed spatula-accessible slides can be withdrawn from the vessel during cultivation without disturbing biofilm formation through an innovative window-flap accessibility mechanism. The vessel and its accessories were three-dimensional (3D) printed by applying a fused deposition modeling technique utilizing biodegradable polylactic acid. Biofilms of clinically relevant bacteria namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were successfully grown in the ESCF and observed through confocal laser scanning microscopy. Advantages of the device include an enhanced surface area for biofilm formation, ease of insertion and removal of microscopic slides, convenient fitting into standard rotary shaker platforms, creation of anoxic/microaerophilic environment inside the vessel as well as the feasibility of pH, dissolved gases, and metabolite measurements in the liquid surrounding the biofilm. The ESCF will find widespread application in medical, industrial, and environmental disciplines.

Antimicrobial peptides (AMPs) are host defense peptides that act against a broad spectrum of microorganisms. AMPs are of high interest as medicinal products, antimicrobial coatings, and for controlling biofilm formation. Applications and research of many AMPs are still hampered by insufficient titers and lack of production platforms that can tolerate high titers of AMPs. Corynebacterium glutamicum is an excellent microbial host for protein secretion and has been barely explored as a host for AMP production. Here, we report the successful production and secretion of two AMPs (amounts of up to 130 mg/L for liquid chromatography peak I [LCI] and 54 mg/L for Psoriasin) by C. glutamicum with low amounts of secreted byproducts.

Recombinant adeno-associated virus (rAAV) vector production is a complex process in which the robust cultivation of human embryonic kidney cells (HEK293) plays a critical role in generating high-quality viral vectors. Tracking the viable cell concentration (VCC) during upstream production is essential for process monitoring and for implementing actions that ensure optimal process management. The advent of inline capacitance probes has introduced a crucial process analytical technology (PAT) tool for real-time VCC measurement. Here, we present the development and application of a method for real-time monitoring of VCC in HEK293-based rAAV vector production. In a first step, BioPAT Viamass probes were used to record capacitance data of individual 10 L rAAV-8 batches within a frequency range of 50 kHz–20 MHz. Based on the capacitance data, a linear single-frequency model and an orthogonal partial least square (OPLS) multifrequency model for VCC prediction were developed. Subsequently, these models were deployed inline, and predictions were exposed into BioPAT MFCS bioprocess control software, enabling real-time VCC monitoring in subsequent rAAV-8 production batches. In addition, the continuous VCC signal was used as input for an exponential cell growth model that was deployed inline to provide accurate real-time forecasting of the transfection time point. To the best of our knowledge, this is the first example of inline deployment of VCC and Time-Till-Transfection predictive models to the bioprocess control system for real-time monitoring and forecasting of these parameters in HEK-cell-based transient rAAV vector production.

For about 100 years, the shake flask has been established for biotechnological cultivations as one of the most important cultivation systems in early process development. Its appeal lies in its simple handling and highly versatile application for a wide range of cell types—from bacteria to mammalian cells. In recent decades, extensive research has been conducted on the shake flask, to not perform processes blindly but to gain a deeper understanding of the various process parameters, phenomena, and their impact on the process. Although the characterization of the shake flask is now well-established in literature, many publications show that this knowledge is often inadequately applied. Therefore, this review provides an overview of the current state of knowledge on various topics related to the shake flask. We first present the key process parameters and their influence on different physical phenomena, such as power input, the largely unknown in-phase/out-of-phase phenomenon, as well as temperature and mass transfer. Then, the most common online monitoring systems that have been established for shake flasks are discussed. Finally, various pitfalls that often arise from inadequate knowledge of handling shake flask cultivations are discussed and guidance on how to avoid them is provided.