Biotechnology Journal最新文献

Organoids have emerged as valuable tools for the study of development and disease. Assembloids are formed by integrating multiple organoid types to create more complex models. However, the process by which organoids integrate to form assembloids remains unclear and may play an important role in the resulting organoid structure. Here, a microfluidic platform is developed that allows separate culture of distinct organoid types and provides the capacity to partially control the geometry of the resulting organoid surfaces. Removal of a microfabricated barrier then allows the shaped and positioned organoids to interact and form an assembloid. When midbrain and unguided brain organoids were allowed to assemble with a defined spacing between them, axonal projections from midbrain organoids and cell migration out of unguided organoids were observed and quantitatively measured as the two types of organoids fused together. Axonal projection directions were statistically biased toward other midbrain organoids, and unguided organoid surface geometry was found to affect cell invasion. This platform provides a tool to observe cellular interactions between organoid surfaces that are spaced apart in a controlled manner, and may ultimately have value in exploring neuronal migration, axon targeting, and assembloid formation mechanisms.

The cover image is based on the Research Article Unlocking the formate utilization of wild-type Yarrowia lipolytica through adaptive laboratory evolution by Qian Chen et al., https://doi.org/10.1002/biot.202400290.

Enzymes that degrade β-glucan play important roles in various industries, including those related to brewing, animal feed, and health care. Csph16A, an endo-β-1,3(4)-glucanase encoded by a gene from the halotolerant, xerotolerant, and radiotrophic black fungus Cladosporium sphaerospermum, was cloned and expressed in Pichia pastoris. Two isoforms (Csph16A.1 and Csph16A.2) are produced, arising from differential glycosylation. The proteins were predicted to contain a catalytic Lam16A domain, along with a C-terminal domain (CTD) of unknown function which exhibits minimal secondary structure. Employing PCR-mediated gene truncation, the CTD of Csph16A was excised to assess its functional impact on the enzyme and determine potential alterations in biotechnologically relevant characteristics. The truncated mutant, Csph16A-ΔC, exhibited significantly enhanced thermal stability at 50°C, with D-values 14.8 and 23.5 times greater than those of Csph16A.1 and Csph16A.2, respectively. Moreover, Csph16A-ΔC demonstrated a 20%–25% increase in halotolerance at 1.25 and 1.5 M NaCl, respectively, compared to the full-length enzymes. Notably, specific activity against cereal β-glucan, lichenan, and curdlan was increased by up to 238%. This study represents the first characterization of a glucanase from the stress-tolerant fungus C. sphaerospermum and the first report of a halotolerant and engineered endo-β-1,3(4)-glucanase. Additionally, it sheds light on a group of endo-β-1,3(4)-glucanases from Antarctic rock-inhabiting black fungi harboring a Lam16A catalytic domain and a novel CTD of unknown function.

Bone tissue engineering offers a promising alternative to stimulate the regeneration of damaged tissue, overcoming the limitations of conventional autografts and allografts. Recently, titanium alloy (Ti) implants have garnered significant attention for treating critical-sized bone defects, especially with the advancement of 3D printing technology. Although Ti alloys have impressive versatility, their lack of cellular adhesion, osteogenic and antibacterial properties are significant factors that contribute to their failure. Hence, to overcome these obstacles, this study aimed to incorporate osteoinductive and antibacterial cue-loaded hydrogels into 3D-printed Ti (3D-Ti) scaffolds. 3D-Ti scaffolds were synthesized using the direct metal laser sintering method and loaded with a gelatin (Gel) hydrogel containing strontium-doped silver nanoparticles (Sr-Ag NPs). Compared with Ag NPs, Sr-doped Ag NPs increased the expression of Runx2 mRNA, which is a key bone transcription factor. We subjected the bioactive 3D-hybrid scaffolds (3D-Ti/Gel/Sr-Ag NPs) to physicochemical and material characterization, followed by cytocompatibility and osteogenic evaluation. The microporous and macroporous topographies of the scaffolds with Sr-Ag NPs showed increased Runx2 expression and matrix mineralization, with potent antibacterial properties. Therefore, the 3D-Ti scaffolds incorporated with Sr-Ag NP-loaded Gel hydrogels favored osteoblast differentiation and antibacterial activity, indicating their potential for orthopedic applications.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

Through iterative rounds of mutation and selection, proteins can be engineered to enhance their desired biological functions. Nevertheless, identifying optimal mutation sites for directed evolution remains challenging due to the vastness of the protein sequence landscape and the epistatic mutational effects across residues. To address this challenge, we introduce MLSmut, a deep learning-based approach that leverages multi-level structural features of proteins. MLSmut extracts salient information from protein co-evolution, sequence semantics, and geometric features to predict the mutational effect. Extensive benchmark evaluations on 10 single-site and two multi-site deep mutation scanning datasets demonstrate that MLSmut surpasses existing methods in predicting mutational outcomes. To overcome the limited training data availability, we employ a two-stage training strategy: initial coarse-tuning on a large corpus of unlabeled protein data followed by fine-tuning on a curated dataset of 40−100 experimental measurements. This approach enables our model to achieve satisfactory performance on downstream protein prediction tasks. Importantly, our model holds the potential to predict the mutational effects of any protein sequence. Collectively, these findings suggest that our approach can substantially reduce the reliance on laborious wet lab experiments and deepen our understanding of the intricate relationships between mutations and protein function.

Natural sesquiterpene are valuable compounds with diverse applications in industries, such as cosmetics and energy. Microbial synthesis offers a promising way for sesquiterpene production. Methanol, can be synthesized from CO₂ and solar energy, serves as a sustainable carbon source. However, it is still a challenge to utilize methanol for the synthesis of value-added compounds. Pichia pastoris (syn. Komagataella phaffii), known for its efficient utilization of glucose and methanol, has been widely used in protein synthesis. With advancements in technology, P. pastoris is gradually engineered for chemicals production. Here, we successfully achieved the synthesis of α-bisabolene in P. pastoris with dual carbon sources by expressing the α-bisabolene synthase gene under constitutive promoters. We systematically analyzed the effects of different steps in the mevalonate (MVA) pathway when methanol or glucose was used as the carbon source. Our finding revealed that the sesquiterpene synthase module significantly increased the production when methanol was used. While the metabolic modules MK and PMK greatly improved carbon source utilization, cell growth, and titer when glucose was used. Additionally, we demonstrated the synthesis of β-farnesene from dual carbon source by replacing the α-bisabolene synthase with a β-farnesene synthase. This study establishes a platform strain that is capable to synthesize sesquiterpene from different carbon sources in P. pastoris. Moreover, it paves the way for the development of P. pastoris as a high-efficiency microbial cell factory for producing various chemicals, and lays foundation for large-scale synthesis of high value-added chemicals efficiently from methanol in P. pastoris.

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.