Biotechnology Journal最新文献

Evaluating the Gibbs–Donnan and volume exclusion effects during protein ultrafiltration and diafiltration (UF/DF) is crucial in biopharmaceutical process development to precisely control the concentration of the drug substance in the final formulation. Understanding the interactions between formulation excipients and proteins under these conditions requires a domain-specific knowledge of molecular-level phenomena. This study developed gradient boosted tree models to predict the Gibbs–Donnan and volume exclusion effects for amino acids and therapeutic monoclonal antibodies using simple molecular descriptors. The models’ predictions were interpreted by information gain and Shapley additive explanation (SHAP) values to understand the modes of action of the antibodies and excipients and to validate the models. The results translated feature effects in machine learning to real-world molecular interactions, which were cross-referenced with existing scientific literature for verification. The models were validated in pilot-scale manufacturing runs of two antibody products requiring high levels of concentration. By only requiring a molecule's biophysicochemical descriptors and process conditions, the proposed models provide an in silico alternative to conventional UF/DF experiments to accelerate process development and boost process understanding of the underlying molecular mechanisms through rational interpretation of the models.

Halomonas elongata thrives in hypersaline environments producing polyhydroxyalkanoates (PHAs) and osmoprotectants such as ectoine. Despite its biotechnological importance, several aspects of the dynamics of its metabolism remain elusive. Here, we construct and validate a genome-scale metabolic network model for H. elongata 153B. Then, we investigate the flux distribution dynamics during optimal growth, ectoine, and PHA biosynthesis using statistical methods, and a pipeline based on shadow prices. Lastly, we use optimization algorithms to uncover novel engineering targets to increase PHA production. The resulting model (iEB1239) includes 1534 metabolites, 2314 reactions, and 1239 genes. iEB1239 can reproduce growth on several carbon sources and predict growth on previously unreported ones. It also reproduces biochemical phenotypes related to Oad and Ppc gene functions in ectoine biosynthesis. A flux distribution analysis during optimal ectoine and PHA biosynthesis shows decreased energy production through oxidative phosphorylation. Furthermore, our analysis unveils a diverse spectrum of metabolic alterations that extend beyond mere flux changes to encompass heightened precursor production for ectoine and PHA synthesis. Crucially, these findings capture other metabolic changes linked to adaptation in hypersaline environments. Bottlenecks in the glycolysis and fatty acid metabolism pathways are identified, in addition to PhaC, which has been shown to increase PHA production when overexpressed. Overall, our pipeline demonstrates the potential of genome-scale metabolic models in combination with statistical approaches to obtain insights into the metabolism of H. elongata. Our platform can be exploited for researching environmental adaptation, and for designing and optimizing metabolic engineering strategies for bioproduct synthesis.

Terminal deoxynucleotidyl transferase (TdT), a unique DNA polymerase that catalyzes the template-free incorporation of nucleotides into single-stranded DNA, has facilitated the development of various oligonucleotide-based tools and methods, especially in the field of template-free enzymatic DNA synthesis. However, expressing vertebrate-derived TdTs in Escherichia coli complicates purification and increases production costs. In this study, N-terminal truncation of TdTs was performed to improve their expression and stability. The results revealed that N-terminal truncation could enhance the expression level of six TdTs. Among the truncated mutants, N-140-ZaTdT and N-140-CpTdT, with 140 amino acids removed, exhibited an increase in protein expression, which was 9.5- and 23-fold higher than their wild-types, respectively. Importantly, the truncation preserves the catalytic function of TdT. Additionally, the T_m values of N-140-ZaTdT increased by 4.9°C. The improved expression of the truncated mutants makes them more suitable for reducing production costs and advancing enzyme engineering.

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

21-Hydroxy-20-methylpregn-4-en-3-one (4-HBC, bisnoralcohol) is a crucial intermediate for the synthesis of steroidal drugs. Significant challenges including by-products formation and poor substrate solubility were still confronted in its main synthetic route by microbial conversion from phytosterol. Construction of a direct bioconversion pathway to 4-HBC and an efficient substrate emulsion system is therefore urgently required. In this study, three novel isoenzymes of 3-ketosteroid-Δ¹-dehydrogenase (KstD) and 3-ketosteroid 9α-hydroxylase (KsH) in Mycobacterium neoaurum were excavated and identified as KstD4, KstD5, and KsHA3. A strain capable of fully directing the synthesis of 4-HBC was metabolically engineered via serial genetic deletion combined with enhanced expression of cholesterol oxidase (ChOx2) and enoyl-CoA hydratase (EchA19). Moreover, a micro-emulsion system combined with soybean oil and hydroxypropyl-β-cyclodextrin improved substrate solubility and bioavailability. In batch fermentation, molar yield of 96.7% with 39.5 g L⁻¹ 4-HBC was obtained from 50 g L⁻¹ phytosterol. Our findings demonstrate the potential for industrial-scale biosynthesis of 4-HBC.

Extracellular vesicles (EVs) operate as chemical messengers that facilitate intercellular communication. Emerging evidence has demonstrated that lung tissue-derived EVs play pivotal roles in pulmonary physiological processes and have potential as biomarkers and therapeutics for lung diseases. Multiple methods have been proposed for the isolation of lung tissue-derived EVs. However, the effects of different tissue pre-treatments on lung EV isolation and subsequent disease biomarker discovery have not yet been comprehensively investigated. In this study, we compared the physical characteristics, recovery yields, and protein compositions of EVs isolated from lung tissues using three methods based on different tissue dissociation principles. Methodologically, the beneficial roles of blood perfusion and gentle meshing were emphasized based on their impact on EV yield and purity. These results demonstrate that different methods enrich distinct subpopulations of EVs that exhibit significant differences in their protein cargo and surface properties. These disparities directly affect the diagnostic detection of marker proteins related to lung diseases, including lung tumors, asthma, and pulmonary fibrosis. Collectively, these findings highlight the variations in EV characteristics resulting from the applied approaches and offer compelling suggestions for guiding researchers in selecting a suitable isolation method based on downstream functional studies and clinical applications.