Biotechnology Progress最新文献

Membrane chromatography devices are a viable alternative to packed-bed resins and enable highly productive purification cascades for monoclonal antibodies and Fc-fusion proteins. In this study, ion exchange and protein A membrane chromatography performances were assessed and compared with their resin counterparts. Protein A dynamic binding capacities were higher than 50 g/L for two of the tested membranes and with a residence time of 0.2 min. For polishing, it was observed that aggregate clearance was generally less performant with membrane separation when compared to resins with similar ligands. However, the comparable yield and increased productivity of membranes could be enough to consider their implementation. In addition, lifetime studies demonstrated that the performance of membranes remained robust over cycles. One hundred cycles were reached for most of the tested membranes with no impact on the process performance nor product quality. Finally, purification cascades were fully operated with membranes, from capture to polishing, reaching good levels of host cells proteins (less than 50 ppm) and aggregates (equal to or less than 1%). The outcome of this study demonstrated that resin chromatography could be fully replaced by membranes for monoclonal antibody and Fc-fusion protein purification processes.

Alkanes are high-energy hydrocarbons that are foreseen as next generation biofuels. Cyanobacteria are known to naturally synthesize C15-C19 alkanes; however, the titers are too low to make this a commercially viable process. Therefore, to leverage these photosynthetic platforms for improved alkane production, here we engineered three novel isolates of Synechococcus elongatus PCC 11801, PCC 11802, and IITB6. The two gene AAR-ADO alkane biosynthesis pathway was constructed by cloning the genes for acyl-ACP reductase (aar) and aldehyde deformylating oxygenase (ado) from S. elongatus PCC 7942 under the regulation of P_rbcL promoter from PCC 7942 and native promoters from PCC 11801 such as P_cpcB300, P_psbAI, and P_psbAIII. The genes were separately cloned under two different promoters, creating a library of the engineered strains. The results indicated that the engineered strains of novel S. elongatus isolates produced significantly higher amounts of alkanes than the model strain PCC 7942. The highest alkane yield achieved was 4.1 mg/gDCW in BG-11, while the highest titer was 31.5 mg/L in 5X BG-11, with an engineered IITB6 strain (P_cpcB300:aar:T_rrnB::P_rbcL:ado:T_Lac). Overall, the study highlights the potential of newly isolated S. elongatus strains as efficient alkane production platforms.

The immobilization of free enzymes is crucial for enhancing their stability in different environments, enabling reusability, and expanding their applications. However, the development of a straightforward immobilization method that offers stability, high efficiency, biocompatibility, and modifiability remains a significant challenge. Silk fibroin (SF) is a good carrier for immobilized enzymes and drugs. Here, we employed urease as a model enzyme and utilized our developed technology called unidirectional nanopore dehydration (UND) to efficiently dehydrate a regenerated SF solution containing urease in a single step, resulting in the preparation of a highly functionalized SF membrane immobilizing urease (UI-SFM). The preparation process of UI-SFM is based on an all-water system, which is mild, green and able to efficiently and stably immobilize urease in the membranes, maintaining 92.7% and 82.8% relative enzyme activity after 30 days of storage in dry and hydrated states, respectively. Additionally, we performed additional post-treatments, including stretching and cross-linking with polyethylene glycol diglycidyl ether (PEGDE), to obtain two more robust immobilized urease membranes (UI-SFMs and UI-SFMc). The thermal and storage stability of these two membranes were significantly improved, and the recovery ratio of enzyme activity reached more than 90%. After 10 repetitions of the enzymatic reaction, the activity recovery of UI-SFMs and UI-SFMc remained at 92% and 88%, respectively. The results suggest that both UND-based and post-treatment-developed membranes exhibit excellent urease immobilization capabilities. Furthermore, the enzyme immobilization method offers a straightforward and versatile approach for efficient and stable enzyme immobilization, while its flexible modifiability caters to diverse application requirements.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.

Human serum albumin (HSA) is currently used as a plasma expander (PE) to increase blood volume during hypovolemic conditions, such as blood loss. However, its effectiveness is suboptimal in septic shock and burn patients due to their enhanced endothelial permeability, resulting in HSA extravasation into the tissue space leading to edema, and deposition of toxic HSA-bound metabolites. Hence, to expand HSA's applicability toward treating patients with compromised endothelial permeability, HSA has been previously polymerized to increase its molecular size thus compartmentalizing the polymerized HSA (PolyHSA) molecules in the vascular space. Previous studies bracketed PolyHSA between 100 kDa and 0.2 μm. In this research, PolyHSA was synthesized at two cross-link densities 43:1 and 60:1 (i.e., molar ratios of glutaraldehyde to HSA) and subsequently fractionated via tangential flow filtration (TFF) into two narrower brackets: bracket A (500 kDa and 0.2 μm) and bracket B (50-500 kDa). PolyHSA within the same size bracket at different cross-link densities exhibited similar solution viscosity, zeta potential, and osmolality but differed in hydrodynamic diameter. At the same cross-link density, the PolyHSA A bracket showed higher viscosity, lowered zeta potential, and a larger hydrodynamic diameter compared with the PolyHSA B bracket while maintaining osmolality. Interestingly, PolyHSA 43:1 B, PolyHSA 60:1 A, and PolyHSA 60:1 B brackets exhibited colloid osmotic pressure similar to HSA, indicating their potential to serve as PEs.

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry.

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.

Bacteriocins are ribosomally synthesized peptides with the innate ability to kill or inhibit growth of other bacteria. In recent years, bacteriocins have received increased interest, as their antimicrobial activity enhances food safety and shelf life by combatting pathogens such as Listeria monocytogenes. They also have application potential as an active pharmaceutical compound to combat multidrug-resistant pathogens. As new bacteriocins continue to be discovered, accelerated workflows for screening, identification, and process development have been developed. However, antimicrobial activity measurement is often still limited with regards to quantification and throughput. Here, we present the use of a non-linear calibration model to infer nisin concentrations in cultivation supernatants of Lactococcus lactis ssp. lactis B1629 using readouts of pHluorin2 fluorescence-based antimicrobial activity assays.