Biotechnology and Bioengineering最新文献

Biological production of 5-aminolevulinic acid (5-ALA) has received growing attention over the years. However, there is the tradeoff between 5-ALA biosynthesis and cell growth because the fermentation broth will become acidic due to the production of 5-ALA. To address this limitation, we engineered an acid-tolerant yeast, Issatchenkia orientalis SD108, for 5-ALA production. We first discovered that the cell growth rate of I. orientalis SD108 was boosted by 5-ALA and its endogenous ALA synthetase (ALAS) showed higher activity than those homologs from other yeasts. The titer of 5-ALA was improved from 28 mg/L to 120-, 150-, and 300 mg/L, by optimizing plasmid design, overexpressing a transporter, and increasing gene copy number, respectively. After redirecting the metabolic flux using the pyruvate decarboxylase (PDC) knockout strain (SD108ΔPDC) and culturing with urea, we increased the titer of 5-ALA to 510 mg/L, a 13-fold enhancement, proving the importance of the newly identified IoALAS with higher activity and the strategic selection of nitrogen sources for knockout strains. This study demonstrates the acid-tolerant I. orientalis SD108ΔPDC has a high potential for 5-ALA production at a large scale in the future.

Filamentous fungi cultivated as biopellets are well established in biotechnology industries. A distinctive feature of filamentous fungi is that hyphal growth and fungal morphology affect product titers and require tailored process conditions. Within the pellet, mass transfer, substrate consumption, and biomass formation are intricately linked to the local hyphal fraction and pellet size. This study combined oxygen concentration measurements with microelectrode profiling and three-dimensional X-ray microtomography measurements of the same fungal pellets for the first time. This allowed for the precise correlation of micromorphological information with local oxygen concentrations of two Aspergillus niger strains (hyperbranching and regular branching). The generated results showed that the identified oxygen-penetrated outer pellet regions exhibited a depth of 90-290 µm, strain-specific, with the active part percentage in the pellet ranging from 18% to 69%, without any difference between strains. Using a 1D continuum diffusion consumption model, the oxygen concentration in the pellets was computed depending on the local hyphal fraction. The best simulation results were achieved by individually estimating the oxygen-related biomass yield coefficient of the consumption term within each examined pellet, with an average estimated value of 1.95 (± 0.72) kg biomass per kg oxygen. The study lays the foundation for understanding oxygen supply in fungal pellets and optimizing processes and pellet morphologies accordingly.

Helicobacter pylori cells undergo chemotaxis toward several small molecules, called chemo-attractants, including urea produced by the epithelial cells of the stomach. The biophysical mechanisms of chemotaxis are not well understood in H. pylori. Here, we developed point sources of urea by encapsulating it in Poly(lactic-co-glycolic acid) or PLGA microbeads for H. pylori chemotaxis studies. Microscopy and Dynamic Light Scattering characterization indicated that the PLGA particles had an average diameter of < 0.8 μm. The particles were relatively stable and had a net negative surface charge. Absorbance measurements indicated that the beads released ~70% of the urea over a 2-week period, with most of the release occurring within the first 24-h period. Varying pH (2.0-7.0) had little effect on the rate of urea release. A diffusion model predicted that such beads could generate sufficient urea gradients to chemotactically attract H. pylori cells. Single-bead single-cell chemotaxis assays confirmed the predictions, revealing that H. pylori continued to be attracted to beads even after most of the urea had been released in the first 24 h. Our work highlights a novel use of PLGA microbeads as delivery vehicles for stimulating a chemotaxis response in H. pylori, with potential applications in bacterial eradication strategies.

1,5-Pentanediol (1,5-PDO) is an important five-carbon alcohol, widely used in polymer and pharmaceutical industries. Considering the substantial energy (ATP and NADPH) requirements of previous pathways, an energy-conserving artificial pathway with a higher theoretical yield (0.75 mol/mol glucose) was designed and constructed in this study. In this pathway, lysine is converted into 1,5-PDO by decarboxylation, two transamination, and two reduction reactions. For the purpose of full pathway construction, 5-aminopetanal reductase and 5-amino-1-pentanol (5-APO) transaminase were identified and characterized. By implementing strategies such as modular optimization of gene expression, enhancing lysine biosynthesis and increasing NADPH supply, the engineered strains were able to produce 1502.8 mg/L 5-APO and 726.2 mg/L 1,5-PDO in shake flasks and 11.7 g/L 1,5-PDO in a 3 L bioreactor. This work provides a new and promising pathway for the efficient production of 5-APO and 1,5-PDO.

Flavin-dependent halogenase (FDH) is highly prized in pharmaceutical and chemical industries for its exceptional capacity to produce halogenated aromatic compounds with precise regioselectivity. This study has devised a multi-enzyme self-assembly strategy to construct an effective and reliable in vitro coenzyme cycling system tailored for FDHs. Initially, tri-enzyme self-assembling nanoclusters (TESNCs) were developed, comprising glucose dehydrogenase (GDH), flavin reductase (FR) and FDH. The TESNCs exhibited enhanced thermal stability and conversion efficiency compared to free triple enzyme mixtures during the conversion of L-Trp to 6-Cl-L-Trp, resulting in a 2.1-fold increase in yield. Subsequently, an ordered co-immobilization of GDH, FR, and FDH was established, further amplifying the stability and catalytic efficiency of the FDH coenzyme cycle system. Compared to the free TESNCs, the immobilized TESNCs demonstrated a 4.2-fold increase in catalytic efficiency in a 5 mL reaction system. This research provides an effective strategy for developing a robust and efficient coenzyme recycling system for FDHs.

Cellular metabolism plays a role in the observed variability of a drug substance's Critical Quality Attributes (CQAs) made by biomanufacturing processes. Therefore, here we describe a new approach for monitoring biomanufacturing processes that measures a set of metabolic reaction rates (named Critical Metabolic Parameters (CMP) in addition to the macroscopic process conditions currently being used as Critical Process Parameters (CPP) for biomanufacturing. Constraint-based systems biology models like Flux Balance Analysis (FBA) are used to estimate metabolic reaction rates, and metabolic rates are used as inputs for multivariate Batch Evolution Models (BEM). Metabolic activity was reproducible among batches and could be monitored to detect a deliberately induced macroscopic process shift (i.e., temperature change). The CMP approach has the potential to enable "golden batches" in biomanufacturing processes to be defined from the internal metabolic activity and to aid in detecting process changes that may impact the quality of the product. Overall, the data suggested that monitoring of metabolic activity has promise for biomanufacturing process control.

Vibegron functions as a potent and selective β₃-adrenergic receptor agonist, with its chiral precursor (2S,3R)-aminohydroxy ester (1b) being crucial to its synthesis. In this study, loop engineering was applied to the carbonyl reductase (EaSDR6) from Exiguobacterium algae to achieve an asymmetric reduction of the (rac)-aminoketone ester 1a. The variant M5 (A138L/A190V/S193A/Y201F/N204A) was obtained and demonstrated an 868-fold increase in catalytic efficiency (k_cat/K_m = 260.3 s^-1 mM^-1) and a desirable stereoselectivity (>99% enantiomeric excess, e.e.; >99% diastereomeric excess, d.e.) for the target product 1b in contrast to the wild-type EaSDR6 (WT). Structural alignment with WT indicated that loops 137-154 and 182-210 potentially play vital roles in facilitating catalysis and substrate binding. Moreover, molecular dynamics (MD) simulations of WT-1a and M5-1a complex illustrated that M5-1a exhibits a more effective nucleophilic attack distance and more readily adopts a pre-reaction state. The interaction analysis unveiled that M5 enhanced hydrophobic interactions with substrate 1a on cavities A and B while diminishing unfavorable hydrophilic interactions on cavity C. Computational analysis of binding free energies indicated that M5 displayed heightened affinity towards substrate 1a compared to the WT, aligning with its decreased K_m value. Under organic-aqueous biphasic conditions, the M5 mutant showed >99% conversion within 12 h with 300 g/L substrate 1a (highest substrate loading as reported). This study enhanced the catalytic performance of carbonyl reductase through functional loops engineering and established a robust framework for the large-scale biosynthesis of the vibegron intermediate.