Peroxisome compartmentalization of a toxic enzyme improves alkaloid production.

Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with cytosolic factors. We found that cytosolic expression of norcoclaurine synthase (NCS), the enzyme that catalyzes the first committed reaction in benzylisoquinoline alkaloid biosynthesis, is toxic in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer. This strategy is achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and products across the peroxisome membrane. We demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing benzylisoquinoline alkaloid titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

Rapid Identification of Disulfide Bonds and Cysteine-Related Variants in an IgG1 Knob-into-Hole Bispecific Antibody Enhanced by Machine Learning.

Bispecific antibodies are regarded as the next generation of therapeutic modalities as they can simultaneously bind multiple targets, increasing the efficacy of treatments for several diseases and opening up previously unattainable treatment designs. Linking two half antibodies to form the knob-into-hole bispecific antibody requires an additional in vitro assembly step, starting with reduction of the antibodies and then reoxidization. Analysis of the disulfide bonds (DSBs) is vital to ensuring the correct assembly, stability, and higher-order structures of these important biomolecules because incorrect disulfide bond formation and/or presence of cysteine-related post-translational modifications can cause a loss of biological activity or even elicit an immune response from the host. Despite advancements in analytical methods, characterization of cysteine forms remains technically challenging and time-consuming. Herein, we report the development of an improved nonreduced peptide map method coupled with machine learning to enable rapid identification of disulfide bonds and cysteine-related variants in an IgG1 knob-into-hole bispecific antibody. The enhanced method offers a fast, consistent, and accurate workflow in mapping-out expected disulfide bonds in both half antibodies and bispecific antibodies and identifying cysteine-related modifications. Comparisons between two versions of the bispecific antibody molecule and analysis of stressed samples were also accomplished, indicating this method can be utilized to identify alterations originating from bioprocess changes and to determine the impact of assembly and postassembly stress conditions to product quality.

Microfluidic bubbler facilitates near complete mass transfer for sustainable multiphase and microbial processing.

A microfluidic device (channels <70 µm) was utilized to create micro-scale bubbles to significantly increase mass transfer efficiency at low flow rates. The convergence of one gas and two liquid channels at a Y-junction generates bubbles via cyclic changes in pressure. At low flow rates, the bubbles had an average diameter of 110 µm, corresponding to a volumetric mass transfer KL a of 1.43 h(-1) . Values of KL a normalized per flow rate showed that the microbubbler had a 100-fold increased transfer efficiency compared to four other commonly used bubblers. The calculated percentage of oxygen transferred was approximately 90%, which was consistent with a separate off-gas analysis. The improved mass transfer was also tested in an algae bioreactor in which the microbubbler absorbed approximately 90% of the CO2 feed compared to 2% in the culture with an alternative needle bubbling method. The microbubbler yielded a cell density 82% of the cell density for the alternative needle tip with an 800-fold lower flow rate (0.5 mL/min versus 400 mL/min) and a 700-fold higher ratio of biomass to fed carbon dioxide. The application of microfluidics may transform interfacial processing in order to increase mass transfer efficiencies, minimize gas feeding, and provide for more sustainable multiphase processes. Biotechnol. Bioeng. 2016;113: 1924-1933. © 2016 Wiley Periodicals, Inc.