Biology subjects

Yano, U.

Publications and source records attributed to Yano, U..

4 recordsLinked to original sources

bioRxiv2026

Improved Biosynthesis of Ethylene Glycol from Xylose in Engineered E. coli Utilizing Two-Stage Dynamic Control

In this study, we employ a two-stage dynamic metabolic control strategy to enhance the NADPH dependent biosynthesis of ethylene glycol from xylose in engineered E. coli. We evaluated the use of metabolic valves to dynamically reduce the enzymes involved in competitive pathways which compete for substrates with ethylene glycol biosynthesis, as well as regulatory pathways aimed at increasing NADPH fluxes. The performance of our initial strains with limits in pathway expression levels was improved by the addition of competitive valves, but not by increases in NADPH flux. In contrast, improving pathway expression levels, led to strains improved significantly by our regulatory valves which improved NADPH flux, but not by the competitive valves. This is consistent with a central hypothesis that faster pathways in and of themselves can compete with other metabolic fluxes by being faster and are better aided by regulatory changes capable of change rates elsewhere in metabolism. In this case in NADPH flux. Lastly, upon scale up to fed-batch bioreactors, our optimized strain, featuring dynamic control of two regulatory valves produced 140 g/L of EG in 70 hours at 92% of the theoretical yield.

synthetic biology↗

bioRxiv2025

Decoupling Central Metabolism from Catabolite Repression Enables Robust Cellulosic Sugar Co-consumption in E. coli

Lignocellulosic biomass is the most abundant and sustainable carbon source for bioproduction, but its efficient utilization is hampered by the heterogeneous mixture of sugars released upon hydrolysis. Most industrial strains consume these mixed sugars sequentially due to strong regulation and cross-inhibition, leading to complex processes and reduced carbon efficiency. To address this, we leverage a novel central metabolism that decouples central carbon flux from native regulatory feedback by employing a Gluconate-Bypass of glycolysis. We demonstrate that the Gluconate-Bypass effectively alleviates feedback regulation in E. coli, enabling co-consumption of glucose and xylose. Further strain engineering leads to the first robust co-utilization of four major lignocellulosic sugars: glucose, xylose, arabinose, and galactose. By decoupling central carbon flux from native regulatory feedback, this architecture provides a feedstock-agnostic platform that maintains high and robust consumption regardless of extreme fluctuations in sugar composition.

synthetic biology↗

bioRxiv2025

Engineering Orthogonal Carbon Dissimilation: A Gluconate Bypass Platform for Robust Stationary-Phase Biomanufacturing

Two-stage bioprocesses which decouple cell growth from product synthesis are an attractive approach to biomanufacturing. However high levels of production in stationary phase cultures often suffer from a progressive decline in metabolism. We demonstrate that in E. coli pyruvate accumulation, an inevitable consequence of high-flux metabolism, acts as a major inhibitor of stationary-phase glucose uptake. We present a novel central metabolism to optimize stationary phase production, a gluconate-bypass, which circumvents this challenge by rerouting carbon flux around glycolysis. This redesign achieves two critical outcomes: first, it decouples glucose uptake from pyruvate inhibition; second, glucose oxidation intrinsically co-generates the NADPH cofactor. Validated using NADPH-dependent L-alanine as a representative model, the GBP creates a self-regulating host that achieved a record titer of 197 g/L with a 1.6-fold extension of production longevity. This work establishes the GBP as a generalizable platform for robust stationary phase biosynthesis.

synthetic biology↗

bioRxiv2023

Precipitation and Extraction Methods for Protein Purification: A Meta-Analysis of Purification Performance and Cost-Effectiveness

For protein drug purification, packed-bed chromatography often remains both the predominant method and a bottleneck for cost and scalability. Accordingly, extensive efforts have been made to develop alternatives, such as precipitation and liquid-liquid extraction. Despite decades of development, such methods have been slow to see adoption in commercial processes. To diagnose the key barriers to implementation and guide future work, we have systematically reviewed studies of protein precipitation and liquid-liquid extraction. We classify the products, methods, and results of 168 publications representing 290 unique purification operations and analyze these operations in terms of both process economics and purification performance. Whereas it is generally assumed that precipitation and extraction methods will have lower costs than chromatography, we find that this is only the case under specific process conditions such as at a large manufacturing scale and low initial sample purity. Furthermore, we find that only a small number of the many precipitation and extraction methods reported to date have shown readiness for implementation in protein drug purification processes. Finally, we identify key factors governing both the economic and purification performance of this class of methods: first, that operating costs are almost entirely predictable by the ratio between the mass of phase-forming materials used and the mass of product protein yielded; second, that use of modern optimization techniques such as Design of Experiments is associated with significantly better purification performance and cost-effectiveness. HighlightsO_LIAlternative separation purification methods are not always cheaper than chromatography C_LIO_LIThe use of a combination of phase separating agents remains largely underexplored/underutilized C_LIO_LILower initial purity and increasing production scale favor phase-separation over chromatography C_LIO_LIThe direct material usage rate is an important predictor of alternative separation cost-effectiveness C_LIO_LICurrent alternative separation method development has largely ignored optimization of direct material usage rate C_LI

bioengineering↗