Chem & Bio Engineering最新文献

Yeasts have long been celebrated for their metabolic prowess, in particular, their ability to transform grain and fruit into valuable food products. In recent decades, humans have exploited them for pharmaceutical applications driven by advances in metabolic engineering and synthetic biology. Through convergence of these disciplines, this study highlights the development of a cell-free protein synthesis (CFPS) platform using Kluyveromyces lactis, a yeast prized for its role in the dairy industry. We present a workflow for preparing K. lactis extracts and incorporate lactose as a sustainable and cost-effective carbon source for biomass generation and as an energy source for CFPS. A semiautomated design-of-experiments (DoE) approach was undertaken, based on a Latin Hypercube experimental design, which tested 128 unique CFPS reaction mix compositions (against a baseline optimized for Pichia pastoris). The optimized reaction mix was validated by the synthesis of two model proteins: green fluorescent protein (deGFP) and Erythropoietin (EPO), which is a clinically relevant therapeutic. We identified conditions with 4-fold improvement in yield with the optimized reaction producing 54 nM of EPO. By integrating lactose-based growth, protein synthesis, and rational optimization strategies, this study sets the scene for developing yeast-based CFPS platforms tailored for diverse applications, from biosensor development to industrially relevant biopharmaceutical production.

As a nonmodel microorganism, Pichia pastoris has unique advantages in recombinant protein expression and natural product biosynthesis. The development of synthetic biology has provided key technical support for the construction of microbial cell factories. However, it faces challenges such as the complexity and diversity of living systems, the necessity for repeated trial and error during research, and the low throughput of manual experiments, which fail to satisfy the demands of various applications. Automated synthetic biotechnology, featured by high throughput, automation, and intelligence, can conduct extensive experiments and expedite the Design-Build-Test-Learn cycle for the construction and optimization of microbial cell factories. This study describes an automated genetic manipulation process for rapid construction of P. pastoris cell factories based on BioFoundry. First, we optimized critical parameters using an automated system, including heat shock temperature, reducing agent, as well as the amounts of donor DNA and sgRNA plasmid, enabling highly efficient heat shock-based transformation and high throughput genome editing in P. pastoris. Specifically, the automated genetic manipulation process enabled the single-site and two-site genome editing efficiency reaching up to 94.6% and 36.3%, respectively. Then, we characterized 96 endogenous promoters in an automated and high throughput manner, including their strengths and time-course features in P. pastoris. Finally, we constructed yeast cell factories using promoters with different strength to produce sesquiterpene α-santalene and α-santalol. Our study provides a reference for the automated genetic manipulation of P. pastoris and other nonmodel yeast species.

Tungstate-based catalysts for olefin metathesis generally suffer from insufficient reaction rates, which require relatively high temperatures for the satisfied activities. This issue is mainly due to the shortage of active WO_x species related to intrinsic low adsorption and poor activation of olefin molecules. Herein, we found that the silanol nests in dealuminated Beta zeolite (DeAl-Beta) were favorable for high dispersion of tungsten species, forming active WO_x species on the zeolite, which was helpful for the adsorption and activation of olefin molecules, thus facilitating the generation of metallocycle intermediates. As a result, the propene yield in the metathesis of ethene and 1-butene over WO_x/DeAl-Beta was 2.3 times higher than that of the tungsten species supported on siliceous Beta zeolite with fewer silanol nests (WO_x/Si-Beta) under the equivalent conditions. A propene yield as high as >50% was achieved by optimizing the silanol nests in the WO_x/DeAl-Beta catalyst, outperforming those in the industrial silica supported tungsten species (W/SiO₂) catalysts reported previously.

The conversion of biomass fermentation liquor has garnered significant attention due to its potential for sustainable chemical production. Particularly, the transformation of an acetone-ethanol mixture, derived from the separation of high-value butanol, into other valuable compounds represents a critical advancement in biorefinery processes. Herein, we present a high-efficiency Zr/Beta zeolite catalyst for the conversion of an acetone-ethanol mixture into propene. Through systematic optimization, the optimal catalyst 5%Zr/Beta achieves a high propene yield (37.8%) with a propene selectivity of 67%. Spectroscopic results reveal that the conversion of acetone and ethanol primarily proceeds via the Meerwein-Ponndorf-Verley (MPV) reduction at Zr sites to form the isopropanol intermediate, followed by acid-catalyzed dehydration to propene facilitated by Si-OH groups. The high propene selectivity is due to the minor side reaction of converting acetone to isobutene, accompanied by the accumulation of cyclic unsaturated aldehydes/ketones and aromatic compounds deposited on the Zr active sites, leading to catalyst deactivation. Additionally, the Zr/Beta catalyst demonstrates good regenerability, which could recover to the initial state after a facile calcination process in air. This work offers a promising approach for the synthesis of propene from a biomass-derived acetone-ethanol mixture, contributing to the development of sustainable catalytic processes for biorefinery applications.