Foldy: An open-source web application for interactive protein structure analysis.

Foldy is a cloud-based application that allows non-computational biologists to easily utilize advanced AI-based structural biology tools, including AlphaFold and DiffDock. With many deployment options, it can be employed by individuals, labs, universities, and companies in the cloud without requiring hardware resources, but it can also be configured to utilize locally available computers. Foldy enables scientists to predict the structure of proteins and complexes up to 6000 amino acids with AlphaFold, visualize Pfam annotations, and dock ligands with AutoDock Vina and DiffDock. In our manuscript, we detail Foldy's interface design, deployment strategies, and optimization for various user scenarios. We demonstrate its application through case studies including rational enzyme design and analyzing proteins with domains of unknown function. Furthermore, we compare Foldy's interface and management capabilities with other open and closed source tools in the field, illustrating its practicality in managing complex data and computation tasks. Our manuscript underlines the benefits of Foldy as a day-to-day tool for life science researchers, and shows how Foldy can make modern tools more accessible and efficient.

Characterization and Diversification of AraC/XylS Family Regulators Guided by Transposon Sequencing.

In this study, we explored the development of engineered inducible systems. Publicly available data from previous transposon sequencing assays were used to identify regulators of metabolism in Pseudomonas putida KT2440. For AraC family regulators (AFRs) represented in these data, we posited AFR/promoter/inducer groupings. Twelve promoters were characterized for a response to their proposed inducers in P. putida, and the resultant data were used to create and test nine two-plasmid sensor systems in Escherichia coli. Several of these were further developed into a palette of single-plasmid inducible systems. From these experiments, we observed an unreported inducer response from a previously characterized AFR, demonstrated that the addition of a P. putida transporter improved the sensor dynamics of an AFR in E. coli, and identified an uncharacterized AFR with a novel potential inducer specificity. Finally, targeted mutations in an AFR, informed by structural predictions, enabled the further diversification of these inducible plasmids.

Maximizing Heterologous Expression of Engineered Type I Polyketide Synthases: Investigating Codon Optimization Strategies.

Type I polyketide synthases (T1PKSs) hold enormous potential as a rational production platform for the biosynthesis of specialty chemicals. However, despite great progress in this field, the heterologous expression of PKSs remains a major challenge. One of the first measures to improve heterologous gene expression can be codon optimization. Although controversial, choosing the wrong codon optimization strategy can have detrimental effects on the protein and product levels. In this study, we analyzed 11 different codon variants of an engineered T1PKS and investigated in a systematic approach their influence on heterologous expression in Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Our best performing codon variants exhibited a minimum 50-fold increase in PKS protein levels, which also enabled the production of an unnatural polyketide in each of these hosts. Furthermore, we developed a free online tool (https://basebuddy.lbl.gov) that offers transparent and highly customizable codon optimization with up-to-date codon usage tables. In this work, we not only highlight the significance of codon optimization but also establish the groundwork for the high-throughput assembly and characterization of PKS pathways in alternative hosts.

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production.

Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.