Probing the Flexibility of an Iterative Modular Polyketide Synthase with Non-Native Substrates in Vitro.

In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates in vitro. BorM5 differentially extends various aliphatic and substituted substrates.

An Oil-Free Picodrop Bioassay Platform for Synthetic Biology.

Droplet microfluidics enables massively-parallel analysis of single cells, biomolecules, and chemicals, making it valuable for high-throughput screens. However, many hydrophobic analytes are soluble in carrier oils, preventing their quantitative analysis with the method. We apply Printed Droplet Microfluidics to construct defined reactions with chemicals and cells incubated under air on an open array.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control.

Engineering a Polyketide Synthase for In Vitro Production of Adipic Acid.

Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design-build-test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid.

Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction.

An enzyme that catalyzes the formose reaction, termed "formolase", was recently engineered through a combination of computational protein design and directed evolution. We have investigated the kinetic role of the computationally designed residues and further characterized the enzyme's product profile. Kinetic studies illustrated that the computationally designed mutations were synergistic in their contributions towards enhancing activity.

Computational protein design enables a novel one-carbon assimilation pathway.

We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway.

Understanding the role of histidine in the GHSxG acyltransferase active site motif: evidence for histidine stabilization of the malonyl-enzyme intermediate.

Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. The ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood.

In vitro analysis of carboxyacyl substrate tolerance in the loading and first extension modules of borrelidin polyketide synthase.

The borrelidin polyketide synthase (PKS) begins with a carboxylated substrate and, unlike typical decarboxylative loading PKSs, retains the carboxy group in the final product. The specificity and tolerance of incorporation of carboxyacyl substrate into type I PKSs have not been explored. Here, we show that the first extension module is promiscuous in its ability to extend both carboxyacyl and non-carboxyacyl substrates.

PR-PR: cross-platform laboratory automation system.

To enable protocol standardization, sharing, and efficient implementation across laboratory automation platforms, we have further developed the PR-PR open-source high-level biology-friendly robot programming language as a cross-platform laboratory automation system. Beyond liquid-handling robotics, PR-PR now supports microfluidic and microscopy platforms, as well as protocol translation into human languages, such as English. While the same set of basic PR-PR commands and features are available for each supported platform, the underlying optimization and translation modules vary from platform to platform.

Narrowing the gap between the promise and reality of polyketide synthases as a synthetic biology platform.

Engineering modular polyketide synthases (PKSs) has the potential to be an effective methodology to produce existing and novel chemicals. However, this potential has only just begun to be realized. We propose the adoption of an iterative design-build-test-learn paradigm to improve PKS engineering.

Perchlorate reduction using free and encapsulated Azospira oryzae enzymes.

Existing methods for perchlorate remediation are hampered by the common co-occurrence of nitrate, which is structurally similar and a preferred electron acceptor. In this work, the potential for perchlorate removal using cell-free bacterial enzymes as biocatalysts was investigated using crude cell lysates and soluble protein fractions of Azospira oryzae PS, as well as soluble protein fractions encapsulated in lipid and polymer vesicles. The crude lysates showed activities between 41 700 to 54 400 U L(-1) (2.

PaR-PaR laboratory automation platform.

Labor-intensive multistep biological tasks, such as the construction and cloning of DNA molecules, are prime candidates for laboratory automation. Flexible and biology-friendly operation of robotic equipment is key to its successful integration in biological laboratories, and the efforts required to operate a robot must be much smaller than the alternative manual lab work. To achieve these goals, a simple high-level biology-friendly robot programming language is needed.