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Author: Babetta (Babs) Marrone, Laboratory Relationship Manager for BETO programs at Los Alamos National Laboratory

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For several years, Los Alamos National Laboratory (LANL) scientists have been taking advantage of the molecular tools that naturally reside within microbial cells. Although microbes use these tools to carry out their metabolism and other life-sustaining processes, scientists can use these tools to produce fuel precursors and bioproduct building blocks. Today, researchers at LANL are further improving this process by adding a biosensor to the microbes that will use light to tell them how efficiently the product is made—thus enabling the researchers to identify cells with increased overall yield.

Smart microbial cell colonies "light up" when enzyme activity is high. Photo: Los Alamos National Laboratory

Smart microbial cell colonies "light up" when enzyme activity is high. Photo: Los Alamos National Laboratory

Engineered microbes are particularly effective due to a versatile metabolism housed in a relatively simple cell structure. One of the challenges, however, is that when microbes are engineered for making a new product, it is difficult to pin-point the best performer in a pool of microbial cells with variable product formation efficiencies. Furthermore, testing the efficiency of microbial cells has always been a low throughput process and ultimately a bottleneck in the Design-Build-Test-Learn (DBTL) cycle for bio-based products.
In order to overcome this challenge of identifying and isolating top performing cells, LANL scientists have leveraged longstanding capabilities in protein design and computational modeling of ligand binding pockets in proteins. This knowledge base allows researchers at LANL to engineer custom biosensors that detect intracellular concentrations of a specific precursor or building block as a target. These protein-based biosensors for small molecules detect the presence of the desired target and respond by accumulating another molecule that physically glows/lights up, enabling researchers to actually visualize the productivity. Furthermore, by coupling this “smart microbial cell” to the high throughput efficiency of a single-cell sorting technique called flow cytometry, the team can successfully evaluate a large number of metabolic designs formulated for improved production of whatever precursor or building block is desired.
Figure 1. (Left to right) Structure based design of an enzyme is used to create a library of diversified genes. Smart microbial cell technology demonstrates the efficiency of enzyme variants by producing a readable fluorescence signal.
With enhanced throughput in testing the designs, this approach can accelerate the DBTL cycle for biomanufacturing. Specifically, this approach is crucial for relieving the bottleneck in the Test step of the DBTL cycle. Not only does the increased throughput in the “test” step help match the “design” and “build” steps, but the capability to produce enormous amounts of data by high throughput evaluation contributes to enhanced learning for subsequent DBTL cycles.
Overall, LANL’s smart microbial cell technology is an advanced platform for high throughput screening for enzyme discovery, design, and evolution. The approach can be translated to screening of metagenomic samples, rational enzyme design, or directed evolution of known enzymes. The technology is adaptable to a single enzyme, or a pathway, or global optimization of an industrial strain.
The microbial biosensor team includes LANL scientists Ramesh Jha, Niju Narayanan, and Taraka Dale (lead). The work was performed under the Agile BioFoundry, a multi-national laboratory effort to expedite biomanufacturing processes (https://agilebiofoundry.org).
Babetta (Babs) L. Marrone
Laboratory Relationship Manager for BETO programs at Los Alamos National Laboratory
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