Lighting the Way to Bioproducts: Smart Microbial Biosensors for Conversion Pathway Design

Bioprose Blog: For several years, Los Alamos National Laboratory (LANL) scientists have been taking advantage of the molecular tools that naturally...

Bioenergy Technologies Office

February 21, 2019

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

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BIOPROSE BLOG

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

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

Dr. Babetta (Babs) L. Marrone is the former Laboratory Relationship Manager for BETO programs at Los Alamos National Laboratory (LANL). She is LANL’s Biofuels Program Manager in the Applied Energy Program Office at LANL and a Senior Scientist in the Bioscience Division.

Babs has a background in cell biology and 30+years of experience in finding innovative solutions in the biosciences. She is currently finishing a 2-year term as Senior Technologist in Residence, partnered with Procter & Gamble, in AMO’s Technologist in Residence Program, with a focus on renewable manufacturing. Marrone participates in several R&D projects sponsored by BETO. She previously led the Ultrasonic Harvesting and Extraction project for the National Alliance for Advanced Biofuels and Bioproducts (NAABB), a large algae biofuels and bioproducts consortium of over 30 institutions sponsored by BETO, from 2010-2013. She was the Director and PI of the NIH-sponsored National Flow Cytometry Resource from 2009-2014; and has led projects sponsored by NIH, LDRD, DOE-BER, DHS, and the FBI.

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Bioproduct Production
Biotechnology
Bioenergy
Advanced Manufacturing Processes
Commercial Implementation