By combining two complementary techniques, University of Wisconsin–Madison scientists have developed a way to efficiently pinpoint genes that help microbes tolerate harsh conditions found in industrial fermentations, technology that could enable breakthroughs in biotechnology.
Microbes, such as bacteria, can turn sugars and other parts of plant fiber into biofuels, drugs, and chemicals. But production is often limited by stressors such as toxic chemicals introduced when plant fibers — or biomass — are broken down, or even in the plants themselves.
Genetic modification – such as turning off or boosting certain genes – can often overcome these limitations, but with thousands of genes, it’s hard to predict which ones will give the cells an advantage.
Traditionally scientists grow “libraries” of mutants — hundreds of thousands of cells each with different genes turned off — in the presence of a chemical. Analyzing the surviving cells can provide clues about which genes improve fitness for that condition.
But this technique, known as chemical genomics, tends to produce false positives, requiring labor-intensive follow-up experiments.
Researchers with the Great Lakes Bioenergy Research Center solved the problem by using two complementary gene perturbation techniques to narrow the field of target genes.
“Traditionally people would do one big screen, and then they would pick a bunch of genes to follow up on, one at a time.... Some fraction of those things aren’t going to work the way that you expect them to," said Amy Enright Steinberger, a postdoctoral researcher in Jason Peters’ lab and co-author of the paper published in the journal mSystems.
“In this case, since we already had two lines of evidence saying that they would behave this way, by the time we got to our follow ups, we weren’t surprised that they worked.”
In the process they discovered a surprising quirk of the microbe they were studying.
“There are two things that are probably the most exciting about this paper,” Steinberger said. “One is the approach that we took... and then the other is the biology that we found using that approach.”
The experiment used an orthogonal approach — meaning techniques that can answer the same question in different ways — by applying two gene perturbation technologies with complementary strengths and weaknesses.
Transposon insertion sequencing (TnSeq) randomly disrupts genes, which is useful for identifying essential genes. But since essential gene disruption kills the cell, it doesn't help explain what the gene does, and it doesn't always work in organisms with multiple genome copies.
CRISPR interference (CRISPRi) works more like a dimmer switch, allowing partial “knockdowns” that keep the cells alive and allow scientists to identify the gene function. But bacterial genes work in clusters, so knocking down the first one can sometimes turn off the whole set.
“That can make CRISPRi a little bit difficult to disentangle which gene is actually responsible,” Steinberger said. “TnSeq is a good complement for that, because it's more precise.”
The researchers tested the approach on Zymomonas mobilis, a promising industrial microbe that’s naturally good at converting sugars into alcohol and can function with or without oxygen (aerobically or anaerobically).
After screening each library against known chemical stressors at sub-lethal concentrations, the researchers calculated chemical-gene scores for each mutation. Positive scores indicate increased fitness, while negative scores represent decreased fitness. They used another calculation to test the ability of one library to accurately predict results from the other.
These methods identified 103 genes with significant scores in at least one chemical and genetic library, but only 31 had significant scores in both. Follow-up experiments on a set of those found no false positives.
“We were able to basically narrow in on the things where they agree the most,” said Jacob Eckmann, a PhD student in Patricia Kiley’s lab who co-authored the paper. “It was a way of trying to find ... the biggest winners rather than sorting through a lot of the other noise.”
In Z. mobilis, the genes identified help make proteins involved in the electron transport chain, a process that cells use to turn food into energy.
The experiment showed that disrupting that pathway improves the microbe’s resistance to chemicals often encountered during fermentation of plant biomass, but it’s not clear how.
So in addition to identifying new gene engineering targets, the findings point to an unexpected function for this electron transport chain, which Eckmann is now investigating.
“This is a fascinating discovery about the central energy-producing components of Zymomonas mobilis,” Eckmann said. “(This pathway) seems to have its fingers in a lot of different systems, and we’re trying to look further into what exactly the architecture of this pathway is and why it seems to be messing with so many different things.”
The researchers say the technique can be applied to other microbes and other chemical-gene interactions, potentially accelerating advances in biomanufacturing, fighting antibiotic resistance, and understanding how microbes use chemical signals to form partnerships that help humans and plants.
“This type of approach is becoming more and more accessible. It used to be that these genome-scale screens were really big and laborious,” Steinberger said. “We’re getting to the point where this could be feasible for any organism that has these technologies available.”