Episode Summary

How does an engineer approach microbial genetics? Wayne Curtis works with microbes of all kinds to optimize metabolic and agricultural systems. Here he discusses his work with Rhodobacter to make biofuels and for membrane protein expression, with Agrobacterium and plant pathogenic viruses to make drought-resistant plants, and with Clostridium and yeast cocultures for lignocellulose digestion.

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Julie’s Biggest Takeaways

Faster isn’t always better, when it comes to engineering microorganisms. Faster growing organisms often require more energy to simply remain alive, a concept known as maintenance (one could call them high-maintenance microbes). For large-scale processes, a slower-growing organism with lower energy requirements for maintenance can be advantageous: this is one reason why the Curtis lab works with Rhodobacter.

Rhodobacter is extremely metabolically flexible: it can grow aerobically on the bench using a sugar medium, anaerobically using photosynthesis, or chemolithotrophically. This flexibility means they can be genetically manipulated on the bench without anaerobic chambers but can be used in a variety of process environments.

When it transitions to anaerobic photosynthesis, Rhodobacter pinches off its membrane to fill its cytoplasm with vesicles. These vesicles increase the total membrane surface area covered with light-harvesting antennae to increase its efficacy of capturing energy, and in fact, the number of vesicles can be influenced by the amount of light exposure to the microbial culture. This ability of Rhodobacter to make many vesicles containing membrane-bound proteins, combined with genetic tractability of the bacterium, can be utililzed to engineer anaerobic photosynthetic membrane-bound protein expression.

Viral gene therapy using genetically engineered plant pathogenic viruses offers benefit for agriculture and many points for biocontainment. The specific requirements of the virus, plant, insect vector, insect vector endosymbiont bacteria, and genetic regulation of the desired construct all confer opportunities to fine-tune this agricultural system. This project is part of a large-scale drought-protection system that the Curtis lab is developing in collaboration with several other groups.

Featured Quotes

“When I think about biology, it’s a matter of where you get your electrons and where you discard them.”

“Whereas a photosynthetic organism will use sunlight and shake up the electrons in breaking up water, some organisms can simply fix carbon just like a photosynthetic organism does, but they need an electron source to do that. In this particular case (of Rhodobacter), they use hydrogen.”

“We use the word fix when we talk about photosynthetic organisms: they ‘fix’ CO2. And by fixing it, what we mean is—it’s not broken—it just needs some electrons to make it useful. Hydrogen provides that. [Rhodobacter] is referred to as chemolithotrophic, but I look at it simply as an organism that has the ability to fix carbon dioxide, just like that done in photosynthesis, but instead of using light, it will use hydrogen that can come from splitting water by electrolysis.”

“If you think about biofuels, you’re making energy—from energy.”

“If you look, there is a microbe that has figured out how to do just about everything...really, microorganisms are incredibly versatile. If there’s just a little tiny niche of electrons to eat somewhere and to crap out somewhere else, they’ll figure it out.”

“In contrast to a class, where you have someone telling you what to learn, in a research setting you have a lot more flexibility. So I’d like to encourage students to look for that, and commit some time. Really take advantage of that unique experience you can get by doing something hands on—and don’t undersell what you can contribute.”

Links for This Episode

History of Microbiology Tidbit

In today’s history of microbiology tidbit, I thought we’d revisit a comment Wayne made about E. coli being engineered to make insulin. E. coli are really the founders of all things genetics, in my mind, because E. coli was used to figure out how DNA is replicated, what a codon is, even what a gene is. It was the biochemist Jacques Manod who said, what’s true for E. coli is true for an elephant, since the DNA code and its coding and uncoding processes turned out to be extremely similar in all living organisms. The heydey of these bacterial genetic discoveries came to a head in the 1970s, as scientists began to use all of these new findings to start to tinker with genomes.

In 1978, a 2-yr old company with 12 employees called Genentech and their collaborators at the private nonprofit City of Hope National Medical Center announced that they had produced insulin produced solely from E. coli in the first demonstration of the power of recombinant DNA technologies. Insulin is a protein hormone required by diabetics to control their blood sugar, and before this advancement, insulin had been purified from porcine or bovine pancreas, the organ that produces insulin. Recombinant technologies were being pursued because they would allow production of human instead of pig insulin, alleviating issues in patients who had allergic reactions to the animal version or who avoided porcine products for religious reasons. Most importantly, it would relieve the need for thousands of pounds of animal pancreas - it takes 8000 pounds of porcine pancreases to make 1 pound of purified insulin. That’s over 23,000 animals! Eli Lilly, who was producing insulin, needed 56 million animals a year to supply patients with insulin, and they were worried that the rate of insulin production from animals wouldn’t keep up with demand. Enter recombinant technologies.

Insulin is coded by two genes in the human genome, and in order to recapitulate the human insulin form, the genes were synthesized chemically. This allowed the scientists to eliminate the introns inherent in the human genes, since bacteria don’t have the molecular machinery to recognize and eliminate these unwanted segments when processing mRNA. Each insulin chain, called A or B, had to be individually expressed and purified from E. coli cultures. The purified proteins could then be chemically combined to add the disulfide linkage found in human insulin. As you likely know, the scientists were able to successfully clone insulin, but the story isn’t as straightforward as ‘they tried it and it worked’ - there were many missteps, and the future of the young company was riding on the results the entire time. Genentech, now a subsidiary company of Roche, has the story on their website, and it recounts the reaction of lead scientists Dave Goeddel and Dennis Kleid’s first moments of success.

Finally, in the early hours of August 21, 1978, Goeddel succeeded in reconstituting the two amino acid chains into one molecule: human insulin. It was an extraordinary moment, not only for Genentech, but for the history of medicine and the future of biotechnology. When Goeddel told the rest of the team the news, they were elated. Kleid likened the feeling to finishing a marathon. “You’re pretty much exhausted when you get to the line, and it takes a while for it to soak in that you actually won the race.”

The story continues from there, however, since the challenge was to have E. coli produce high yields of insulin protein, and the initially cloned human genes yielded low protein concentrations. In the subsequent years, expression was optimized and recombinant insulin was made available in 1982, sold as Humulin through an agreement between Genentech and Eli Lilly. What a difference 40 years makes! This makes me appreciate the heavy lifting done by scientists like those at Genentech whose technological breakthroughs made possible the undergraduate cloning projects Wayne discussed assigning students in his lab. Just think - the type of project that kickstarted a multibillion dollar company might today have been an undergraduate project! Sometimes I find these types of comparisons mind blowing.

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