Researchers, the pharmaceutical industry and industry in general want bacteria and fungi to produce a cornucopia of various proteins and enzymes. However, the bacteria do not always cooperate, and researchers have now mapped out how they react to being used in biotechnology.
Bacteria and fungi have enormous potential in biotechnology, but this has not been exploited optimally.
Similar to yeast that produce insulin, bacteria and fungi can potentially produce thousands of different proteins that may be useful in research, pharmaceuticals and industry in general.
The trick to obtaining drugs, proteins to prevent cancer or new enzymes for laundry detergent is to determine how to get bacteria or fungi to produce something they cannot use.
Genes can be inserted, but bacteria or fungi do not always cooperate and produce large quantities of a given protein. Instead, they often resist strongly.
A new research project has identified how Escherichia coli reacts to being used (or misused) in biotechnology.
“This study focuses on the heart of biotechnology: how to optimally get bacteria and fungi to make foreign proteins they cannot use. For example, yeast cannot use insulin, but we still want them to produce as much as possible. This requires understanding what happens inside the bacteria or fungi when we persuade them to make proteins that we want but they cannot use,” says Bernhard Palsson, CEO, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.
The research has been published in Metabolic Engineering.
Major benefits by making bacteria better at producing proteins
People involved in biotechnology know that bacteria and fungi can react very differently to having to produce proteins. They even react differently to producing different types of proteins.
This can present a challenge in getting E. coli to produce a lot of a given protein, such as BRCA1 protein, which are known from breast cancer and may be of great research interest because they are a target for developing drugs to combat breast cancer.
The E. coli should make as much BRCA1 protein as possible for two main reasons.
• Fewer bacteria and less growth medium are needed to make the required amount of protein.
• The purification process is much easier if 40% of the proteins in a bacterium are BRCA1 instead of only 2%.
“Increasing productivity or understanding why it is so low can strongly influence how to get bacteria and fungi to make proteins,” says Bernhard Palsson.
Like only listening to the second violins in a symphony
To determine what prevents the bacteria from producing huge quantities of protein, Bernhard Palsson and his colleagues developed a method to find out what the bacteria do when they produce proteins and the barriers to doing this when they rebel.
The method is to feed data to a computer on the bacteria’s overall synthesis of proteins: the transcriptome.
An artificial intelligence algorithm can extract signals from the transcriptome and eavesdrop on and analyse the underlying mechanisms that make the final protein product look the way it does.
“The algorithm can extract and select individual signals from a complicated pattern. This is like listening to an entire symphony and solely extracting on the signal from the second violins,” says Bernhard Palsson.
Finding stress signals in the transcriptome
The biotechnology researchers did not use the algorithm to listen to violins, cellos or tambourines but instead used it to listen to stress signals in the E. coli.
For example, these stress signals may be related to changes in pH, oxidative stress or temperature changes.
“This enables us to determine which stress signals are amplified when we try to get bacteria to produce foreign proteins,” explains Bernhard Palsson.
Various barriers prevent bacteria from making proteins
The researchers inserted 45 genes into E. coli and thus caused them to produce 45 different proteins they did not usually produce.
The researchers also analysed the transcriptome and found that the bacteria had five broad reaction patterns to being exploited in biotechnology.
• Fear versus greed. This reaction is known across biology and causes the bacteria to shut down many biological processes when being asked to produce foreign proteins, producing little of the desired product.
• Protecting metalloproteins. Another reaction is being hyperprotective about proteins with metals in them. Many proteins need metals to function, such as haemoglobin, so if bacteria are forced to make proteins with metals in them, they react by retaining the metals in their own proteins rather than using them for the proteins people want them to make.
• Dysregulating chaperones. Getting many proteins to function properly requires folding by chaperones, which are proteins that help other proteins become functional. However, the researchers found that the E. coli often reacted to the presence of foreign proteins by knocking out the chaperones so that they could not fold these proteins the way that they should be folded.
• Dysregulating amino acid biosynthesis. The E. coli must use amino acids to make proteins, and when genes are inserted to get the bacteria to produce foreign proteins they often have to use amino acids they do not produce in large quantities themselves. They must therefore change how they biosynthesize amino acids. The cell can react to the demand to make foreign proteins by producing more or fewer of the desired amino acids.
• Uncharacterized reaction. Some of the E. coli reaction patterns could not be characterized.
“This last group can clearly become an independent field of research,” says Bernhard Palsson.
Enormous potential in biotechnology
Bernhard Palsson says that the new insight can potentially improve the bioproduction of proteins for a wide range of purposes.
The biggest problem is getting the bacteria to produce proteins efficiently, because various barriers prevent this.
However, if researchers can better understand these barriers and modulate them, they can get E. coli to produce a lot more of certain proteins.
“Their reaction can be changed genetically or by changing the growth medium. Tricking the bacteria into producing just 10% more is often a huge benefit,” says Bernhard Palsson.
“Independent component analysis of E. coli’s transcriptome reveals the cellular processes that respond to heterologous gene expression” has been published in Metabolic Engineering. Co-author Bernhard Palsson is the CEO of the Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby.