Date: 28th January 2021
The ribosome represents a promising avenue for synthetic biology, but engineering efforts have been hindered due to their complex and indispensable nature to the cell. They function as a micro-machine for making proteins, catalysing the biosynthesis of the complete cellular proteome. Now, scientists have a developed a high-throughput method for engineering ribosomes, which can measure and optimise the ribosomes’ ability to catalyse protein production, offering a new platform to screen antibiotics for ribosome-specific inhibition.
Synthetic biology researchers almost solely use parts from the E.coli ribosome when engineering new macromolecules. However, heterologous ribosomes, which comprise of rRNA and ribosomal proteins (r-proteins), derived from different micororganisms may offer new opportunities for the discovery or engineering of novel translational functions. Such ribosomes have already been evaluated in E.coli via complementation of genomic ribosome deficiency mutants, but this fails to guide the engineering of the many refractory ribosomes.
Now a team scientists, led by Ahmed Badran, from Broad Institute of MIT and Harvard, US, have used orthogonal translation, a technique that forces the ribosome to exclusively translate a researcher-defined transcript, such as green fluorescent protein (GFP), creating an assay that allows the researcher to easily determine which bacterial ribosomes function in E.coli. The team then used the assay to improve the function of refractory ribosomes and developed universal engineering rules for orthogonal translation that extended to any reporter protein.
The first question the team wanted to address was why some ribosomes are refractory in E.coli. So they developed a high-throughput orthogonal translation assay. Here, they used previously described orthogonal ribosome–mRNA pairs, where the anti-ribosome binding site (RBS) of the rRNA was engineered to exclusively translate a researcher defined transcript, here the reporter mRNA GFP, which bore the complementary RBS. This cannot be translated by the wild type ribosomes, creating an orthogonal pool of ribosomes (O-ribsomes) whose function was very easily determine by GFP reporter expression.
The team first engineered the O-antiRBS of 21 heterologous rRNAs closely related to E.coli and known to be capable of complementing the E.coli ribosome mutant, in addition 13 phylogenetically more divergent rRNAs were also engineered . Using the assay, they discovered that the ribosomes derived from bacteria closely related to E.coli could readily translated the GFP however, GFP translation fell markedly with phylogenetic distance from E. coli.
The observed relationship between heterologous orthogonal translation activity and phylogenetic distance from E. coli suggested to the team that certain elements may have sufficiently diverged to restrict efficient ribosome activity in E. coli. Therefore, to improve the function of the refractory ribosomes the team introduced or replaced key RNA and proteins, such as complementary O-rRNA intergenic sequences from the host E.coli and a subset of r-proteins from the original cell.
Replacement of intergenic sequences for moderately divergent O-rRNAs substantially increased GFP expression, and some of the non-functional O-rRNAs also now yielded robust GFP. However, little change in activity was seen from the most divergent group suggesting other factors were required.
Here, the r-protein complementation further enhanced heterologous ribosome activity of this group. In a set of experiments they team discovered that complementation with only a small number of cognate r-proteins could have substantial effects on the divergent heterologous ribosome function in E. coli.
The interface between rRNA and r-proteins is subject to extensive coevolution and divergence between related organisms, using this information and their data the researchers developed a set of universal engineering rules for orthogonal translation. These were tested and could be extended to any reporter protein.
The team here have developed a high-throughput method for engineering heterologous ribosomes that can be used to measure and optimise ribosome activity and hence protein production. The team will use this platform to engineer new kinds of ribosomes that generate proteins with novel properties and this is the first step towards that aim.
The majority of antibiotic classes currently in clinical use act by inhibiting ribosome function. With antibiotic resistance rising to dangerously high levels, and new resistance mechanisms emerging, it threatens our ability to treat common infectious diseases and minor injuries.
At least 700,000 people die each year due to drug-resistant diseases and it is estimated by 2050 that such diseases could cause 10 million deaths per year. The development of new technologies is absolutely crucial in fighting against this devastating crisis. Steps are being made, with the likes of CRISPR- Cas13a-based antibacterial nucleocapsids being developed as therapeutic agents against antimicrobial-resistant bacteria, or novel computational approaches to search the genome of human microbiota for genes that encode new antibiotic drug-like molecules.
However, the development of a new method to rapidly test new antibiotics will be a valuable tool for antibiotic research. Furthermore, the ability to target only ribosomes of specific human pathogens, but not commensal bacteria, with greatly help the limit of broad resistance acquisition. It is hoped this technology will accelerate the discovery, the design and testing of new antibiotics, and offer hope against increasing antibiotic resistance. The team also plan to use the technology to investigate other biotechnological applications of engineered ribosomes.
From a broader view the platform will expand the ribosome toolkit – from the current almost exclusively E.coli – to ribosomes from other species, using them in new applications, and accelerating new discoveries.
For more information please see the press release from the Broad Institute of MIT and Harvard
Kolber, N. S., R. Fattal, S. Bratulic, G. D. Carver and A. H. Badran (2021). “Orthogonal translation enables heterologous ribosome engineering in E. coli.” Nature Communications 12(1): 599.