Revolutionizing Cellular Factories: Harnessing Ribosomal Translation Machine for Novel Molecule Biosynthesis

Researchers at the University of California, Berkeley, affiliated with the National Science Foundation Center for Genetically Encoded Materials (C-GEM), are modifying cellular ribosomes to produce advanced and diverse polymers. By introducing new building blocks into these polymers, their aim is to generate innovative biomaterials, enzymes, and drugs. This groundbreaking approach could lead to the development of unprecedented materials, such as a hybrid polymer blend of spider silk and nylon.

Synthetic biologists have been increasingly adept at engineering yeast or bacteria to produce valuable chemicals that go beyond the typical capabilities of microbes, including fuels, fabrics, and pharmaceuticals.

However, a consortium of chemists from multiple universities is pursuing a more ambitious objective: reengineering the cell’s peptide-manufacturing machinery, specifically the ribosomes responsible for assembling proteins, in order to produce intricate polymer chains that surpass the current limits achievable in cells or test tubes.

With a budget of $20 million, this research initiative, based at the University of California, Berkeley, has made significant advancements toward its goal. Three new papers, published in Nature Chemistry and ACS Central Science, address three major challenges: reprogramming cells to supply the ribosome with building blocks other than the standard alpha-amino acids that compose all proteins today, predicting the most suitable building blocks, and modifying the ribosome to incorporate these novel building blocks into polymers.

The long-term objective of the National Science Foundation Center for Genetically Encoded Materials (C-GEM) is to achieve full programmability of the translation system. By introducing mRNA instructions and new building blocks other than the alpha-amino acids currently used, the ribosome would have the capacity to produce an infinite variety of molecular chains. These chains could serve as the foundation for novel biomaterials, enzymes, and even pharmaceuticals.

To gain insights into this process, the researchers conducted molecular dynamics simulations using a model system based on cryo-electron microscopy studies of the E. coli ribosome’s structure. The simulations revealed the regions of the ribosome involved in bonding amino acids or other monomers, while transfer RNAs (tRNAs) delivered novel monomers to be incorporated into polymers. These findings provide valuable knowledge for advancing the field.

The published papers serve as the starting point for developing a comprehensive strategy to reengineer cellular synthetic machinery for the synthesis of unprecedented polymers, including bio-polymers and circular polymers known as peptide macrocycles, with various anticipated and unforeseen applications.

“The objective of C-GEM is to biosynthesize molecules that have never been produced within a cell and are designed to possess unique properties. These tools could have broad applications for polymer chemists, medicinal chemists, and biomaterials scientists, enabling the creation of customized materials with novel functions,” stated Alanna Schepartz, director of C-GEM and a distinguished chair and professor at UC Berkeley’s Department of Chemistry and Molecular and Cell Biology.

For instance, one possibility would be programming the ribosome to synthesize a hybrid polymer combining the strength of spider silk, one of nature’s toughest proteins, with nylon, a synthetic polymer typically produced in chemical reactors. Although spider silk can already be generated in genetically engineered microbes, the technology being developed by C-GEM could enable these microbes to produce an infinite range of polymers that merge the characteristics of silk and nylon, offering chemists new and unique materials. Additionally, this technology could enhance the heat resistance of protein-like polymers beyond what is observed in natural proteins.

An exciting aspect of a programmable ribosome machine capable of synthesizing polymers is the opportunity to evolve these polymers to optimize their functions, similar to how proteins have evolved over millions of years to enhance cellular and organismal fitness.

“While protein polymers have evolved on our planet for billions of years, their diversity has been limited to the same 20 amino acids. If we can establish a system that allows for the evolution of polymers that have never existed in nature, it would provide a platform for anyone with a creative idea to evolve a polymer tailored to their needs,” explained Jamie Cate, professor of chemistry and molecular and cell biology at UC Berkeley.

This approach builds upon the concept of directed evolution, for which Frances Arnold, a UC Berkeley alumna, was awarded the 2018 Nobel Prize in Chemistry.

“Our objective goes beyond what Frances Arnold accomplished with directed evolution, which focused on proteins. We aim to establish a method for evolving polymers that have never before existed in nature,” Cate added.

Engineering an entirely new ribosome system is a complex task. In all cells, ribosomes, acting as nanomachines, follow instructions from messenger RNA (mRNA) molecules, which are copies of the genetic code stored in DNA. They assemble proteins by linking amino acids together. Remarkably, these linear protein chains spontaneously fold into precise 3D structures, ready to perform their designated functions as enzymes, structural components, or regulators within the cell.

Ten years ago, retooling such a complex nanomachine seemed insurmountable. However, the persistence of Alanna Schepartz in pursuing this project led to the establishment of C-GEM, which is currently in its third year of its initial five-year funding cycle.

One of the center’s key objectives is to provide the ribosome with building blocks, known as monomers, other than the standard alpha-amino acids. To achieve this, the C-GEM team focused on enzymes responsible for loading amino acid monomers onto transfer RNA (tRNA), which transports amino acids to the ribosome. Each tRNA is encoded to carry a specific one of the 20 amino acids.

In a recent Nature Chemistry publication, co-authored by Schepartz and graduate students Riley Fricke and Cameron Swenson, the team identified a family of tRNA synthetases capable of loading tRNA with four different non-alpha-amino acids. One of these non-standard monomers is a precursor that can be used to assemble polyketide-like molecules, including antibiotics like erythromycin and tetracycline.

“We identified enzymes capable of loading tRNAs with monomers that possess structural differences from anything previously loaded onto tRNA. One of these monomers can serve as a precursor for the synthesis of polyketide-like molecules. Scientists have been striving for decades to reengineer polyketide synthase modules to create libraries of natural products. These studies have provided valuable insights into the intricacy of these modules, but engineering them has been exceedingly challenging,” explained Schepartz.

The study demonstrated that the native ribosome in the bacterium E. coli readily accepted these novel monomers, showcasing the potential to incorporate chemically distinct building blocks into protein polymers.

Addressing the challenge of how to incorporate non-alpha-amino acid monomers into polymers, a second paper published in ACS Central Science employed cryogenic electron microscopy (cryo-EM) to investigate detailed structures of three related monomers, none of which were alpha-amino acids, bound to the E. coli ribosome. These insights shed light on how these monomers bind, albeit less efficiently than amino acids. They also provide valuable hints for modifying the monomers or the ribosome to enhance the ribosome’s ability to use them for building novel polymers.

In a third paper, published in Nature Chemistry, the research team, including lead author Zoe Watson, used cryo-EM to analyze the structure of the E. coli ribosome while it bound to normal alpha-amino acids. Collaborating with Schrödinger Inc., a San Diego-based computational modeling company, the researchers conducted metadynamic simulations to understand which non-natural monomers would react in the ribosome’s catalytic center and which would not.

Schepartz and Cate emphasized the importance of ensuring that all modifications made to the ribosomal system are compatible with living cells, enabling the production of new polymers without disrupting the routine protein production necessary for life.

“Our aim is to develop enzymes, synthetases, and ribosomes that can function effectively within cells since scalability hinges on their successful application in a cellular context. Achieving this goal requires robust ribosomes, exceptional enzymes, and a deep understanding of the chemical intricacies of these complex molecular machines. It is a challenging problem, but an exciting one. Moreover, it allows us to expose students and postdoctoral researchers to groundbreaking scientific research,” said Schepartz.

The research mentioned in the article was made possible by funding primarily provided by the National Science Foundation under grant CHE 2002182. The project involves various contributors, including Matthew Francis from UC Berkeley, Scott Miller from Yale University, Abhishek Chatterjee from Boston College, Bhavana Shah and Zhonqi Zhang from Amgen Inc., and Sarah Smaga, managing director of C-GEM. Schepartz is affiliated with the Chan Zuckerberg Biohub and the California Institute for Quantitative Biosciences (QB3), while Cate is a member of the Innovative Genomics Institute. Both Schepartz and Cate are faculty scientists at Lawrence Berkeley National Laboratory.

Frequently Asked Questions (FAQs) about ribosome reprogramming

More about ribosome reprogramming

• Nature Chemistry – Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo
• ACS Central Science – Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome
• Nature Chemistry – Atomistic simulations of the Escherichia coli ribosome provide selection criteria for translationally active substrates
• UC Berkeley News – Hijacking Cellular Factories: Retooling the Ribosomal Translation Machine to Biosynthesize Molecules