Not So Giant Steps On Tuning Protein Production and Diminished Genomes

Summary

An abundance of new information and methods is available in the fields of molecular biology and genome engineering. The proper understanding of all this information is key to fully unlock the potential of microbial biotechnology. In this thesis we review the most relevant new findings and the state-of-the-art of two specialized fields: protein expression and CRISPR-based template-free genome editing, with a focus on prokaryotic DNA repair pathways. Additionally, three experimental studies are outlined and discussed on the topics.

The fields of molecular biology and genome engineering have witnessed remarkable progress throughout the recent years, a wealth of new information having been created that has significantly advanced scientific understanding. Two areas that stand out in biotechnology and that are going to be dealt with in this thesis are protein expression and CRISPR-based template-free genome editing.

Protein expression has been a focal point of research, with recent studies aiming to optimize the production of proteins. The utilization of big data has provided comprehensive insights into the factors influencing protein production. Notably, a strong correlation between mRNA stability and translation elongation rates has been observed in eukaryotes. Additionally, novel tools have enabled the experimental determination of RNA secondary structures in vivo, contributing to a better understanding of translation elongation. However, deciphering the specific effects of individual sequence features on overall protein production efficiency remains challenging due to the intricate interplay of multiple factors. Machine learning methods offer promise in addressing this complexity, but they may lack a full understanding of the underlying biology, leading to optimization without complete comprehension.

The field of membrane protein expression has seen significant strides, particularly in E. coli. The introduction of a translational-tuning system, the Bicistronic Design (BCD) elements, has enabled fine-tuning of membrane protein production. By combining two ribosome binding site (RBS) sites in the same mRNA, hindering secondary structures during translation initiation are prevented, resulting in enhanced membrane protein levels. This approach, combined with a standardized library of BCD elements, offers a potential solution for high-level production of functional membrane proteins, benefiting membrane protein purification studies and synthetic biology projects.

Another area of substantial growth is CRISPR-based genome editing, specifically in template-free repair pathways. Non-homologous end joining (NHEJ) and alternative end joining (AEJ) have been recognized as critical pathways for DNA repair in prokaryotes. Leveraging these pathways with CRISPR-Cas tools reveals various strategies for genome editing, including gene inactivation, sparse DNA insertion, and genome minimization. The choice between NHEJ and AEJ depends on specific applications, with each pathway offering distinct advantages. Future research in this area is essential to fully understand the mechanisms and outcomes of prokaryotic non-templated DNA repair pathways.

Template-free genome editing has shown great potential in R. sphaeroides, where certain colonies can escape CRISPR-Cas9 targeting without exogenous DNA repair templates, as revealed by data from our previous experiments. The identification of high-fidelity repair of the chromosome through homology-directed repair (HDR) with the RecA protein paves the way for further understanding fundamental processes that rule phenomena such as CRISPR-Cas9 and HDR-based genome editing efforts. Nevertheless, careful consideration is required when using certain compounds, such as 5-fluorouracil (5-FU), for screening mutants due to their genotoxic properties, as we demonstrate and discuss extensively.

In the pursuit of genome minimization, E. coli MG1655 has been a focal point. Utilizing a novel type I-C CRISPR system involving the Cas3 nuclease, the editing process has been successful in generating deletions in the lacZ gene. However, challenges in stacking deletions have been encountered, necessitating further refinement of the methodology. The development of a multiplex loci PCR (MLPCR) method has improved monitoring capabilities during iterative genome editing, facilitating the study of non-essential regions for deletion.

In conclusion, the recent advances in molecular biology and genome engineering have opened up exciting opportunities for scientific exploration. The insights gained from studying protein expression and CRISPR-based genome editing have ample implications across various disciplines, from biotechnology to synthetic biology.