Genome Engineering, Morten Nørholm

The Genome Engineering Group contributes to the CFB overall goals by focusing on talent development, developing molecular tools for accelerating cell factory design, and creating specific bacterial cell factories for production of proteins and biochemicals. The group focuses on two different but highly complementary themes:

1. Development of synthetic biology tools and standards for microbial genome engineering

 

Despite its dominant role as a model organism in molecular microbiology for more than a century, precision genome engineering of Escherichia coli has only recently been enabled, mainly by the phage recombination systems and CRISPR-based tools. In a next phase, it is the vision of the Genome Engineering to contribute significantly to two aspects that generally are recognized as being highly important within synthetic biology: automation and standardisation of molecular biology and genome engineering.

 

In close collaboration with the largest initiative for synthetic biology standards, the European-based BioRoBoost (http://standardsinsynbio.eu), we will work on concepts for standardising synthetic biology. Furthermore, we will experimentally develop a standardised bacterial genome engineering platform, enabling more predictive cell factory engineering.

Finally, we will optimise and simplify the current state-of-the-art molecular tools thereby enabling both small-scale experiments in academic research teams, as well as large-scale and industrial automation of synthetic biology workflows. Beyond Escherichia coli, the Genome Engineering Group is moving into other industrially relevant organisms such as Bacillus subtilisAspergilli, and Pichia Pastoris with the intention of improving industrial-scale production of enzymes and pharmaceutical proteins. 

 

 

2. Fundamental understanding of global gene regulation and genome organisation

 

When designing robustly performing cell factories for industrial use, it is essential to minimise the metabolic burden caused by rewiring of genetic circuits and the use of antibiotics in the recombinant strains. For this reason, genetic constructs are typically integrated directly into the genomes in the microbial production host. However, little is currently understood about the interplay of global gene regulation and genome organisation with synthetic integrated genetic constructs. This is particular true when it comes to growth-phase differences, spatio-temporal effects, and under industrial-scale fermentation conditions.

 

Using optimised and standardised synthetic biology approaches, the Genome Engineering Group will explore the performance of genetic constructs in different genome locations and growth phases. This will be supplemented with studies in mutant genetic backgrounds with rewired global gene regulation and genome structure. Using e.g. FLOWseq, which is an established state-of-the-art methodology at the CFB, we will identify the sequence determinants of promoters that makes them more or less robust to changes in the genomic structure and regulation.

As a complementary approach, we will exploit our expertise in adaptive laboratory evolution in the dormant state of bacteria. We believe that studies under dormancy are underappreciated in molecular biology and that much can be learned from studying evolution in this phase. Recent findings in the lab indicate that mutations evolved under dormancy are highly informative for exploring molecular mechanisms. Thus, the evolution of global gene regulation and genome structure will be studied by adaptive evolution of mutants that are perturbed in relevant genetic loci.

 

In summary, we are interested in understanding and engineering gene expression on a global scale and favour two different but highly complementary experimental approaches: on one hand we use bottom-up synthetic biology to design cell factories, on the other hand we are informed by perturbing natural genetic circuits and studying how microbes adapt.

The complex 3D packaging of the genome inside a living cell raises the question how spatial distances and the gene locus specifically influence expression patterns. Whether the positioning of genes along the bacterial chromosome is non-random has long been discussed and is well-documented in eukaryotic systems. With the recent advance in synthetic biology tools, flow cytometry, proteomics and next generation sequencing, we are at CFB in a perfect position to explore this at an unprecedented level. Furthermore, while previous studies have identified important parameters of genome topology and regulation, these have focused largely on gene expression in the exponential growth phase. Ironically, exponential growth is only a minor fraction in the lifespan of a microorganism. We therefore expect to obtain new highly fundamental insights by taking a different approach.

 

Videos that show some of our work: 
Challenges in genome engineering and the SEGA | Carolyn Bayer, Technical University of Denmark

 

Contact

Morten Helge Hauberg Nørholm
Senior Researcher
DTU Biosustain
+45 45 25 80 26