Reconstructing life

As the knowledge of genes, proteins, and other biological components has developed, so has the interest in studying the interaction between them in order to understand complete biological systems. Here, the systems biology approach provides concepts and tools. The ultimate goal is to rebuild nature on the computer and set up a complete mathematical model of living cells in silico. Such models may then, for instance, be used to optimize the biotechnological production of value-added chemicals by microorganisms.

The research focuses on mathematically modeling the dynamics, regulation, and control of metabolic networks. In particular, we are interested in studying structural, i.e. topological properties of complex metabolic networks and how these structures give rise to metabolic functions. We develop computational tools to predict optimized microbes for biotechnological applications. Most of the experimental work is carried out in collaboration with other research groups within acib.

 

Latest publications

Costs of ribosomal RNA stabilization affect ribosome composition at maximum growth rate

Diana Széliová, Stefan Müller, and Jürgen Zanghellini. 2024. Commun Biol 7, 196. doi.org/10.1038/s42003-024-05815-4

Abstract: Ribosomes are key to cellular self-fabrication and limit growth rate. While most enzymes are proteins, ribosomes consist of 1/3 protein and 2/3 ribonucleic acid (RNA) (in E. coli).

Here, we develop a mechanistic model of a self-fabricating cell, validated across diverse growth conditions. Through resource balance analysis (RBA), we explore the variation in maximum growth rate with ribosome composition, assuming constant kinetic parameters.

Our model highlights the importance of RNA instability. If we neglect it, RNA synthesis is always cheaper than protein synthesis, leading to an RNA-only ribosome at maximum growth rate. Upon accounting for RNA turnover, we find that a mixed ribosome composed of RNA and proteins maximizes growth rate. To account for RNA turnover, we explore two scenarios regarding the activity of RNases. In (a) degradation is proportional to RNA content. In (b) ribosomal proteins cooperatively mitigate RNA instability by protecting it from misfolding and subsequent degradation. In both cases, higher protein content elevates protein synthesis costs and simultaneously lowers RNA turnover expenses, resulting in mixed RNA-protein ribosomes. Only scenario (b) aligns qualitatively with experimental data across varied growth conditions.

Our research provides fresh insights into ribosome biogenesis and evolution, paving the way for understanding protein-rich ribosomes in archaea and mitochondria.

Sulfate limitation increases specific plasmid DNA yield in E. coli fed-batch processes

Mathias Gotsmy, Florian Strobl, Florian Weiß, Petra Gruber, Barbara Kraus, Jürgen Mairhofer, and Jürgen Zanghellini. 2023. Microb Cell Fact 22, 242. doi.org/10.1186/s12934-023-02248-2

Abstract: Plasmid DNA (pDNA) is a key biotechnological product whose importance became apparent in the last years due to its role as a raw material in the messenger ribonucleic acid (mRNA) vaccine manufacturing process. In pharmaceutical production processes, cells need to grow in the defined medium in order to guarantee the highest standards of quality and repeatability. However, often these requirements result in low product titer, productivity, and yield.

In this study, we used constraint-based metabolic modeling to optimize the average volumetric productivity of pDNA production in a fed-batch process. We identified a set of 13 nutrients in the growth medium that are essential for cell growth but not for pDNA replication. When these nutrients are depleted in the medium, cell growth is stalled and pDNA production is increased, raising the specific and volumetric yield and productivity. To exploit this effect we designed a three-stage process (1. batch, 2. fed-batch with cell growth, 3. fed-batch without cell growth). The transition between stage 2 and 3 is induced by sulfate starvation. Its onset can be easily controlled via the initial concentration of sulfate in the medium.

We validated the decoupling behavior of sulfate and assessed pDNA quality attributes (supercoiled pDNA content) in E. coli with lab-scale bioreactor cultivations. The results showed an increase in supercoiled pDNA to biomass yield by 29% upon limitation of sulfate.

In conclusion, even for routinely manufactured biotechnological products such as pDNA, simple changes in the growth medium can significantly improve the yield and quality.

Highlights

  • Genome-scale metabolic models predict growth decoupling strategies
  • Sulfate limitation decouples cell growth from pDNA production
  • Sulfate limitation increases the specific supercoiled pDNA yield by 33% and the volumetric productivity by 13%.
  • We propose that sulfate limitation improves the biosynthesis of over 25% of naturally secreted products in E. coli.

Optimizing VLP production in gene therapy: Opportunities and challenges for in silico modeling

Leopold Zehetner, Diana Széliová, Barbara Kraus, Michael Graninger, Jürgen Zanghellini, and Juan A. Hernandez Bort. 2023. Biotechnology J. doi.org/10.1002/biot.202200636

Abstract: Over the past decades, virus-like particle (VLP)-based gene therapy (GT) evolved as a promising approach to cure inherited diseases or cancer. Tremendous costs due to inefficient production processes remain one of the key challenges despite considerable efforts to improve titers. This review aims to link genome-scale metabolic models (GSMMs) to cell lines used for VLP synthesis for the first time. We summarize recent advances and challenges of GSMMs for Chinese hamster ovary (CHO) cells and provide an overview of potential cell lines used in GT. Although GSMMs in CHO cells led to significant improvements in growth rates and recombinant protein (RP)-production, no GSMM has been established for VLP production so far. To facilitate the generation of GSMM for these cell lines we further provide an overview of existing omics data and the highest production titers so far reported.

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