# Variability in subpopulation formation propagates into biocatalytic variability of engineered Pseudomonas putida strains

## TL;DR

This study shows that differences in subpopulations within genetically identical bacterial cultures can lead to variability in biocatalytic performance.

## Contribution

The study is the first to demonstrate that intra-population variability causes clonal variability in genetically identical cultures.

## Key findings

- Subpopulations with differing protein contents were observed in individual cultures.
- Clonal variability in biocatalytic activity correlates with subpopulation sizes.
- Clonal variability is not linked to plasmid copy number but depends on regulatory systems and host strains.

## Abstract

Pivotal challenges in industrial biotechnology are the identification and overcoming of cell-to-cell heterogeneity in microbial processes. While the development of subpopulations of isogenic cells in bioprocesses is well described (intra-population variability), a possible variability between genetically identical cultures growing under macroscopically identical conditions (clonal variability) is not. A high such clonal variability has been found for the recombinant expression of the styrene monooxygenase genes styAB from Pseudomonas taiwanensis VLB120 in solvent-tolerant Pseudomonas putida DOT-T1E using the alk-regulatory system from P. putida GPo1. In this study, the oxygenase subunit StyA fused to eGFP was used as readout tool to characterize the population structure in P. putida DOT-T1E regarding recombinant protein content. Flow cytometric analyses revealed that in individual cultures, at least two subpopulations with highly differing recombinant StyA-eGFP protein contents appeared (intra-population variability). Interestingly, subpopulation sizes varied from culture-to-culture correlating with the specific styrene epoxidation activity of cells derived from respective cultures (clonal variability). In addition, flow cytometric cell sorting coupled to plasmid copy number (PCN) determination revealed that detected clonal variations cannot be correlated to the PCN, but depend on the combination of the regulatory system and the host strain employed. This is, to the best of our knowledge, the first work reporting that intra-population variability (with differing protein contents in the presented case study) causes clonal variability of genetically identical cultures. Respective impacts on bioprocess reliability and performance and strategies to overcome respective reliability issues are discussed.