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.
Highlights
The use of whole-cell biocatalysts is a promising approach for applications in industrial biotechnology and is favored over the use of isolated enzymes, when host intrinsic cofactor regeneration, degradation of reactive oxygen species, and/or continuous synthesis of instable enzymes are required (Schmid et al, 2001; Woodley, 2006; Leak et al, 2009; Faber, 2011; Schrewe et al, 2013)
The interaction between the host-specific regulatory network and the regulatory system used for recombinant gene expression appeared to play a crucial role, giving evidence for an alk-regulatory system-related extrinsic type of noise as the cause for the observed clonal variability (Lindmeyer et al, 2015). We investigated if such clonal variability in recombinant gene expression involves a variable subpopulation structure or relies on a random distribution of a large range of expression levels among individual cells, and if and to what extent such variability is related to plasmid copy number (PCN)
StyAB is composed of an oxygenase (StyA)-eGFP, encoded on the plasmid pA-EGFP_B and expressed under control of the alk-regulatory system, was characterized regarding its catalytic styrene epoxidation performance in whole microbial cells by means of resting-cell activity assays
Summary
The use of whole-cell biocatalysts is a promising approach for applications in industrial biotechnology and is favored over the use of isolated enzymes, when host intrinsic cofactor regeneration, degradation of reactive oxygen species, and/or continuous synthesis of instable enzymes are required (Schmid et al, 2001; Woodley, 2006; Leak et al, 2009; Faber, 2011; Schrewe et al, 2013). Phenotypic heterogeneity often arises from stochastic variations in regulatory network operation and can be linked to variations in the amounts of intracellular compounds involved in gene expression such as transcription factors or inhibitors (Ryall et al, 2012) These variations are attributed to two different types of noise: extrinsic and intrinsic noise (Elowitz et al, 2002). Intrinsic noise does not depend on regulatory predispositions but results from stochastic events, e.g., the frequency of collision/contact between the individual functional units involved in gene expression (inducer, promotor, regulator) Both intrinsic and extrinsic noise jointly contribute to phenotypic heterogeneity, whereas extrinsic noise was shown to make a greater contribution to total gene expression variability than intrinsic noise (Elowitz et al, 2002; Kaern et al, 2005; Avery, 2006)
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