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

Keywords: Pseudomonas putida; alk-regulatory system; clonal variability; flow cytometry; fluorescent reporter; intra-population variability; phenotypic heterogeneity; plasmid copy number.

Figure 1

Growth, expression, and specific styrene epoxidation activity profiles of P. taiwanensis VLB120 (pA-EGFP_B) (A) and P. taiwanensis VLB120 (pStyAB) (B) grown on citrate as sole carbon source. Cells were cultivated in M9* medium and induced with 0.025% (v/v) DCPK after 3 h of growth. Samples for resting-cell activity, specific fluorescence, and SDS-Page analyses were taken at regular time intervals (every 2 h) after induction. For determination of the specific styrene epoxidation activity, cells were harvested, resuspended in KPI buffer containing 0.5% (v/v) citrate, and supplied with 1.5 mM styrene to start the reaction. Growth rates [h⁻¹] were determined during exponential phase (dashed line). Yields on citrate were determined after inoculation and at the time point of citrate depletion. Error bars represent standard deviations of two independent assays.

Figure 2

Clonal variability in specific styrene epoxidation activity and styA-eGFP expression levels for P. putida DOT-T1E (A) and P. putida KT2440 (B) both harboring pA-EGFP_B. Specific styrene epoxidation activities and expression levels were determined via resting-cell activity assays and specific eGFP fluorescence, respectively. Exemplary results for 8 independent clones are shown. Cultures were induced with 0.025% (v/v) DCPK for 4 h. SDS-PAGE analyses and induction kinetics of respective clones are shown in Figures S6, S7. Average specific styrene epoxidation activities M_w [U g${}_{\text{CDW}}^{-1}$], average specific fluorescence values [OD${}_{450}^{-1}$], coefficients of variation c_ν [%], and experimental errors σ_exp [%] given correspond to 21 and 8 tested cultures of P. putida DOT-T1E (pA-EGFP_B) and P. putida KT2440 (pA-EGFP_B), respectively.

Figure 3

(A) Comparison of clonal variability in specific styrene epoxidation activity and specific eGFP fluorescence with P. putida DOT-T1E (pA-EGFP_B), P. putida KT2440 (pA-EGFP_B), and P. taiwanensis VLB120 (pA-EGFP_B) expressing styA-eGFP under control of the alk-regulatory system. Variations among cultures are displayed via the coefficient of variation c_ν. Dark gray areas represent calculated experimental errors σ_exp [%]. For P. putida KT2440 and P. taiwanensis VLB120 harboring pA-EGFP_B, the c_ν represented by a bar was determined via specific styrene epoxidation activity and specific eGFP fluorescence measurements performed for 8 separate cultures originating from 8 freshly transformed colonies. For P. putida DOT-T1E (pA-EGFP_B), c_ν determination was based on 21 separate cultures. (B) Correlation of specific activities of 21 independent P. putida DOT-T1E (pA-EGFP_B) cultures with respective specific eGFP fluorescence. The correlation of activity and fluorescence is divided in two sections showing a different interdependence. Black squares represent the 4 colonies which were further analyzed via flow cytometry and digital PCR for PCN determination.

Figure 4

Comparison of growth behavior (A,B) and variability in StyA-eGFP levels (C,D) upon expression under the control of the alk- (A,C) and the lac- (B,D) regulatory systems in P. putida DOT-T1E. Results for 4 representative cultures are shown each for P. putida DOT-T1E (pA-EGFP_B) and P. putida DOT-T1E (pA-EGFP_B_lac) (see text for details on clone selection). Cells were cultivated in M9^* medium supplement with 0.5% (w/v) citrate. Induction was initiated at 0 h by the addition of 0.025% (v/v) DCPK or 1 mM IPTG for the alk- or the lac-based expression, respectively. The specific fluorescence was determined via eGFP fluorescence measurement and normalization to the cell concentration. Error bars give the standard deviations of two independent fluorescence measurements.

Figure 5

Subpopulation formation of recombinant P. putida DOT-T1E regarding styA-eGFP expression determined via flow cytometry. (A) Flow cytometry analysis of cultures visualized in Figure 4 expressing the fusion construct styA-eGFP under control of the alk- (left) and the lac-regulatory systems (right) before, 4 h after, and 8 h after induction. Populations are split into non-fluorescing (red), low- to medium-fluorescing (white) and high-fluorescing (blue) subpopulations. (B) Bar chart correlating the percentage of cells showing high eGFP fluorescence and respective specific styrene epoxidation activities of the whole population after 4 h of induction.

Figure 6

Average PCN per cell after 0 h, 4 h and 8 h of induction of P. putida DOT-T1E recombinants. One thousand cells originating from the 8 respective cultures visualized in Figure 5 expressing the fusion construct styA-eGFP under control of the alk- (A) and the lac- (B) regulatory systems were sorted according to defined gates (Figure S9) for fluorescing (eGFP+) and non-fluorescing (eGFP−) cells. PCNs were determined in quadruplicates for each subpopulation and are presented as an average value with standard deviations.