Displaced by Deceivers: Prevention of Biosensor Cross-Talk Is Pivotal for Successful Biosensor-Based High-Throughput Screening Campaigns

Abstract

Transcriptional biosensors emerged as powerful tools for protein and strain engineering as they link inconspicuous production phenotypes to easily measurable output signals such as fluorescence. When combined with fluorescence-activated cell sorting, transcriptional biosensors enable high throughput screening of vast mutant libraries. Interestingly, even though many published manuscripts describe the construction and characterization of transcriptional biosensors, only very few studies report the successful application of transcriptional biosensors in such high-throughput screening campaigns. Here, we describe construction and characterization of the trans-cinnamic acid responsive transcriptional biosensor pSenCA for Escherichia coli and its application in a FACS based screen. In this context, we focus on essential methodological challenges during the development of such biosensor-guided high-throughput screens such as biosensor cross-talk between producing and nonproducing cells, which could be minimized by optimization of expression and cultivation conditions. The optimized conditions were applied in a five-step FACS campaign and proved suitable to isolate phenylalanine ammonia lyase variants with improved activity in E. coli and in vitro. Findings from this study will help researchers who want to profit from the unmatched throughput of fluorescence-activated cell sorting by using transcriptional biosensors for their enzyme and strain engineering campaigns.

Keywords: directed evolution; fluorescence-activated cell sorting; library screening; product sensing; protein engineering; transcriptional biosensor.

Figure 1

trans-Cinnamic acid biosensor pSenCA. (A) Schematic of the sensor principle. Upon binding of supplemented CA, the HcaR regulator undergoes conformational changes that enable binding to the target promoter P_hcaE and activation of hcaE′ and eyfp expression. (B) Dose–response plot, CA (circles) or pHCA (filled circles) were supplemented extracellularly in eight different concentrations ranging from 1 to 1000 μM. The biosensor response after 24 h is shown as fold change in specific EYFP fluorescence in comparison to the background fluorescence (no inducer). Error bars represent standard deviations calculated from three biological replicates. CA, trans-cinnamic acid; pHCA, p-coumaric acid; EC5, inducer concentration that results in 5% of maximal fold induction; EC95, inducer concentration resulting in 95% of maximal fold induction.

Figure 2

Influence of induction of heterologous xal_Tc expression with 13 μM l-Ara or 130 μM l-Ara on biosensor response and CA production in E. coli pSenCA pBAD-xal_Tc. After cultivation for 3 h in the presence of different l-Ara concentrations, either 3 mM l-Phe (circles and triangles) or 3 mM CA (squares) was added. Samples were taken at four time points depicted by dotted lines (A) Specific fluorescence is shown (EYFP fluorescence × biomass formation^–1, arbitrary units). (B) CA concentration in E. coli culture supernatants. (C) FACS measurement of EYFP fluorescence of 62 000 representative single cells in the histogram representation. Abbreviations: CA, trans-cinnamic acid; l-Ara, l-arabinose; and l-Phe, l-phenylalanine.

Figure 3

EYFP fluorescence of various mixed cultures of trans-cinnamic acid producing and nonproducing E. coli strains during cytometric analysis. All cultures were started using different inoculums (iOD₆₀₀) as indicated. All cultivations were performed in the in the presence of 13 μM l-arabinose for induction of heterologous xal_Tc expression and 3 mM l-Phe as XAL-substrate always added 3 h after starting each cultivation. FACS measurements were performed 5 h after substrate addition. Strains: CA⁺, E. coli pSenCA pBAD-xal_Tc; CA^–, E. coli pSenCA pBAD.

Figure 4

Stepwise enrichment of improved CA producers from a xal_Tc library using biosensor-based FACS-screening. The continuous orange graph depicts the development of the CA titer of the xal_Tc library at the culture level relative to the starting variant E. coli pSenCA pBAD-xal_Tc (black line). The dashed orange graph depicts the CA titer development in a control experiment, in which another positive sorting (fluorescence) instead of the negative sorting (no fluorescence) was performed. The dark gray shading depicts the CA-titer range of one standard deviation from the Xal_Tc starting variant, whereas light gray shading depicts the CA-titer range of three standard deviations. Histograms show the fluorescence distribution of the cultures of each cultivation step without induction of heterologous gene expression (no l-Ara) or substrate addition (no l-Phe) (histogram without pattern), with supplementation of 13 μM l-Ara and 3 mM l-Phe (vertical lines) and 130 μM l-Ara and 3 mM l-Phe added (horizontal lines). Conditions of each step leading to the eventually isolated xal_Tc variants are highlighted in bright orange relative to the conditions only used as controls that are shown in dim orange. Gray boxes visualize the respective sorting gate set in each step: Step 1–4, positive sorts (isolation of the top 5% fluorescing events); Step 5, negative sort (isolation of the lower 5% fluorescing events). Cultivations for CA titer determination were always performed in triplicates, error bars depict the respective standard deviations. The dotted line depicts development of CA production for an additional round of positive sorting in step five (Supplementation of 13 μM l-Ara), which was performed in parallel for comparison. Abbreviations: CA, trans-cinnamic acid; l-Ara, l-arabinose; l-Phe, l-phenylalanine.

Figure 5

(A) trans-Cinnamic acid production of 183 FACS-isolated Xal_Tc variants. Presented data are means of three cultivations and error bars depict standard deviations. The dark gray shading depicts the CA-titer range of one standard deviation from the Xal_Tc starting variant, whereas light gray shading depicts the CA-titer range of three standard deviations. (WT cultivated as biological triplicates). Variants selected for a more detailed analysis are highlighted in orange. (B), CA (left) and pHCA (right) production of 19 strains selected during the initial characterization (Figure 5A), after retransformation of the respective pBADxal_TcEc plasmid. All cultivations were performed in biological triplicates, error bars depict the standard deviation. Abbreviations: CA, trans-cinnamic acid; pHCA, p-coumaric acid.