Development and optimization of N‐acetylneuraminic acid biosensors in Bacillus subtilis

Abstract

Transcriptional factor (TF)‐based metabolite‐responsive biosensors are important tools for screening engineered enzymes with desired properties and for the dynamic regulation of biosynthetic pathways. However, TF‐based biosensor construction is often constrained by undesired effects of TF‐binding site sequence insertion on gene expression and unpredictable optimal TF expression levels. In the present study, a stepwise TF‐based biosensor construction approach was developed using an N‐acetylneuraminic acid (NeuAc) biosensor for Bacillus subtilis, as a case study. Specifically, 12 promoters with various strengths were selected as the first promoter library. Next, binding site sequences for the NanR were inserted into various positions of the selected promoter sequences to develop the second promoter library, resulting in 6 engineered promoters containing TF‐binding site sequences (NanO), without major effects on promoter strength. NanR expression cassettes with different expression levels were further integrated to construct the biosensor library, yielding 9 NeuAc biosensors with efficient repression in the absence of NeuAc. Finally, biosensor activation was characterized by testing fold changes in expression levels of the green fluorescent protein reporter in the presence of NeuAc in vivo, which revealed 61‐fold activation when NeuAc was present. The NeuAc biosensor developed in this study can be used for screening engineered enzymes for enhanced NeuAc biosynthesis in B. subtilis.

Inducible Population Quality Control of Engineered Bacillus subtilis for Improved N-Acetylneuraminic Acid Biosynthesis

“…Under the control of this biosensor, gfp expression was inhibited without NeuAc and was activated in the presence of NeuAc (Figure 1a), but this biosensor showed the almost same output in the presence of 0.1 to 2 g/L of NeuAc. 31 Therefore, the NeuAc-responsive biosensor was used to analyze the distribution of the producing and nonproducing cell subpopulations in NeuAc synthesis performance. We next introduced the NeuAc-responsive biosensor 31 into NeuAc-producing strain No.…”

“…31 Therefore, the NeuAc-responsive biosensor was used to analyze the distribution of the producing and nonproducing cell subpopulations in NeuAc synthesis performance. We next introduced the NeuAc-responsive biosensor 31 into NeuAc-producing strain No. 1, generating strain No.…”

“…1 with 2.15 g/L of NeuAc titer, which was constructed by overexpressing the key genes related to NeuAc synthesis and blocking the competitive and branched pathways. , Metabolite-responsive biosensor-based flow cytometry has been broadly used to reveal cell-to-cell variation . Therefore, we used a NeuAc-responsive biosensor, consisting of the transcriptional factor NanR and promoter veg105 (P veg105 ) with the NanR-binding site, as well as the gfp reporter gene, to reveal the distribution of cell subpopulations in NeuAc synthesis performance using flow cytometry. Under the control of this biosensor, gfp expression was inhibited without NeuAc and was activated in the presence of NeuAc (Figure a), but this biosensor showed the almost same output in the presence of 0.1 to 2 g/L of NeuAc .…”

Engineering of Synthetic Transcriptional Switches in Yeast

“…Besides, Umeno et al argued that TFs are intrinsically evolvable; therefore, mutants that are responsive to non-native compounds can be reached within a few directed evolution cycles (89). Indeed, dozens of new or evolved genetic switches (biosensors) based on bacterial TFs have been reported within the last 2 years (1,90,91,92,93,94,95,96,97,98). Library creation guided by machine-learning technology combined with deep mutagenesis and extensive sequencing technology will further accelerate the discovery of novel TF mutants (98).…”

Special issue (67:4): Synthetic and engineered enzymes for biocatalysis and biotransformation

“…The results reported by Gul et al [17] demonstrate the successful biosensing of epichlorohydrin as a model analyte, using halohydrin dehalogenase as a biological recognition element. Transcriptional factor-based N-acetylneuraminic acid (NeuAc) biosensors in Bacillus subtilis were developed by Zhang et al [18]. They also propose that the best NeuAc biosensor may be used for screening engineered enzymes and enhancing NeuAc biosynthesis.…”