(228bq) Hybrid Promoter Engineering for the Development of Novel Promoters Inducible By Lignin Derived Phenolics

Lignin valorization is viewed as a key for the development of a cost effective lignocellulosic biorefinery, and synthetic biology tools would play an important role in the construction of an efficient chassis towards this goal. In this work, we have employed hybrid promoter engineering approach for the construction of higher strength phenolics inducible promoters. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, PemrAB that was naturally inducible by phenolics. The strength of the promoters was interrogated by using mCherry as the reporter, and the engineered promoters were found to be stronger than the native promoter. In the presence of vanillin, the strains RIF02, RIF03 and RIF04, with the engineered promoters P_vtac, P_vtrc, and P_vtic had their fluorescence increase by 4.6, 3.0, and 1.5 fold respectively in comparison to RIF01 that had the native promoter PemrAB. The promoters were also observed to be highly specific towards vanillin and further promoter engineering efforts can increase the specificity and promoter strength beyond the current levels. Flow cytometry was employed to identify heterogeneity if present in the cell population. We observed the emergence of a sub-population constituting the healthy and dividing cells especially in the cultures that were induced with 5mM vanillin. The size of this sub-population remained about the same regardless of the promoter employed and it lay between 24% and 27% of the total cell population. The analysis also uncovered that this smaller sub population of cells were primarily responsible for the increased expression that was observed with the engineered promoters. The study also reveals that to achieve higher density of the heterologous proteins, efforts must be laid not only in increasing the expression level of genes but also in maintaining a healthy cell population in the presence of phenolics.