Directed Evolution of Terpene Synthases Using High-Throughput Colorimetric Screening Based on Substrate Consumption

Terpenoids are one of the largest groups of natural products with huge structural diversity and molecular functions including pharmaceuticals, fragrance, synthetic rubber and diesel/jet fuels. Integrated efforts by synthetic biologists have dramatically improved the production levels of valuable terpenoids in heterogonous hosts such as microbes or plants. This improvement has primarily been achieved by metabolic engineering efforts, reactor designs, and host breeding, but not by engineering the catalytic capacity of terpene synthases (TPSs). This is despite that TPSs are generally slow enzymes (k_cat values typically range from 1 min^-1 to 1 sec^-1). Given the maturation of metabolic engineering of isoprenoid pathways, the major limiting factor in the total production levels of terpene compounds has been shifting to the TPSs.

TPSs catalyze complex and multi-step carbon-carbon bond formation reactions including carbocation formation, rearrangements, and quenching of reactive intermediates into various terpene structures. The complexity of these processes renders rational engineering of terpene synthases nearly impossible. In addition, forward engineering of TPSs have been severely limited due to the lack of reliable screening/selection systems for TPSs: both substrates and products of TPSs are colorless, diverse in structure, and volatile. A systematic and high-throughput screening of TPS activities should greatly accelerate the advancement of the forward engineering of TPS.

In this study, we developed a high-throughput colorimetric assay for TPS based on substrate consumption. Here, the expression of active TPSs results in decreased building blocks for carotenoid biosynthesis, thereby reducing the pigmentation of the host cells. We show that this strategy enables selection for various types of TPSs and/or isoprenyl transferases, irrespective of their product types.

Using this method, we attempted to the forward engineering of TPSs. Various TPSs for monoterpnene, sesquiterpene, and diterpene, were subjected to error-prone PCR to generate large pool of TPS variants. The resultant TPS libraries were then transformed into E. coli cells harboring either of C₃₀- or C₄₀-carotenoid pathway enzymes. In order to obtain TPS mutants with improved cellular activity, the colonies with least pigmentation were selected. The selected mutants thus obtained were then re-screened to eliminate false positives from them, and were analyzed for their performance in the cell harboring another plasmids for elevating the precursor supply. The production level of terpene was higher for TPS variants when compared with the cells expressing the wild type, indicating our screening method indeed isolate the TPS variants with elevated cellular activity.

In this talk, we present a series of screening constructs specially made for isolating active variants of monoterpene, sesquiterpene, and diterpene synthases. Also presented are our current efforts to make specific/selective screen for the size-variant TPSs.