An automated pipeline for the screening of diverse monoterpene synthase libraries

Abstract

Monoterpenoids are a structurally diverse group of natural products with applications as pharmaceuticals, flavourings, fragrances, pesticides, and biofuels. Recent advances in synthetic biology offer new routes to this chemical diversity through the introduction of heterologous isoprenoid production pathways into engineered microorganisms. Due to the nature of the branched reaction mechanism, monoterpene synthases often produce multiple products when expressed in monoterpenoid production platforms. Rational engineering of terpene synthases is challenging due to a lack of correlation between protein sequence and cyclisation reaction catalysed. Directed evolution offers an attractive alternative protein engineering strategy as limited prior sequence-function knowledge is required. However, directed evolution of terpene synthases is hampered by the lack of a convenient high-throughput screening assay for the detection of multiple volatile terpene products. Here we applied an automated pipeline for the screening of diverse monoterpene synthase libraries, employing robotic liquid handling platforms coupled to GC-MS, and automated data extraction. We used the pipeline to screen pinene synthase variant libraries, with mutations in three areas of plasticity, capable of producing multiple monoterpene products. We successfully identified variants with altered product profiles and demonstrated good agreement between the results of the automated screen and traditional shake-flask cultures. In addition, useful insights into the cyclisation reaction catalysed by pinene synthase were obtained, including the identification of positions with the highest level of plasticity, and the significance of region 2 in carbocation cyclisation. The results obtained will aid the prediction and design of novel terpene synthase activities towards clean monoterpenoid products.

Alternative metabolic pathways and strategies to high-titre terpenoid production inEscherichia coli

“…255 Many of these approaches can be accelerated using high-throughput automated assembly, screening and MS analysis. 256,257 Consequently, new bacterial terpenoid biosynthetic pathways will be identied and utilised in future high-titre production in E. coli or other bacterial hosts.…”

“…This might involve the engineering of enzymes that are more resistant to feedback control, or enzymes with improved catalytic efficiency, or new specicities towards desired target activities/products. 77,137,168,173,186,198,257,285,286 The use of alternative and articial pathways with this ever-expanding enzyme resource and pathway engineering tools should extend current capabilities, thereby allowing high-titre production of a wide range of terpenoids in E. coli and other microbial species.…”

“…Amongst others, developments in high-throughput GC-QTOF, which measures terpenoids from the small volumes of multi-well plates, is potentially an attractive approach. 257 Balancing central metabolism precursors, replacing the carbon source, modifying the energy and reducing power balance in the cell, and other approaches will also need to be explored alongside the engineering of new biosynthetic pathways. Redirecting carbon ux and balancing precursor levels are known to increase terpenoid levels, [294][295][296][297][298] as is enhanced availability of essential cofactors such as ATP and NADPH.…”

Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids

“…Selecting residues for modification from plasticity regions of plant MTPSs or according to the results of molecular dynamics simulations, the functional changes of substitutions can be more predictable [ 101 ]. Furthermore, customizing the Design–Build–Test–Learn cycle by high-throughput screening method for monoterpenoids, such as developing an automatic pipeline [ 102 ], can significantly enhance the efficiency and fuel the application of plasticity engineering of plant MTPSs for microbial production of terpenoids.…”

Robotics for enzyme technology: innovations and technological perspectives

“…In another work, Leferink and coworkers developed an automated system that was applied for screening libraries of plant monoterpene cyclases/synthases (mTC/S) expressed in E. coli. The screening system explored robotic liquid handling platforms coupled to GC-MS (Leferink et al 2019). Recently, various tools of machine learning, artificial neural network (ANN), genetic algorithmartificial neural network (GA-ANN), and other similar tools are being combined with high-throughput screening methods and explored in various enzyme engineering protocols (Kumar et al 2019;Mandeep et al 2021;Saini et al 2020).…”