Background

       Directed evolution within a biological organism requires an appropriate chassis to be established. Yeast, as eukaryotic chassis, has been widely used in many fields in synthetic biology due to its high secretion capacity, high growth rate and many other properties. However, yeast used in industry is often limited to a few species, such as S. cerevisiae, and the research of some nonconventional yeast species is often lacking. Additionally, during the development of yeast chassis, the properties of some enzymes may not meet the requirements of industrial production, which often limits the industrial application and requires methods such as directed evolution for optimization. Besides, when producing substances such as monoterpenes that require complex detection methods, the complexity of measuring their yield complicates the evaluation of the effectiveness of directed evolution.

       Kluyveromyces marxianus and Yarrowia lipolytica are two nonconventional yeast strains with unique industrial characteristics. K. marxianus can efficiently utilize lactose as carbon source and exhibits rapid growth, making it ideal for industrial fermentation. It also has a long history of safe usage in dairy products, qualifying it for the production of pharmaceuticals and food-grade proteins. Meanwhile, Y. lipolytica has a high metabolic flux of acetyl-CoA and NAD(P)H, which can be converted through the endogenous mevalonate pathway to produce geranyl pyrophosphate (GPP). It also forms oil droplets that can store lipophilic terpenes and reduce their cytotoxicity to the yeast. We focus on these two nonconventional yeasts, hoping to establish a chassis for continuous directed evolution and high-throughput screening for difficult-to-detect substances.

       After constructing the cell factory, in order to obtain a higher yield strain, we tried to perform continuous directed evolution of the synthase of the target product. In a traditional directed evolution experiment, at least some of these steps are carried out manually, necessitating substantial investment of researcher time and lengthy evolution campaigns, which typically take months or longer to complete. Continuous evolution, by contrast, automates all of these steps, greatly reducing the generation time and the effort required from the researcher during evolution. These advantages enable large sequence-space searches, iterative improvements, and dramatic changes in phenotype over practical time scales. Continuous evolution also enables the exploration of longer and more numerous evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies.

       In order to overcome the difficulties in high-throughput screening of difficult-to-detect product-producing strains in directed evolution, we tried to construct biosensors to measure their production in real time and high-throughput, so as to reflect the directed evolution effect of producing enzymes. Biosensor is an effective method for high-throughput and real-time detection of endogenous substance synthesis. They are important links between catalytic synthesis and directed evolution. For example, the traditional detection method of monoterpenes relies on gas chromatography-mass spectrometry (GC-MS), which is costly and low-throughput, and GC-MS cannot meet the needs of screening high-yield strains. In order to achieve high-throughput endogenous detection of monoterpenes, the development of biosensors has become a common strategy. Here, we constructed a biosensor sensitive to monoterpene borneol in Y. lipolytica chassis, which facilitates the subsequent directed evolution of borneol synthase.