Description

       In our project, we constructed two production platforms for difficult-to-detect products with continuous directed evolution in two nonconventional yeasts, namely, Kluyveromyces marxianus-tagatose production platform and Yarrowia lipolytica-monoterpene production platform. In addition, we constructed biosensor systems sensitive to the target product in two platforms to achieve high-throughput screening.


       For Y. lipolytica chassis, it has a high metabolic flux of acetyl coenzyme A and NAD ( P ) H, which can be converted into geranyl pyrophosphate through the endogenous mevalonate pathway, and this yeast can form oil droplets, which can store lipophilic terpenoids and reduce its cytotoxicity to yeast. Based on these two characteristics, we selected three monoterpenes, nerol, borneol, and linalool, as the target products of the Y. lipolytica cell factory. Using the endogenous MVA pathway of Y.lipolytica PO1f strain, we only introduced the enzymes that synthesize these three monoterpenes into it, and obtained nerol synthesis strain PLN1, borneol synthesis strain PBE1, and linalool synthesis strain PAE1.
       For the K. marxianus chassis, based on its efficient utilization of lactose as carbon source and its ability to grow rapidly at high temperatures, we chose tagatose, a high-value rare sugar with huge potential market and can be used as a sugar substitute, as the target product of this cell factory. Tagatose synthesis requires galactose as a precursor. We used lactose as substrate and knocked out the gene encoding galactose kinase in the yeast chassis by homologous recombination to accumulate intracellular galactose. We constructed strains containing two types of tagatose synthesis pathways. The first type is one-step isomerization from D-galactose to D-tagatose, and the second type is the conversion from galactose to tagatose by reduction and dehydrogenation. It is worth mentioning that we obtained three different galactose reductases from three organisms, and screened strains that can produce tagatose more efficiently by constructing them into the reduction-dehydrogenation pathway.

       After obtaining the K. marxianus-tagatose production chassis and the Y. lipolytica-monoterpene production chassis, we introduced the continuous directed evolution system nyEvolvR to achieve the directed evolution of synthase, thereby obtaining higher yield. The nyEvolvR system integrates the specificity of the CRISPR / Cas9 system and the mutant activity of the error-prone DNA polymerase. The method uses a designed guide RNA (gRNA) of about 20 nucleotides for precise targeting. The nyEvolvR technology consists of two key components: enhanced nick Cas9 (enCas9) and error-prone DNA polymerase I, which are fused together. The mechanism of the nyEvolvR system is very simple: gRNA guides enCas9 to the target motif, forming a gap. Subsequently, the error-prone DNA polymerase I fills the gap by introducing random base substitution, resulting in mutations in the target gene region, which facilitates continuous directed evolution.

       Finally, in order to evaluate the effect of continuous directed evolution by measuring the improvement of the difficult-to-detect products yield after introducing nyEvolvR system, we constructed biosensors specific to the target products on two platforms respectively. In Y. lipolytica, we constructed a borneol biosensor. We used the allosteric transcription factor CamR manipulation system from Pseudomonas to heterologously express the inhibitory operon CamR in Y. lipolytica. The CamR binding sequence was introduced between the TATA box and the transcription start site (TSS) of the endogenous strong promoter pTEF1, thereby transforming the promoter into a borneol-inducible promoter. Then we used hrGFP as a reporter gene to evaluate the promoter efficiency by measuring the fluorescence intensity per OD600 (RFU/ OD600). In K. marxianus, we designed a tagatose biosensor. We found a gene cluster that controls the metabolism of tagatose in E. coli Nissle 1917 (EcN). Since the opening and closing of the gene cluster are induced by tagatose, we speculate that the gene cluster must contain regulatory factors that can bind tagatose and regulate gene expression. Therefore, we attempted to transplant the entire EcN gene cluster into the K. marxianus-tagatose production platform in order to achieve efficient endogenous detection of tagatose. In order to achieve this goal, we cleverly used eGFP to replace a non-essential gene in the gene cluster that can be complemented by endogenous enzymes, so that the induction of the gene cluster by tagatose can be measured by fluorescence intensity.


Fig.1. Schematic diagram of the integrated directed evolution platform for difficult-to-detect substances. (a) Construction and directed evolution principles of the EvolvR system in nonconventional yeast. (b) Efficient detection of difficult-to-detect substances through biosensor. (c) The working principles of biosensor.

       In the end, we have constructed a directed evolution platform capable of targeting various challenging chemical substrates, and achieved high-throughput endogenous detection through biosensors. Furthermore, we attempted directed evolution tests on the synthesis enzymes of two high-value-added products, borneol and tagatose, to validate the functionality of our system. In doing so, we hope to establish a research paradigm for other difficult-to-detect substances, based on a directed evolution platform combining biosensors and cell factories.
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