Li Guangjian,Liang Hui,Gao Ruichen,Qin Ling,Xu Pei,Huang Mingtao,Zong Min-Hua,Cao Yufei,Lou Wen-Yong
Microbial biosynthesis has become the leading commercial approach for large-scale production of terpenoids, a valuable class of natural products. Enhancing terpenoid production, however, requires complex modifications on the host organism. Recently, a two-step isopentenol utilization (IU) pathway relying solely on ATP as the cofactor has been proposed as an alternative to the mevalonate (MVA) pathway, streamlining the synthesis of the common terpenoid precursors. Herein, we find that isopentenol inhibits energy metabolism, leading to reduced efficiency of the IU pathway in Saccharomyces cerevisiae . To overcome this, we engineer an IU pathway-dependent (IUPD) strain, designed for growth-coupled production. The IUPD strain is compelled to enhance the ATP supply, essential for the IU pathway, and incorporates a high-throughput screening method for enzyme evolution. The refined IU pathway surpasses the MVA pathway in synthesizing complex terpenoids. Our work offers valuable insights into developing growth-coupled strains adapted to efficient natural product synthesis.
Jiawei Li,Mario Roque Huanca Nina,Xiaoyan Zhang,Yunpeng Bai
Poly(ethylene terephthalate) (PET) and phthalate esters (PAEs) are used extensively as plastics and plasticizers. Enzymatic degradation of PET and PAEs has drawn great attention in recent years; however, evolution of PET- and PAE-degrading enzymes is still a big challenge, partly because of the lack of an effective screening method to detect phthalic acid (PA) and terephthalic acid (TPA), which are the main hydrolysis products of PAEs and PET. Here, by directed evolution of a promiscuous transcription factor, XylS from Pseudomonas putida, we created two novel variants, XylS-K38R-L224Q and XylS-W88C-L224Q, that are able to bind PA and TPA and activate the downstream expression of a fluorescent reporter protein. Based on these elements, whole-cell biosensors were constructed, which enabled the fluorimetric detection of as little as 10 μM PA or TPA. A PAE hydrolase, GoEst15, was preliminarily engineered using this new biosensor, yielding a mutant GoEst15-V3 whose activity toward dibutyl phthalate (DBP) and p-nitrophenyl butyrate was enhanced 2.0- and 2.5-fold, respectively. It was shown that 96.5% DBP (5 mM) was degraded by GoEst15-V3 in 60 min, while the wild-type enzyme degraded only 55% DBP. This study provides an effective screening tool for directed evolution of PAE-/PET-degrading enzymes.
危险品化学品经营许可证（带存储）