Jiancong Ni,Shengxiu Huang,Weiqiang Yang,Qiaoling Chen,Zhenyu Lin
Glycolysis, a pivotal step in yeast metabolism, plays an indispensable role as a carbohydrate utilization process crucial for cellular survival. Developing advanced technologies to elucidate this fundamental physiological process holds significant scientific implications. Electrochemiluminescence (ECL) imaging exhibits the advantage of negligible background interference and facilitates straightforward visualization, thereby conferring significant value in biomolecular observation. In this study, we present an ECL imaging method for investigating yeast metabolism by utilizing the endogenetic NADH as an efficient coreactant for ECL generation. The yeast glycolysis process drives the conversion of NAD+ to NADH, resulting in enhanced ECL response as well as the increased brightness of ECL images that can be used for quantification of yeast activity. There was a linear correlation between the reciprocal of both the gray value of ECL image and yeast concentration within the range of 6.25 × 106 – 6.25 × 108 CFU/mL. Due to the highly efficient coreactant behavior of NADH, our method demonstrated excellent selectivity with minimal interference. Furthermore, we employed this approach to investigate some toxic inhibitors on yeast metabolism, yielding reliable results. This ECL imaging method not only avoids the use of additional coreactants but also provides a sensitive and intuitive approach for monitoring yeast metabolism, demonstrating great potential in revealing various complex biological processes.
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.
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