Department of Chemical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan.
Faculty of Chemistry, University of Science, Vietnam National University Ho Chi Minh City, Ho Chi Minh City 72711, Vietnam.
ACS Synth Biol. 2020 May 15;9(5):1138-1149. doi: 10.1021/acssynbio.0c00006. Epub 2020 Apr 28.
FDCA (2,5-furandicarboxylic acid) can be enzymatically converted from HMF (5-hydroxymethylfurfural). S12 is promising for FDCA production, but generating stable S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and λ-Red recombineering enabled one-step gene integration with high efficiency and frequency, and simultaneously replaced endogenous genes in all chromosomes. Using this approach, we generated two stable S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert 50 mM HMF to ≈42-43 mM FDCA in 24 h. Cosupplementation of MnO and CaCO to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating and (HMF transporter) genes further improved FDCA production, enabling the cells to convert 250 mM HMF to 196 mM (30.6 g/L) FDCA in 24 h. This study implicates the potentials of CRISPR for generating stable S12 strains for FDCA production.
FDCA（2,5-呋喃二甲酸）可通过酶促转化从 HMF（5-羟甲基糠醛）生成。S12 是生产 FDCA 的有前途的菌株，但由于其多倍体性和缺乏基因组工程工具，因此难以生成稳定的 S12。在这里，我们表明，CRISPR 和 λ-Red 重组酶系统的结合能够实现高效且高频的一步基因整合，同时在所有染色体中替换内源性基因。使用这种方法，我们生成了两个稳定的 S12 菌株，它们表达 HMF/糠醛氧化还原酶（HMFH）和 HMF 氧化酶（HMFO），均能够在 24 小时内将 50mM HMF 转化为约 42-43mM FDCA。在培养基中补充 MnO 和 CaCO3 可大大提高细胞对 HMF 的耐受性，并提高 FDCA 的产量。共整合 和 （HMF 转运蛋白）基因进一步提高了 FDCA 的产量，使细胞能够在 24 小时内将 250mM HMF 转化为 196mM（30.6g/L）FDCA。这项研究表明，CRISPR 具有生成用于 FDCA 生产的稳定 S12 菌株的潜力。
