期刊
ACS SYNTHETIC BIOLOGY
卷 9, 期 5, 页码 1138-1149出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00006
关键词
Pseudomonas putida S12; FDCA; HMF; CRISPR; genome engineering; polyploid
资金
- Ministry of Science and Technology [MOST 107-2221-E-007-046-MY3, 107-2622-8-007-015, 107-3017-F-007-002, 108-3017-F-007-003, 108-2622-8-007-016, 108-2622-E-007-012-CC1]
- Frontier Research Center on Fundamental and Applied Sciences of Matters, from Featured Areas Research Center Program within Ministry of Education, Taiwan [MOE 108QR001I5]
FDCA (2,5-furandicarboxylic add) can be enzymatically converted from HMF (5-hydroxymethylfurfural). Pseudomonas putida S12 is promising for FDCA production, but generating stable P. putida S12 is difficult due to its polyploidy and lack of genome engineering tools. Here we showed that coupling CRISPR and lambda-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 P. putida S12 strains expressing HMF/furfural oxidoreductase (HMFH) and HMF oxidase (HMFO), both being able to convert SO mM HMF to approximate to 142-43 mM FDCA in 24 It Cosupplementation of MnO2 and CaCO3 to the medium drastically improved the cell tolerance to HMF and enhanced FDCA production. Cointegrating HMFH and HMFT1 (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 P. putida S12 strains for FDCA production.
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