Metabolic engineering of Schizosaccharomyces pombe via CRISPR-Cas9 genome editing for lactic acid production from glucose and cellobiose

Metabolic Engineering Communications

Seiya Takayama¹  Aiko Ozaki¹  Tsutomu Tanaka¹  Rie Konishi²  Takuya Matsumoto²  Akihiko Kondo²  Mayumi Kishida²  Chisako Otomo² 

[1] Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1, Rokkodaicho, Nada, Kobe 657-8501, Japan;Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan;

关键词: Schizosaccharomyces pombe;    Genome editing;    Lactic acid;    CRISPR-Cas9 system;   

DOI  :  10.1016/j.meteno.2017.08.002

来源: DOAJ

【 摘 要 】

Modification of the Schizosaccharomyces pombe genome is often laborious, time consuming due to the lower efficiency of homologous recombination. Here, we constructed metabolically engineered S. pombe strains using a CRISPR-Cas9 system and also demonstrated D-lactic acid (D-LA) production from glucose and cellobiose. Genes encoding two separate pyruvate decarboxylases (PDCs), an L-lactic acid dehydrogenase (L-LDH), and a minor alcohol dehydrogenase (SPBC337.11) were disrupted, thereby attenuating ethanol production. To increase the cellular supply of acetyl-CoA, an important metabolite for growth, we introduced genes encoding bacterial acetylating acetaldehyde dehydrogenase enzymes (Escherichia coli MhpF and EutE). D-LA production by the resulting strain was achieved by expressing a Lactobacillus plantarum gene encoding D-lactate dehydrogenase. The engineered strain efficiently consumed glucose and produced D-LA at 25.2 g/L from 35.5 g/L of consumed glucose with a yield of 0.71 g D-LA / g glucose. We further modified this strain by expressing beta-glucosidase by cell surface display; the resulting strain produced D-LA at 24.4 g/L from 30 g/L of cellobiose in minimal medium, with a yield of 0.68 g D-LA / g glucose. To our knowledge, this study represents the first report of a S. pombe strain that was metabolically engineered using a CRISPR-Cas9 system, and demonstrates the possibility of engineering S. pombe for the production of value-added chemicals.

【 授权许可】

Unknown