Ogataea (Hansenula) polymorpha, a representative methylotrophic yeast, can efficiently assimilates methanol, and thus is considered as a promising chassis host for efficient methanol biotransformation. 

However, difficulties in convenient and precise genome editing in O. polymorpha limits its metabolic engineering toward industrial application.

Recently, a research group led by Prof. ZHOU Yongjin from the Dalian Institute of Chemical Physics of the Chinese Academy of Sciences achieved efficient and precise genome editing in methylotrophic yeasts.

They developed novel genetic tools for precise genome editing in O. polymorpha by enhancing high homologous recombination (HR) rates, and revealed a strong competition between HR and non-homology end joining (NHEJ).

With these efficient tools, O. polymorpha was harnessed for the first production of fatty alcohols from both glucose and methanol.

This work was published in iScience on Feb. 9.

Recombination machinery engineering for precise genome editing in methylotrophic yeast Ogataea polymorpha (Image by GAO Jiaoqi) 

CRISPR/Cas9 system was constructed and optimized by fine regulation of Cas9 and expression of sgRNA, which achieved efficient genome editing with positive rates up to 90%. Then the dynamical regulation of key gene KU80 in NHEJ pathway and enhanced expression of HR-related proteins (Rad51, Rad52, Sae2) increased HR rates to 60~70%.

"This HR up-regulated system has been applied for scarless gene deletion, homologous integration of large fragments and in vivo assembly of multiple fragments, which enables the first production of fatty alcohols in O. polymorpha," said Prof. ZHOU.

This study was supported by National Natural Science Foundation of China, Dalian Science and Technology Innovation Funding, DMTO and BioChE-X Research Grant of DICP, CAS.