BioChem - DOS

Improved ssDNA recombineering for rapid and efficient pathway engineering in Corynebacterium glutamicum

Journal of chemical technology and biotechnology

Su, Tianyuan; Jin, Haiying; Zheng, Yi; Zhao, Qian; Chang, Yizhao; Wang, Qian; Qi, Qingsheng

<P><B>Abstract</B></P><P><B>BACKGROUND</B></P><P><I>Corynebacterium glutamicum</I> is an important microorganism for the industrial production of high value&#8208;added compounds. Therefore, powerful genetic engineering tools are required for metabolic engineering of <I>C. glutamicum</I>.</P><P><B>RESULTS</B></P><P>In this study, the application of ssDNA recombineering was extended to realize efficient and rapid genetic engineering of <I>C. glutamicum</I>. The efficiency of one&#8208;step ssDNA recombineering reached 60% without the need for an additional selective marker. Multi&#8208;nucleotide modifications and strain iterative engineering were also achieved. As evidence of its potential in metabolic engineering, a base substitution mutation of aspartokinase T311I was introduced to relieve the feedback inhibition involved in L&#8208;lysine biosynthesis while simultaneously investigating the combinatory effect of adjacent residues in one step. Iterative engineering enabled rapid mutation of 11 nucleotides in the RBS/initiation codon region to improve lysine production 100&#8208;fold compared with the wild&#8208;type organism.</P><P><B>CONCLUSIONS</B></P><P>CRISPR&#8208;Cas9&#8208;assisted ssDNA recombineering was developed for genome editing of <I>C. glutamicum</I> with high efficiency. This strategy contributes to the toolbox available for genetic engineering of <I>C. glutamicum</I> and boosts the strain engineering for more industrial applications. &copy; 2018 Society of Chemical Industry</P>

2018

John WileySons, Ltd

0268-2575

1097-4660

93

12

pp.3535-3542

10.1002/jctb.5726

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART90533420

CRISPR/Cas9; ssDNA Recombineering; Corynebacterium glutamicum; metabolic engineering