Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis
The methylotrophic bacterium Methylorubrum extorquens AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered...
Saved in:
Published in | Applied microbiology and biotechnology Vol. 104; no. 10; pp. 4515 - 4532 |
---|---|
Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
Berlin/Heidelberg
Springer Berlin Heidelberg
01.05.2020
Springer Springer Nature B.V |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | The methylotrophic bacterium
Methylorubrum extorquens
AM1 holds a great potential of a microbial cell factory in producing high value chemicals with methanol as the sole carbon and energy source. However, many gene functions remain unknown, hampering further rewiring of metabolic networks. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been demonstrated to be a robust tool for gene knockdown in diverse organisms. In this study, we developed an efficient CRISPRi system through optimizing the promoter strength of
Streptococcus pyogenes
–derived deactivated
cas9
(
dcas9
). When the
dcas9
and sgRNA were respectively controlled by medium P
R/tetO
and strong P
mxaF-g
promoters, dynamic repression efficacy of cell growth through disturbing a central metabolism gene
glyA
was achieved from 41.9 to 96.6% dependent on the sgRNA targeting sites. Furthermore, the optimized CRISPRi system was shown to effectively decrease the abundance of exogenous fluorescent protein gene
mCherry
over 50% and to reduce the expression of phytoene desaturase gene
crtI
by 97.7%. We then used CRISPRi technology combined with 26 sgRNAs pool to rapidly discover a new phytoene desaturase gene
META1_3670
from 2470 recombinant mutants. The gene function was further verified through gene deletion and complementation as well as phylogenetic tree analysis. In addition, we applied CRISPRi to repress the transcriptional level of squalene-hopene cyclase gene
shc
involved in hopanoid biosynthesis by 64.9%, which resulted in enhancing 1.9-fold higher of carotenoid production without defective cell growth. Thus, the CRISPRi system developed here provides a useful tool in mining functional gene of
M. extorquens
as well as in biotechnology for producing high-valued chemicals from methanol.
Key points
Developing an efficient CRISPRi to knockdown gene expression in C1-utilizing bacteria
CRISPRi combined with sgRNAs pool to rapidly discover a new phytoene desaturase gene
Improvement of carotenoid production by repressing a competitive pathway |
---|---|
Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0175-7598 1432-0614 |
DOI: | 10.1007/s00253-020-10543-w |