Gene targets for engineering osmotolerance in Caldicellulosiruptor bescii
Abstract Background Caldicellulosiruptor bescii , a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities...
Saved in:
Published in | Biotechnology for biofuels Vol. 13; no. 1 |
---|---|
Main Authors | , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Netherlands
Springer Science + Business Media
13.03.2020
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | Abstract
Background
Caldicellulosiruptor bescii
, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst.
Results
Two strains of
C. bescii
showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions—introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the
fruR/cra
gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the
fruR
/
cra
-deficient strain, including a substitution mutation in
dnaK
, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner.
Conclusions
Two systems able to effect tolerance to elevated osmolarities in
C. bescii
are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the
dnaK
chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of
fapR
) and the challenges (in the case of
fruR/cra
). |
---|---|
Bibliography: | USDOE Office of Science (SC), Biological and Environmental Research (BER) AC05-00OR22725 |
ISSN: | 1754-6834 1754-6834 |