The Zymomonas mobilis regulator hfq contributes to tolerance against multiple lignocellulosic pretreatment inhibitors.
BACKGROUND
Zymomonas mobilis produces near theoretical yields of ethanol and recombinant strains are candidate industrial microorganisms. To date, few studies have examined its responses to various stresses at the gene level. Hfq is a conserved bacterial member of the Sm-like family of RNA-binding proteins, coordinating a broad array of responses including multiple stress responses. In a previous study, we observed Z. mobilis ZM4 gene ZMO0347 showed higher expression under anaerobic, stationary phase compared to that of aerobic, stationary conditions.


RESULTS
We generated a Z. mobilis hfq insertion mutant AcRIM0347 in an acetate tolerant strain (AcR) background and investigated its role in model lignocellulosic pretreatment inhibitors including acetate, vanillin, furfural and hydroxymethylfurfural (HMF). Saccharomyces cerevisiae Lsm protein (Hfq homologue) mutants and Lsm protein overexpression strains were also assayed for their inhibitor phenotypes. Our results indicated that all the pretreatment inhibitors tested in this study had a detrimental effect on both Z. mobilis and S. cerevisiae, and vanillin had the most inhibitory effect followed by furfural and then HMF for both Z. mobilis and S. cerevisiae. AcRIM0347 was more sensitive than the parental strain to the inhibitors and had an increased lag phase duration and/or slower growth depending upon the conditions. The hfq mutation in AcRIM0347 was complemented partially by trans-acting hfq gene expression. We also assayed growth phenotypes for S. cerevisiae Lsm protein mutant and overexpression phenotypes. Lsm1, 6, and 7 mutants showed reduced tolerance to acetate and other pretreatment inhibitors. S. cerevisiae Lsm protein overexpression strains showed increased acetate and HMF resistance as compared to the wild-type, while the overexpression strains showed greater inhibition under vanillin stress conditions.


CONCLUSIONS
We have shown the utility of the pKNOCK suicide plasmid for mutant construction in Z. mobilis, and constructed a Gateway compatible expression plasmid for use in Z. mobilis for the first time. We have also used genetics to show Z. mobilis Hfq and S. cerevisiae Lsm proteins play important roles in resisting multiple, important industrially relevant inhibitors. The conserved nature of this global regulator offers the potential to apply insights from these fundamental studies for further industrial strain development.
Yang
Shihui
S
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA. brownsd@ornl.gov
Pelletier
Dale A
DA
Lu
Tse-Yuan S
TY
Brown
Steven D
SD
D000085:Acetates / Q000633:toxicity; D000900:Anti-Bacterial Agents / Q000633:toxicity*; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001547:Benzaldehydes / Q000633:toxicity; D005662:Furaldehyde / Q000031:analogs & derivatives / Q000633:toxicity; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D008031:Lignin / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D016601:RNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D013312:Stress, Physiological; D017355:Zymomonas / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
D000085:Acetates; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001547:Benzaldehydes; D016601:RNA-Binding Proteins; C036909:lignocellulose; C008046:5-hydroxymethylfurfural; D008031:Lignin; C100058:vanillin; D005662:Furaldehyde
J Proteome Res. 2008 Aug;7(8):3319-28
18590317
Nucleic Acids Res. 1998 Jan 1;26(1):73-9
9399804
Appl Biochem Biotechnol. 1998 Spring;70-72:161-72
9627380
Appl Environ Microbiol. 2006 Jan;72(1):890-900
16391131
Nat Biotechnol. 2009 Oct;27(10):893-4
19816441
Microbiology (Reading). 2008 Mar;154(Pt 3):989-994
18310045
Mol Microbiol. 2004 Mar;51(6):1525-33
15009882
Mol Microbiol. 2003 Nov;50(4):1111-24
14622403
Plasmid. 1987 Jul;18(1):46-53
2962214
PLoS Genet. 2008 Aug 22;4(8):e1000163
18725932
Plasmid. 2008 Jul;60(1):45-52
18501964
Microbiol Sci. 1984 Sep;1(6):133-6
6444136
Gene. 1995 Dec 1;166(1):175-6
8529885
Appl Microbiol Biotechnol. 2003 Dec;63(3):258-66
13680206
Appl Biochem Biotechnol. 2000 Spring;84-86:343-56
10849801
Biotechniques. 1999 May;26(5):824-6, 828
10337469
J Mol Biol. 1990 Oct 5;215(3):403-10
2231712
RNA. 2007 Dec;13(12):2213-23
17959927
Appl Biochem Biotechnol. 2007 Apr;137-140(1-12):847-58
18478439
Arch Microbiol. 1995 Mar;163(3):195-204
7778976
FEBS Lett. 2006 Sep 18;580(21):5084-8
16950260
Adv Biochem Eng Biotechnol. 2007;108:263-88
17522816
Proc Natl Acad Sci U S A. 2010 Jun 8;107(23):10395-400
20484677
BMC Genomics. 2009 Jan 20;10:34
19154596
Appl Biochem Biotechnol. 2001 Spring;91-93:133-46
11963842
Appl Biochem Biotechnol. 1999 Sep;81(3):193-203
10652785
Mol Microbiol. 1994 Jul;13(1):35-49
7984093
Nucleic Acids Res. 2003 Jan 1;31(1):216-8
12519985
Appl Environ Microbiol. 1984 Jan;47(1):198-200
6364969
Bacteriol Rev. 1977 Mar;41(1):1-46
16585