title: Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate.
abstract: BACKGROUND
  Complete conversion of the major sugars of biomass including both the C5 and C6 sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to 'hydrolysate toxicity,' a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate.
  
  
  RESULTS
  A turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several 'sub-strains' were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanol yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3's improved performance.
  
  
  CONCLUSIONS
  We have adapted/evolved Z. mobilis strain 8b for enhanced tolerance to the toxic compounds present in corn stover hydrolysates. The adapted strain SS3 has higher xylose utilization rate and produce more ethanol than the parent strain. We have identified transcriptional changes which may be responsible for these phenotypes, providing foundations for future research directions in improving Z. mobilis as biocatalysts for the production of ethanol or other fuel precursors.
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Mohagheghi
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Linger
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Shihui
authors.initials: S
authors.lastname: Yang
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Holly
authors.initials: H
authors.lastname: Smith
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: Dowe
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: National Renewable Energy Laboratory, National Bioenergy Center, 15013, Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Philip T
authors.initials: PT
authors.lastname: Pienkos
keywords: Adaptation; Next-generation sequencing (NGS); Pretreated corn stover; Turbidostat; Xylose; Zymomonas mobilis
references.citation: Appl Biochem Biotechnol. 2003 Spring;105 -108:69-85
references.pmid: 12721476
references.citation: Nat Biotechnol. 2009 Oct;27(10):893-4
references.pmid: 19816441
references.citation: Biotechnol Biofuels. 2012 Feb 27;5:8
references.pmid: 22369467
references.citation: Biotechnol Biofuels. 2013 Jul 09;6(1):99
references.pmid: 23837621
references.citation: J Biotechnol. 2009 Dec;144(4):259-67
references.pmid: 19683550
references.citation: BMC Genomics. 2009 Jan 20;10:34
references.pmid: 19154596
references.citation: Curr Opin Microbiol. 2009 Jun;12(3):223-30
references.pmid: 19467921
references.citation: BMC Microbiol. 2010 May 07;10:135
references.pmid: 20459639
references.citation: Microbiology. 2008 Dec;154(Pt 12):3609-23
references.pmid: 19047729
references.citation: Appl Biochem Biotechnol. 2005 Spring;121-124:923-34
references.pmid: 15930571
references.citation: J Ind Microbiol Biotechnol. 2010 Oct;37(10):993-1021
references.pmid: 20835748
references.citation: Biotechnol Bioeng. 2011 Sep;108(9):2046-52
references.pmid: 21455937
references.citation: Proc Natl Acad Sci U S A. 2010 Jun 8;107(23):10395-400
references.pmid: 20484677
references.citation: Front Microbiol. 2014 May 22;5:246
references.pmid: 24904559
references.citation: Science. 1995 Jan 13;267(5195):240-3
references.pmid: 17791346
references.citation: Nucleic Acids Res. 2012 Aug;40(15):e115
references.pmid: 22730293
references.citation: Biotechnol Biofuels. 2012 Aug 13;5(1):60
references.pmid: 22888758
references.citation: Biotechnol Biofuels. 2014 Sep 30;7(1):140
references.pmid: 25298783
references.citation: J Microbiol Biotechnol. 2010 Jul;20(7):1156-62
references.pmid: 20668411
references.citation: Biotechnol Biofuels. 2014 Feb 18;7(1):23
references.pmid: 24548527
references.citation: Nucleic Acids Res. 2015 Jan;43(Database issue):D204-12
references.pmid: 25348405
references.citation: Biotechnol Biofuels. 2014 Feb 01;7(1):19
references.pmid: 24485299
references.citation: PLoS One. 2013 Jul 16;8(7):e68886
references.pmid: 23874800