title: Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose.
abstract: BACKGROUND
  An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to β-glucosidases (bG) and cellobiose-specific PTS permease.
  
  
  RESULTS
  We have engineered and evolved a cellobiose-negative and xylose-negative C. glutamicum that utilizes cellobiose as sole carbon and co-ferments cellobiose and xylose. NGS-genomic and DNA microarray-transcriptomic analysis revealed the multiple genetic mutations for the evolved cellobiose-utilizing strains. As a result, a consortium of mutated transporters and metabolic and auxiliary proteins was responsible for the efficient cellobiose uptake. Evolved and engineered strains expressing an intracellular bG showed a better rate of growth rate on cellobiose as sole carbon source than did other bG-secreting or bG-displaying C. glutamicum strains under aerobic culture. Our strain was also capable of co-fermenting cellobiose and xylose without a biphasic growth, although additional pentose transporter expression did not enhance the xylose uptake rate. We subsequently assessed the strains for simultaneous saccharification and fermentation of cellulosic substrates derived from Canadian Ponderosa Pine.
  
  
  CONCLUSIONS
  The combinatorial strategies of metabolic engineering and adaptive evolution enabled to construct C. glutamicum strains that were able to co-ferment cellobiose and xylose. This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels.
authors.affiliation: Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea. jungseok4698@gmail.com.
authors.forename: Jungseok
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Wood Science, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada. jack.saddler@ubc.ca.
authors.forename: Jack N
authors.initials: JN
authors.lastname: Saddler
authors.affiliation: Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea. yum@kist.re.kr.|Department of Clean Energy and Chemical Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea. yum@kist.re.kr.
authors.forename: Youngsoon
authors.initials: Y
authors.lastname: Um
authors.affiliation: Clean Energy Research Center, Korea Institute of Science and Technology (KIST), Hwarangro 14-gil 5, Seongbuk-gu, Seoul, 02792, Republic of Korea. hmwoo@kist.re.kr.|Department of Clean Energy and Chemical Engineering, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon, 34113, Republic of Korea. hmwoo@kist.re.kr.|Green School (Graduate School of Energy and Environment), Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea. hmwoo@kist.re.kr.
authors.forename: Han Min
authors.initials: HM
authors.lastname: Woo
mesh_terms: D002475:Cellobiose / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods; D014994:Xylose / Q000378:metabolism*
chemical_list: D002475:Cellobiose; D014994:Xylose