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Functional analysis of arabinofuranosidases and a xylanase of Corynebacterium alkanolyticum for arabinoxylan utilization in Corynebacterium glutamicum

  • Applied genetics and molecular biotechnology
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Abstract

Xylooligosaccharides (XOSs) and arabinoxylooligosaccharides (AXOSs) are major oligosaccharides derived from arabinoxylan. In our previous report, Corynebacterium glutamicum was engineered to utilize XOSs by introducing Corynebacterium alkanolyticum xyloside transporter and β-xylosidase. However, this strain was unable to consume AXOSs due to the absence of α-l-arabinofuranosidase activity. In this study, to confer AXOS utilization ability on C. glutamicum, two putative arabinofuranosidase genes (abf51A and abf51B) were isolated from C. alkanolyticum by the combination of degenerate PCR and genome walking methods. Recombinant Abf51A and Abf51B heterologously expressed in Escherichia coli showed arabinofuranosidase activities toward 4-nitrophenyl-α-l-arabinofuranoside with k cat values of 150 and 63, respectively, with optimum at pH 6.0 to 6.5. However, Abf51A showed only a slight activity toward AXOSs and was more susceptible to product inhibition by arabinose and xylose than Abf51B. Introduction of abf51B gene into the C. glutamicum XOS-utilizing strain enabled it to utilize AXOSs as well as XOSs. The xylI gene encoding a putative xylanase was found upstream of the C. alkanolyticum xyloside transporter genes. A signal peptide was predicted at the N-terminus of the xylI-encoding polypeptide, which indicated XylI was a secreted protein. Recombinant mature XylI protein heterologously expressed in E. coli showed a xylanase activity toward xylans from various plant sources with optimum at pH 6.5, and C. glutamicum recombinant strain expressing native XylI released xylose, xylobiose, xylotriose, and arabino-xylobiose from arabinoxylan. Finally, introduction of the xylI gene into the C. glutamicum AXOS-utilizing strain enabled it to directly utilize arabinoxylan.

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  1. Research Institute of Innovative Technology for the Earth, 9-2, Kizugawadai, Kizugawa-shi, Kyoto, 619-0292, Japan

    Takayuki Kuge, Akira Watanabe, Satoshi Hasegawa, Haruhiko Teramoto & Masayuki Inui

  2. Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma-shi, Nara, 630-0101, Japan

    Takayuki Kuge & Masayuki Inui

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  1. Takayuki Kuge
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  2. Akira Watanabe
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Correspondence to Masayuki Inui.

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Kuge, T., Watanabe, A., Hasegawa, S. et al. Functional analysis of arabinofuranosidases and a xylanase of Corynebacterium alkanolyticum for arabinoxylan utilization in Corynebacterium glutamicum . Appl Microbiol Biotechnol 101, 5019–5032 (2017). https://doi.org/10.1007/s00253-017-8280-y

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