Engineering a thermoregulated intein-modified xylanase into maize for consolidated lignocellulosic biomass processing

Journal name:
Nature Biotechnology
Volume:
30,
Pages:
1131–1136
Year published:
DOI:
doi:10.1038/nbt.2402
Received
Accepted
Published online

Abstract

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall–degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.

At a glance

Figures

  1. Development of the thermoregulated intein-modified XynB.
    Figure 1: Development of the thermoregulated intein-modified XynB.

    (a) Candidate validation on diagnostic agar plates with phage plaques. XynB, native XynB; pBluescript, no xylanase; Ser158, iXynB variant with the native Tth intein; Ser158-3108, iXynB variant with R51G mutation; Thr134-1101, S+2V;P+3 double mutation. (b) Thermoregulation of iXynB. Cell lysate (biological replicates n = 12) of representative iXynB variants, pBluescript and XynB after heat pretreatment at 37 °C (blue bar) or 59 °C (red bar) for 4 h. Ser158 variants (19, 3103, 3108, 3110, 30, 319 and 342), intein mutation R51G; Thr134 variants (1 and 195), C-terminal extein insertion (P+3); Thr134 variants (1101, 1007, 1065, 1001 and 1108), double mutation (S+2V;P+3); S158-2, intein mutation (S124P; V245D); T134-171, intein mutation (R185H) and C extein mutation (P+3 insertion; L+3W; G+4A). Error bars, mean ± s.d.

  2. Dynamics of iXynB activation and splicing.
    Figure 2: Dynamics of iXynB activation and splicing.

    (a) iXynB activation. Bacterial lysate of representative iXynB variants, pBluescript and native XynB were assayed in triplicate after heat pretreatment at designated time and temperatures (y axis scaled by taking XynB initial activity as 100%). (b) iXynB splicing. Representative western blot of variant Ser158-3108 after heat pretreatment of lysate at designated temperatures and times. Antiserum against XynB was used. iXynB and XynB are ~39 KD and 86 KD, respectively.

  3. Amino acid residues that modulate thermoregulated intein splicing.
    Figure 3: Amino acid residues that modulate thermoregulated intein splicing.

    (a) The numbering corresponds to the intein from Cys1 to Asn423, amino acids in the N-terminal extein are indexed backward from −1, and the amino acids in the C-terminal extein are indexed upward from +1. Thr134 and its neighboring extein sequences are shown in blue, Ser158 and its neighboring extein sequences are shown in green, and key amino acids (R51, P71 and S+2) are shown in red. (b) A structural model of the Tth intein, showing close proximity of the key amino acids (R51 and P71) to splicing junctions (C1 and N423). R51 is 3.3 Å to C1 and P71 is 5.6 Å to N423. Average c-c bond length is 1.5 Å.

  4. xynB and ixynB seed development.
    Figure 4: xynB and ixynB seed development.

    (a) Morphology of representative seeds from transgenic xynB, ixynB and nontransgenic AxB. All seeds were photographed at the same magnification (×1). (b) Biomass of seeds from three cobs each of xynB, ixynB and wild-type (WT, AxB). Error bars, mean ± s.d.

  5. Glucose and xylose yields from processing xynB and ixynB stover.
    Figure 5: Glucose and xylose yields from processing xynB and ixynB stover.

    (a) xynB, ixynB and nontransgenic (AxB) stover were processed and assayed using enzyme cocktails that either included (FCt) or excluded (Ct-Xyl) xylanase. Glucose from three T0 events was measured by HPLC, and the percentage of sugar yield was calculated by comparison with glucan content. (b,c) Corn stover of transgenic ixynB.T0 (n = 3) and its progeny ixynB.T1 (n = 5) was processed together with wild-type (AxB) stover (n = 5) and assayed after treatment with FCt. Glucose (b) and xylose (c) yields were measured by HPLC and theoretical yield (TY) was calculated by comparison with glucan and xylan content. Error bars, mean ± s.d.

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Author information

Affiliations

  1. Agrivida, Inc., Medford, Massachusetts, USA.

    • Binzhang Shen,
    • Xueguang Sun,
    • Xiao Zuo,
    • Taran Shilling,
    • James Apgar,
    • Mary Ross,
    • Oleg Bougri,
    • Vladimir Samoylov,
    • Matthew Parker,
    • Elaina Hancock,
    • Hector Lucero,
    • Benjamin Gray,
    • Nathan A Ekborg,
    • Dongcheng Zhang,
    • Jeremy C Schley Johnson,
    • Gabor Lazar &
    • R Michael Raab

Contributions

B.S. conceived, developed and performed protein engineering experiments, and led enzyme development and data analysis. X.S. and X.Z. constructed genes, and screened and characterized variants. T.S., M.R. and M.P. validated variants. J.A. conducted structural analysis and modeling. O.B. and V.S. oversaw and conducted plant transformation. E.H., H.L., B.G. and N.A.E. conducted plant analysis. D.Z. and J.C.S.J. oversaw and conducted processing experiments. G.L. and R.M.R. managed the overall project, helped design experiments, organized efforts and contributed intellectually. B.S. and R.M.R. wrote the paper.

Competing financial interests

This research was funded by Agrivida, Inc.

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