Coexpression of a β-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome

Abstract

Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCECaldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

Keywords: Caldicellulosiruptor; biomass deconstruction; consolidated bioprocessing; xylanase; β-d-xylosidase.

FIG 1

Strain construction flow chart. The A. cellulolyticus E1 and Acel_0180 genes were inserted into the JWCB18 chromosome at chromosomal integration sites one (CIS1) and CIS2, respectively. The plasmids with (pSKW28) and without (pJGW07) the TM_0076 gene from T. maritima then were introduced into the JWCB18, JWCB52, and JWCB87 strains.

FIG 2

Confirmation of β-d-xylosidase expression and activity in C. bescii. (A) SDS-PAGE analysis of concentrated extracellular proteins (10 μg). (B) Zymogram analysis of concentrated extracellular proteins (15 μg) using 0.3 mM MUX as a substrate for detecting protein bands with β-d-xylosidase activity. (C) Zymogram analysis of concentrated extracellular proteins (15 μg) using 5 mM MUG as a substrate for detecting protein bands with β-d-glucosidase activity. M, prestained SDS-PAGE standards, broad range (Bio-Rad Laboratories); 1, JWCB82 (parental strain); 2, JWCB73 (E1-expressing strain); 3, JWCB95 (E1 + Tm_0076-expressing strain).

FIG 3

Chromosomal integration of the Acel_0180 xylanase gene into the C. bescii genome. (A) A depiction of the chromosomal location and integration event of the Acel_0180 expression cassette. SP, signal peptide; GH10, a Family 10 glycoside hydrolase; CBM3, a Family 3 carbohydrate-binding module; CBM2, a Family 2 carbohydrate-binding module. (B) Agarose gel showing PCR products amplified using primers SK65 and SK66 annealing to regions outside the site of integration in the parent strain, JWCB52 (ΔpyrFA+ E1), 2.2 kb (lane 1); the newly constructed strain JWCB87 (ΔpyrFA+ E1 + Acel_0180), 4.4 kb (lane 2); no template PCR control (lane 3); and NEB 1-kb DNA ladder (lane M).

FIG 4

Effects of expression of Tm_0076 β-d-xylosidase and Acel_0180 xylanase on the activity of the C. bescii exoproteome. (A) The enzyme was incubated at 65°C or 75°C for 10 min in reaction buffer containing 5 mM pNP-X, 20 mM MES buffer (pH 5.5), 1 mM DTT, 1 mM CaCl₂, and 1 mM MgCl₂. (B and C) Relative enzymatic activity of the extracellular fraction of C. bescii strains on oat spelt and birchwood xylans. Activity of extracellular protein (25 μg/ml concentrated protein) on oat spelt and birchwood xylans was measured after 12 h of incubation at 65°C (B) or 75°C (C). JWCB82, the parent strain used in these experiments; JWCB73, the E1 expression strain; JWCB95, the E1 expression strain containing Tm_0076; JWCB102, the E1 and Acel_0180 expression strain; JWCB103, the E1 and Acel_0180 expression strain containing Tm_0076. Results are means from triplicate experiments, and error bars indicate standard deviations.

FIG 5

Growth of C. bescii strains on cellobiose (A), xylan substrates (B), Avicel (C), or Avicel plus oat spelt xylan (D). (A) Growth as measured by OD at 680 nm. (B) Viable cell numbers after 36 h of cultivation on xylan substrates. (C and D) Growth of recombinant strains on Avicel without (C) and with (D) the addition of xylan. JWCB82, the parent strain used in these experiments; JWCB73, the E1 expression strain; JWCB95, the E1 expression strain containing Tm_0076; JWCB102, the E1 and Acel_0180 expression strain; JWCB103, the E1 and Acel_0180 expression strain containing Tm_0076. Results are the means from duplicate experiments, and error bars indicate standard deviations.