Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose

Abstract

Background: The Caldicellulosiruptor bescii genome encodes a potent set of carbohydrate-active enzymes (CAZymes), found primarily as multi-domain enzymes that exhibit high cellulolytic and hemicellulolytic activity on and allow utilization of a broad range of substrates, including plant biomass without conventional pretreatment. CelA, the most abundant cellulase in the C. bescii secretome, uniquely combines a GH9 endoglucanase and a GH48 exoglucanase in one protein. The most effective commercial enzyme cocktails used in vitro to pretreat biomass are derived from fungal cellulases (cellobiohydrolases, endoglucanases and a β-d-glucosidases) that act synergistically to release sugars for microbial conversion. The C. bescii genome contains six GH5 domains in five different open reading frames. Four exist in multi-domain proteins and two as single catalytic domains. E1 is a GH5 endoglucanase reported to have high specific activity and simple architecture and is active at the growth temperature of C. bescii. E1 is an endo-1,4-β-glucanase linked to a family 2 carbohydrate-binding module shown to bind primarily to cellulosic substrates. We tested if the addition of this protein to the C. bescii secretome would improve its cellulolytic activity.

Results: In vitro analysis of E1 and CelA shows synergistic interaction. The E1 gene from Acidothermus cellulolyticus was cloned and expressed in C. bescii under the transcriptional control of the C. bescii S-layer promoter, and secretion was directed by the addition of the C. bescii CelA signal peptide sequence. The vector was integrated into the C. bescii chromosome at a site previously showing no detectable detrimental consequence. Increased activity of the secretome of the strain containing E1 was observed on both carboxymethylcellulose (CMC) and Avicel. Activity against CMC increased on average 10.8 % at 65 °C and 12.6 % at 75 °C. Activity against Avicel increased on average 17.5 % at 65 °C and 16.4 % at 75 °C.

Conclusions: Expression and secretion of E1 in C. bescii enhanced the cellulolytic ability of its secretome. These data agree with in vitro evidence that E1 acts synergistically with CelA to digest cellulose and offer the possibility of engineering additional enzymes for improved biomass deconstruction with the knowledge that C. bescii can express a gene from Acidothermus, and perhaps other heterologous genes, effectively.

Fig. 1

Synergistic effect of purified C. bescii CelA and A. cellulolyticus E1 enzymes in vitro. Avicel hydrolysis by C. bescii CelA (black squares) at an enzyme loading of 15 mg/g, A. cellulolyticus E1 (black dots) at an enzyme loading of 4 mg/g, and a mixture of C. bescii CelA and A. cellulolyticus E1 (red triangles) at an enzyme loading of 11 and 4 mg/g, respectively.

Fig. 2

Chromosomal integration of the E1 gene into the C. bescii genome. a A diagram of native E1 protein: SP, signal peptide; a family 5 endoglucanase; and CBM2, a family 2 carbohydrate-binding module/domain. The black bar beneath the diagram represents the portion of the pDCW175 construct derived from Acel_0614. b A depiction of the chromosomal location and integration event of the E1 expression cassette/secretion cassette. c Agarose gel showing PCR products amplified using primers DC477 and DC478 annealing to regions outside the site of integration in the newly constructed strain JWCB052 ΔpyrFA ldh::ISCbe4 Δcbe1::P_S-layer acel0614 E1 containing the E1 expression cassette, 4.079 kb (lane 2) and the parent strain, JWCB018 ΔpyrFA ldh::ISCbe4 Δcbe1, 2.440 kb (lane 3); DNA MW standards (lane 1); no template PCR control (lane 4).

Fig. 3

Confirmation of E1 expression and secretion in C. bescii using Western blot analysis. Concentrated extracellular proteins (10 µg) were electrophoresed in a 15 % gradient Mini-Protean TGX gel (BIO-RAD) and electro-transferred to a PVDF membrane (ImmobilonTM-P; Millipore). The membrane was then probed with an E1 monoclonal antibody. Truncated version of E1 produced in Streptomyces lividans (lane 1); parent strain JWCB018 ΔpyrFA ldh::ISCbe4 Δcbe1 grown at 75 °C (lane 2); E1 expression strain JWCB052 ΔpyrFA ldh::ISCbe4 Δcbe1::P _S-layer acel0614 (E1) grown at 75 °C (lane 3), 70 °C (lane 4), and 65 °C (lane 5); MW, MagicMark™ molecular weight marker (Invitrogen).

Fig. 4

Growth of the wild type, parent strain (JWCB018), and E1 expression strain (JWCB052) on cellobiose. Growth of the wild type (black circles); JWCB018, ΔpyrFA ldh::ISCbe4 Δcbe1 (blue squares); and JWCB052, ΔpyrFA ldh::ISCbe4 Δcbe1::P _S-layer acel0614 (E1) (red triangles) strains on cellobiose. a Growth at 65 °C and b growth at 75 °C.

Fig. 5

Relative quantification of enzymatic activity of the extracellular fraction of C. bescii expressing E1 (Acel0614) on Avicel and carboxymethylcellulose. Carboxymethylcellulose (CMC) or Avicel was used as substrate at either 65 or 75 °C. a Activity of the extracellular fraction (25 µg/mL concentrated protein) on CMC from the parent strain JWCB018 ΔpyrFA ldh::ISCbe4 Δcbe1 (black), the E1 expression strain, JWCB052 ΔpyrFA ldh::ISCbe4 Δcbe1::P _S-layer acel0614 (E1) (grey), and no enzyme control (white). b Activity of the extracellular fraction (25 µg/mL of concentrated protein) on Avicel from the parent strain JWCB018 (black), the E1 expression strain, JWCB052 (grey), and no enzyme control (white).