Recombinant Bacillus subtilis that grows on untreated plant biomass

Abstract

Lignocellulosic biomass is a promising feedstock to produce biofuels and other valuable biocommodities. A major obstacle to its commercialization is the high cost of degrading biomass into fermentable sugars, which is typically achieved using cellulolytic enzymes from Trichoderma reesei. Here, we explore the use of microbes to break down biomass. Bacillus subtilis was engineered to display a multicellulase-containing minicellulosome. The complex contains a miniscaffoldin protein that is covalently attached to the cell wall and three noncovalently associated cellulase enzymes derived from Clostridium cellulolyticum (Cel48F, Cel9E, and Cel5A). The minicellulosome spontaneously assembles, thus increasing the practicality of the cells. The recombinant bacteria are highly cellulolytic and grew in minimal medium containing industrially relevant forms of biomass as the primary nutrient source (corn stover, hatched straw, and switch grass). Notably, growth did not require dilute acid pretreatment of the biomass and the cells achieved densities approaching those of cells cultured with glucose. An analysis of the sugars released from acid-pretreated corn stover indicates that the cells have stable cellulolytic activity that enables them to break down 62.3% ± 2.6% of the biomass. When supplemented with beta-glucosidase, the cells liberated 21% and 33% of the total available glucose and xylose in the biomass, respectively. As the cells display only three types of enzymes, increasing the number of displayed enzymes should lead to even more potent cellulolytic microbes. This work has important implications for the efficient conversion of lignocellulose to value-added biocommodities.

Fig 1

B. subtilis displays minicellulosomes that assemble on the cell surface. (A) Schematic of the B. subtilis minicellulosome. The scaffoldin protein (Scaf) contains type I cohesin modules from C. thermocellum (t), C. cellulolyticum (c), and R. flavefaciens (f); a family 3 carbohydrate binding module (CBM); and a cell wall sorting signal (CWSS) that enables it to be anchored to the cell wall. All enzymes are derived from C. cellulolyticum. These include the family 9 glycoside hydrolase (GH) enzyme fused to the R. flavefaciens type I dockerin module (Cel9E), the family 48 GH enzyme fused to the C. thermocellum type I dockerin module (Cel48F), and the family 5 GH enzyme fused to the C. cellulolyticum type I dockerin module (Cel5A). (B) Immunoblots of the cell fractions demonstrating assembly of minicellulosomes containing one, two, or three distinct cellulases. Cell wall (CW) and secreted protein (Sec) fractions were isolated from minicellulosome-displaying cells (three cellulases, strain TDA17; two cellulases [Cel9E and Cel48F], TDA14; one cellulase [Cel5A], TDA11) and cells that could only secrete the enzyme because the sortase and scaffoldin were not present (strain TDA18). Data for the Cel9E, Cel48F, and Cel5A fusion enzymes are shown.

Fig 2

Quantification and saturation level determination of surface-displayed Scaf protein. (A) Immunoblots of the cell wall fraction demonstrating that the Scaf proteins displayed on the cell surface are entirely bound by cellulases. Cells of strain TDA17 were induced for SrtA, Scaf, and cellulase expression (Expressed). The cells were then collected and incubated with excess amounts of E. coli purified Cel5A, Cel9E, and Cel48F. Addition of purified enzymes (Purified) did not increase the intensity of the bands corresponding to Cel5A, Cel9E, and Cel48F. Negative-control strains TDA18 and TDA19 were unable to bind any cellulases, while positive-control strain TDA10 demonstrates the ability of E. coli purified enzymes to bind to cell-displayed Scaf. (B) Cel5A-associated activity on CMC was used to determine the amount of Scaf displayed per cell. TDA10 cells were induced to display covalently anchored Scaf (solid squares), followed by incubation with increasing amounts of E. coli purified Cel5A. After a washing step, the cell-associated Cel5A activity was measured; each cell is capable of displaying ∼150,000 Scaf molecules. Negative-control strain TDA19, which is unable to successfully display Scaf (open squares), had negligible Cel5A-associated activity.

Fig 3

B. subtilis displaying minicellulosomes grows on dilute acid-pretreated biomass. (A) Growth of minicellulosome-displaying B. subtilis on dilute acid-pretreated corn stover (TDA17, open squares). Cells grew to densities similar to those of cells cultured in the presence of glucose (solid squares, culture in glucose). Growth on biomass requires that the minicellulosome be attached to the peptidoglycan, as cells lacking SrtA and Scaf failed to grow even though the cellulase enzymes were produced and secreted (strain TDA18, solid diamonds). Wild-type BAL2238 cells also failed to grow on biomass (data not shown). (B) CFU measurements of cells grown on biomass and glucose. Symbols and growth conditions are as described for panel A. CFU/ml measurements are reported. (C) Growth of strains of B. subtilis displaying two cellulases (strain TDA14, open triangles; strain TDA15, shaded triangles; strain TDA16, solid triangles) or one cellulase (strain TDA13, open circles; strain TDA12, shaded circles; strain TDA11, solid circles) on dilute acid-pretreated corn stover. Cells had a noticeable lag phase and were unable to reach cell densities similar to those of cells cultured in the presence of glucose (solid squares).

Fig 4

Cells displaying minicellulosomes efficiently hydrolyze dilute acid-pretreated biomass. (A) Amount of insoluble dilute acid-treated corn stover remaining after incubation with minicellulosome-displaying azide-treated cells (strain TDA17, open boxes). Strain TDA18, which only secretes the three cellulases, was unable to degrade biomass (solid diamonds). In this procedure, after incubation with azide-treated cells, the insoluble residual biomass was washed to remove bound cells, dried, and weighed. (B) Amount of insoluble dilute acid-pretreated corn stover remaining after incubation with strains of B. subtilis displaying one cellulase (strain TDA11, solid circles; strain TDA12, shaded circles; strain TDA13, open circles) or two cellulases (strain TDA14, open triangles; strain TDA15, shaded triangles, strain TDA16, solid triangles).

Fig 5

Cells displaying minicellulosomes containing three enzymes efficiently release soluble glycan and xylan from pretreated biomass and are comparable to commercially available cellulase cocktails. (A) Soluble reducing sugars released from dilute acid-pretreated corn stover by cells displaying minicellulosomes (strain TDA17, open squares) and cells that only secrete the enzymes (strain TDA18, solid diamonds) and the results of applying a CTec2/HTec2 cellulase enzyme mixture produced by Novozymes Inc. to the biomass (crosses). Checkered squares represent data from cells displaying minicellulosomes that were supplemented with beta-glucosidase. (B) Data are identical to those shown in panel A but record the concentration of soluble glucose released. (C) Data are identical to those shown in panel A but record the concentration of soluble xylose released.

Fig 6

Cells displaying minicellulosomes containing three enzymes grow on untreated corn stover, straw, and switch grass. (A) Growth curves of minicellulosome-displaying B. subtilis cultured with untreated corn stover (open squares), straw (solid squares), and switch grass (shaded squares). In each assay, cells were grown on M9 salts and 0.5% (wt/vol) untreated biomass. Strain TDA18, which only secretes the enzymes, could not grow on corn stover (solid diamonds), straw (shaded diamonds), or switch grass (open diamonds). (B) Reducing sugars released by minicellulosome-displaying azide-treated cells (strain TDA17). Sugars released from corn stover (open squares), switch grass (shaded squares), and straw (solid squares) are shown. Solid diamonds are data from control strain TDA18 cultured with untreated corn stover, which produced only small amounts of soluble sugar (similar data, not shown, were obtained with straw and switch grass). (C) Data are identical to those shown in panel B but report the concentration of soluble glucose released from untreated biomass. (D) Data are identical to those of panels B and C but report the concentration of soluble xylose released.