Assembly and Activity of Engineered Minicellulosomes

Qi Xu, John Baker, Bill Michener, Roman Brunecky, Bill Adney, Shi-you Ding and Michael E. Himm

Part A: Optimization of reaction conditions for C. thermocellum cellulaseTh f ll i bi ti f b ff d H l td f i f C th ll ll l ti it

Part E. Activities of engineered minicellulosomes -- Effects of beta-glucosidase

The following combinations of buffer and pH were evaluated for use in assay of C. thermocellum cellulase activityon crystalline cellulose (1 mg/mL total protein acting against 1 % Avicel at 40o C, with 300 mM NaCI, 1 mM CaCI2in all assay mixtures):• 50 mM Tris, pH 7.0• 50 mM Tris, pH 7.0, 300 mM imidazole• 20 mM MES, pH 6.0• 20 mM acetateThe results (Figure 1) showed that native C. thermocellum cellulases were more active at pH 6.0 (20 mM MES) and

at pH 5.0 (20 mM acetate). In our further work, cellulosome and minicellulosome activities were assayed at pH 6.0in 20 mM MES (with NaCl and CaCl2 as noted above), because some recombinant cellulosomal enzymes were not

stable at pH 5.0.(Note: C. thermocellum cellulase was prepared from the total protein of cell-free broth of C. thermocellum, ATCC

27405, grown on 0.7 % Avicel.)

Goals of our project: (1) Elucidate the mechanism of biomassdegradation by C. thermocellum cellulosomes; (2) construct activeengineered minicellulosomes mainly using the recombinant C.thermocellum enzymes; (3) modify C. thermocellum to degrade

biomass more efficiently.

Table 1. Overexpression in E. coli of all fifty-four C. 

thermocellum genes that encode cellulosomal enzymes related to 

biomass degradation

Avicelase activity of the engineered minicellulosome from Part D(1) was determined, either aadded fungal beta-glucosidase (20 μg/ml). Reaction conditions: Please see Part C. The resuminicellulosomes.• The engineered minicellulosome showed some activity, but its activity was half of that of C. thand Table 4).• Beta-glucosidase helped to increase the glucose production, but had little effect on total conver

Table 4. Ratio of cellobiose and glucose converted by native and min

Percentage of 

cellobiose (%) 

Total Conversion of 

avicel (%) 

Cellulases

Module structure Solubility 

CBM3b-GH5-Doc1 

GH5-CBM6-FN3-Doc1 

GH5-Doc1 GH5-Doc1 GH5-Doc1 

GH8-Doc1

CBM4-Ig-GH9-2(Fn3)-CBM3b-Doc1

CBM4-Ig-GH9-Doc1

Ig-GH9-Doc1 

GH9 CBM3 CBM3b D 1

SolubleSolubleSolubleSolubleSolublePartially solubleSolubleSolublePartially solubleP i ll l bl

Part B: Comparison of C. thermocellum and fungal cellulasesThe following three samples were used for activity assaying against 1 % avicel:• Native cellulosomes (Cth, see the Part A)• Total cell-free protein from culture broth of a new species of Trichoderma (Tne)

• GC220---commercial enzyme mixture produced from T. reesei (Tre)(Note: reaction conditions as described in Part A)The following conclusions could be drawn from Figure 2:• Fungal cellulase activity (Tne: 61.7 % and Tre: 54.8 % conversion) was stronger than that of C.

thermocellum (31.8 %).

• After a lengthy (155-hour) digestion the major product of digestion by fungal enzymes was glucose,

whereas products of C. thermocellum cellulase digestion contained slightly more cellobiose than glucose

(Table 2).

S h dl hi h ll d i h ll i d ll d d

Cth MC 

MC+Beta-G

53.115.377.3 

31.8

13.9

13.5

Figure 1. The Effect of Reaction Conditions on Activity of Native 

Cellulosome

2025

Figu

121416

e l (% )

Part C: Library of cellulosomal enzymes for assembly of engineered minicellulosomes:• All fifty-four of the C thermocellum cellulosomal genes assigned as being related to biomass degradation Xylanases

GH9-CBM3c-CBM3b-Doc1GH9-CBM3c-CBM3b-Doc1

GH9-CBM3c-Doc1 GH9-CBM3c-Doc1 GH9-CBM3c-Doc1 GH9-CBM3c-Doc1 GH9-CBM3c-Doc1 GH9-CBM3c-Doc1 GH9-CBM3c-Doc1 

GH9-Doc1GH9-Doc1GH48-Doc1

Partially soluble

Partially solubleSolubleSolublePartially solubleSolublePartially solublePartially solubleNot determinedPartially solubleInclusion bodiesPartially soluble

• Somewhat unexpectedly, higher cellodextrins, such as cellotriose and cellotetraose, were not detected at

digestion times ranging from 14 to 155 hours apparently.

Table 2. Ratio of cellobiose and glucose converted by cellulases

Cth Tne Tre

Cellobiose (%) Glucose (%)

53.1

95.0

5.7

5.0

46.9

94.3

05

1015

0 10 20 30 40 50 60

Digestion Time (hour)

Conversion of Avicel (%)

50 mM Tris, pH 7.0

02468

10

0 20 

Conversion of Avic e

Table 3. All C. thermocellum cellulosomal cellulases and their activities on MUC

GH5 GH8 GH9 GH48

Number in genome 7 1 16 1

All fifty four of the C. thermocellum cellulosomal genes assigned as being related to biomass degradation,

including cellulases, hemicellulases, pectinases and multifuctional modules, have been cloned andoverexpressed in E. coli (Table 1). Forty-six of these were soluble or partially soluble, and we are workingon making all 54 enzymes soluble.

• The activities of 23 purified recombinant cellulases have been assayed, and 16 of them have apparent

activity on MUC (4-methylumbelliferyl-beta-D-cellobioside)(Table 3). Other hemicellulases

CBM22-GH10-Doc1 CBM22-GH10-Doc1 GH11-CBM4-Doc1-CE4 

GH16-Doc1 GH18-Doc1 CBM-GH26-Doc1 

GH26-Doc1 

GH30-CBM6-Doc1 GH81-Doc1 GH53-Doc1 

SolubleSolubleSolubleSolubleSolubleSolubleSolubleSolublePartially solublePartially soluble

P t ti l id

50 mM Tris, pH 7.0, 300 mM imidazole20 mM MES, pH 6.020 mM acetate, pH 5.0

Figure 2. Comparison of cellulase activi

species

70

Number active on MUC

Number soluble

0

7 17 81

14 1

Putative glycosidases

Xyloglucanhydrolase

Putative carbohydrate esterases

GH2-CBM6-Doc1 GH39-2(CBM6)-Doc1 

GH43-CBM6-Doc1 

GH43-CBM13-Doc1 

GH43-2(CBM6)-Doc1 

GH74-Doc1

Fn3-CE12-Doc1-CBM6-CE12 

CE3 CE3 D 1

Inclusion bodiesInclusion bodiesSolubleInclusion bodiesSolubleSolublePartially solublePartiall sol ble

Part D. Assembly of engineered cellulosomesOur first goal is to construct active, engineered (“mini-“) cellulosomes for efficient degradation of crystalline cellulose. Two methods have been designed and employed for the assembly of engineered minicellulosomes:(1) In order to do activity-screening of recombinations of various fusion enzymes for new engineeredminicellulosomes from our cellulosomal enzyme library a truncated cipA with 9 cohesins of the same type (without

30405060

ersion of Avicel (%)

Putative pectinases

Multifunctional components

CE3-CE3-Doc1

Doc1-CE6 CE1-CBM6-Doc1 

GH28-Doc1 PL1-Doc1-CBM6 PL1-Doc1-CBM6-PL9 PL10-UN-Doc1 Doc1-CBM6-PL11 

CBM30-Ig-GH9-GH44-Doc1-UN

GH26 GH5 CBM9 D 1

Partially solubleSolublePartially solublePartially solubleSolubleSolubleInclusion bodiesInclusion bodiesSolubleS l bl

minicellulosomes from our cellulosomal enzyme library, a truncated cipA with 9 cohesins of the same type (withoutX domain and dockerin II) and 9 recombinant enzymes with that type of dockerin, applied in equal molar loadings,were used for undirected (by the experimenter) assembly of engineered minicellulosomes. Assay of their activity isin progress.(2) In a more directed, engineered exercise, a chimeric scaffoldin with 6 cohesins taken from six different specieswas used for the specifically-ordered assembly of 6 C. thermocellum enzymes, each of which had been fused to ad ki di t f th h i

2 3 4 5 6 89 7

CBM3a

1

GH5-A GH5-B GH5-C GH48 GH9-A GH9-B GH9-C GH9-D GH9-E

0

1020

0 20 40 60 80 100 

Digestion Time (hour)

Conve

(Tne) % conversion to cellobiose and glucose (Tne) % 

GH26-GH5-CBM9-Doc1

GH30-GH54-GH43-Doc1 

GH54-Doc1-GH43 GH54-GH43-Doc1 

CE1-CBM6-Doc1-GH10 

CBM22-GH10-CBM22-Doc1-CE1 

GH5-Doc1-CE2 

Soluble

Partially solubleInclusion bodiesPartially solublePartially solublePartially solublePartially soluble

dockerin corresponding to one of the cohesins.

GH9-C

CBM3a

GH5-A GH48 GH5-B GH9-A GH9-B

(Tne) % conversion to cellobiose and glucose (Tne) % (Tne) % conversion to glucose (Cth) % (Cth) % conversion to cellobiose (Cth) % (Tre) % conversion of cellobiose and glucose (Tre) % (Tre) % conversion of glucose

NREL is a national laboratory of the U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, operated by the Alliance for Sustainable Energy, LLC. • NREL/PO-270-46338

Presented at the BEST Retreat Meeting, 21-24 June 2009, Asheville, North Carolina