Incorporation of fungal cellulases in bacterial minicellulosomes yields viable, synergistically acting celluloytic complexes

Artificial designer minicellulosomes comprise a chimeric scaffoldin that displays an optional cellulose-binding module (CBM) and bacterial cohesins from divergent species which bind strongly to enzymes engineered to bear complementary dockerins. Incorporation of cellulosomal cellulases from Clostridium cellulolyticum into minicellulosomes leads to artificial complexes with enhanced activity on crystalline cellulose, due to enzyme proximity and substrate targeting induced by the scaffoldin-borne CBM. In the present study, a bacterial dockerin was appended to the family 6 fungal cellulase Cel6A, produced by Neocallimastix patriciarum, for subsequent incorporation into minicellulosomes in combination with various cellulosomal cellulases from C. cellulolyticum. The binding of the fungal Cel6A with a bacterial family 5 endoglucanase onto chimeric miniscaffoldins had no impact on their activity toward crystalline cellulose. Replacement of the bacterial family 5 enzyme with homologous endoglucanase Cel5D from N. patriciarum bearing a clostridial dockerin gave similar results. In contrast, enzyme pairs comprising the fungal Cel6A and bacterial family 9 endoglucanases were substantially stimulated (up to 2.6-fold) by complexation on chimeric scaffoldins, compared to the free-enzyme system. Incorporation of enzyme pairs including Cel6A and a processive bacterial cellulase generally induced lower stimulation levels. Enhanced activity on crystalline cellulose appeared to result from either proximity or CBM effects alone but never from both simultaneously, unlike minicellulosomes composed exclusively of bacterial cellulases. The present study is the first demonstration that viable designer minicellulosomes can be produced that include (i) free (noncellulosomal) enzymes, (ii) fungal enzymes combined with bacterial enzymes, and (iii) a type (family 6) of cellulase never known to occur in natural cellulosomes

A diverse set of family 48 bacterial glycoside hydrolase cellulases created by structure-guided recombination

Sequence diversity within a family of functional enzymes provides a platform for elucidating structure–function relationships and for protein engineering to improve properties important for applications. Access to nature's vast sequence diversity is often limited by the fact that only a few enzymes have been characterized in a given family. Here, we recombined the catalytic domains of three glycoside hydrolase family 48 bacterial cellulases (Cel48; EC 3.2.1.176) – Clostridium cellulolyticum CelF, Clostridium stercorarium CelY, and Clostridium thermocellum CelS – to create a diverse library of Cel48 enzymes with an average of 106 mutations from the closest native enzyme. Within this set, we found large variations in properties such as the functional temperature range, stability, and specific activity on crystalline cellulose. We showed that functional status and stability were predictable from simple linear models of the sequence–property data: recombined protein fragments contributed additively to these properties in a given chimera. Using this, we correctly predicted sequences that were as stable as any of the native Cel48 enzymes described to date. The characterization of 60 active Cel48 chimeras expands the number of characterized Cel48 enzymes from 13 to 73. Our work illustrates the role that structure-guided recombination can play in helping to identify sequence–function relationships within a family of enzymes by supplementing natural diversity with synthetic diversity

Development of an: E. coli strain for one-pot biofuel production from ionic liquid pretreated cellulose and switchgrass

Biological production of chemicals and fuels using microbial transformation of sustainable carbon sources, such as pretreated and saccharified plant biomass, is a multi-step process. Typically, each segment of the workflow is optimized separately, often generating conditions that may not be suitable for integration or consolidation with the upstream or downstream steps. While significant effort has gone into developing solutions to incompatibilities at discrete steps, very few studies report the consolidation of the multi-step workflow into a single pot reactor system. Here we demonstrate a one-pot biofuel production process that uses the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) for pretreatment of switchgrass biomass. [C2C1Im][OAc] is highly effective in deconstructing lignocellulose, but nonetheless leaves behind residual reagents that are toxic to standard saccharification enzymes and the microbial production host. We report the discovery of an [C2C1Im]-tolerant E. coli strain, where [C2C1Im] tolerance is bestowed by a P7Q mutation in the transcriptional regulator encoded by rcdA. We establish that the causal impact of this mutation is the derepression of a hitherto uncharacterized major facilitator family transporter, YbjJ. To develop the strain for a one-pot process we engineered this [C2C1Im]-tolerant strain to express a recently reported d-limonene production pathway. We also screened previously reported [C2C1Im]-tolerant cellulases to select one that would function with the range of E. coli cultivation conditions and expressed it in the [C2C1Im]-tolerant E. coli strain so as to secrete this [C2C1Im]-tolerant cellulase. The final strain digests pretreated biomass, and uses the liberated sugars to produce the bio-jet fuel candidate precursor d-limonene in a one-pot process

Thermobifida fusca exoglucanase Cel6B is incompatible with the cellulosomal mode in contrast to endoglucanase Cel6A

Cellulosomes are efficient cellulose-degradation systems produced by selected anaerobic bacteria. This multi-enzyme complex is assembled from a group of cellulases attached to a protein scaffold termed scaffoldin, mediated by a high-affinity protein–protein interaction between the enzyme-borne dockerin module and the cohesin module of the scaffoldin. The enzymatic complex is attached as a whole to the cellulosic substrate via a cellulose-binding module (CBM) on the scaffoldin subunit. In previous works, we have employed a synthetic biology approach to convert several of the free cellulases of the aerobic bacterium, Thermobifida fusca, into the cellulosomal mode by replacing each of the enzymes’ CBM with a dockerin. Here we show that although family six enzymes are not a part of any known cellulosomal system, the two family six enzymes of the T. fusca system (endoglucanase Cel6A and exoglucanase Cel6B) can be converted to work as cellulosomal enzymes. Indeed, the chimaeric dockerin-containing family six endoglucanase worked well as a cellulosomal enzyme, and proved to be more efficient than the parent enzyme when present in designer cellulosomes. In stark contrast, the chimaeric family six exoglucanase was markedly less efficient than the wild-type enzyme when mixed with other T. fusca cellulases, thus indicating its incompatibility with the cellulosomal mode of action