A cellulosomal double-dockerin module from Clostridium thermocellum shows distinct structural and cohesin-binding features.

Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components.

Cryptic diversity of cellulose-degrading gut bacteria in industrialized humans.

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

Structure of the transcription open complex of distinct σI factors.

Bacterial σI factors of the σ70-family are widespread in Bacilli and Clostridia and are involved in the heat shock response, iron metabolism, virulence, and carbohydrate sensing. A multiplicity of σI paralogues in some cellulolytic bacteria have been shown to be responsible for the regulation of the cellulosome, a multienzyme complex that mediates efficient cellulose degradation. Here, we report two structures at 3.0 Å and 3.3 Å of two transcription open complexes formed by two σI factors, SigI1 and SigI6, respectively, from the thermophilic, cellulolytic bacterium, Clostridium thermocellum. These structures reveal a unique, hitherto-unknown recognition mode of bacterial transcriptional promoters, both with respect to domain organization and binding to promoter DNA. The key characteristics that determine the specificities of the σI paralogues were further revealed by comparison of the two structures. Consequently, the σI factors represent a distinct set of the σ70-family σ factors, thus highlighting the diversity of bacterial transcription.

Essential autoproteolysis of bacterial anti-σ factor RsgI for transmembrane signal transduction.

Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from Clostridium thermocellum, which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis. The RsgI-based autocleavage site was located at a conserved Asn-Pro motif between the ß1 and ß2 strands in the periplasmic domain. This cleavage was demonstrated to be essential for subsequent regulated intramembrane proteolysis to activate the cognate SigI, in a manner similar to that of autoproteolysis-dependent activation of eukaryotic adhesion G protein-coupled receptors. These results indicate the presence of a unique prevalent type of autoproteolytic phenomenon in bacteria for signal transduction.

Carbohydrate Depolymerization by Intricate Cellulosomal Systems.

Cellulosomes are multi-enzymatic nanomachines that have been fine-tuned through evolution to efficiently deconstruct plant biomass. Integration of cellulosomal components occurs via highly ordered protein-protein interactions between the various enzyme-borne dockerin modules and the multiple copies of the cohesin modules located on the scaffoldin subunit. Recently, designer cellulosome technology was established to provide insights into the architectural role of catalytic (enzymatic) and structural (scaffoldin) cellulosomal constituents for the efficient degradation of plant cell wall polysaccharides. Owing to advances in genomics and proteomics, highly structured cellulosome complexes have recently been unraveled, and the information gained has inspired the development of designer-cellulosome technology to new levels of complex organization. These higher-order designer cellulosomes have in turn fostered our capacity to enhance the catalytic potential of artificial cellulolytic complexes. In this chapter, methods to produce and employ such intricate cellulosomal complexes are reported.

Self-assembly of a dimeric avidin into unique higher-order oligomers.

The dimeric avidin family has been expanded in recent years to include many new members. All of them lack the intermonomeric Trp that plays a critical role in biotin-binding. Nevertheless, these new members of the avidins maintain the high affinity towards biotin. Additionally, all of the dimeric avidins share a very unique property: namely, the cylindrical oligomerization in the crystal structure. The newest member described here, agroavidin from the agrobacterium, Rhizobium sp. AAP43, shares their important structural features. However, the affinity of agroavidin towards biotin is lower than all other members of the avidin family, due to the presence of phenylalanine instead of a conserved tyrosine in the biotin-binding site. Mutating this phenylalanine into tyrosine regenerated the high affinity, which emphasizes the importance of this particular tyrosine residue. Another unique feature that distinguishes agroavidin from the other dimeric avidins is that it does not produce oligomers in its crystal structure. In order to understand the factors that promote oligomerization in dimeric avidins, we exchanged the C-terminal region of agroavidin with that of hoefavidin that produced octamers. This exchange resulted in a decamer rather than an octamer. This unusual outcome demonstrates the impact of the C-terminal region on the ability to produce oligomers. The decameric assembly of agroavidin expands the avidin-biotin toolbox even further and could well pave the path into new biotin-based technologies. Moreover, uncovering the factors that induce dimeric avidins into oligomeric assemblies may aid in better understanding the general molecular determinants that promote oligomerization.

Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies.

The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.

Deciphering Cellodextrin and Glucose Uptake in Clostridium thermocellum.

Sugar uptake is of great significance in industrially relevant microorganisms. Clostridium thermocellum has extensive potential in lignocellulose biorefineries as an environmentally prominent, thermophilic, cellulolytic bacterium. The bacterium employs five putative ATP-binding cassette transporters which purportedly take up cellulose hydrolysates. Here, we first applied combined genetic manipulations and biophysical titration experiments to decipher the key glucose and cellodextrin transporters. In vivo gene inactivation of each transporter and in vitro calorimetric and nuclear magnetic resonance (NMR) titration of each putative sugar-binding protein with various saccharides supported the conclusion that only transporters A and B play the roles of glucose and cellodextrin transport, respectively. To gain insight into the structural mechanism of the transporter specificities, 11 crystal structures, both alone and in complex with appropriate saccharides, were solved for all 5 putative sugar-binding proteins, thus providing detailed specific interactions between the proteins and the corresponding saccharides. Considering the importance of transporter B as the major cellodextrin transporter, we further identified its cryptic, hitherto unknown ATPase-encoding gene as clo1313_2554, which is located outside the transporter B gene cluster. The crystal structure of the ATPase was solved, showing that it represents a typical nucleotide-binding domain of the ATP-binding cassette (ABC) transporter. Moreover, we determined that the inducing effect of cellobiose (G2) and cellulose on cellulosome production could be eliminated by deletion of transporter B genes, suggesting the coupling of sugar transport and regulation of cellulosome components. This study provides key basic information on the sugar uptake mechanism of C. thermocellum and will promote rational engineering of the bacterium for industrial application. IMPORTANCE Highly efficient sugar uptake is important to microbial cell factories, and sugar transporters are therefore of great interest in the study of industrially relevant microorganisms. Clostridium thermocellum is a lignocellulolytic bacterium known for its multienzyme complex, the cellulosome, which is of great potential value in lignocellulose biorefinery. In this study, we clarify the function and mechanism of substrate specificity of the five reported putative sugar transporters using genetic, biophysical, and structural methods. Intriguingly, the results showed that only one of them, transporter B, is the major cellodextrin transporter, whereas another, transporter A, represents the major glucose transporter. Considering the importance of transporter B, we further identified the missing ATPase gene of transporter B and revealed the correlation between transporter B and cellulosome production. Revealing the mechanism by which C. thermocellum utilizes cellodextrins will help pave the way for engineering the strain for industrial applications.

Metaproteome plasticity sheds light on the ecology of the rumen microbiome and its connection to host traits.

The arsenal of genes that microbes express reflect the way in which they sense their environment. We have previously reported that the rumen microbiome composition and its coding capacity are different in animals having distinct feed efficiency states, even when fed an identical diet. Here, we reveal that many microbial populations belonging to the bacteria and archaea domains show divergent proteome production in function of the feed efficiency state. Thus, proteomic data serve as a strong indicator of host feed efficiency state phenotype, overpowering predictions based on genomic and taxonomic information. We highlight protein production of specific phylogenies associated with each of the feed efficiency states. We also find remarkable plasticity of the proteome both in the individual population and at the community level, driven by niche partitioning and competition. These mechanisms result in protein production patterns that exhibit functional redundancy and checkerboard distribution that are tightly linked to the host feed efficiency phenotype. By linking microbial protein production and the ecological mechanisms that act within the microbiome feed efficiency states, our present work reveals a layer of complexity that bears immense importance to the current global challenges of food security and sustainability.

Mapping the deformability of natural and designed cellulosomes in solution.

BACKGROUND: Natural cellulosome multi-enzyme complexes, their components, and engineered 'designer cellulosomes' (DCs) promise an efficient means of breaking down cellulosic substrates into valuable biofuel products. Their broad uptake in biotechnology relies on boosting proximity-based synergy among the resident enzymes, but the modular architecture challenges structure determination and rational design. RESULTS: We used small angle X-ray scattering combined with molecular modeling to study the solution structure of cellulosomal components. These include three dockerin-bearing cellulases with distinct substrate specificities, original scaffoldins from the human gut bacterium Ruminococcus champanellensis (ScaA, ScaH and ScaK) and a trivalent cohesin-bearing designer scaffoldin (Scaf20L), followed by cellulosomal complexes comprising these components, and the nonavalent fully loaded Clostridium thermocellum CipA in complex with Cel8A from the same bacterium. The size analysis of Rg and Dmax values deduced from the scattering curves and corresponding molecular models highlight their variable aspects, depending on composition, size and spatial organization of the objects in solution. CONCLUSIONS: Our data quantifies variability of form and compactness of cellulosomal components in solution and confirms that this native plasticity may well be related to speciation with respect to the substrate that is targeted. By showing that scaffoldins or components display enhanced compactness compared to the free objects, we provide new routes to rationally enhance their stability and performance in their environment of action.

Combinatorial assembly and optimisation of designer cellulosomes: a galactomannan case study.

BACKGROUND: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. RESULTS: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. CONCLUSION: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

Nanoscale resolution of microbial fiber degradation in action.

The lives of microbes unfold at the micron scale, and their molecular machineries operate at the nanoscale. Their study at these resolutions is key toward achieving a better understanding of their ecology. We focus on cellulose degradation of the canonical Clostridium thermocellum system to comprehend how microbes build and use their cellulosomal machinery at these nanometer scales. Degradation of cellulose, the most abundant organic polymer on Earth, is instrumental to the global carbon cycle. We reveal that bacterial cells form 'cellulosome capsules' driven by catalytic product-dependent dynamics, which can increase the rate of hydrolysis. Biosynthesis of this energetically costly machinery and cell growth are decoupled at the single-cell level, hinting at a division-of-labor strategy through phenotypic heterogeneity. This novel observation highlights intrapopulation interactions as key to understanding rates of fiber degradation.

Sas20 is a highly flexible starch-binding protein in the Ruminococcus bromii cell-surface amylosome.

Ruminococcus bromii is a keystone species in the human gut that has the rare ability to degrade dietary resistant starch (RS). This bacterium secretes a suite of starch-active proteins that work together within larger complexes called amylosomes that allow R. bromii to bind and degrade RS. Starch adherence system protein 20 (Sas20) is one of the more abundant proteins assembled within amylosomes, but little could be predicted about its molecular features based on amino acid sequence. Here, we performed a structure-function analysis of Sas20 and determined that it features two discrete starch-binding domains separated by a flexible linker. We show that Sas20 domain 1 contains an N-terminal ß-sandwich followed by a cluster of α-helices, and the nonreducing end of maltooligosaccharides can be captured between these structural features. Furthermore, the crystal structure of a close homolog of Sas20 domain 2 revealed a unique bilobed starch-binding groove that targets the helical α1,4-linked glycan chains found in amorphous regions of amylopectin and crystalline regions of amylose. Affinity PAGE and isothermal titration calorimetry demonstrated that both domains bind maltoheptaose and soluble starch with relatively high affinity (Kd ≤ 20 µM) but exhibit limited or no binding to cyclodextrins. Finally, small-angle X-ray scattering analysis of the individual and combined domains support that these structures are highly flexible, which may allow the protein to adopt conformations that enhance its starch-targeting efficiency. Taken together, we conclude that Sas20 binds distinct features within the starch granule, facilitating the ability of R. bromii to hydrolyze dietary RS.

Wilavidin - a novel member of the avidin family that forms unique biotin-binding hexamers.

Nature's optimization of protein functions is a highly intricate evolutionary process. In addition to optimal tertiary folding, the intramolecular recognition among the monomers that generate higher-order quaternary arrangements is driven by stabilizing interactions that have a pivotal role for ideal activity. Homotetrameric avidin and streptavidin are regularly utilized in many applications, whereby their ultra-high affinity toward biotin is dependent on their quaternary arrangements. In recent years, a new subfamily of avidins was discovered that comprises homodimers rather than tetramers, in which the high affinity toward biotin is maintained. Intriguingly, several of the respective dimers have been shown to assemble into higher-order cylindrical hexamers or octamers that dissociate into dimers upon biotin binding. Here, we present wilavidin, a newly discovered member of the dimeric subfamily, forming hexamers in the apo form, which are uniquely maintained upon biotin binding with six high-affinity binding sites. Removal of the short C-terminal segment of wilavidin resulted in the presence of the dimer only, thus emphasizing the role of this segment in stabilizing the hexamer. Utilization of a hexavalent biotin-binding form of avidin would be beneficial for expanding the biotechnological toolbox. Additionally, this unique family of dimeric avidins and their propensity to oligomerize to hexamers or octamers can serve as a basis for protein oligomerization and intermonomeric recognition as well as cumulative interactions that determine molecular assemblies.

Coordinated ß-glucosidase activity with the cellulosome is effective for enhanced lignocellulose saccharification.

Consolidated bio-saccharification (CBS) technology employs cellulosome-producing bacterial cells, rather than fungal cellulases, as biocatalysts for cost-effective production of lignocellulosic sugars. Extracellular ß-glucosidase (BGL) expression in the whole-cell arsenal is indispensable, due to severe cellobiose inhibition of the cellulosome. However, high-level BGL expression in Clostridium thermocellum is challenging, and the optimal BGL production level for efficient cellulose saccharification is currently unknown. Herein, we obtained new CBS biocatalysts by transforming BGL-expressing plasmids into C. thermocellum, which produced abundant BGL proteins and hydrolyzed cellulose effectively. The optimal ratio of extracellular BGL-to-cellulosome activity was determined to be in a range of 5.5 to 21.6. Despite the critical impact of BGL, both excessive BGL expression and its assembly on the cellulosome via type I cohesin-dockerin interaction led to reduced cellulosomal activity, which further confirmed the importance of coordinated BGL expression with the cellulosome. This study will further promote industrial CBS application in lignocellulose conversion.

NMR chemical shift assignments of a module of unknown function in the cellulosomal secondary scaffoldin ScaF from Clostridium thermocellum.

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.

Novel clostridial cell-surface hemicellulose-binding CBM3 proteins.

A novel member of the family 3 carbohydrate-binding modules (CBM3s) is encoded by a gene (Cthe_0271) in Clostridium thermocellum which is the most highly expressed gene in the bacterium during its growth on several types of biomass substrates. Surprisingly, CtCBM3-0271 binds to at least two different types of xylan, instead of the common binding of CBM3s to cellulosic substrates. CtCBM3-0271 was crystallized and its three-dimensional structure was solved and refined to a resolution of 1.8 Å. In order to learn more about the role of this type of CBM3, a comparative study with its orthologue from Clostridium clariflavum (encoded by the Clocl_1192 gene) was performed, and the three-dimensional structure of CcCBM3-1192 was determined to 1.6 Šresolution. Carbohydrate binding by CcCBM3-1192 was found to be similar to that by CtCBM3-0271; both exhibited binding to xylan rather than to cellulose. Comparative structural analysis of the two CBM3s provided a clear functional correlation of structure and binding, in which the two CBM3s lack the required number of binding residues in their cellulose-binding strips and thus lack cellulose-binding capabilities. This is an enigma, as CtCBM3-0271 was reported to be a highly expressed protein when the bacterium was grown on cellulose. An additional unexpected finding was that CcCBM3-1192 does not contain the calcium ion that was considered to play a structural stabilizing role in the CBM3 family. Despite the lack of calcium, the five residues that form the calcium-binding site are conserved. The absence of calcium results in conformational changes in two loops of the CcCBM3-1192 structure. In this context, superposition of the non-calcium-binding CcCBM3-1192 with CtCBM3-0271 and other calcium-binding CBM3s reveals a much broader two-loop region in the former compared with CtCBM3-0271.

Discovery and mechanism of a pH-dependent dual-binding-site switch in the interaction of a pair of protein modules.

Many important proteins undergo pH-dependent conformational changes resulting in "on-off" switches for protein function, which are essential for regulation of life processes and have wide application potential. Here, we report a pair of cellulosomal assembly modules, comprising a cohesin and a dockerin from Clostridium acetobutylicum, which interact together following a unique pH-dependent switch between two functional sites rather than on-off states. The two cohesin-binding sites on the dockerin are switched from one to the other at pH 4.8 and 7.5 with a 180° rotation of the bound dockerin. Combined analysis by nuclear magnetic resonance spectroscopy, crystal structure determination, mutagenesis, and isothermal titration calorimetry elucidates the chemical and structural mechanism of the pH-dependent switching of the binding sites. The pH-dependent dual-binding-site switch not only represents an elegant example of biological regulation but also provides a new approach for developing pH-dependent protein devices and biomaterials beyond an on-off switch for biotechnological applications.

Surface Display of Designer Protein Scaffolds on Genome-Reduced Strains of Pseudomonas putida.

The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host. In this work, we report an essential step in this endeavor-a display of designer enzyme-anchoring protein "scaffoldins", encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were optimally delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric ß-glucosidase and fluorescent proteins. Our results not only highlight the value of cell surface engineering for presentation of recombinant proteins on the envelope of Gram-negative bacteria but also pave the way toward designer cellulosome strategies tailored for P. putida.

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level.

The multi-enzyme cellulosome complex can mediate the valorization of lignocellulosic biomass into soluble sugars that can serve in the production of biofuels and valuable products. A potent bacterial chassis for the production of active cellulosomes displayed on the cell surface is the bacterium Lactobacillus plantarum, a lactic acid bacterium used in many applications. Here, we developed a methodological pipeline to produce improved designer cellulosomes, using a cell-consortium approach, whereby the different components self-assemble on the surface of L. plantarum. The pipeline served as a vehicle to select and optimize the secretion efficiency of potent designer cellulosome enzyme components, to screen for the most efficient enzymatic combinations and to assess attempts to grow the engineered bacterial cells on wheat straw as a sole carbon source. Using this strategy, we were able to improve the secretion efficiency of the selected enzymes and to secrete a fully functional high-molecular-weight scaffoldin component. The adaptive laboratory process served to increase significantly the enzymatic activity of the most efficient cell consortium. Internal plasmid re-arrangement towards a higher enzymatic performance attested for the suitability of the approach, which suggests that this strategy represents an efficient way for microbes to adapt to changing conditions.