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Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

ABSTRACT: BACKGROUND: The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes crystalline cellulose. Enzymes interact with protein scaffolds via type 1 dockerin/cohesin interactions, while scaffolds in turn bind surface anchor proteins by means of type 2 dockerin/cohesin interactions, which demonstrate a different binding specificity than their type 1 counterparts. Recombinant chimeric scaffold proteins containing cohesins of different specificity allow binding of multiple enzymes to specific sites within an engineered complex. RESULTS: We report the successful display of engineered chimeric scaffold proteins containing both type 1 and type 2 cohesins on the surface of Lactococcus lactis cells. The chimeric scaffold proteins were able to form complexes with the Escherichia coli ?-glucuronidase fused to either type 1 or type 2 dockerin, and differences in binding efficiencies were correlated with scaffold architecture. We used E. coli ?-galactosidase, also fused to type 1 or type 2 dockerins, to demonstrate the targeted incorporation of two enzymes into the complexes. The simultaneous binding of enzyme pairs each containing a different dockerin resulted in bi-enzymatic complexes tethered to the cell surface. The sequential binding of the two enzymes yielded insights into parameters affecting assembly of the complex such as protein size and position within the scaffold. CONCLUSIONS: The spatial organization of enzymes into complexes is an important strategy for increasing the efficiency of biochemical pathways. In this study, chimeric protein scaffolds consisting of type 1 and type 2 cohesins anchored on the surface of L. lactis allowed for the controlled positioning of dockerin-fused reporter enzymes onto the scaffolds. By binding single enzymes or enzyme pairs to the scaffolds, our data also suggest that the size and relative positions of enzymes can affect the catalytic profiles of the resulting complexes. These insights will be of great value as we engineer more advanced scaffold-guided protein complexes to optimize biochemical pathways.

SUBMITTER: Wieczorek AS

PROVIDER: S-EPMC3542058 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

Wieczorek Andrew S AS Martin Vincent J J VJ

Microbial cell factories 20121215

<h4>Background</h4>The microbial synthesis of fuels, commodity chemicals, and bioactive compounds necessitates the assemblage of multiple enzyme activities to carry out sequential chemical reactions, often via substrate channeling by means of multi-domain or multi-enzyme complexes. Engineering the controlled incorporation of enzymes in recombinant protein complexes is therefore of interest. The cellulosome of Clostridium thermocellum is an extracellular enzyme complex that efficiently hydrolyzes ...[more]

PMID: 23241215

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Project description:Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin-dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be "purified" via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin-dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome's total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.

Project description:The sulfur-containing amino acids methionine and cysteine play an important role in food industry. These amino acids are used to confer a sulfur smell or meat-related aroma to food products. Besides their use as food additives, methionine and cysteine participate in flavor formation in dairy fermentations. For instance, the characteristic aroma of Cheddar cheeses is derived from methionine. Therefore, bacterial strains with the ability to overproduce and secrete these amino acids are relevant for the food industry. In addition, the quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods such as high-performance liquid chromatography. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors. The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aim to develop biosensors to detect methionine and cysteine, which are produced and secreted by wild-type Lactococcus lactis strains. We employed two strategies to create L. lactis biosensors, the first one is based on the methionine auxotrophy of this bacterium and the second strategy is based on a cysteine-responsive promoter. The characterization of the biosensors showed their specific response to the presence of these amino acids. Subsequently, we applied the methionine biosensor to quantify the presence of methionine in bacterial supernatants of wild-type L. lactis that naturally secretes methionine to benchmark the performance of our biosensors. The methionine biosensor responded linearly to the amounts of methionine present in the bacterial supernatants, i.e., the increases in the biosensor cell densities were proportional to the amounts of methionine present in the supernatants. The biosensors developed in this study tackle the limitations of amino acid quantification and the selection of strains with secretion of amino acids. These biosensors may eventually be used for screening of engineered strains to increase methionine and cysteine production, and may facilitate the detection of these amino acids in complex food matrices.

Dataset Information

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

Publications

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis.

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