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Cellulosomes and designer cellulosomes: why toy with N ature?

2016; Wiley; Volume: 9; Issue: 1 Linguagem: Inglês

10.1111/1758-2229.12489

1758-2229

Edward A. Bayer,

Polysaccharides and Plant Cell Walls

Originally, neither by design nor by chance was the cellulosome discovered over three decades ago (Lamed et al., 1983b). Dogma at the time proclaimed that cellulose was hydrolyzed by blends of simple free enzymes, the cellulases, produced in quantity by cellulolytic fungi. Historically, research in this area was spearheaded by the discovery of the filamentous fungus, Trichoderma reesei, first isolated in the Solomon Islands in the 1940s during World War II, owing to its disastrous consequences on the canvass army tents in the tropics (Reese et al., 1950; Reese, 1976). From there, decades of research on fungal cellulases ensued, but no Crystal Ball would foresee the existence of the multi-enzyme complex in bacteria. So scientists searched for single cellulases in bacteria, but in the relentless, cellulolytic anaerobe, Clostridium thermocellum, none could be found. In our early work on this bacterium, we chose a different path and let science take the lead (Bayer et al., 1983; Lamed et al., 1983a). While disregarding dogma, our experiments led us faithfully to the discovery of the cellulosome. Controversy followed: some supportive, and others not! Some of our colleagues lauded us for a significant discovery, whereas others claimed that the cellulosome was not a complex of purpose, but only a loose aggregate of enzymes. Nevertheless, in time, dedicated experimentation proved otherwise. The enzymes were demonstrated conclusively to self-assemble onto a scaffoldin subunit, and the definitive bi-modular cohesin–dockerin interaction was shown to govern cellulosome assembly. The Lego-like, high-affinity binding of these two complementary modular counterparts sparked a second phase in cellulosome research. This time, a Crystal Ball did come into play. In 1994, we were asked by the editor of Trends in Biotechnology to look into the future and predict where the cellulosome field might lead. In prophesizing the future, we thus envisaged the advent of designer cellulosome technology (Bayer et al., 1995). For cellulosomes are like molecular Legos, where each subunit is composed of a multiplicity of distinct protein modules, each characterized by its own individual fold and biological activity. In true Lego-like fashion, we wanted to take the cellulosomes apart and put their component parts back together to form artificial complexes of our design. After this initial proposal of designer cellulosome technology, we were left with the following dilemma: It's nice to propose something, but then you have to do it somehow! At the time, genetic engineering methodologies were not as developed, convenient or prevalent as they are today, and we, as protein chemists, were not very knowledgeable in their practice. In short, continued experiment and development were essential, but we were not capable of doing it! We had a lot to learn! But persistence paid off, and it took us seven years to master the approach and publish the first simplistic examples of designer cellulosomes, with a lot of help from our friends (Fierobe et al., 2001)! Now, 15 years later, our students produce them at will and with great enthusiasm. In many respects, the early attempts were primary examples of synthetic biology in its infancy. Since within a given bacterial species, the various scaffoldin-borne cohesins all recognize the numerous dockerin-containing enzymes in a similar manner, we could not control their reassembly into a complex of predetermined enzyme content. And control is imperative, if we want to examine experimentally the mechanism of cellulosome action. Moreover if we could control enzyme incorporation, we could, perhaps, manufacture cellulosomes of enhanced performance to generate large quantities of sugars from cellulosic biomass en route to biofuel production. Maybe we could thus improve on what Mother Nature has provided us. So, can we do better than Nature? Maybe we can even explore the unnatural by funneling designer cellulosome technology into directions that was of no interest or intent to Mother Nature. If we could graft dockerin modules onto foreign macromolecules, e.g., other types of enzymes, affinity agents or structural components, we could thus adopt the cellulosome as a general platform for nanobiotechnological application. To control the assembly process, we would have to prepare chimaeric scaffoldins that contain cohesins of divergent specificity (derived from different bacterial species), and attach dockerins of matching specificity to the enzymatic components. And this we learned to do! So why do we want to toy with Nature? Well, as scientists we like to play, and as scientists, we like to control! You don't believe me? Then come take a look at my students! Today, it's hard for me to find a student who wants to work on a topic other than designer cellulosomes. Yes, it's fun, but it's also challenging and sometimes exasperating. But we're up for the challenge, and some frustration here and there is not a bad thing! As scientists, we like to solve problems. We have to! So where are we now, and where do we want to go? Let's look again into our Crystal Ball and see what we can see… . Along the way, we and others have achieved major milestones, and today designer cellulosome technology is indeed flourishing. Significant achievements have thus been made, from exploring novel cellulosome geometries (Mingardon et al., 2007) to incremented expansion in the number and types of enzymes that can be integrated therein (Moraïs et al., 2012; Stern et al., 2016). Our capacity to prepare designer cellulosomes of precision content has advanced enormously. We have demonstrated over and over again that incorporation of cellulolytic enzymes into a cellulosome serves to increase their performance. We have forced designer cellulosomes to do things that native cellulosomes are incapable of doing (Arfi et al., 2014; You and Zhang, 2014; Davidi et al., 2016)! However, the experimental work necessary for fabricating designer cellulosomes is still laborious, requiring experience, dedication and finesse. Although serious progress has been made, there is much to do before it becomes a routine approach for broad application. Of course we have far to go if we want to contribute substantially to the current goals of renewable energy programs. Even more pertinent, though, is the relative lack of progress in the peripheral areas that would render designer cellulosome technology as a general nanobiotechnological tool. If we again go back the two decades and look into the Crystal Ball we used at the time, we had indeed envisioned a broader view of the potential of designer cellulosome technology. Of course it was all imaginary at the time, but we then stated that 'we cannot foresee all possible applications, given our own limited imagination and/or knowledge'. We considered the potential of the system unlimited and that 'successful implementation will be directly dependent on the needs and imagination of the user'. Some things never change!