This year, GreatBay_SCIE sets out to construct a cellulosome complex in order to degrade cellulose and PET present in fabrics at a faster rate. Cellulosomes are multi-enzyme complexes consisting of a scaffolding protein and two types of complementary recognition modules known as “dockerin” and “cohesin”. The cellulosome allows enzymes to work synergistically, which makes cellulosomes more efficient in degrading cellulose than any other free enzyme system. We aim to express cellulases, namely endoglucanase (TrEGIII), exoglucanase (CBHII), beta-glucosidase (NpaBGS), cellulase boosters (TaLPMO and MtCDH), and scaffold proteins (CipA and OlpB) with E.coli and Kluyveromyces marxianus.
To achieve the goal of degrading a more comprehensive range of materials in clothes, we successfully expressed PETase. For the future, we planned to fuse PETase with a dockerin protein so that PETase could bind to our scaffold, which would improve the efficiency of PETase.
We also express ferritin in our host E.coli so that we can recycle the cellulosome complex and thus be more sustainable and cost-effective. It is noticeable that we have come up with an original approach to verifying ferritin expression.
Here are the contributions we have achieved:
A New Approach for Verifying The Expression of Ferritin
We have invented a new approach for verifying the expression of ferritin by using RFP as a reporter molecule. This method is capable of assisting any future teams who intend to prove the production of ferritin.
The gene coding for RFP was fused with a plasmid that provided resistance to Chloramphenicol, while the gene coding for ferritin was connected to a plasmid that provided resistance to Kanamycin. We transformed the two plasmids coding for RFP and ferritin to DH5α.
A solid medium with both Chloramphenicol and Kanamycin was used to culture the DH5α so that we could select the DH5α containing both plasmids. The DH5α was transferred into conical flasks for enlarged cultivation. When the OD reached 0.1, we induced the expression of RFP with rhamnose. The culture was left to grow until the OD reached 0.6, at which we induced the expression of ferritin with IPTG and added Mohr salt as an iron source. Then, we resuspended the DH5α with tris HCL and placed the DH5α into Petri dishes with strong magnets.
After leaving DH5α with the magnets overnight, we placed the Petri dish under a blue light and saw through a filter that filtered out blue and purple light. We found that the DH5α, which emitted red light, was concentrated around the magnet, showing that ferritin was successfully expressed. We found that a high rhamnose concentration allowed better observation since it induced more RFP.
Refinement of The Yeast Toolkit for Kluyveromyces marxianus
We refined the yeast toolkit by making the commonly used yeast toolkit available for Kluyveromyces marxianus.
Kluyveromyces marxianus (K. marxianus) is a non-conventional yeast that is not widely studied. However, its physiological and metabolic characteristics show great potential in biotechnological applications. Therefore, a more comprehensive yeast toolkit for K. marxianus is of significant importance.
We expanded the yeast toolkit proposed by Michael E. Lee. The toolkit was designed for Saccharomyces cerevisiae. To make the parts compatible with K. marxianus, we designed the Lac4 vector, which contained a pKmK.C1 origin, KanR selection marker, and Lac4 promoter, that could be successfully expressed in K. marxianus. We changed the origin into the pKmK.C1 origin for K. marxianus. This allowed the vector plasmid to be replicated within K. marxianus. We used KanR as the selection marker for K. marxianus and proved that it was an effective selection marker for K. marxianus.
In addition, we changed the alpha factor pre-pro secretion leader (Mα) for S. cerevisiae to the Mα for K. marxianus. With the help of the Mα for K. marxianus, we were able to directly extract the desired proteins from the supernatant. This eliminated the need for performing cell lysis, thus making the production of cellulases more cost-effective and convenient.
## Using eforRed as A Reporter Molecule
We have developed a novel detection experiment for the interaction between cohesin I and dockerin I, and cohesin II and dockerin II, using eforRED as a reporter molecule.
We constructed several E.coli expression vectors for type I dockerin domain ligated with an eforRed domain, type II dockerin domain ligated with an eforRed domain, antigen domain ligated with an eforRed domain, respectively, and E.coli expression vector ligated with an eforRed protein. The successful binding of cohesins to dockerins was indicated by the red light emitted by eforRed. When type I dockerin with eforRed binds to the type I cohesins without eforRed, the residue displayed a red light after centrifugation. When the binding of eforRed-containing type I dockerin and the non-eforRed-containing cohesin was unsuccessful, the residue did not demonstrate the red light as the eforRed-ligated type I cohesins were left in the supernatant. The same happened to the type II dockerins ligated with eforRed and the type II cohesins without eforRed. This allowed us to visualize and verify the binding of cohesins to dockerins. This approach is helpful to any future teams who intend to test for the interaction between cohesins and dockerins (view the engineering success page for more details).
To achieve the closed-loop recycling of Natural and Artificial Fabrics into their corresponding monomers, we have developed a multi-module, multi-functional, and high-biosafety fabric degradation bioreactor for the project Fabrevivo. The construct includes a fabric pre-treatment module, two detachable, interconvertible fermentation modules, and one autoclave module. This model is designed for the complete bio-degradation and closed-loop recycling of natural cotton fibres, artificial polyester fibres, and cotton-polyester blends.
Figure 1 An overview of our multi-module Cellulose-PET degradation Bioreactor. The whole design includes a pre-treatment model (top-left), two fermentation modules (bottom-left), and a separate autoclave module (right). The untreated fabrics enter the machinery through the large, cylindrical blades of the pre-treatment module.
Figure 2 The longitudinal section of our whole model. The autoclave(left) functions for the final sterilization of our product of degradation. The pre-treatment module(top-right) consists of two large cylindrical blades, which reduce the surface area of the substrate, and a pre-treatment tank for chemical pre-treatment. The two fermentation modules(bottom-right), one loaded with scaffoldin and Cellulase-expressing K.marxianus strains, serves for the degradation of cellulose to glucose, whilst the second, loaded with scaffoldin and PETase-expressing K.marxianus strains, serves for the degradation of Polyethylene terephthalic acid (PET).
The first module - the fabric pre-treatment module - consists of two large cylindrical blades and a chemical pre-treatment tank with built-in electric-heating apparatus. The untreated, useless, and contaminated commercial fabrics are initially sliced into small-sized threads to increase the surface area for chemical treatment and cellulosome-based enzymatic degradation. The treatment tank, with a constant core temperature of 75℃, is filled with hydrogen peroxide or other dye-removing agents and then treated with a dilute sulfuric acid solution to thoroughly remove the acetyl groups on the cellulose acetate fibres. This treatment removes most of the dyes and impurities from the fabric from our desired degradation target.
Figure 3 The 3D structure of the pre-treatment module. The two intersecting cylindrical blades (opaque black) slice the target of degradation into minuscule threads, facilitating the subsequent chemical pre-treatment(the first transparent tank) and enzymatic degradation(the two transparent fermentation modules). A core temperature of 75℃ is maintained in the pre-treatment tank, with dilute sulfuric acid, H2O2 or other dye-removing agents filled within the space.
The two fermentation modules - the cellulose degradation module and PET degradation module, are comprised of a cylindrical fermentation chamber with a cone-shaped bottom surface equipped with an electro-magnetic ring, an electric-heating apparatus which maintains the core temperature of the modules to 50℃, and a six-bladed shutter which controls the movement of substances in and out of the module. The first module (cellulose fermentation module) is designed for completely degrading natural fibres and hosts the scaffoldin-expressing K.marxianus strains and cellulase-expressing K.marxianus strains. The second module (PET fermentation module) is designed for completely degrading synthetic fibres (PET) and hosts the scaffoldin-expressing K.marxianus strains and PETase-expressing K.marxianus strains. The high temperature within the container will attenuate the glucose-consuming activity of the K.marxianus strains. Thus the end-product of cellulose digestion can be mostly protected from microbial consumption.
Figure 4 An outer view of the two fermentation modules(lustrous-silver). The upper fermentation module contains the scaffoldin K.marxianus strains and cellulase K.marxianus strains. Together they express the protein components of a fully functional membrane-anchored cellulose-degrading cellulosome complex, whilst the second module contains K.marxianus strains that express the protein components of a functional PET-degrading cellulosome-based complex. Both modules contain a six-bladed shutter-like opening(bottom-middle) on their base, which controls the flux of degradation products and degradation intermediates through the different modules.
Figure 5 A longitudinal section of the two fermentation modules(opaque-black). Both modules have an inclined bottom with an angle of 10° towards the centre. This inclination allows the ferritin-expressing magnetic sensitive K.marxianus strains to migrate away from the central exit and be attracted to the electromagnets in the periphery of the modules, minimizing the loss of K.marxianus in each round of fermentation. The core temperature of both modules is maintained at 50℃, which is in the optimal temperature range of PETases and Cellulases. These two modules' presence and relative position could be modified based on the type of degradation material.
To avoid the loss of K.marxianus in each cycle of degradation, the peripheral circular electromagnet powers on at the end of each recycling period and attracts the ferritin-expressing magnetic sensitive K.marxianus to the periphery of the cylindrical fermentation container, further away from the shutter exit in the middle. After most of the K.marxianus yeast is immobilized on the gradient of the cone-shaped bottom of the container via electro-magnetism, the six-bladed shutter automatically opens and delivers the reacted products to the next chamber. The two fermentation modules are interconvertible, so their combination or position could be switched based on the type of untreated fabric for recycling. When the target of degradation is purely constituted of natural fibres, a single cellulose-degradation module will be sufficient; when polyester is the only substance present in the untreated mass, a PET degradation module should be deployed. For the complete degradation of Cotton-polyester blends or other combinations of natural and synthetic fibres, two modules should be simultaneously applied.
Figure 6 The transverse section of the degradation consortium on the plane of a fermentation module. When a degradation cycle is completed, and all the insoluble substrates are converted into their target soluble products, the electromagnet chunks on the periphery(lustrous silver) will be powered on and attract magnetic-sensitive K.marxianus yeasts along the inclined bottom plane. After most of the yeasts have been localized to the periphery, away from the central exit, the six-bladed mechanical shutter will automatically open, delivering the degradation product to the next module of this degradation consortium.
Figure 7 & 8 A closer look into the 3D structure and the functioning mechanism of the six-bladed shutter ( transverse section on the bottom side). After complete degradation of the substrate AND the immobilization of K.marxianus yeasts on the inclined slope, the controller of this shutter (protruded outward) will automatically rotate for 30℃ and open the six-bladed shutter, analogous to the mechanism of a camera shutter.
The last module of this construct - the autoclave module, with a core temperature of 121℃, is applied in the end to sterilize the degradation product. Although K.marxianus only has a Biosafety Level of BSL-1, strict commercial or industrial usage protocols must be applied. The end-product of degradation: glucose, ethylene glycol, and terephthalic acid, could be further separated via HPLC. The former could be used to make new bacterial celluloses (BC), while the latter could synthesize new artificial fibres via chemical polymerization.
Figure 9 The 3D longitudinal section of the autoclave module. After the end-product of degradation is settled in the perforation basket(middle), which is hovered from the pressurized container via a circular steel scaffold, the core temperature of this module will be increased up to 121℃, the sterilizing temperature. The pressure of this apparatus is controlled by two air valves(top).
