Kraig Biocraft Laboratories Inc (KBLB)

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Proteins: The next generation of industrial materials
By: Kenji Higashi & Junichi Sugahara

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The development of synthetic protein materials that mimic naturally occurring proteins such as spider silks and insect tendons will offer solutions to the limitations and environmental impact made by the petrochemical plastics that revolutionized the manufacturing industry in the previous century. Spiber Inc. has demonstrated capabilities to create products with synthetic spider silk, and is poised to redefine 21st-century manufacturing with technologies that will enable industrial-scale production of high-performance industrial materials.

In the natural world, proteins are the building blocks of life. Some of these proteins are enzymes that enable chemical reactions, and some are used as structural components of cells and organisms, forming organs such as skin. When we look around the natural world, we see proteins that have evolved over 3.8 billion years to possess astounding functions. A good example is a spider’s dragline silk, which is made mainly of a protein called fibroin. Spiders use their dragline silk as a lifeline when they move through the air. Dragline silks spun by Caerostris darwini spiders have exhibited1 tensile strength (the maximum force that can be applied before rupture) of 1.6 GPa and toughness (a value indicating the amount of energy absorbed before rupture) of 354 MJ/m3. This is 14 times the toughness of carbon fibre — the strongest fibre to have been developed by mankind. If a spider’s lifeline were to break, the probability of the spider dying would increase. But thicker thread would waste the spider’s limited resources. The dragline silks that have evolved are those that have allowed the synthesis of the strongest, toughest threads while using a spider’s resources efficiently.

In the early 20th century, scientists embarked on a new era of polymer material development with the invention of petrochemical plastics. Researchers have been synthesizing new polymers and developing materials ever since, so that today we are surrounded by synthetic fibres, resins, rubber and other materials born of the union between petroleum and chemistry. Carbon fibre-reinforced resins have begun to replace metal materials in vehicle and electronic appliance bodies to reduce their weight. A spider’s dragline silk has a specific gravity of 1.3, making it about 70% lighter than carbon fibre. If we could use spider silk as a structural material in cars, aeroplanes and appliance casings, not only could we expect even further reductions in weight, but also dramatic improvements in shock absorption performance, which would translate into more economical and durable products, and greater safety for users.

Other areas in which synthetic proteins promise to enable new levels of functionality include lighter-weight and more impact-resistant protective clothing, such as bulletproof vests, and supple, highly biocompatible material for medical applications, such as surgical sutures, artificial blood vessels, wound dressings and cell culture scaffolds.

Spider silk is not the only protein that offers amazing possibilities. Resilin, found at the base of insects’ limbs and wings, has incredible elastic properties. When it needs to escape from a predator, the tiny spittlebug (or froghopper) can jump as high as 70 times its own height2. Resilin, the protein that makes this possible, has been known to exhibit3 resilience of 99.2%. In other words, it rebounds with 99.2% of the force that is applied to it. No conventional general-purpose rubber material exhibits such a high-energy storage rate. Resilin is also very durable. The resilin found at the base of the wings of a fruit fly (Drosophila melanogaster) withstands repetitive wing flapping from the time the fly matures into adulthood until it dies, without ever being replaced, even though a fly’s wings are estimated4 to flap at 720,000 cycles h-1. Resilin appears promising as a new type of rubber material for use in industrial applications and in implanted medical devices such as artificial heart valves and artificial tendons.

Other high-performance natural proteins include the egg-sac silk spun by one type of spider that can be stretched more than spandex, with a rupture elongation of 750%5. And squid beak is made of a compound material consisting of polysaccharide and proteins, making it one of the hardest organic materials known to man. It has a stiffness that is approximately three times more than synthetic commodity resins such as polycarbonate or polyphenylene ether6.

Humans have long been aware of the outstanding properties of many natural proteins but have not yet been able to use spider silk, resilin or similar structural proteins as manufacturing materials because, unlike wool or silkworm silk, we have not been able to obtain them in sufficient quantities. However, as a result of remarkable developments in biotechnology, this is about to change. We now have the tools we need to decode the genetic information of natural proteins and mass-produce them using recombinant systems.

Setting up Spiber Inc.

Spiber Inc. was established in 2007 to achieve industrial commercialization of synthetic spider silk and other high-performance protein materials. So far Spiber Inc., whose roots are in research conducted at the Institute for Advanced Biosciences at Keio University, has been financed mainly by venture capitalists and grants from the Japanese government. Among the various technologies that Spiber Inc. has been researching and developing are technologies for mass-producing recombinant structural proteins using microbes, fibre spinning and other material processing technologies, and technologies for applying materials in the manufacture of final products.

Spiber Inc. has adopted a highly systematic approach to fibre development. Researchers start by designing new recombinant genes in line with hypotheses aimed at balancing the mechanical properties of fibres against fermentation productivity, and artificially synthesizing the genes using genetic engineering techniques. Next, the genes are introduced into host microbes and cultured under various fermentation conditions and using various nutrient mixtures for maximum protein production. The protein is then separated from the host microbes, refined into a purified polymer solution called dope, and spun into fibres by applying various spinning techniques and conditions. Each time a new recombinant protein is produced, its productivity and fibre properties are examined and recorded, resulting in a database of knowledge founded on hypothesis-driven trial and error. The insights gained from these iterations are incorporated into each ensuing generation of genetic design.

Producing spider silk protein using fermentation

It is impossible to obtain industrially significant amounts of spider silk by cultivating and ‘milking’ spiders. Spiders are highly territorial and aggressively cannibalistic. Moreover, they only eat live prey. In addition, a single spider produces several types of thread. So even if it were possible to successfully cultivate a large number of spiders, it would still not be possible to consistently harvest thread with identical quality.

Given these hurdles, scientists around the world have been searching for ways to mass-produce spider silk using genetic engineering by introducing fibroin genes into a variety of potential hosts, which include goats, tobacco, yeast and Escherichia coli. However, none of these attempts have yet achieved a level of productivity that is remotely sufficient for industrial application. Of all the methods tested, those using microbes were found to be the most efficient. Kazuhide Sekiyama and Junichi Sugahara, then undergraduate students who later co-founded Spiber Inc., began by extracting genes from Nephila clavata (Golden orb-web spiders) caught in the shrubbery around the campus of Keio University. They introduced these genes into host microbes in a university laboratory, but were initially only able to produce quantities of recombinant fibroin weighed in milligrams. It is extremely difficult to get microbes to efficiently produce a protein whose molecular structure is as large and complex as that of spider silk.

Researchers seeking to improve productivity when cultivating recombinant proteins will usually test microbes to find the most efficient hosts and fine-tune culturing conditions to optimize fermentation processes. The Spiber Inc. team succeeded in boosting productivity to extraordinary levels because it didn’t stop at finding highly efficient hosts and fermentation processes but also devel-oped methods of optimizing the genetic codes of recombinant proteins. Based on published reports, Spiber Inc. has been the only research team in the world to succeed using this approach.

As a result, Spiber Inc. has continued to gradually raise productivity. The company has synthesized almost 500 variations of designed fibroin genes, and has improved productivity by a factor of many thousands since it was founded.

Turning proteins into workable materials

Even the best technologies for producing spider silk protein would not be enough to make the industrial use of artificial spider silk a reality. Once produced, the protein must be dissolved in a safe and economical solvent and subjected to optimal spinning processes to produce high-performance fibre.

Just as a spider spins out thread from its abdomen using liquid protein secreted from its silk glands, protein obtained through fer-mentation is dissolved in liquid, extruded through tiny holes, and processed into fibres. Fibre-making processes entail the determina-tion of precise parameters for solvent blends, spinning mechanisms, solidification methods and drafting processes. Each choice has a critical impact on the physical properties and durability of the fibre produced. Spiber Inc. and its business partners have developed optimal equipment and processes for spinning their QMONOS™ brand synthetic fibres, which are made with spider silk-inspired recombinant protein polymers. By conducting trial-and-error experiments aimed at finding optimal spinning conditions, the company has succeeded in producing fibre that is equivalent to natural spider silk in toughness and far stronger than other synthetic spider-silk fibre reported to date.

In addition to developing fibre, Spiber Inc. is cultivating production technologies for a variety of materials including films, gels, sponge and nanofibre non-woven cloth made from protein polymers, and tough resin-fibre compounds made by post-processing fibre. These efforts include finding optimal molecular designs for each material format and optimizing processing conditions.

As it works to improve productivity and performance through iterative design — prototyping — feedback cycles, Spiber Inc.
is simultaneously building its knowledge of the mechanisms by which molecular design affects productivity and material perfor-mance. The company is close to being able to freely design proteins with specific characteristics based on molecular design. In other words, it will be able to supply tailor-made fibres to manufacturers’ requirements. Customers will be able to say, for example, “We want a fibre with x strength and y elasticity”, and Spiber Inc. will be able to oblige. Varying both molecular design and processing conditions should make it possible to produce a virtually infinite range of materials.

Application in industrial products

In May 2013, Spiber Inc. used its proprietary elemental technologies to create a dress using QMONOS™ fibres, proving its ability to make an industrial product from artificially synthesized protein material. In November 2013, Spiber Inc. and its partner, Kojima Industries Corporation, backed by funding from the Japanese government, built a prototyping studio with facilities for fermenting, refining and spinning. Spiber Inc.’s groundbreaking prototyping studio, with maximum annual production capacity of 1,000kg, is the world’s first facility in which protein is produced by microbial fermentation, processed into usable material, and developed into prototypes of final products under one roof. In spring 2015, Spiber Inc. plans to inaugurate a next-generation pilot line that will serve as a model for scaling up processes already developed in the lab and prototyping studio. Within a few years, the company plans to enable this facility to handle a wide range of production scales.

The benefits of sustainability

Using new synthetic versions of naturally occurring proteins promises other benefits to man in addition to the introduction of ultra high-performance consumer products. For centuries, man has used new materials to help trigger revolutionary industrial devel-opments that have shaped the structure of human society. The invention of ceramic technologies around 10,000 BC enabled people to produce a variety of containers by freely forming clay. Around 3,000 BC, people living in Mesopotamia learned to make bronze tools, leading to dramatic innovations in building, farming and hunting implements that took advantage of the strength of bronze. From around 300 BC the Romans began the widespread use of concrete, which for the first time enabled them to design buildings and infrastructure without limitations caused by the shapes of raw materials such as stone or brick. The 20th century saw the rise of petrochemical industries kicked off by Leo Baekeland’s invention of Bakelite, the first synthetic thermosetting plastic, and the invention of nylon by Wallace Carothers. A wide variety of petroleum-derived synthetic fibres, synthetic resins and synthetic rubbers — including polyester and vinyl — were developed in rapid succession. Low-cost sup-plies of these materials have come to support modern industrialized societies.

Most of the 4.2 billion tons of petroleum consumed worldwide each year is used for fuel. Some 230 million tons, or 5%, is used to produce petrochemical-derived polymer materials. It is clear that the existing social structure — dependent as it is on dwindling petroleum resources — is problematic for various reasons. One reason is that petroleum supplies will eventually run out. The 1.2 billion inhabitants of industrialized OECD countries consume 50% of the petroleum resources currently used by the world’s 7.3 billion people. The world population is still growing, and is expected to reach 9 billion in 2050. If people in developing countries begin to use petroleum the way it is currently used in industrialized countries, petroleum consumption will increase dramatically.

Our planet’s oil reserves have been estimated at 240 billion tons. At the current pace of consumption, these reserves should remain available for several decades, and if we consider other reserves that are not currently available for exploitation due to technical or economic factors, petroleum resources may not be depleted for another several centuries. But in either case, it is clear that our descendants will need to find sustainable, alternative resources to replace petroleum. Another problem is that reliance on scarce resources has historically led to wars. As long as world economies depend on non-renewable resources, these violent conflicts cannot be fundamentally resolved.

A number of initiatives around the world aim to solve these global problems. In the field of energy production, a great deal of research aims to free humanity from the yoke of petroleum dependence by developing sustainable alternative fuels like biodiesel and bioethanol as well as cleaner methods of electric power generation using solar power, wind power, hydropower and geothermal power. No doubt the success of such efforts will lead to increasing replacement of fossil fuels. Similarly, there are a number of initiatives aimed at producing industrial materials such as polylactic acid, polyhydroxyalkanoate, biopolypropylane and bio-nylon from corn, sugar cane and other biomass feed stocks. Looking at the long-term future, it is likely that biomass-derived synthetic polymers will replace petroleum-derived synthetic polymers.

Spiber’s vision of the future

The potential design space for creating new synthetic protein materials is huge. Whereas conventional industrial polymers typically consist of chains of one or at the most two types of monomers, each link of a protein can draw from a group of 20 amino acids. This offers a tremendous degree of control over the final material’s structure and properties. Through the ages, the process of evolution has driven dynamic changes in the structures and functions of living beings. By designing new synthetic proteins, we will be able to create materials that are tailor-made at the molecular level to suit users’ specific needs for characteristics such as strength, elasticity, hydrophilic or hydrophobic properties, ultraviolet ray resistance, biocompatibility and resin compatibility. The range of properties that proteins can express are far broader than those of petrochemical polymers such as nylons or polyesters, and the range of poten-tial applications could touch every scene of our daily life and every area of industry.

Biomass-derived polymer materials that have been developed so far resemble petro-leum-derived polymers in the sense that they require polymerization by means of a chemical process. They also resemble existing materials in that they require dedicated production equipment designed specifically for each type of material produced. On the other hand, a single microbe-based fermentation process can be used to produce an enormous range of protein polymers by simply changing the genes that provide the code for each target protein. In a single fermentation plant, infinite varieties of materials could be manufactured through a biochemical process from a single type of biomass feed stock. This constitutes an epoch-making change. It means that the reductions in cost that can be achieved by making large quantities of a single type of material can be achieved when making small quantities of a variety of types of material as long as the overall volume is large. Once a large-scale production system for making structural protein materials has been constructed, it may be possible to introduce tens of thousands of tons of a new material to the market within a year of conceiving the molecular design for that new material. It is unlikely that there is any category of material other than proteins for which this could be true. This capability will have a dramatic impact on the life cycles of industrial materials. And this in turn will bring about a major paradigm shift in manufacturing. Instead of looking at a material and considering what can be made from it, people will be free to imagine a product they want to make and then design a material from which to make it.

In light of the industrial significance of structural proteins described above, structural proteins will constitute more than a small portion of the biomass-derived synthetic polymers that will be used in the future. Spiber Inc. aims to replace some 30% of worldwide synthetic polymer production with structural protein materials by the end of this century. Following stone, ceramics, bronze, iron, glass, concrete and plastics, proteins — natural materials that are now being developed in new ways — may be the next material to spark a revolution in industry.


References:
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