Companies Developing Modified Microorganisms for Manufacture of Biofuel Enzymes

Of all the industrial sectors I’ll be profiling in this blog, the industrial enzyme sector is by far the most mature, and the one that is to the greatest extent already a critical part of today’s transportation fuel industry. It also benefits from being part of a larger, longstanding sector of the bio-economy that for decades has been producing pure or partially-purified preparations of enzymes for use in various industries primarily including food and beverage processing. 

Enzymes are the protein catalysts that drive virtually all the biochemical reactions that take place in living cells: enzymes and the genes that encode them are the key to almost all the genetic modifications that are discussed in this blog. Like most proteins, each enzyme in every living cell is encoded by a single gene (although technically, many enzymes are built of multiple subunits, and each subunit is usually encoded by its own gene). Much of the genetic modification discussed elsewhere in this blog is directed towards transferring genes into desired “host” organisms to give them the ability to express enzymes not naturally found in that host, giving the host new biochemical powers (particularly when multiple enzyme-encoding genes are spliced into the host). However, the companies producing enzymes for industrial use follow a different strategy. These companies seek to identify or create microbial strains that can synthesize very high levels of a given enzyme (so-called “overexpression”), which are often secreted from the host organism, but which in any case are purified after the microbes are fermented at large scale. Pure or partially pure preparations of such enzymes are then sold as commercial products for various industrial uses. Historically, enzyme manufacturers have relied on naturally occurring or classically mutated strains, but more recently these companies have turned to genetic engineering to enable the creation of host organisms capable of high level expression of desired enzymes. 

Enzymes have been produced and sold for various industrial purposes for decades, and today industrial enzymes represent a major sector of the life sciences industry. Market estimates place annual global revenues as approaching $3 billion in the early years of the current decade (a 2007 BCC Research report estimated the 2007 market at $2.3 billion, and projected an increase to over $2.7 billion by 2012; and a report from Global Industry Analysts, Inc. estimated that the world market will exceed $2.9 billion by 2012; there are also estimates that are even higher). The sector of this market that includes the enzymes used in biofuel production is harder to estimate, but is widely considered to be one of the more rapidly growing segments of the industry. 

Industrial enzymes are currently an integral part of most existing cellulosic ethanol production processes. Unlike sugar-based ethanol feedstocks like sugar cane and corn, cellulosic biomass has a more complex molecular structure, composed not only of cellulose, which is a simple homopolymer of glucose molecules, but also of hemicellulose  (heteropolymers of 6- and 5-carbon sugars) and lignins. Few if any naturally-occurring ethanol-fermenting organisms possess the ability to break down these more complex molecules, and so it is common in most existing ethanol plants for the cellulosic feedstock to be pretreated using a combination of enzyme preparations to degrade the long polymers into sugars that are more readily utilized by natural ethanol-fermenting organisms. 

At one time, the cost of enzymes made up a significant percentage of the overall cost of cellulosic ethanol – reportedly as much as 50% of the production cost, at a time when cellulosic ethanol cost $5-10 per gallon (most observers feel that the target production price for cellulosic to be competitive with corn ethanol is between $1-2 per gallon). The advances that have been made by the companies described below and by other researchers have substantially cut the cost of manufacturing enzymes for cellulosic ethanol, to the point where new product announcements by Novozymes and Genencor in February 2010 (described below) brought widespread speculation that enzyme costs were starting to get low enough to realistically expect these target costs to be met early in the decade.  

The market for biofuel enzymes is currently dominated by big players – companies that have been in the enzyme business for decades. However, some of the companies profiled below are smaller, start-up companies or are otherwise new to the industrial enzyme industry. It seems clear that the larger companies have a substantial edge in the market, due to their existing infrastructure, sales force, knowledge and experience with the market, and other advantages. Often, smaller companies in any given market would be more prone to innovation than would the larger companies, and while that may be true here, it should be said that some of the established companies, particularly Danisco Genencor and Novozymes, have top-notch molecular biologists on their research staffs and conduct cutting edge research that smaller companies may have trouble rivaling. Although there may be an ongoing role in the market for niche players, it is likely that the larger companies will continue to dominate for the foreseeable future.

An intriguing question, however, is whether the need for enzyme pretreatment of cellulosic feedstocks will ever be supplanted by the increased use of some of the more versatile microorganisms that are being developed that will express exogenous enzymes enabling the strains to digest complex carbohydrates on their own. It is surely the long-term hope of many of the microorganism developers that this will take place, resulting in process cost savings by altogether avoiding the need for added enzyme preparations.  However, my gut feeling is that this won’t happen for quite some time, if at all, for several reasons. First, it is too early to know how soon such improved microbes may become widely available, and widely accepted, for use in cellulosic biofuel production. Second, we can anticipate that the demand for cellulosic ethanol will grow dramatically in the coming years, due to the Renewable Fuel Standards in the U.S. and similar aggressive government policies elsewhere in the world, meaning that production capacity may expand rapidly enough so that there is room in the market for both the more versatile bugs as well as traditional microorganisms which will continue to need to be supplemented by enzyme pretreatment of cellulosic biomass.

The following are the companies that are manufacturing enzymes for use in biofuel production. 

  • AB Enzymes
  • Danisco/Genencor
  • DSM
  • Dyadic International
  • Infinite Enzymes*
  • Iogen
  • Medicago*
  • Novozymes
  • Proteus
  • Syngenta*
  • Zymetis

Most of these companies are using microorganisms, enhanced using both traditional methods and through biotechnology, to produce enzymes. However, the companies identified with an asterisk are using modified plant species as the platform to produce enzymes for use in biofuel production, and these companies will be profiled in a later section of the blog. 

The following are brief profiles of companies that are developing engineered or modified microorganisms for the production of industrial enzymes for biofuel production and other uses. These profiles have been adapted or excerpted from company websites and/or other publicly available information, and I don’t assume any liability for the accuracy, comprehensiveness or use of the information. 

AB Enzymes GmbH is one of the world’s oldest and best known companies producing industrial enzymes. AB Enzymes is a member of the ABF Ingredients group of companies, which focuses on high value ingredients for both food and non-food applications. AB Enzymes manufactures and sells enzymes for industrial applications worldwide, including food enzymes for bakery and beverages, enzymes for animal feed, textile technology and the pulp and paper industry. 

AB Enzymes’ focus in the biofuel market is in supplying enzymes to support the use of lignocellulosic biomass to produce transportation fuels. The company believes that abundant forms of low value agricultural residues such as ricestraw, cornstover, wood chips, wheatstraw and baggase will increasingly need to be used for this purpose. The company sees the key challenges to include the availability of low-cost substrate and the reduction of the processing costs of cellulosic biomass, and that  a significant part of this effort will come from the reduction in costs of enzymatic saccharification.  Currently, enzyme dosages and thereby enzyme costs are significantly higher for cellulosic ethanol compared to ethanol production from starch, and such costs need to be reduced dramatically over the next few years to enable success in the use of lignocellulosic biomass for ethanol production.

The wide variety of feedstocks and processes require a tailor-made enzyme approach. AB Enzymes offers this “tailor-made” service which will enable the industry to speed up development processes at a reduced cost. AB Enzymes has been successful in the discovery and production of enzymes for the breakdown of lignocellulosic substrates, using Trichoderma strains as the production host of choice for this application. 

Danisco US Inc., Genencor Division is a leading industrial biotechnology company that develops and markets innovative enzymes and bio-based products. Originally formed in the 1980s as a joint venture between Genentech and Corning Glassworks, Genencor is now a division of the Danish company Danisco. Genencor discovers, develops, manufactures, and delivers eco-friendly, efficient enzyme product solutions for the agri processing, cleaning and textiles, food and feed, consumer, and industrial markets, and is also developing innovative technologies for the biofuels, biodefense, and biosafety industries. The company has recognized expertise and longstanding experience in protein and pathway engineering. The company’s two-pronged strategy includes sales of products through its commercial enzyme business as well as its joint venture with DuPont – DuPont Danisco Cellulosic Ethanol LLC (discussed in an earlier entry of this blog). 

Genencor was one of the first companies to develop expertise in cellulosic ethanol enzyme technology, and the company says it was the first company to come to the market with a large-scale enzyme for biomass-to-ethanol conversion, with the introduction of Accellerase in 2007. In February 2009, Genencor launched Accellerase 1500 enzyme complex, for improved economics and higher yields. Genencor added  three products to its Accellerase enzyme line for smaller scale process developers: Accellerase XY, XC and BG.  

In February 2010, Genencor announced the availability of a new enzyme in its Accllerase line. This product,  Accellerase® DUET, is the latest generation in the company’s line of enzymes used to convert biomass into sugars, a critical step in the production of cellulosic ethanol and other advanced biofuels and biochemicals from non-food feedstocks. The company claims that with improved overall hemicellulase activity, Accellerase® DUET builds on the advances in beta-glucosidase and cellulase activity previously made by Accellerase® 1500. These additional improvements allow Accellerase® DUET to achieve higher sugar and biofuel yields, often at 3-fold lower dosing, and to be feedstock- and pretreatment- flexible. Accellerase® DUET employs a whole broth formulation, which provides nutrients for fermentative organisms and lowers the chemical load introduced into our customers’ processes. Higher performance at lower dose will lead to significant improvements in enzyme cost in use for producers, which is critical to enable the cellulosic biofuels industry. 

Royal DSM N.V. is a publicly listed company, founded in 1902, that creates innovative products and services in life sciences and materials sciences. DSM’s products and services are used globally in a wide range of markets and applications, in end markets that include human and animal nutrition and health, personal care, pharmaceuticals, automotive, coatings and paint, electrical and electronics, life protection and housing. DSM has annual net sales of EUR 9.3 billion and employs some 23,500 people worldwide. The company is headquartered in the Netherlands, with locations on five continents. 

DSM is a leading producer of numerous enzymes, in particular for the food market. With its extensive expertise and know-how in the field of enzyme technology, DSM is increasingly able to replace its chemical process steps by enzymatic processes. These are more efficient, sustainable and cleaner. DSM is a major practitioner of white (industrial) biotechnology, Among its capabilities are the use of advanced micro-organisms, including microbial biocatalysts and an ethanologen capable of co-fermenting C5 and C6 sugars; and enzymatic hydrolysis, e.g. using cellulases and hemicellulases. DSM says that it does not produce any genetically modified products. Although genetically modified micro-organisms are sometimes used in production, the end product is always identical to the natural product. 

Dyadic International, Inc. is a global biotechnology company with the groundbreaking technology that it says “brings nature to the marketplace”. Dyadic is focused on the discovery, development, and manufacturing of novel products derived from the DNA of complex living organisms – including humans – found in the earth’s biodiversity. Using its integrated technology platform, Dyadic develops biological products such as proteins, enzymes, polypeptides and small molecules for applications in large segments of the agricultural, industrial, bioenergy, chemical and biopharmaceutical industries. The company utilizes a proprietary host organism that allows the rapid discovery and expression of eukaryotic genes which can then be used to manufacture unique biological products for commercial applications. 

Dyadic’s C1 integrated technology platform is expected to enable researchers to identify, select and analyze novel enzymes best suited to convert biomass materials into biofuels. The company believes that its technology may play a significant role in the development of biofuels such as ethanol at costs competitive to oil prices, thereby reducing subsidies and ultimately expanding consumers’ use of these renewable energy sources. Dyadic is using this technology platform along with other proprietary technology to develop biofuels from agricultural byproducts such as corn stover and wheat straw, and the company is using this technology for itself and others. Among its enzyme products for biofuel processing are AlternaFuel® 100P,  a powdered fungal hemicellulase enzyme complex, and AlternaFuel® 200P, a powdered fungal cellulase enzyme complex, both produced from Trichoderma longibrachiatum, that can be used for the degradation of various lignocellulosic biomass substrates for conversion of biomass to glucose. 

Dyadic has a non-exclusive license agreement with Abengoa Bioenergy for certain Dyadic patent rights and know-how relating to its C1 Technology Platform. The license agreement provides for facility fees and royalties to be paid to Dyadic upon commercialization.  In November 2008: Dyadic and Codexis announced a license agreement covering use of Dyadic’s C1 expression system for large-scale production of enzymes in certain fields including biofuels and chemical and pharmaceutical intermediate production. The agreement includes an upfront payment by Codexis of $10 million provided that certain performance criteria are satisfied. Additional financial terms were not disclosed.

Iogen Corporation is a biotechnology firm specializing in cellulosic ethanol.  Iogen also develops, manufactures and markets enzymes used to modify and improve the processing of natural fibers within the textile, animal feed, and pulp and paper industries. Iogen owns and operates a large-scale state-of-the-art enzyme manufacturing facility in Ottawa, Canada. Iogen’s line of enzymes for cellulosic ethanol production is currently being used in the company’s demonstration plant, which has been operational at its Ottawa headquarters since 2004. At full capacity the plant is designed to process about 20-30 tonnes per day of feedstock, and to produce approximately 5,000 – 6,000 liters of cellulosic ethanol per day. But the company’s enzymes are also expected be available for sale in conjunction with technology licenses for the cellulosic ethanol facilities in the future. 

Iogen’s major partner in its ethanol business is Royal Dutch Shell, which first invested in Iogen in 2002. Shell subsequently increased its ownership stake in Iogen’s technology to 50 percent in 2007. Under an expanded agreement with Shell, Codexis will optimize the efficiency of Iogen Energy’s cellulosic ethanol catalysts, as well as developing new to convert biomass directly into green gasoline or green diesel. In October 2008, Iogen announced that it had commenced shipments of a 47,000 gallon cellulosic ethanol order from Shell. In January 2010, the company announced that its cellulosic ethanol production in 2009 topped 581,000 liters, more than doubling the firm’s 2008 fuel production, and surpassing the one million liter mark in cumulative production since 2004. Iogen is currently planning to construct a larger capacity plant a former pulp mill in Prince Albert, Saskatchewan, with financial assistance from the provincial and federal governments.  Iogen representatives have said that they hoped to launch the 23 Mgy plant north of Saskatoon by 2011, using wheat straw and other cellulose as feedstock. Iogen is backed in the venture by Shell, Goldman Sachs and Petro Canada. 

Iogen appears to be the only company that has submitted notifications to Environment Canada under the Canadian Environmental Protection Act for the manufacture of biofuel enzymes using engineered microorganisms (I’ll have more to say about these regulations and their impact on biofuel production in later entries in the blog). These notifications include the following: 

  • NSN (New Substance Notification) #6823: Commercial production of a novel thermophilic xylanase enzyme by the genetically engineered strain Trichoderma longibrachiatum RM4-100.
  • NSN# 11017: Trichoderma reesei 1391A, expressing of a novel xylanase II enzyme with enhanced thermal stability and a selectable marker.
  • NSN # 11909: Commercial production of a β-glucosidase enzyme by genetically engineered Trichoderma reesei P59G.
  • NSN # 12961: Commercial production of a novel thermophilic and alkalophilic xylanase II (xln2) enzyme by genetically engineered Trichoderma reesei P210A.
  • NSN # 13912: Commercial production of a thermophilic/ alkalophilic xylanase II enzyme by genetically engineered strain Trichoderma reesei P345A.

Novozymes A/S says it is the world leader in what it calls “bioinnovation”, creating biological solutions to improve its customers’ businesses. With over 700 products used in 130 countries, Novozymes’ bioinnovations improve industrial performance and safeguard the world’s resources by offering superior and sustainable solutions for tomorrow’s ever-changing marketplace. Novozymes claims to offer the leading technology platform for biofuel production.

Among relevant developments since 2008, Novozymes has launched several new enzyme preparations, including the Cellic product family, the company’s first commercial enzymes for cellulosic bioethanol; Spirizyme Ultra and Spirizyme HS for saccharification, which are expected to  provide yield increases for starch bioethanol production; and Liquozyme SC 4X for liquefaction, for use in  starch bioethanol production. In early 2009, Novozymes broke ground on a new $200 million production facility in Blair, Nebraska, to meet demand for enzymes for the production of first and second generation bioethanol. 

In February 2010, Novozymes launched its newest products in the Cellic product family, which it says are the first commercially viable enzymes to allow the production of biofuel from agricultural waste, and which might enable cellulosic biofuel to be a cost-competitive alternative to gasoline.  Novozymes says that its new Cellic® CTec2 enzymes enable the biofuel industry to produce cellulosic ethanol at a price below US $2.00 per gallon for the initial commercial-scale plants that are scheduled to be in operation in 2011. This cost is on par with gasoline and conventional ethanol at the current US market prices. The company says that Cellic CTec2 has been proven to work on many different feedstock types, including corn cobs and stalks, wheat straw, sugarcane bagasse, and woodchips.

The company says that extraordinary advances in enzyme development have reduced the enzyme cost for cellulosic ethanol by 80% over the past two years so that enzyme costs are now down to approximately 50 cents per gallon of cellulosic ethanol. Novozymes has allocated unprecedented resources to the project, and the company has also received development grants totaling $29.3 million from the US Department of Energy.

Novozymes has partnered with leading companies in the biofuel industry, such as POET, Greenfield Ethanol, Inbicon, Lignol, ICM, M&G, CTC, COFCO, Sinopec, and PRAJ to help accelerate process technology development and implementation. Coupled with further improvements in enzyme efficiency, Novozymes expects the cost to produce cellulosic biofuel to be further reduced. 

Protéus is a biotechnology company that focuses on the discovery, engineering and manufacturing of proteins of industrial interest, and on the development of innovative protein-based bioprocesses. The efficiency of Proteus’ technology platform has been demonstrated by the successful track record of the company in the life science industry, including healthcare, fine and specialty chemicals, environment and bioenergy. 

Proteus creates bio-based solutions to efficiently convert biomass into energy with dramatically lower energy inputs and net carbon emissions.  The goal is to improve and accelerate the conversion of biomass from a variety of origins, including lignocellulosic feedstock from industrial by-products, agricultural waste, municipal waste, or activated sludge into bioenergy. 

Because of the recalcitrance of biomass to hydrolysis, enhancing and accelerating the conversion of these raw materials into fermentable material is the core challenge for sustainable production of cellulosic fuels. Proteus (i) improves the productivity of existing bio-based processes by improving the performance of the enzymes involved in biomass cracking into fermentable material and (ii) creates and fine-tunes competitive natural or engineered biological cocktails that fit the specific composition of the renewable feedstock available and the characteristics of the energy-producing processes of its clients. 

Among its products are bespoke enzymes for SHF (Separate Enzymatic Hydrolysis and Fermentation), SSF (Simultaneous Saccharification and Fermentation) and for engineering CBP-enabling microorganisms (Consolidated BioProcessing). In January 2009, Proteus announced an agreement with Syngenta to develop enzymes for cellulosic ethanol production.  Syngenta said that the agreement with Proteus will allow it to accelerate its development of optimized enzymes based on Syngenta’s core competency in plant expression. 

Zymetis, Inc. is a biotechnology company dedicated to developing novel enzyme products for the emerging bio-refining industry. Derived from unique organisms, Zymetis products are designed to achieve lower costs, improved yields and higher manufacturing efficiencies in the conversion of cellulosic biomass to usable sugars. Currently, the company is developing the Ethazyme® family of industrial enzymes for use in the production of fuel–grade ethanol from biomass.  This appears to be a change in emphasis for the company, which initially stated its intentions of commercializing the microorganisms themselves as biofuel catalysts. 

Zymetis was formed in 2006 to commercialize technologies discovered by Dr. Steve Hutcheson, professor of cell biology and molecular genetics, and Dr. Ron Weiner, Professor Emeritus, from the University of Maryland. Zymetis genetically modified a rare, cellulose-eating bacterium isolated from the Chesapeake Bay to break down and convert cellulose into sugars for conversion to ethanol. In its first commercial-scale trial in 2008,  the company ran the modified microbe through a series of tests in large fermenters and found that it was able to convert one ton of cellulosic plant fiber into sugar in 72 hours.

According to the company’s website, Zymetis is offering a wide range of specialty enzymes and custom research services. The Ethazyme® family provides flexible and cost–effective enhancements and alternatives for existing enzymatic strategies for biomass saccharification. Ethazyme® products, once optimized, are effective on virtually any biomass and can be tailored to most production processes. Zymetis is currently working with consumer and industrial wastes such as waste paper, agricultural wastes such as corn cob and corn stover, timber sources such as paper mill waste and hybrid poplar and emerging fuel crops such as switchgrass.

In January 2010, the company raised $757,464 out of a total equity offering of $765,000. In February 2010, Zymetis was awarded a grant from the University of Maryland’s Maryland Industrial Partnerships Program of $108,085 to further development of low-cost solvent systems to reduce the crystallinity of native cellulose, reducing the need for enzymes in biomass digestion for the production of ethanol and other biofuels.

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in patents, technology licensing, industrial biotechnology regulatory affairs, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation are available at www.slideshare.net/djglass99.

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