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Impact of Biotechnology on Chemical Industry

Impact of Biotechnology on Chemical Industry

Appeared in Chemical Weekly in 2006 

Introduction

The chemical industry has come under increasing pressure to make chemical production more eco-friendly due to its reliance on fossil resources, its environmentally damaging production processes and its toxic byproducts and waste. The recently adopted REACH (registration, evaluation and authorization of chemicals) regulation (law entered into force from 1st June 2007) demands all produced or imported chemicals be registered and tested while the Restriction on Hazardous Substances (RoHS) Directive prohibits or restricts the use of the most dangerous chemicals in electronic equipment. The sustainability of the chemical industry requires an integrated strategy that takes into account safety, health and environmental benefits with technological and economic objectives.

From Hydrocarbon Economy to Carbohydrate Economy

For the United States chemical industry, around 98% of all chemicals produced in excess of 4 million kg/yr are produced from petroleum and natural gas. The rapid rise in the costs of these fossil fuels has placed the United States chemical industry at a competitive disadvantage. Petroleum refineries are source for spectrum of products and services. 67% of refined petroleum goes for transportation needs, while 7% finds its way into chemical manufacturing. $255 billion in value is added to the economy by converting petroleum into plastic, rubber, and chemicals. Benzene which is the primary building block derived from petroleum serves as starting material for most of the chemicals.

If biotechnological route is used and biorefinery concept implemented the chemicals will be produced with glucose as the starting material obtained of course from cheaper natural raw materials. The economics of chemical manufacture from petroleum refineries is threatened with increasing shortage and rising prices of petroleum. Biorefineries can repair the economic situation. Incremental use of agricultural produces for chemical manufacture will also provide domestic alternate markets for agricultural produces when exports are declining. In a sense what is true for U.S. is also true for rest of the world to certain extent.

Companies that have been building up their biotech capabilities will have advantages while those who are slow on adopting biotech capabilities may miss out on some long-term opportunities. Value will be derived through the creation of new business opportunities, such as developing synthetic compounds that were previously inaccessible through classical chemistry, adding value to processes by shortening time to market, reducing process costs by cutting out steps in synthesis or vastly increasing yields, and through the use of cheaper raw materials, for example, making the transition from oil to corn and, ultimately, corn stover.

Although biomass as a feedstock is potentially cost-competitive to petroleum-based feedstocks, there are still some challenges ahead. A major shift to biomaterials and biofuels has to be assessed in terms of availability of farmland and water and competing claims upon these resources. Even use of genetic engineering, requires systemic ecological evaluation processes.

A switch to biomass would require building an entirely new value chain, with massive capital investments and collaboration among different players. Furthermore, technological advances in the enzymatic conversion of cellulose are still needed for commercial application.

Challenges exist, making processes of the industrial scale. With a few exceptions, most applications have been for low-volume, high-value products, such as drugs and fine chemicals. Exceptions include ethanol and fructose produced by fermentation at more than a million tons per year and costing less than $1.00 per kg. But for bulk chemical production, whole cells or enzymes need further engineering to raise productivity and stability and to lower the cost of production.

Green chemistry and White biotechnology:

The concept of ‘green chemistry’ was introduced in the early 1990s by the US Environmental Protection Agency, in order ‘to promote chemical technologies that reduce or eliminate the use or generation of hazardous substances in the design, manufacture and use of chemical products’. Its guiding rule is prevention rather than cure. Green chemistry is currently associated with the 12 principles formulated by Paul Anastas and John Warner, which advocate a decrease in the environmental impact of a chemical product by considering aspects of its entire life cycle – from raw material to product use and fate.

Green Chemistry field includes: modifying engineering practices, the development of new catalytic processes, the design of new Green chemicals and materials, use of sustainable resources, modification of existing chemical processes, use of biotechnology alternatives and bioremediation. Examples of these are using renewable feedstock, selective catalysts and alternative, increased reaction specificities, non-toxic solvents; high atom efficiency; minimizing risks, minimizing waste generation and reducing energy consumption; and design of safer and biodegradable chemicals.

Some drivers for development of Green Chemistry include: high toxicity or other environmental impacts (like ozone depleting substance) of some chemicals, trend towards product takeback and recycling of chemicals, new discoveries in toxicological research relating to disruption of endocrine systems, synergistic interactions among toxins, and heightened vulnerability during childhood and pregnancy, and technical breakthrough in substituting industrial enzymes, biomaterials.

Though green chemistry is primarily aimed at environment protection, it also makes economical sense, due to considerable savings on wastewater treatment, energy use, and use of organic carbon resources instead of fossil stocks.

Significant share of today’s oil production is needed for synthetic chemistry. In view of the exhausting fossil fuels sources alternative energy sources and new sources for building blocks for chemical synthesis have to be developed. Biocatalytic processes will be required for this.

Biocatalytic processes can be subdivided into –

Biotransformation reactions in which a reaction precursor, renewable or petrol-based, is converted to the desired product, and

Fermentations that use the carbon source for de novo product synthesis, usually from a renewable carbon source

White biotechnology, also called industrial biotechnology, is at a relatively early stage in the chemical industry. But it has the potential as a key driver to the industry’s future. Industrial applications of biotechnology today include bio-feedstock that replace fossil fuel, bioprocesses such as fermentation for vitamin production, biocatalysis in active pharmaceutical ingredient production and other applications in textiles and leather, animal feed, pulp and paper, energy, metals, minerals and waste processing. Ethanol accounts for roughly $15 billion; various fine chemicals/pharmaceuticals, $7.5 billion; amino acids (e.g. lysine and glutamic acid), $4 billion; citric acid, $2.5 billion; enzymes, $2 billion; and vitamin C, $1 billion. Smaller products include vitamins B2 and B12, xanthan gum, lactic acid and a number of flavors and fragrances.

Industrial biotechnology can provide the process tools for bio-based production of chemicals. The chemical industry in Europe, which contributes ~28% of the demand for chemicals in the world, has identified industrial biotechnology as a key emerging technology area.

There are sufficient amounts of biomass to generate about 40 percent of bulk chemicals, according to McKinsey & Company estimates. The potential environmental benefits of shifting to bio-feedstock are substantial. If we look at greenhouse gas emissions alone, savings due to white biotech could potentially account for up to 20 percent of the global Kyoto target. This places white biotech among the key technologies for sustainable industrial production.

The pace of applications of biotechnology in chemical industry quickens as biocatalysts are becoming more stable, yields are rising and public reaction is demanding that industry develop safe, environment friendly and sustainable products.

The new industrial revolution is quietly underway, as biotechnology brings innovation in the chemical industry. In a challenging environment where companies struggle to achieve efficiency and cost savings while maintaining an environmentally sound operation, biotechnology may provide new strategy for value creation. Value creating potential of biotechnology in chemical market is $160 billion.

Compared to the about 135 billion tons of total reserve of mineral oil worldwide, 170 billion tons of biomass are the annual reserve for future industrial development. For production of all types of chemicals this means that future processes will more and more need the application of new technologies to be developed in-between the disciplines of White Biotechnology, including (bio)chemical process engineering and process development, Green Chemistry, and Cleaner Production & Zero Emission Technologies.

Industrial biotechnology, in Europe, is already used for the manufacture of several commodity and specialty chemicals. Biocatalysis has more commonly been directed towards the production of high-value products for the fine chemicals and pharmaceutical industries. According to a recent survey, 22 out of 38 large-scale asymmetric syntheses already incorporate biocatalysis. The production of chemicals using industrial biotechnology is often able to meet several of the green chemistry principles, particularly reduced energy consumption and waste generation, selective catalysis, and biodegradable products. White biotechnology with increased energy efficiency finally can reduce production costs.

Furthermore, it can replace multi-step chemical synthesis with a single step involving low energy and less material input, and even enables the synthesis of products that are not possible chemically. Organic synthesis using biocatalysis has been possible by performing reactions in predominantly water-free media, or in water–organic biphasic systems. Owing to concerns regarding volatile organic carbon emissions, there is a development towards replacing the organic solvents used as reaction media with alternatives such as supercritical carbon dioxide and ionic liquids or, preferably, solvent-free media.

Life Cycle Assessment

First developed around 30 years ago, the Life Cycle Assessment (LCA) concept is now a valuable tool in the chemical industry. It is used to compare and benchmark the performance of a product against several competing, alternative processes and products, and to find hot spots in the life cycle that might require performance improvements. In the eco-efficiency analysis used by BASF, equal weight is given to the LCA as to the costs of the product, which include the costs during the production, the usage, and for the disposal or recycling of the spent product.

Market Potential:

The firm McKinsey & Company pinpoints the value of the entire chemical market at roughly $1,000 billion in 2000, and, assuming a 2-5% annual growth rate in the sector, expects the market to be worth about $ 1,400 billion in 2010. In total, about 20 percent–approximately $280 billion–of the entire chemical market could be affected by biotech. By this time more than 10% of polymers would involve biotechnology in either monomer production or polymerization. By segment, biotechnology has the potential to represent 60 percent of fine chemicals sales in 2010 (or $90 million), 10 percent to 15 percent of polymer sales ($370 million) and 10 percent to 15 percent of bulk chemical sales ($380 million) and specialty chemicals at between zero and 50 percent. Thus, a McKinsey study has indicated that the market share of industrial biotechnology will strongly increase in all areas by 2010, and strongly increase even further afterwards. The penetration speed will depend mainly on a number of factors such as the prices for crude oil and agricultural raw materials, technological developments and the political will to support and structure this new technology.

Depending on whether the uptake is fast or slow, the chemical industry could generate additional added value of up to [euro] 11 billion to [euro] 22 billion per annum by 2010, according to McKinsey & Company. Of this total, between [euro] 5 billion to [euro] 10 billion would be from additional revenues generated by new products and value-added processes, and between [euro] 6 billion and [euro] 12 billion would be generated from cost reductions for raw materials and processing, combined with smaller-scale investment in fermentation plants.

Except for United States, Germany is a strong center carrying out research and development and production of chemicals by biotransformations. Experts estimate the value of biologically manufactured products in the global chemical industry at 50 billion euros. BASF, Degussa, Henkel, Wacker are the companies in Germany which use White biotechnology for chemical production.

It is clear that leading chemical companies such as BASF, DuPont, Dow, Hoechst, Rhône Poulenc, Novartis and Monsanto are reorienting themselves to take advantage of the increasingly important role of biotechnology. The important message to others is that companies that can understand the strategic production pathways and exploit these avenues through biotechnology R&D will be the companies that grow and prosper throughout the coming decades.

19% of the energy is used for the production of polymers and plastics. Therefore their sustainable production is the biggest challenge.

Advantages of Biotechnology in Chemical Production:  

(a)   Cleaner production since fewer wastes will be generated. It can eliminate environmental concerns over the disposal of chemical processing wastes.

(b)   Increased product yield.

(c)   Reaction steps will be reduced, usually compressed into one synthesis and one, isolation step in a biotech process. The outcome is a 75% saving in capital equipment costs and a 50% cut in operation cost.

(d)   Cost-effective routes and new chemical entities possible.

(e)   Low-cost raw materials. Use of cellulose and biomass will reduce the cost. We will see the first bio-refineries in a few years.

(f)    Innovative, can provide new strategy for value creation.

(g)   No by-products generated which, are having undesirable colour or odour.

(h)   High regio- stereo-seletivity of biocatalytic reactions.

(i)     Using plant biomass, as feedstock means feedstock grows, and it consumes CO2 — one of the greenhouse gases.

(j)     Use of plant biomass if successfully done will provide primary feedstock as well as energy. Today at least 5 billion kilograms of commodity chemicals are produced annually in the United States using plant biomass as the primary feedstock.

(k)   Unlike many chemical reactions that require very high temperatures and pressures, reactions using biological molecules work best ambient temperatures under 100°F, atmospheric pressure and water-based solutions. Therefore, manufacturing processes that use biological molecules can lower the amount of energy needed to drive reactions.

 

Examples of Applications

(1) Bioprocess for the Production of S-chloropropionic Acid

Industrial biotechnology is increasingly applied in the synthesis of conventional pesticides. The synthesis of S-chloropropionic acid, an intermediate product in the synthesis of chiral phenoxypropionate herbicides (2,000 t / year) serves as an example. These herbicides recently became important and industrial biotechnology is particularly suitable for their synthesis. One starts with racemic chloropropionic acid that contains a mixture of R- and S- forms, whereby only the S-form results in an active herbicide. With the help of an R-specific dehalogenase enzyme from Pseudomonas bacteria, only the R-form is converted that can be separated and recycled. Another bioprocess starts from glucose that is converted to D-lactic acid using fermentation. The D-lactic acid is then chlorinated chemically to S-chloropropionic acid.

(2) Bio-colorants

Bio-colorants are increasingly produced by industrial biotechnology, in particular when these are employed for food, pharmaceutical or cosmetic applications. These substances can often be made by both chemical synthesis and industrial biotechnology, with comparable production costs. Bio-colorants produced by biotechnology have an important marketing advantage because consumers disapprove of synthetic substances.

β-Carotene (or provitamin A) is produced by organic synthesis, extraction from roots as well as by fermentation with the fungus Blakeslea trispora. Optically active hydroxycarotenoids such as zeaxanthin and astaxanthin are important as animal and human food, and are mainly used in the fish and animal feed industry. The pink pigment astaxanthin is added to the feed of sea-farm raised salmon to obtain the beautiful pink salmon meat. In nature, salmons get the pigment from their natural diet. Until recently, astaxanthin was mostly synthetically produced, via a complex synthesis route using a combination of chemical and enantio-selective bioconversion steps. Recently, there has been growing interest in the direct fermentative synthesis of this pigment with the help of the red yeast Xanthophyllomyces rhodozyma, because the synthetic variant has been criticised in view of the fact that it differs slightly from natural astaxanthin. A blue food pigment – phycocyanin – is produced in Japan with the cyanobacterium Spirulina sp. Also the orange red food/drink pigment – monascin – is produced with the fungus Monascus purpureus via a fermentation process.

(3) L-Carnitin is a vitamin-like natural component in animal tissues that stimulates the lipid metabolism. Initially, L-carnitin was produced via chemical synthesis, but now it is entirely made through a fermentation process, starting from renewable raw materials. The L-carnitin obtained is very pure and is increasingly used. People and animals use L-carnitin as a food supplement to stimulate their fat metabolism (more energy, less fat synthesis and more growth).

carnitine Production 

Fig. 1 – L-Carnitine Production by Biotechnological Route (Lonza)

Table 1 – Comparison of L-Carnitine production by Chemical and Biotechnological Route

   

Chemical Production

Biotechnological Production

1 Waste for incineration in tons per ton of L-Carnitine

4.5

0.5

2 TOC in the waste water in kg per ton of L-Carnitine

750

360

3 Waste water in m3 per ton of L-Carnitine

220

40

4 Salts in tons per ton of L-Carnitine

3.3

0.8

 

(4) Flavor / Fragrance Chemicals

Most natural flavor/fragrance chemicals are heavily dependent on plant and animal origins. However, the quality and the supply of traditional natural flavor/fragrance chemicals are somewhat limited. Viable alternative and innovative ways to synthesize flavor and fragrance chemicals include biotechnological routes, i.e., microbial fermentation and plant tissue culture.

Microorganisms are being used to produce aroma chemicals. The ability to produce aroma chemicals by microbial fermentation may supplement and enhance the quality of plant-based flavor/fragrance chemicals. Microbial biotransformation and biosynthesis of flavor and fragrance chemicals offer the potential benefits of producing optically active isomers which often have marked differences in flavor and fragrance quality and sensory intensity.

Recent developments of commercialized processes to produce and/or to biotransform natural precursors into valuable flavor/fragrance chemicals via microbial metabolic pathways include the following;

1) Production of Tuberose lactone (a new GRAS chemical) via hydroxylation of unsaturated fatty acids and limited β-oxidation of the hydroxylated fatty acids;

2) Production of chirally active (R)-styrallyl acetate by regioselective reduction of acetophenone to styrallyl alcohol and subsequent esterification; and

3) de novo synthesis of chirally pure (+)-jasmonic acid and subsequent esterification to methyl jasmonate.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the characteristic aroma component of the vanilla pod and is used in a broad range of flavors for foods, confectionery, and beverages; as a fragrance ingredient in perfumes and cosmetics; and for pharmaceuticals. The main production of vanillin is done via chemical synthesis from guaiacol and lignin. The increasing customer-led demand for natural flavors has induced growing interest in producing vanillin from natural raw materials by biotransformation, which can then be regarded as a natural aroma chemical.

A method for biotransformation of eugenol to vanillin was developed in 1991, (patent application EP0405197), based on a new Pseudomonas sp. strain, HR199, which degrades eugenol via coniferyl alcohol (4-hydroxy-3-methoxycinnamyl alcohol), coniferyl aldehyde (4-hydroxy-3-methoxycinnamyl aldehyde), ferulic acid, vanillin, vanillic acid (4-hydroxy-3-methoxybenzoate), and protocatechuic acid (3,4-dihydroxybenzoate).

Other approaches were based on the biotransformation of ferulic acid to vanillin by the white-rot fungus Pycnoporus cinnabarinus or the gram-positive bacterium Amycolatopsis sp. strain HR167. Amycolatopsis sp. strain HR167 is used for the biotechnological production of vanillin (patent application EP0761817). To use eugenol as a cheap resource to provide ferulic acid for this biotransformation, the industrially approved bacterium Ralstonia eutropha H16 was genetically modified to convert eugenol to ferulic acid, quantitatively.

Haarmann & Reimer (H&R) who are leaders in the flavour field have begun production of natural version of vanillin. H&R has developed a fermentation process in which ‘eugenol’ the natural aroma chemical obtained from number of plant extracts, including clove oil is used. Eugenol is converted to ferulic acid by microbial oxidation and then ferulic acid is converted to vanillin by an oxidative degradation.

The German company BASF recently started with the microbial synthesis of 4-decalactone, a peach aroma. It is based on a fermentation process with the yeast Yarrowia lipolytica, whereby 12-0H-19 octadecenic acid is released from ricinus oil and subsequently metabolized to the desired 4-decalactone.

Unilever in England makes the butter aroma, R-d-dodecanolide, starting from 5-ketododecane acid with the help of baker’s yeast as the biocatalyst. Butyric acid and its ethylesters have been obtained by fermentation since a long time and are used in cheese aroma, fruit aroma.

(5) Antibiotics

Safer, cleaner and more cost-effective processes also can be realized through biocatalysis and biotransformations. In the early 1980s, DSM began exploring biocatalysis for synthesis of antibiotics such as cephalosporins. A key step is coupling a nucleus–7-amino-3-deacetoxycephalosporanic acid (7-ADCA)–to a side chain. The chemical coupling required very low temperatures (–50 to –60 °C) and chemical protection and activation of various groups to minimize side reactions. Biocatalysis reduced waste by 67%, use of organic reagents by 80%, use of organic solvents by 82%, use of steam by 60%, and use of liquid nitrogen by 100%.

Biochemic (Austria), since 1995 uses bioprocess involving two enzymes which work at room temperature. Solvents use is completely eliminated. Load of toxic wastes required to process by incineration reduced from 31 kg per 1 kg of 7ACA to 0.3 kg per 1 kg of 7ACA.

Since 2000, more than 400 patents on use of microorganisms, parts of microorganisms, or enzymes to produce higher purity specialty chemicals have been issued. Biocatalysis and biotransformation for fine chemicals production is expected to grow as companies like Codexis and Diversa make available enzymes and microorganisms that enable breakthroughs in chemical manufacturing.

Fermentation strains improved by gene shuffling are now in commercial processes. One such process is Pfizer’s production of the antibiotic doramectin; another is DSM’s production of the antibiotic precursor 7-ADCA. In both cases, an enzyme in the metabolic pathway had been limiting production. Gene shuffling provided better genes that were then incorporated into the fermentation organism.

The power of genome shuffling was demonstrated by researchers who, in one year, obtained a fermentation strain for making the antibiotic tylosin that is as productive as the current strain being used by Eli Lilly, which had taken two decades to generate. Eli Lilly and Codexis now are collaborating to improve Eli Lilly’s fermentations.

The complex traditional process of Cephalexin production involved 10 steps. Biotechnological process which is combination of fermentation and enzymatic process has been developed. The process generates >70 percent less polluted wastewater than the synthetic version. Biotechnological process developed by DSM uses 65% less chemicals, 65% less energy and bring about overall cost reduction of 50%. After developing the process in the late-1990s, DSM invested in a new facility at Delft, the Netherlands, to house the one-step process. The site came on stream in 2001 and product made using the green route was launched that year.

(6) Acrylamide Production

Mitsubishi Rayon produces acrylamide from acrylonitrile with the help of an immobilised bacterial enzyme nitrile hydratase. Acrylamide is then polymerised to the conventional plastic polyacrylamide. This process was one of the first large-scale applications of enzymes in the bulk chemical industry and replaces the conventional production process that uses sulphuric acid and inorganic catalysts.

The enzymatic process has clear advantages with respect to the chemical alternative. The efficiency of enzymatic conversion leads to less waste, higher yields and significantly lower energy consumption with consequently reduced CO2 production, as indicated in table 2.

Degussa Company has several thousand tons per year of biocatalytic acrylamide production in Perm, Russia, for water treatment applications. Its care specialties unit uses biocatalysis to produce fatty acid-derived esters and ceramides for personal care applications, and its oligomers/silicones unit produces silicone acrylates as paint additives.

Degussa has also developed enzymatic process for making a polyglycerine ester that it will use as an active ingredient in deodorant applications. This ester is chemically attainable only by a multistep process using protecting groups, an option that’s too difficult and expensive to be worthwhile.

About 10% of Degussa’s nearly $450 million annual R&D budget is spent on biotech research. The company’s Project House Biotechnology biocatalyst initiative has about $18 million to spend over three years. And the company is acquiring the biocatalysis and catalyst research activities of Aventis Research & Technologies.

 

 

Table 2 – Comparison of chemical and bioprocess for Acrylamide Production

 

    Chemical process Bioprocess
   

 

 

1 Reaction temperature

70°C

0 – 15°C

2 Single-pass reaction yield

70 – 80 %

100 %

3 Acrylamide concentration

30 %

48 – 50 %

4 Product concentration

necessary

not required

5 Energy demand (steam and electricity-demand in MJ/kg acrylamide)

1.9

0.4

6 CO2 production (kg CO2/kg acrylamide)

1.5

0.3

 

Surprisingly, the main reason for introducing the biotechnological route was the much better product quality. No undesired polymerization occurs when the biotechnological route is employed, resulting in a purer acrylamide that can be better polymerized for high-quality applications. Today, about 100,000 tons of acrylamide are produced yearly via this method in Japan and other countries.

 Enzymatic conversion of nitriles and amides

Fig. 2 – Enzymatic conversion of nitriles and amides

Acrylic monomers can be converted to Acrylamide, Acrylic acid, Metacrylates, N-substituted acrylamide, Polyacrylamides, Flocculants

(7) Vitamins

Vitamin B2 ((riboflavin, 4,000 t/y)), was produced using a multiple-step synthesis process until 1990. Then researchers and developers at BASF established a single-step fermentation process based on soy oil that offered key advantages over the old petrochemical process. Waste was reduced by 95 percent, CO2 emissions by 30 percent, VOC emissions into air decreased by 50%, Emissions to water reduced by 66%. Solid waste consists of 100% biomass. Resource consumption reduced by 60 percent. As a result, the cost of producing vitamin B2 decreased by 40 percent. Reduction of non-renewable resources is by 80%. Energy use is equal.

Just 5 percent of vitamin B2 was produced using a biotech route in 1990. In 2002, 75 percent of production was biotechnology-based. BASF produces vitamin B-2 with the aid of the fungus Ashbya gossypii. In collaboration with the University of Salamanca, researchers at the company have succeeded in increasing the productivity of the microorganism by 20%. BASF is among the leaders in biocatalysis as a major producer of vitamin B2 and the amino acid lysine. Traditional chemical-biotechnological synthesis route of Vitamin B2 production was 8-step process while biotechnological process of BASF is single step fermentation. The conventional process consisted of the synthesis of the building block D-ribose by fermentation with Bacillus bacteria, followed by a sequence of chemical reactions to obtain riboflavin.

This combined synthesis route has been recently replaced by the complete biotechnological synthesis of riboflavin in one single fermentation step with the help of bacteria, yeast or fungi (respectively by Roche, ADM and BASF). The productivity of these fermentation processes is so high that the product already crystallizes out during the fermentation itself. The production cost of the new biotechnological process is 40 % lower than the conventional process.

Another example is the synthesis of vitamin C (ascorbic acid) that is made conventionally via the Reichstein-Grüssner synthesis, a synthesis process starting with glucose and consisting of one fermentation step and 5 chemical steps. Cerestar/BASF recently developed a new process in which a fermentation process takes over the greatest part of the chemical steps. The new synthesis route consists of one fermentation step and two simple chemical steps (via 2- keto-L-gluconic acid). Moreover, work is on for a fully biotechnological route that will convert glucose to vitamin C in a single fermentation step.

In the fine chemical sector, Lonza has developed a biotechnological route, starting with 3-cyanopyridine to nicotinamide (niacin or vitamin B3), nicotinic acid and 6-hydroxynicotinic acid. Now, these intermediate products are made via industrial biotechnology.

(8) Glycerol to Propanediol

Compared to traditional polyester (2GT), the polymer polytrimethylene terephthalate (3GT) has improved properties. Yet commercialization has been slow because of the high cost of making trimethylene glycol (3G), one of 3GT’s monomers. Some naturally occurring yeast converts sugar to glycerol, while a few bacteria can change glycerol to 3G. Through recombinant DNA technology, an alliance of scientists from DuPont and Genencor International has created a single microorganism with all the enzymes required to turn sugar into 3G. This breakthrough is opening the door to low-cost, environmentally sound, large-scale production of 3G. The eventual cost of 3G by this process is expected to approach that of ethylene glycol (2G). The 3G-fermentation process requires no heavy metals, petroleum or toxic chemicals. All liquid effluent is easily and harmlessly biodegradable. What’s more, 3GT can readily undergo methanolysis, a process that reduces polyesters to their original monomers. Post-consumer polyesters can thus be repolymerized and recycled indefinitely.

Sorona 3GT is a new polyester synthetic fiber produced by DuPont. 1,3-propanediol is one of the monomers for the production of this polymer. It is made by fermentation from renewable raw materials, i.e. glucose, derived from corn. In a collaborative project between Genencor and DuPont, an E. coli production strain has been equipped with 4 foreign genes from other microorganisms. As a result, the recombinant production organism converts glucose to 1,3- propanediol, which it naturally does not produce. This is example of so-called “metabolic engineering”. The monomer is usually produced from the petrochemical raw materials ethylene oxide or acrolein via conventional chemical synthesis, but can now also be produced at comparable cost using biotechnology from renewable resources. The new polymer Sorona is mainly used as a synthetic fibre in the textile industry. It is not biodegradable and is thus a conventional plastic in that sense.

Dupont (Wilmington, DE, USA), the company that invented nylon, has for many years been developing a polymer based on 1,3-propanediol (PDO), with new levels of performance, resilience and softness. Adding an environmentally responsible dimension to the production, Dupont’s polymerization plant in Decatur, Illinois (USA) has now successfully manufactured PDO from corn sugar, a renewable resource. Sorona®, is more environmentally friendly and has improved characteristics.

The overall goal of the present project is the synthesis of biodegradable polyesters from two main monomers, 1,3-propanediol and succinate (produced by fermentation from renewable sources) and from other dicarboxylic acids (like terephtalic acid) used as auxiliary monomers to modified the chemical and physical properties of the polyester. The main advantage of this approach compared to polyhydroxyalkanoates or polylactides (two biodegradable polyesters that can be produced from renewable sources) is the possibility that the physical properties of the polyester can be easily modified to meet the manufacturing specifications for the final article made from the plastic.

(9) Adipic acid

E.coli can be used to convert D-Glucose to adipic acid. When perfected, this method will eliminate the need for benzene feedstock for phenol production. Over 2 million tons of adipic acid is currently produced using benzene feedstock. Biocatalytic transformation of glucose to 3-dehydroshikimate (DHS) yields the basic building block, from which adipic acid, catechol, gallic acid are produced. Thus the petrochemical route to these key chemicals can be avoided.

(10)  Production Process for Rare Sugars

Biochemical methods, usually microbial or enzymatic, are suitable for the production of unnatural or rare monosaccharides.

 Table 3 – Bioprocesses for rare sugars

 

No. Rare or Unnatural Sugar From By Organism / Enzyme
         
1 D-Arabitol D-glucose fermentation Candida famata R28
2 D-xylulose D-arabitol fermentation Acetobacter aceti IFO 3281
3 D-lyxose D-xylulose Enzymatically L-ribose isomerase (L-RI)
4 L-ribulose Ribitol Bioconversion Acetobacter aceti IFO 3281
5 L-xylulose L-ribulose Epimerization D-tagatose 3-epimerase
6 L-lyxose L-Ribose isomerization  
7 L-ribose andL-arabinose ribitol   Acetobacter aceti IFO 3281 and isomerization using L-RI and L-arabinose isomerase (L-AI) respectively
8 Other pentoses     Cell or enzyme bioconversions

 

In spite of the demand for these rare sugars, their commercial availability, application or usefulness is negligible as they are expensive to prepare and unavailable in nature.

Table 4 – Importance of rare carbohydrates

 

No.

Sugar or Derivative

Use

     
1 Several modified nucleosides derived from L-sugars potent antiviral agents and also usable in antigens therapy
2 Derivatives of rare sugar Anti-hepatitis B virus and Anti-(HIV) agents
3 L-monosaccharides anti-neoplastic characteristics, useful in cancer therapy
4 L-arabinose Prevents postprandial hyperglycemia in diabetic patients
5 L-arabinose derivative – 2-deoxy-2-fluoro-5-methyl-b-Larabinofuranosyl uracil (L-FMAU) Potent anti-HBV (Hepatitis B Virus) and anti-EBV (Epstein-Barr virus) agent
6 L-arabinose Platelet-activating factors, like 2,3-O-isopropylidene-snglycerol, can be produced
7 L-arabinose L-arabitol could be obtained using Candida entomaea and Pichia guilliermondii.
8 Rare sugars as low-calorie sweeteners, are well tolerated by diabetics, absence of an objectionable aftertaste
9 D-tagatose low caloric carbohydrate sweeteners and bulking agent
10 D-sorbose building blocks for the synthesis of interesting natural and biological active products
11 L-fructose Inhibitors of various glycosidase, Mixture of L- and D-fructose kills ants and house flies
12 L-Glucose Antineoplastic activity, can be used in cancer therapy
13 D-allose for the treatment of chronic myeloid leukemia, can reduce thrombus formation during postoperative period in combination with other anticlotting drugs
14 L-ribose For the production of two rare aldohexoses, L-allose and L-altrose.  Use as a potent anti-Hepatitis B virus (HBV) and anti-Epstein Barr virus (EBV) agents

 

(11) Biosynthesis of Unnatural Amino Acids

Solid-phase peptide synthesis (SPPS) is a straightforward method for incorporation of unnatural amino acids. The size of the peptides produced is of less than 50 amino acid length. However, chemoselective ligations, allow the stitching together of peptide fragments or adding peptides to biosynthetically produced proteins. Recently these techniques, have allowed the production of large proteins incorporating unnatural amino acids.

Multi-site incorporation of unnatural amino acids is particularly useful in the construction of protein-based biomaterials.

An artificial extracellular matrix (aECM) protein, designed as a synthetic vascular graft material, which incorporates paraazidophenylalanine (pN3Phe) has been produced for the purpose of photochemical cross-linking. The construct incorporates an endothelial cell-binding domain from fibronectin and a structural motif derived from elastin, a natural structural protein within the vasculature. This aECM construct was designed to avoid two problems commonly seen with synthetic vascular grafts, failure due to modulus mismatch and thrombosis resulting from the failure to form an endothelial cell lining. Incorporation of pN3Phe allows for cross-linking, via photolytic formation of the reactive nitrene, needed for the construct to form a cohesive vessel with the proper modulus able to withstand the pulsatile stress of the vasculature. Photochemical crosslinking is advantageous because it avoids the use of chemical crosslinkers which can cause difficulties with graft production and acceptance.

Photodecomposition of the arylazide also enables this protein to be used as a negative type photoresist. Photopatterning spun aECM films armed with pN3Phe provides a novel method of forming bioactive protein patterns, which is useful in a variety of biotechnologies.

(12) Application to Hair Dye Industry

Enzyme assisted organic synthesis may be applied in hair dye industry. The business of hair dye industry worldwide is over $2 billion. The first step is oxidation of p-phenylenediamine to benzoquinonediimine (BQI) by H2O2. BQI reacts with couplers to form coloured species and / or trimerize. This reaction is key part of colour development in hair and fur dyeing. Japanese researchers have found that uricase (urate oxidase UOD) in presence of uric acid and oxygen can be used. Large-scale commercial production of high purity UOD is now possible with recombinant DNA technology. Thus, direct use of H2O2 can be avoided by formulating UOD and uric acid in a suspension so that uric acid is continuously supplied in low concentration.

(13) Amino acids

Researchers at Wacker-Chemie GmbH in Germany have developed a fermentation process that avoids multi-step chemical synthesis and produces semi-synthetic L-amino acids. This process is more economical than traditional methods since it uses glucose, a relatively low-cost raw material.

NSC Technologies (A Monsanto Company) has developed a biotech process for asymmetric biosynthesis of D-phenylalanine and D-tyrosine. The amino acids are synthesized using d-transaminase in a batch process. Instead of using isolated transaminase enzyme, microorganisms that produce the enzyme are used. Keto-acid substrate, amino donor and the useful microorganisms are put together in the reactor. The reaction yields the desired amino acid and by-product acetolactate, which quickly degrades to acetoin. A single batch yields 1 ton of the product. The process is substantially cost-effective than conventional resolution methods.

L-Phenylalanine is yet another amino acid, taking part in the synthesis of L-aspartame. Aspartame is an artificial sweetener that is 200 times sweeter than sugar. It is used in many foodstuffs, such as “light” beverages. Worldwide, around 15,000 tons of aspartame are produced each year at an approximate world market price of 35 €/kg. The initial synthetic process for aspartame was based on chemical synthesis.

But, in the mean time, it is now strongly based on industrial biotechnology. For example, the most important building blocks, L-phenylalanine and L-aspartic acid, are produced by fermentation and biocatalysis, respectively. The Holland Sweetener Company uses enzymatic technology to connect the two building blocks: the amino acids phenylalanine and aspartic acid are very specifically linked to one another by the bacterial enzyme thermolysine. After that, a few more chemical steps are needed to obtain the sweetener aspartame.

(14) Erythorbic acid or iso-ascorbic acid

Erythorbic acid or iso-ascorbic acid is an anti-oxidant used in food. It is made by a fermentation process from glucose (Pfizer, Roquette). It is a chemical analog of vitamin C, but has no vitamin action. By fermentation with bacteria, glucose is almost quantitatively converted to 2-keto gluconic acid that is then chemically cyclised to erythorbic acid.

(15) Organic acids and oleochemicals

Acetic acid, succinic acid and some oleochemicals might be among the next bulk chemicals to become “bio.” The key to low-cost production here is the shift towards waste biomass as a feedstock for fermentation, which would substitute for petroleum-based feedstocks. Ethylene, (by far largest volume chemical), could be produced economically by fermentation. For ethylene, biomass could become a cost-competitive feedstock when crude oil is roughly at $20 to $25 per barrel and other renewable resources, such as wheat, would only be a comparable alternative if crude oil is between $50 to $60 per barrel.

(16) Organo-fluorine compounds

The replacement of chemical synthesis by fermentation processes highlights the attractive potential of genetically modified organisms that can produce bioactive organo-fluorine compounds by fermentation.

(17) Glyoxylic acid

Dupont has developed a biocatalytic process for glyoxylic acid by enzymatic oxidation of glycolic acid using whole cells of recombinant methyltrophic yeast. Conventional process involved nitric acid oxidation of acetaldehyde. Lonza’s process for 5-methylpiperazine 2-carboxilic acid is also another example of biotrnasformation.

(18) Yeast Fermentation Produces L-Dopa

Several useful pharmaceuticals including L-DOPA, a valuable medicinal compound could potentially be made by use of yeast. L-DOPA is one hydroxyl group (one cactus enzyme) away from tyrosine; it is one further aromatic hydroxyl group and three methyl groups and a decarboxylation away from mescaline. Yeast transformed with only one of the cactus enzymes would produce L-DOPA. Biologically manufactured L-DOPA would be hundreds of times less costly than that which is currently made by pharmaceutical companies using expensive traditional methods which, unlike enzymatic synthesis, create a toxic waste stream. Enzymatic synthesis is totally clean and extremely efficient. Also enzymes almost always produce a single enantiomeric species, though that is not a consideration here.

 (19) Taxol

Under EPA’s Green Chemistry Program, the Presidential Green Chemistry Challenge awards are given in five categories each year. These categories are: alternative synthetic pathways; alternative solvents and reaction conditions; designing safer chemicals; academics; and small business.

Bristol-Myers Squibb Company won the Alternative Synthetic Pathways Award for the development of a green synthesis for Taxol® manufacture via plant cell fermentation (PCF) and extraction. The new process replaces an existing route requiring 11 chemical transformations and 7 isolation steps that used 13 solvents and 13 organic reagents and materials. The biotransformation relies of plant cell cultures rather than the twigs and leaves of the European yew, allowing for year-round harvest and elimination of solid biomass waste. During its first five years, the PCF process will eliminate approximately 32 metric tons of hazardous chemicals and other materials.

 (20) Indigo Dye

In Indigo manufacture, preparation of N-phenylglycine is still not a standardized process and also generates lot of wastes. In a major development, in manufacture of this dye, in Mitsui Toatsu process the indole is selectively oxidised by alkylhydroperoxide in presence of Mo catalyst. A subsequent development by Genencor reports insertion of gene of Pseudomonas putida in E.coli. The resulting strain was able to convert tryptophan into indigo dye via indole.

 (21) Epoxidized Oils and Fatty Acids

There are potential alternative routes for the production of epoxidized oils or fatty acids. The industrial process used today involves the Prileshajev epoxidation reaction, in which a peracid is used for oxygen transfer to the double bonds in the fatty acid chains. The peracid is usually formed in situ from hydrogen peroxide and acetic or formic acid, using a strong mineral acid or ion-exchange resin as catalyst. However, this process can also be performed in a milder and more selective way using a lipase as a catalyst. This chemo–enzymatic process has also been performed under solvent-free conditions, adding further environmental benefits and, in fact, addressing almost all of the above-mentioned principles of green chemistry.

Furthermore, fewer coloured epoxidation products are obtained using the enzymatic route, which is indicative of the mild process conditions. The major limitation of the process, however, is the low stability of the enzyme in the presence of the peroxide. For an economical process, the lipase stability has to be improved, for example, by protein engineering. Another possibility would be to use a mono-oxygenase that can catalyse epoxidation directly from molecular oxygen and hence circumvent the use of hydrogen peroxide.

(22) Statin Side Chains

Diversa has developed a new route to statin side chains via DERA (2-deoxyribose-5-phosphate aldolase) enzymes. These aldolases break down the sugar in DNA.

Enzymatic route for statin side chain 

Fig. 3 – Enzymatic route for statin side chain 

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