Synthetic biology in industrial biotechnology
This article outlines the role of synthetic biology in current industrial biotechnology production platforms, and gives insights into possible further developments. I will begin by summarizing why and how synthetic biology contributes to industrial biotechnology up to certain extent already today, paying particular attention to applications in bioeconomy. Then, I will illustrate the situation by presenting three company cases of industrial biotechnology platforms: Solazyme, Evolva, and Amyris.
1 Synthetic biology; between biology and engineering
Synthetic biology is a rapidly developing multidisciplinary field between life sciences and engineering. It is characterized by the mindset of designing and constructing living systems up from the DNA level, according to specific needs. The extent of this redesign can vary greatly: In a so-called top-down approach, only parts of existing biological systems are modified, whereas bottom-up approach aims at designing completely new systems and even organisms. While the latter approach is largely at an early developmental stage, applications involving top-down synthetic biology are currently reaching commercial scales. The range of potential applications includes pharmaceuticals, chemicals, fuels, food and agriculture, among others. In industrial biotechnology, synthetic biology contributes to creating microbial “cell factories”; that is, production platforms that are tailor-made for the manufacturing of a desired products, and for the given process conditions. With these developments, it is even envisioned that in the future, biological synthesis could offer an alternative for a chemical synthesis for a wide range of chemicals, which are currently produced from fossil oil. Further, synthetic biology could deliver characteristics and functions to chemicals, which are completely new or are difficult to obtain using traditional chemical synthesis. 1,2
As a relatively new approach, the definition and actual ‘novelty’ of synthetic biology remains somewhat disputed. To summarize, on the one hand the development of synthetic biology is indeed strongly linked with advances in its backbone sciences – especially molecular genetics, metabolic engineering, systems biology and bioinformatics. On the other hand, the novelty comes in combining principles and tools of engineering to the modification of living systems, which before has been rather limited in complexity and has been based on trial and error. 3–5 That is, by trying to integrate the core principles of engineering – design, construction and analysis – to biotechnology, biological functions could firstly be traced into the level of DNA “building blocks”. Secondly, it is becoming more understandable how these DNA building blocks can be combined into novel desired functions in a cell, such as a wider range of products, or improved yields and predictability for the bio-based production. 1 This requires tools for the design of cellular systems according to the set specifications, the use of modular genetic components (genetic libraries) for the generation of the novel biological systems, and the development of chassis organisms (minimal cells) as the basic unit upon which to construct these systems.3,5 Using computer programing terms: the DNA, RNA and proteins function as “hardware”, whereas “software” includes design tools for biological systems, such as gene transfer tools.
2 Synthetic biology and industrial biotechnology
Industrial biotechnology is seen as a vital enabler for bioeconomy, in allowing a broader use of biological processes in the manufacturing of bio-based products. Recently, a number of commercial and near-commercial cases have appeared within industrial biotechnology, where synthetic biology principles and tools are utilized in microbe-based production platforms. Hence, the early pioneer of synthetic biology applications, the production artemisinin acid for malaria drugs (see Amyris platform below), has been accompanied with flavor and fragrance chemicals and industrial chemicals, among others. This is taking place predominantly on the top-down basis, involving both synthetic biology and metabolic engineering. The metabolic pathways of industrial microbes such as yeast cells are modified to yield a product that the cell would not naturally produce, with the help of both synthetic biology and metabolic engineering. That is; instead of solely improving yields of naturally producing organisms (classical strain improvement), as well as instead of constructing completely synthetic cells (bottom-up synthetic biology). 2
There are several reasons for this recent expansion. For instance, bio-based manufacturing and product markets are becoming more established, and are poised for rapid growth in the future. Further, there is already some history of accomplishments and failures of applying synthetic biology, which makes it easier to evaluate the benefits and uncertainties. 1 A very significant enabler has been the increased capability and decreased costs of generating, sharing and interpreting massive amounts of genetic data 5,6. Consequently, the US National Research Council’s (NRC) publication Industrialization of Biology 6 estimates that markets of synthetic biology based industrial chemicals, for instance, will expand 15 – 25 % annually in the foreseeable future. In the US context, a number of synthetic biology startups have emerged, accompanied with rapid uptake and interest by established firms. 6
While the main focus in synthetic biology applications so far has been in pharma chemicals and other high value products, bio-based manufacturing is projected to expand in scale and scope – in both high-value and high-volume products 1,5,7. Indications of this are observable in companies’ product portfolios. For short-term revenue, they concentrate on high-value and relative low-volume products. However, the platforms may also allow the production of more bulky products such as hydrocarbon fuels, if or when this becomes commercially feasible. 1. In fact, a few companies already have had joint venture projects with global fuel corporations and polymer producers, for instance. Yet, there are still challenges particularly related to high volume and low value bio-compounds, which generally have less competitive advantage to existing commercial alternatives. The NRC’s report 6 lists the following challenges as indispensable to address: securing the availability of feasible and sustainable feedstocks, improving yields in biomass-based fermentation processes, improving basic understanding of one-carbon compound fermentation, developing robust microbial strains that allow flexibility with different feedstock and products, and developing measurements for metabolic pathway functions and cellular physiology.
3 Synthetic biology in industrial biotechnology platforms
This section outlines current situation, remaining challenges, and future prospects in industrial biotechnology platforms, highlighting some of the current and prospected roles that synthetic biology may have regarding central elements in the platforms: industrial microbes and production process, feedstocks, and products. Finally, three case examples are presented: The microalgal fermentation platform of Solazyme, and the yeast fermentation platforms of Evolva and Amyris.
3.1 Microbes, the “cell factories”
The cornerstone in industrial biotechnology is an organism that is capable of producing the given compound sufficiently for economic viability. Achieving this requires not only firm understanding of the growth parameters and metabolism of the given organism, but often also reconstruction and further optimisation of its metabolic pathways. For example, a yeast’s genome can be modified to produce more valuable compounds instead of ethanol, by inserting genes from a plant that naturally produces that compound. Often at the beginning the produced amounts are small, but can be improved by further modifications of the yeasts’ own genes to ensure a high yield of the desired product. 1,2 Synthetic biology may provide a range of new tools for pathway modification, such as for the engineering of enzymes catalysing metabolic reactions with respect to their rate and specificity. Along with these tools, also more developed, rapid and practical methods for measuring the metabolic functions and cellular physiology are needed. 1,6
Different types of microbes can function as platform organisms in industrial biotechnology, including yeasts fermenting sugar compounds into end-products, bacteria (e.g. via fermentation; or in case of cyanobacteria via photosynthesis), as well as microalgae via photosynthesis (phototrophic algae) or fermentation (heterotrophic algae). Much of the interest has been centred on the yeasts, like the “baker’s yeast” Saccharomyces cerevisiae, that are already very well-known and adapted for industrial conditions 2. In addition to being applied in existing platforms, synthetic biology may contribute in domesticating a range of new production organisms, by offering design tools for developing robust, predictable and flexible microbial strains for industrial conditions. An essential issue and challenge with this regard is to maintain these characteristics in large-scale fermentation environments and over long periods of time. 1,2,6
In longer term, the so-called cell-free biosystems may offer an alternative paradigm for bio-manufacturing in some application areas. For example, a platform based on modified enzymes, without the use of actual living cells, could have advantages in terms of e.g. higher yields, flexibility, and robustness. 1,6,8
In most cases, the feedstock used in today’s (near)commercial production are so called first-generation feedstocks. That is, the industrial microbes such as yeast get their energy from sugars of “edible” plants, such as sugar cane and corn. In certain cases this is already perceived as a more sustainable alternative – to petroleum-based production, or to the use of more endangered plant species. In many cases, the mid-term focus is however on the second generation feedstock; that is, cellulosic biomass such as straw, corn stover, forest residues, and other waste and residual biomasses that put less pressure on food production and cultivable land area. Many companies are already testing and piloting production based on cellulosic feedstock (see Solazyme below). Challenges still remain in increasing the availability, utility, and reliability, economics, and sustainability of cellulosic feedstocks in a large scale 1,6.
In longer term, potential is seen in one-carbon compounds, such as carbon dioxide and methane, as feedstock for industrial microbes – provided that this becomes feasible at same efficiency rates than with plant sugars 1,6,9. Examples of this development include Lanzatech, which develops bacterium-based fermentation of waste industrial gases into ethanol and further into fuels and chemicals. Development is also continuing with algae and cyanobacteria, converting carbon dioxide into targeted products through photosynthesis (see for example Joule).
3.3 Platform compounds and final products
All bio-based compounds in nature originate from a limited set of precursor compounds, as there are just 12 precursor metabolites in the central carbon metabolism of organisms 2. Insertion of a synthetic pathway into an industrial microbe will cause changes in the drain of precursor metabolites, to which the cell has not been adapted. Initially, this results in low yields of the desired precursor metabolite. However, once those yields are successfully increased with further pathway optimisation, the platform is more easily modifiable for further synthetic pathways starting from the desired precursor metabolite. For instance, when a yeast cell is developed to convert plant sugars into the precursor Acetyl-CoA instead of producing ethanol, it provides a platform for a range of further platform chemicals, such as different terpene compounds, which in turn can be processed into a spectrum of end products 2 (see Amyris platform).
What comes to specifying the end product, the case examples below illustrate well the current situation. In shorter term, the high-value products dominate that offer alternatives to endangered or unstable supply chains from the plant- or animal-based alternatives. Towards the mid and long term, more bulky products replacing use of petrochemical alternatives are added to the picture, with a number of projects like joint ventures already taking place. In some cases, also the quality of microbe-based end products is stated to be superior to the alternative.
4 Case examples
Figure 1 describes the main features of microbial biotechnology platform of the company Solazyme. While it has some case-specific features, it nevertheless provides an overview of general elements of biotechnology platforms, making it illustrative also for the other case companies.
Figure 1. Solazyme’s microalgal fermentation platform for algal oil –based products. The numbers in red are added to the original picture for clarification, with further description provided below for each number.
Raw materials (1)
Solazyme’s current feedstock is mostly Brazilian sugarcane, supplemented with U.S. corn. Their algae platform also allows a more flexible use of different feedstocks, including cellulosic feedstocks. The company’s “mid-term” goal is in transforming into cellulosic feedstocks once the conversion technologies and logistics becomes commercially feasible.
Evolva’s yeast platform uses sugars from starch material, typically corn or wheat.
Amyris’ feedstock sources are sugar cane, corn, and beet molasses.
Industrial microbe (2) and production process (3)
Solazyme’s microalgae are heterotrophic. That is; instead of converting CO2 into biomolecules with sunlight via photosynthesis, these algae are able to ferment variety of plant-based sugars into algal oils. This also means, that they can be grown in dark tanks. Many algae have naturally high oil concentration, which in case of Solazyme is further increased up to over 80% with genetic engineering. This involves introduction of plant genes with desired properties, making changes in microalgae’s existing genes such as shutting off production of an undesired oil component. In addition to the algal oil production, Solazyme produces whole algae products, with non-engineered algae
Evolva’s platform is based on modified “baker’s yeast” Saccharomyces cerevisiae, fermented in closed containers. The yeast’s genome is modified by inserting genes from plants (combinatorial genetics), for the production of the the end product that is traditionally obtained from those plants. Also the yeast’s own genes can be modified (pathway optimisation) for example for increasing the yield. For instance, 14 of the 18 genes needed for the production of resveratrol are already in the yeast, whereas the remaining 4 genes are added from plants that produce resveratrol. In addition, Evolva has proprietary technologies for optimising transporters (proteins for pumping the product out of the yeast cell), glycosylation (e.g. for attaching functional sugar parts to the products), and cytochrome 450 enzymes (e.g. for improving efficiency of production pathways).
Amyris’ platform is also based on fermentation by modified yeast Saccharomyces cerevisiae. Instead of producing ethanol, Amyris’ engineered yeasts convert plant sugars into higher-value hydrocarbon molecules. Amyris uses synthetic biology for the engineering of their yeast strains, and for screening desirable yeast cells from a large number of cells. Their technology development focuses on identifying new molecules for production, creating new yeast strains capable of producing the target molecule, increase the yield of microbial strains through genetic modification and fermentation process improvement, and translating these steps from laboratory to commercial production.
From platform compounds (4) into final products (5)
Solazyme’s oil-rich algal biomass is first dried, followed by mechanical pressing to release the oils. The algal oil provides an alternative raw material for instance for the use of plant and animal fats, and petrochemical-derived oils. The fatty acids from algal oil can be further processed into variable products. The leftover algal biomass is also usable for many purposes, such as for combusting into energy production.
Solazyme’s product portfolio includes industrial products (e.g. surfactants, detergents, and hydraulic fluids), personal care (e.g. skin care, and detergents for home care), and food products (e.g. omega oils). The company has also had joint ventures for developing drop-in alternatives for diesel fuels, as well as jet and marine fuels. Solazyme’s whole algae products, which are produced from non-modified algae, are used for example in vegan food products due to their high oil and protein content.
It was recently announced that Solazyme will change its name to TerraVia, and will focus on food, nutrition, and specialty ingredients in its product portfolio.
Many of Evolva’s products belong to the diverse compound group of terpenes. Terpene platform could enable a wide variety of bio-based products in the future, including medicine and high-quality biofuels. Evolva’s current product portfolio focuses on alternatives for high-value plant-based products, whose supply is challenged for different reasons. The end markets are in nutrition, personal care, and flavours & fragrance markets, and agriculture (under development; crop protection).
Examples of the challenged supply chains include the saffron plant (to be launched in 2016), whose production and price changes drastically from year to year, and the sandalwood tree, which has become endangered due to overharvesting. The strategy is different e.g. in case of stevia (to be launched during 2016), whose composition will differ from plant-based alternative, for improved taste. In case of vanillin, Evolva targets it as an alternative to petrochemically produced vanillin, rather than to plant-based vanilla.
Evolva has a number of ongoing research activities with external partners, and several yet unspecified product families in the development pipeline.
Amyris’ product portfolio is based on a number of platform chemicals. Most of their products derive from yeast strains, that are optimised to produce Acetyl-Co-A, and from thereon different terpene compounds. These include farnesene, which is Amyris’ first commercial platform molecule. Target markets for Amyris’ farnesene include cosmetics (emollients replacing use of olive oil or shark liver oil), biodegradable solvents, polymers for replacing petroleum-derived components in tyres (under development), pharmaceuticals, flavours & fragrances, lubricants, and fuels. The fuels are produced in partnership with Total: their renewable diesel currently fuels public transit buses in Brazil, whereas high-performance jet fuel is underway for initial commercial flights in Europe.
Apart from farnesene, Amyris is developing a platform for another terpene, isoprene, for the production of renewable polymers together with Michelin and Braskem. Apart from terpene compounds, Amyris is evaluating opportunities for a muconic acid platform, a precursor for plastics.
Amyris’ first collaboration was the creating of yeast-based production of artemisinic acid. It is a precursor of anti-malarial drug artemisinin, for which the supply of the plant-based alternative is insufficient. The work was funded by the Gates Foundation, and the yeast strain is made available for pharma company Sanofi on a royalty-free basis. Amyris has several ongoing research activities with different partners, such as building libraries of microorganisms for producing pharmaceutical compounds, and collaboration with DARPA for developing high-performance materials.
The information presented in this article is available at the companies’ websites (www.solazyme.com; www.evolva.com; www.amyris.com); particularly at investor materials that hyperlinked here for: Solazyme, Evolva, and Amyris. Figure 1 from: https://commons.wikimedia.org/wiki/File:Platform.png.
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