Improving biodiesel production through a dynamic sensor regulator system

Module Schematic

FadR/Acyl-CoA Gene Regulation Schematic:

Figure 1: Gene regulation by FadR, the product of the naturally occurring biosensor gene fadR, was tested prior to insertion in the E. coli genome. The promoter sequence that allowed for optimal efficiency was determined by testing a series of 20 strains of E. coli. Each strain contained a different promoter sequence, all with varying FadR binding strength, taken from a promoter library. These promoters were placed upstream of a gene coding for red fluorescent protein (rfp). Each strain was subsequently exposed to the same concentration of fatty acids. Optimal promoter strength and dynamic range, found in strains W and Y were then selected based on how fluorescent each strain was. The same test was conducted for FAEE production using the same promoters, with a similar result: strains W and Y produced the most FAEE.

Behavior of Dynamic Sensor-Regulator System

The production of fatty acid ethyl ester (FAEE) from glucose in the Dynamic Sensor-Regulator System (DSRS) in E. coli was separated into three distinct yet overlapping modules which used both native and synthetic biological pathways. Module A incorporates a naturally occurring fatty acid synthase gene as well as a synthetic cytoplasmic thioesterase gene (tesA gene) which together take Acetyl-CoA and process it in a series of steps to produce fatty acids. Module B includes incorporates two synthetic genes, pyruvate decarboxylase gene (pdc gene) and an alcohol dehydrogenase gene (adhB gene), that together convert pyruvate to ethanol. Lastly, Module C contains an acyl-CoA synthase (fadD gene) and a wax-ester synthase (aftA gene) that condense ethanol and acyl-CoA into FAEE.

Also incorporated into each of these three modules was a promoter sequence that is bound by the gene product of another native E. coli gene (fadR) that codes for a fatty-acid sensing protein. In the absence of free fatty acids within the cell, this protein, designated FadR, binds to the promoter sequences PmodB and PmodC. These promoter sequences are located just upstream of the pdc and adhB genes and aftA and fadD genes respectively. FadR acts as a master regulator in the DSRS as it prevents the other modules from producing their outputs without significant levels of fatty acid. This regulation, though not required, is extremely beneficial to the system as it prevents bottlenecks or production of unnecessary cellular products throughout the process. This is specifically important when considering Module B: ethanol can be dangerous for the system as a whole as it is toxic to E. coli at high concentrations. Once significant levels of fatty acid accumulate within the medium, they bind to FadR, releasing it from the promoter sites, allowing for ethanol and acyl-CoA to be produced and converted to FAEE. Another beneficial aspect of this system is that FadR is also released from PmodB and PmodC by acyl-CoA, making this a positive feedback reaction, allowing for FAEE to quickly be produced once fatty acids begin to accumulate within the medium as they too can be toxic to E. coli at high concentrations.

Replication of the DNA Sequence used in the DSRS System

The DNA sequence for Escherichia coli was first published in 1997 and is publicly available to anyone who wanted to use it for research or replicate this work. In addition, the article provides the following figures that show the pieces of DNA that they changed to produce the DSRS system. However, the entire system that they created is not in the article; if one wanted the entire sequence for this system they would have to contact the authors, which is possible through a corresponding author online. This creates a barrier to future research because access to the system is restricted, and it is possible that the authors could chose to copyright it or otherwise not disclose it to others doing research. This would severely slow the speed at which more DSRS systems can be developed and used to increase yields of biodiesel and other products.

Biological Context for the DSRS System

In this process, Escherichia coli is in order to create the A2A FAEE biosynthetic E. coli strain. E. Coli is being used instead of other raw plant material such as corn or switchgrass because using it “cuts down expensive steps that are otherwise needed to break down cell walls of woody plants…” It is said to be much more effective than using plant matter because it takes up less acreage than growing say, corn to be made into biodiesel, which also requires farming and watering, etc. According to Keasling, who co-authored the research,

“This work shows that we can reduce one of the most expensive parts of the biofuel production process, the addition of enzymes to depolymerize cellulose and hemicellulose into fermentable sugars,” says Jay Keasling, CEO of JBEI and leader of this research. “This will enable us to reduce fuel production costs by consolidating two steps – depolymerizing cellulose and hemicellulose into sugars, and fermenting the sugars into fuels – into a single step or one pot operation."

Cost Effectiveness: Building, Producing, and Potentially Selling the System

A recently developed biosynthetic strain of E. Coli has been developed that covnerts glucose into FAEE biodiesel at 9.4% of the theoretical maximum. This is called the A2A E. Coli strain. The problem with the Escheria Coli was that the A2A strain was unstable and produced “toxic intermediates”. The cell metabolism could not be regulated, creating bottleneck effects or the production of unnecessary proteins. That is where the DSRS came in, stabilizing the cell metabolism and increasing titer and yield. Although this process does increase the yield of biodiesel to 28% (compared to 9.4% without the DSRS), there is a cost associated with implementing the DSRS. Several steps were taken in this process, including construction of FA biosensors, cell culture fluorescence measurement, construction and characterization of hybrid fatty-acid regulated promoters, construction of DSRS into FAEE biosynthetic pathway, and FAEE production and analysis. The question is whether or not this process is inexpensive enough to be worth the cost in order to increase production threefold. “The need for new transportation fuels that are renewable and can be produced in a sustainable fashion has never been more urgent. Scientific studies have consistently shown that liquid fuels derived from plant biomass are one of the best alternatives if a cost-effective means of commercial production can be found.” Because the market for new fuels is high, the cost of production of biofuels with the DSRS system is likely to lower quickly due to the market forces of supply and demand. Information on the current costs of the production of the DSRS system was not available in the report not through the corresponding author.

Clean and green biodiesel fuel production

With the development of the DSRS, biodiesel can be synthetically produced using the bacteria E. coli to convert glucose into fuel that can be used in conventional methods, such as diesel engines in automobiles or diesel powered generators for industrial, commercial, and even home uses. This system is a greener fuel alternative than both traditional diesel, a fractional distillate of petroleum oil, and the methods used for traditional biodiesel production, which uses high-energy agricultural commodities, such as soybeans or corn in order to produce fuel. Furthermore, unlike petroleum-based diesel, biodiesel is biodegradable and creates relatively little air pollution.

Renewable Transportation Fuels, Capability for Sustainable Production, and Cost Effective Commercial Production

Since the DSRS is able to metabolically control the expression of genes affecting levels of production of biodiesel, “the result in one demonstration was a threefold increase in the microbial production of biodiesel from glucose.” This increasingly efficient (and thus cost effective) production greatly encourages wide-scale commercial production of this biodiesel fuel. Nevertheless, it is important to note that “high productivities, titers and yields are essential for microbial production of these chemical products to be economically viable, particularly in the cases of biofuels and low-value bulk chemicals.”

DSRS Applicability to Microbial Production of Chemical Products

Fatty acids, the basis of biodiesel production because of their high-energy molecular content in plant cells, are also widely utilized raw materials for chemical products such as lubricants, solvents, and surfactants because of the relative ease of microbial manipulation and production concerning fatty acids. Furthermore, with further research, it is predicted that the DSRS could eventually produce chemical products not necessarily based in fatty acids.

DSRS Applicability to Biosynthetic Metabolic Pathways

The final result of the DSRS showed that the stability of the FAEE biosynthetic pathway was increased and the metabolism of host cells was balanced. Furthermore, it was said that “Given the large number of natural sensors available, our DSRS strategy can be extended to many other biosynthetic pathways to balance metabolism, increase product titers and yields, and stabilize production hosts,” Zhang says. “It should one day be possible to dynamically regulate any metabolic pathway, regardless of whether a natural sensor is available or not, to make microbial production of commodity chemicals and fuels competitive on a commercial scale.”

The Future of the Dynamic Sensor-Regular System

Zhang and the team that developed the DSRS stated that " we are aiming to develop more DSRS systems, focusing not only on the production of diesel fuel, but expanding the focus to other biobased fuels and chemicals." 3 the DSRS’s usefulness using a ligand-responsive transcription factor can allow for replication of design systems for biosensors and regulatory systems in other types of molecules and for other types of metabolic pathways; this is facilitated because there exists a “large pool of natural ligand-responsive transcription factors.” In the same manner, heterologous ligand-responsive transcription factors in organisms besides that of E. coli (used primarily in DSRS testing) can be grafted in the host of production and potentially utilized to regulate the production of fatty acid based products.

Benefits

(5) As one of the few alternative fuels to petroleum in the United States to complete the goals of the EPA health effects testing, based (primarily on emissions testing), biodiesel stands without threat to human health. When used conventionally in diesel engines, biodiesel doesn’t produce the high amounts of unburned hydrocarbon and carbon monoxide gases that traditional diesel produces; in fact, according to the US Department of Energy, carbon dioxide emissions are reduced 78.5% in the overall production and use of biodiesel in place of petroleum diesel, especially important because of carbon dioxides association with global climate change as a greenhouse gas.

Moreover, unlike the clean exhaust of biodiesel, the exhaust of traditional diesel engines has been attributed to asthma, bronchitis, and even lung cancer if inhaled regularly, such as for children that ride a school bus regularly. But even short-term exposure can cause irritation and inflammation, especially exacerbating existing allergies or asthma symptoms. According to biodiesel.org, "biodiesel emissions have decreased levels ofa ll target polycyclic aromatic hydrocarbons (PAH) and nitrited PAH compounds...[which] have been identified as potential cancer causing compounds. Furthermore, if inadvertently consumed orally, biodiesel is relatively safe; according to biodiesel.org, “the acute oral LD50 (lethal dose) is greater than 17.4 g/Kg body weight. By comparison, table salt is nearly 10 times more toxic.”

Also, as a skin irritant, undiluted biodiesel was shown to produce less irritation than a four percent soap and water solution when tried in a 24-hour human patch test. Furthermore, in terms of storage and handling, biodiesel as a comparative advantage with petroleum because of its relatively high flash point of 200 °F (93 °C), compared to 125 °F (52 °C) flash point of petroleum; high flash points indicate the capacity of storage at higher temperatures before substance vapor can be made to combust (or ignite) in air, meaning temperature control of biodiesel isn’t as of great concern for mass production, shipping, and storage. Furthermore, if the efficiency of DSRS systems in the production of diesel fuel continues, it will readily address the issue of environmental sustainability in the United States. Compared to more traditional biodiesel production methods that use farmland to grow agricultural commodities (like soybeans or corn) for use as biofuel, the synthetic production of biodiesel with the aid of E. coli (or other easily modified bacteria) is environmentally conservative because it does not require hundreds of acres of land for crop growth or produce the same levels of excess waste, such as crude glycerol (known as glycerin) generated which is cost-prohibitive to refine for practical use in food, pharmacy, or soap. Moreover, biodiesel is significantly more biodegradable than traditional petroleum diesel, decomposing about four times faster at a composition rate of over 85% within 28 days, about the same rate as dextrose sugar.

Furthermore, with the use of the DSRS system, wide-scale production of biodiesel will help energy security in the future. Considering that about half of all petroleum used in the United States is imported, the risk of being at the mercy of market forces, including “trade deficits, supply disruption, and price changes” is already extremely high; and as the prices of imported fuel continue to rise, it will become even more important to substitute in biodiesel for traditional fuel sources. Thus, for countries with high per person fuel consumption, such as the United States, commercial use of biodiesel with the DSRS or other efficient dynamically regulating systems would help to free themselves from dependence on unpredictable political foreign relationships and the economic forces to which these relationships are tied.

Another benefit to the DSRS production of biodiesel is the provision of domestic jobs (in the United States) in this industry. Already, over 50, 000 jobs have been created in the US biodiesel industry; even more than that, it is estimated that for every 100 million gallons of biodiesel produced, at least 16 jobs will be created and almost 1.5 billion dollars will be added to the gross domestic product of the United States. Nevertheless, a disclaimer must be made that this prediction necessitates a stable, successful commercial biodiesel industry, although this goal is not far from reality because biodiesel can easily be employing in existing diesel engines.

Detriments

Unlike traditional biodiesel production, this new method of biodiesel production using the DSRS and modified bacteria does not work in conjunction with farmers to grow high-energy commodity crops that may be converted into biodiesel. Thus, if the demand for commodity crops such as corn or soybeans is greatly reduced as the DSRS is increasingly adapted for efficiency, it may effectively take away the steady flow of income on which many US farmers rely in order to make a living and eventually displace many farmers from their current jobs.

Furthermore, traditional biodiesel production from soybean oil generates more energy than needed to produce the biodiesel itself; specifically, for every joule of every input to produce a gallon of biodiesel, over three units of energy are gained. This advantage may be reduced with the synthetic production of biofuel using DSRS because of the absence of agricultural commodities in production.

Ethical Implications

Since the production of cost effective and environmentally friendly fuels has been a hot button issue for many years, there may be an issue regarding competing financial interests regarding the employment of this method of fuel production. Running parallel to the debate concerning intellectual property, this revolutionary synthetic biology discovery may be patented. Although the research into this system was conducted with the U.S Department of Energy’s Joint BioEnergy Institute and supported in part by the National Science Foundation through the Synthetic Biology Engineering Research Center (SynBERC), there still remains legitimate concern regarding intellectual property rights, especially because of the millions or even billions of dollars at stake if this technology is adapted on a larger scale. But if this information is patented but researchers or companies desire to use this technology, the application and licensing fees may deter smaller, well-meaning teams from accessing, utilizing, or improving on this technology. Thus, even though the patenting of this information may encourage investment and spur competitive advantage, further research may still be greatly slowed and the implementation of the DSRS system in other fields besides in the production of biofuels may not be explored if funding is limited.

Furthermore, the DSRS may detract from small scale bio-energy production (through the growth of crops) in poor, rural communities because this synthetic biology technique only requires modified bacteria, rather than agricultural commodities, for production of biofuel; thus, the DSRS could effectively take away jobs from those who most need them and instead place money in the pockets of wealthy and powerful scientists and businessmen. Nevertheless, because DSRS doesn’t employ food crops, it doesn’t lead the threat of endangering local food security or displace local populations from their land (from which they feed themselves). Along the same lines, DSRS produced biodiesel reduces the destruction and degradation of ecosystems, which might otherwise be destroyed to make room for farmland for the production of high energy crops.