Team:Tianjin/Project/Background

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= Call for Biofuels, especially Alkanes=
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= 1. Call for Biofuels, especially Alkanes =
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<p> Biofuels which have a closed CO<sub>2</sub> cycle[1] and don’t require expensive, complex chemical processing, are recognized as promising replacements for diesel fuels in the fields of energy and environment. Among them, fatty-acid-derived alkanes have many advantages over other biofuel compounds, such as high caloric value and low carbon emission[2], which means that they could be an ideal replacement for diesel fuels.</p>
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<p> Nowadays, scientist have put great attentions on biofuels, hoping to find a solution to energy crisis and climate change. Compared with fossil fuels, biofuels are renewable, and biofuel can be used indefinitely without any net carbon emissions [1]. Therefore, biofuels are promising candidates for mitigating dependence on diesel fuels.</p>
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<b>Figure 1.</b>&nbsp; Comparison of carbon cycles of fossil fuels and biofuels </div>
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<p>Among all kinds of biofuels, alkanes stands out because of their excellent properties. First, alkanes have higher energy density, for example, enthalpy of combustion of pentadecane is approximately -47.0 MJ/kg compared with -29.7 MJ/kg for ethanol [2]. Then, compared with ethanols with a freezing point of -114℃,which alkanes have a higher freezing point of about -3~19℃, so they are more likely to be compatible with existing engines as well as transport and storage infrastructure. Besides, they can serve as drop-in replacement for fossil fuels.</p>
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= Pathway of Alkane Biosynthesis=
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= 2. Biosynthesis of alkanes=
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<p> Pathways of long-chain alkane sythesis in microbes has been studied. Two enzymes, acyl-ACP reductase(AAR) and aldehyde decarbonylase(ADC), were heterologously expressed in E.coli to reduce fatty acyl-ACPs to corresponding aldehydes and then convert them to alkanes[3]. Fatty aldehydes can also be produced from fatty acids and fatty acyl-CoAs, catalyzed by acyl-CoA reductases(ACR) and carboxylic acid reductase(CAR) respectively, which have been identified in many species[4,5].</p>
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<th> Enzymes </th>
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<b>Figure 2.</b>&nbsp; Typical alkane biosynthesis pathways </div>
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<td> Acyl-ACP Reductase </td>
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<td> Synechococcus elongatus PCC7942</td>
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<td> Aldehyde Decarbonylase </td>
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<td> Nostoc punctiforme PCC73102</td>
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<p> The pathway of alkane sythesis in microbes has been studied, and heterologous expression in E.coli of the two genes encoding the two key enzymes named NPDC and AAR has been reported, which successfully turns Fatty Acyl-ACP into alkane molecules of certain chain lengths, making it possible for microbes to produce alkanes. In the strain with the highest alkane productivity, alkane titers were over 300 mg/liter using a modified mineral medium, and more than 80% of the hydrocarbons were found outside the cells[3].</p>
 
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= Limitations of Directed Evolution=
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=3. Sensing & Detecting alkanes=
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<p> Directed evolution through random mutagenesis, followed by high-throughput selection has made it possible to improve alkane yield or pathway efficiency. However, it can be a daunting task, because there remain many constraints on throughput imposed during the standard directed evolution workflow (library construction, transformation, and screening). We consider screening and selection as rate-limiting steps in directed evolution efforts for alkane overproduction. Therefore, in the production of large chain alkanes, it is crucial for there to be a sensitive and high-throughput screening device for comparing production rates in engineered strains of E. coli.</p>
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<p> In the research about alkane biosynthesis, productivity of microbes and profile of alkanes produced are basic information that need to be acquired. In the process of directed evolution towards high producing bacteria, a selection strategy that relates alkane productivity with cell growth rate is crucial. In large-scale alkane production, it’s important to monitor the producing process at any time. To achieve all these goals, we need to detect and analyse alkanes in samples both qualitatively and quantitively.</p>
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<p>However, current methods of alkane detection have many limits. First, extracting the product from original samples and operating high-tech equipments such as GC-MS makes the analysis process quite costly, time-consuming and laborious. Second, to select out target strains in directed evolution, commonly used screening tools are inherently low throughput. What's more, the current methods can hardly perform real-time in vivo detection for industrial production. We’re looking forward to developing an alkane sensor without these limits.</p>
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= Difficulties of Selection =
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<b>Figure 3.</b>&nbsp; GC-MS, a high-tech equipment often used in alkane detection </div>
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<p> Conspicuous products can be accurately measured using standard high-throughput colorimetric and fluorometric assays. Alkanes, however, like many small molecules, have no smell or fluorescence, or essential for growth, and they cannot be readily transformed into compounds possessing these properties[4]. Furthermore, commonly used screening tools like chromatography-mass spectrometry methods are inherently low throughput, with screenable library sizes generally limited to less than 103 variants. Therefore, the improved strains are beyond the reach of a general screening tool and cannot be readily obtained[5].</p>
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<p> To develop an alkane sensor, we need to find a mechanism which can respond to alkanes, our target products. In nature, many oil-degrading prokaryotes such as<i> P. putida</i> Gpo1, <i>Alcanivorax borkumensis</i> SK2 and<i> Acinetobacter baylyi</i> ADP1 have gene circuits responding to alkanes(alkS-alkB from<i> P. putida</i> Gpo1, alkS-alkB1 from <i>Alcanivorax borkumensis</i> SK2[6], and alkR-PalkM from <i>Acinetobacter baylyi </i>ADP1[2]). Among them, alkR-PalkM circuit in<i> Acinetobacter baylyi</i> ADP1 has remained untouched in iGEM competition. AlkR responds to a broad range of alkanes with carbon chain length from C7 to C36, and it’s the only bioreporter that is able to detect alkane with carbon chain length greater than C18[7]. Additionally, although side products such as fatty alcohol whose structure is similar to alkane can also combine with alkR, their combined compounds will inhibit PalkM, which makes this mechanism specifically suitable for alkane synthesis pathway.
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<p> One strategy deserving special attention is the use of transcription factors which have long been used to construct whole-cell biosensors for the detection of environmental pollutants, but remain largely untranslated toward library screening and directed evolution purposes. Transcription factors regulate a promoter’s transcriptional output in response to a small-molecule ligand so it’s possible to report on in vivo small-molecule production.</p>
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<p> Screening methods utilizing transcription factors possess many ideal characteristics.The transcription factor-promoter pair can be user-selected to encode for fluorescent or growth-coupled responses, and there’s no need for downstream synthetic chemistry or in vitro manipulation [4]. Therefore,with the design of biosensors, we can transform intracellular alkane molecules without a conspicuous phenotype into detectable signal outputs.</p>
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= Application of High-throughput Selection=
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= References =
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<p>[1] Yan Kung, Weerawat Runguphan, and Jay D. Keasling. “From Fields to Fuels: Recent Advances in the Microbial Production of Biofuels.” ACS Synth. Biol. 2012, 1, 498−513</p>
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<p> [2] Mathew A Rude and Andreas Schirmer.“New microbial fuels: a biotech perspective.” Current Opinion in Microbiology 2009, 12:274–281</p>
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<p> [3] Andreas Schirmer et al. “Microbial Biosynthesis of Alkanes.” Science 2010: Vol. 329 no. 5991 pp. 559-562 </p>
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<p> In conclusion, utilizing high-throughput screening to select out desirable mutations is an important step in directed evolution and pathway improvement, which largely increases the library and saves labour and time. In our project, we are designing a selection module for alkanes so that we can use irrational modification to optimize the pathway of alkane synthesis.</p>
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<p> [4] Rebecca M Lennen and Brian F Pfleger “Microbial production of fatty acid-derived fuels and chemicals” Current Opinion in Biotechnology 2013, 21:1–10</p>
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<p> [5] M. Kalim Akhtar, Nicholas J. Turner, and Patrik R. Jones. “Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities.” PNAS January 2, 2013,vol. 110, no. 1, 87–92</p>
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<p> [6] Rekha Kumari, Robin Tecon, Siham Beggah et al.(2011) “Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.” Environmental Microbiology 13(10), 2808–2819</p>
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<p> [7] Zhang, Dayi, et al.(2012) "Whole-cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills." Microbial Biotechnology 5.1: 87-97 </p>
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<p>[1] Yan Kung et al. (2012) “From Fields to Fuels: Recent Advances in the Microbial Production of Biofuels.” ACS Synth. Biol. 1, 498−513</p>
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<p>[2] Mathew A Rude, Andreas Schirmer et al.(2009) “New microbial fuels: a biotech perspective.” Current Opinion in Microbiology 12:274–281</p>
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<p>[3] Andreas Schirmer, Mathew A. Rude et al.(2010) “Microbial Biosynthesis of Alkanes.” Science Vol. 329 no. 5991 pp. 559-562 </p>
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<p>[4] Jeffrey A. Dietrich, Adrienne E. McKee, Jay D. Keasling et al.(2010) “High-Throughput Metabolic Engineering: Advances in Small-Molecule Screening and Selection.” Annu. Rev. Biochem. 79:563–90</p>
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<p>[5] Jina Yang1, Sang Woo Seo1, Sungho Jang et al.(2013) “Synthetic RNA devices to expedite the evolution of metabolite-producing microbes.” Nature Communications DOI: 10.1038/ncomms2404</p>
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Revision as of 15:16, 22 October 2013

Background

Contents

1. Call for Biofuels, especially Alkanes



Nowadays, scientist have put great attentions on biofuels, hoping to find a solution to energy crisis and climate change. Compared with fossil fuels, biofuels are renewable, and biofuel can be used indefinitely without any net carbon emissions [1]. Therefore, biofuels are promising candidates for mitigating dependence on diesel fuels.

Figure 1.  Comparison of carbon cycles of fossil fuels and biofuels

Among all kinds of biofuels, alkanes stands out because of their excellent properties. First, alkanes have higher energy density, for example, enthalpy of combustion of pentadecane is approximately -47.0 MJ/kg compared with -29.7 MJ/kg for ethanol [2]. Then, compared with ethanols with a freezing point of -114℃,which alkanes have a higher freezing point of about -3~19℃, so they are more likely to be compatible with existing engines as well as transport and storage infrastructure. Besides, they can serve as drop-in replacement for fossil fuels.


2. Biosynthesis of alkanes



Pathways of long-chain alkane sythesis in microbes has been studied. Two enzymes, acyl-ACP reductase(AAR) and aldehyde decarbonylase(ADC), were heterologously expressed in E.coli to reduce fatty acyl-ACPs to corresponding aldehydes and then convert them to alkanes[3]. Fatty aldehydes can also be produced from fatty acids and fatty acyl-CoAs, catalyzed by acyl-CoA reductases(ACR) and carboxylic acid reductase(CAR) respectively, which have been identified in many species[4,5].

Figure 2.  Typical alkane biosynthesis pathways


3. Sensing & Detecting alkanes



In the research about alkane biosynthesis, productivity of microbes and profile of alkanes produced are basic information that need to be acquired. In the process of directed evolution towards high producing bacteria, a selection strategy that relates alkane productivity with cell growth rate is crucial. In large-scale alkane production, it’s important to monitor the producing process at any time. To achieve all these goals, we need to detect and analyse alkanes in samples both qualitatively and quantitively.

However, current methods of alkane detection have many limits. First, extracting the product from original samples and operating high-tech equipments such as GC-MS makes the analysis process quite costly, time-consuming and laborious. Second, to select out target strains in directed evolution, commonly used screening tools are inherently low throughput. What's more, the current methods can hardly perform real-time in vivo detection for industrial production. We’re looking forward to developing an alkane sensor without these limits.

Figure 3.  GC-MS, a high-tech equipment often used in alkane detection

To develop an alkane sensor, we need to find a mechanism which can respond to alkanes, our target products. In nature, many oil-degrading prokaryotes such as P. putida Gpo1, Alcanivorax borkumensis SK2 and Acinetobacter baylyi ADP1 have gene circuits responding to alkanes(alkS-alkB from P. putida Gpo1, alkS-alkB1 from Alcanivorax borkumensis SK2[6], and alkR-PalkM from Acinetobacter baylyi ADP1[2]). Among them, alkR-PalkM circuit in Acinetobacter baylyi ADP1 has remained untouched in iGEM competition. AlkR responds to a broad range of alkanes with carbon chain length from C7 to C36, and it’s the only bioreporter that is able to detect alkane with carbon chain length greater than C18[7]. Additionally, although side products such as fatty alcohol whose structure is similar to alkane can also combine with alkR, their combined compounds will inhibit PalkM, which makes this mechanism specifically suitable for alkane synthesis pathway.


References



[1] Yan Kung, Weerawat Runguphan, and Jay D. Keasling. “From Fields to Fuels: Recent Advances in the Microbial Production of Biofuels.” ACS Synth. Biol. 2012, 1, 498−513

[2] Mathew A Rude and Andreas Schirmer.“New microbial fuels: a biotech perspective.” Current Opinion in Microbiology 2009, 12:274–281

[3] Andreas Schirmer et al. “Microbial Biosynthesis of Alkanes.” Science 2010: Vol. 329 no. 5991 pp. 559-562

[4] Rebecca M Lennen and Brian F Pfleger “Microbial production of fatty acid-derived fuels and chemicals” Current Opinion in Biotechnology 2013, 21:1–10

[5] M. Kalim Akhtar, Nicholas J. Turner, and Patrik R. Jones. “Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities.” PNAS January 2, 2013,vol. 110, no. 1, 87–92

[6] Rekha Kumari, Robin Tecon, Siham Beggah et al.(2011) “Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.” Environmental Microbiology 13(10), 2808–2819

[7] Zhang, Dayi, et al.(2012) "Whole-cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills." Microbial Biotechnology 5.1: 87-97

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