Team:TU Delft/Project/alkane-degradation/parts


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BioBricks, the making of

Two BioBricks are the aim as final constructs for this sub-project. These BioBricks, implemented in E.coli, will be able to degrade medium (C5-13) and long chain (C15-36) alkanes respectively, and utilize them as a C-source. The degradation will occur through the route illustrated in figure 1.

Figure 1 – Schematic description of the alkane degradation pathway with the corresponding genes.

To create these BioBricks, a number of genes were synthesized and combined with promoters and RBSs from the BioBrick distribution plate. Combination of these genes resulted in the following BioBricks (the intermediate have also been submitted to the registry).

BBa_K398014 - Short-chain (C5-13) Alkane degradation

The short-chain degrading BioBrick contains the AlkB gene cluster from ‘’Gordonia sp. TF6’’[1]. This will facilitate the initial step of the degradation of C5-13 alkanes as well as that of C5-8 cycloalkanes. It is expected that the in-house mechanism of E.coli will be able to further degrade the products of this pathway. The gene cluster is formed by the genes for alkB2 (alkane 1-monooxygenase), rubA3 (rubredoxin), rubA4 (rubredoxin) and rubB (rubredoxin reductase). alkB2 is a non-haem diiron monooxygenase membrane protein, reported for several genus and species. This monooxygenase oxidizes n-alkanes to the respective n-alkanols and requires three soluble electron-transfer proteins, rubredoxin (rubA3 & rubA4) and rubredoxin reductase (rubB).

BBa K398014 AlkB2 RubA3 RubA4 RubR.png

BBa_K398022 - Long-chain (C15-36) Alkane degradation

The long-chain degrading BioBrick contains three genes, forming a degradation pathway form alkanes to alkanoic acids. For the first step in this pathway ladA will be used[2], a long-chain alkane monooxygenase from ‘’Geobacillus thermodenitrificans’’ NG80-2. This gene facilitates the conversion of long-chain alkanes (up to at least C36) to their corresponding primary alcohols. Because of the length of the alkanes (and thus also of the pathway intermediates) the BioBrick will also contain an additional ADH and ALDH Bacillus thermoleovorans B23[3-4]. These genes will facilitate the conversion of these long-chain intermediates (alkanols & alkanals). TUDelft BBa K398022.png


[1] Fujii, T., Narikawa, T., Takeda, K., Kato, J., Biotransformation of various alkanes using the Escherichia coli expressing an alkane hydroxylase system from Gordonia sp. TF6. Bioscience, biotechnology, and biochemistry, 68(10) 2171-2177 (2004) [2] Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao. Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. Journal of molecular biology, 376: 453–465 (2008) [3] Tomohisa Kato, Asuka Miyanaga, Mitsuru Haruki, Tadayuki Imanaka, Masaaki Morikawa & Shigenori Kanaya. Gene Cloning of an Alcohol Dehydrogenase from Thermophilic Alkane-Degrading Bacillus thermoleovorans B23. Journal of Bioscience and Bioengineering 91(1):100-102 (2001) [4] Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa. Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23. Extremophiles 14:33-39 (2010)