From 2010.igem.org

Alkane degradation

Aim

Formation of BioBricks for the degradation of n-alkanes to n-alkanols followed by the conversion to n-alkanals and finally n-alkanoic acids. These biobricks will be implemented in Escherichia coli K12 and characterized and evaluated on their alkane degrading capabilities.

Proposed method

The first sub-project will be the degradation of alkanes; here we will attempt to create an Escherichia coli K12 strain capable of converting medium (C5-13) and long chain alkanes (C¬15-36) into alkonoic-acids (figure 1). This will be accomplished by implementing specific genes into the strain using the “BioBrick” method. Once alkonoic-acids have been formed E.coli can further utilize them by converting them through β-oxidation.

Figure 1 Steps and associated enzymes for the degradation of alkanes to alkanoic acids

Step 1: AlkB, from Alkanes to Alkanols (C5-13)

Based on: 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)

The alkB gene cluster from Gordonia sp. TF6 facilitates the initial step of the degradation of C5-13 alkanes as well as that of C5-8 cycloalkanes. The 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).

The necessary genes, alkB2, rubA3, rubA4 and rubR, will be synthesized, and the appropriate RBS’s and promoters that will be used are existing BioBricks. The transformed cells will be cultivated in shake-flasks of 250ml. The selection criterion for RBS and Promoter for each construction will depend on the results obtained during the RBS characterization experiments. The final construct will contain the alkB gene cluster, the necessary promoters and RBS’s on a pSB3C5 plasmid.

Step 2: LadA, from Alkanes to Alkanols (C15-36)

Based on: 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)

LadA, a long-chain alkane monooxygenase from Geobacillus thermodenitrificans NG80-2, facilitates the conversion of long-chain alkanes (up to at least C36) to the corresponding primary alcohols. It will be used complementary to the alkB2 cluster described in step 1.

The synthesized LadA sequence, and appropriate RBS and promoter from the BioBrick kit (existing biobricks) will be combined into one Biobrick. The transformed cells will be cultivated in shake-flasks of 250ml. The selection criterion for RBS and Promoter for each construction will depend on the results obtained during the RBS characterization experiments. The final construct will contain the LadA, the necessary promoters and RBS’s on a pSB3T5 plasmid.

Step 3: ADH-ALDH, from Alkanols to Alkanals followed by Alkanoic Acids

Based on: 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) and 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)

ADH, an alcohol dehydrogenase isolated from Bacillus thermoleovorans B23, is capable of converting n-alkanols into the corresponding n-alkanal, the second step in the biodegradation of alkanes. From this same microorganism we will also be using ALDH, an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids, which can then be further degraded through β-oxidation.

The synthesized ADH and ALDH sequences, as well as the LadA insertion from step 2, and appropriate RBS’s and promoter from the BioBrick kit (existing biobricks) will be combined into one Biobrick. The transformed cells will be cultivated in shake-flasks of 250ml. The selection criterion for RBS and Promoter for each construction will depend on the results obtained during the RBS characterization experiments. The final construct will contain the LadA, ADH, ALDH and the necessary promoters and RBS’s on a pSB3T5 plasmid.

Step 4: Combination & Final Construct

As the fourth step for the alkane degradation sub-part we will transform E.coli with both created plasmids, (1) the pSB3C5-AlkB BioBrick and (2) the pSB3T5-LadA-ADH-ALDH BioBrick. This should result in a strain that is capable of converting alkanes to alkanoic acids, which can then be further degraded by the β-oxidation pathway that is already present in E.coli.

Step 5: Characterization

Strains:

• AlkB: E.coli Top10/014C
• LadA: E.coli Top10/016T
• LadA-ADH-ALDH: E.coli Top10/021T
• AlkB-LadA-ADH-ALDH: E. coli Top10/014C - 021T
• Negative control: E.coli Top10
• Positive control: Pseudomonas putida OCT1

Characterization

The final step of the alkane degradation sub-project will be the characterization of the product strain (step 4), as well as that of the “in-between” strains created in steps 1, 2 and 3 (4 strains in total). The positive control will be an organism that can already degrade alkanes naturally, either Pseudomonas putida. The negative control will be an unmodified E.coli K12 without alkane degrading capabilities. The strains will be grown on low concentrations (1% v/v) of certain hydrocarbons, a choice still has to made between octane, decane, undecane, dodecane, hexadecane, heptadecane, icosane and cyclohexane. Growth curves will be followed using OD-600nm measurements and alkane concentrations as well as any products will be analyzed using gas chromatography. Next to growth-phase kinetics, enzyme activities within cell extracts will be measured.