Part:BBa_K3742000
fadl P450
In this part, we designed and constructed a plasmid containing FadL gene and P450 BM3 gene sequences. After the plasmid was introduced into the FadD gene-deficient E. coli cells in the laboratory, the cells achieved increased absorption of exogenous long-chain fatty acids (LCFA). Then, hydroxylation reaction was carried out to make the host cells produce more hydroxyl fatty acids.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1108
- 1000COMPATIBLE WITH RFC[1000]
The outer membrane protein FadL of Escherichia coli is a necessary carrier for transporting exogenous fatty acids, which helps LCFA to cross the outer membrane, especially for transporting fatty acids with 10-18 carbons [1] [2] .However, there is also a FadD gene in Escherichia coli, which can catalyze the synthesis of long-chain fatty acyl-CoA (LCFA-CoA), and then participate in β-oxidation and be consumed under the catalysis of FadE. The specific process is shown in fig.2, in which FadR gene plays a regulatory role in the whole process [5].
Our objective was to hydroxylate LCFA which had already entered into the membrane. Thus, the presence of the FadD gene would hinder the experiment. By consulting the relevant literature, we found that Jin H and his team have done two experiments to prove that knockout of FadD gene and overexpression of Fadl in Escherichia coli can increase the production of hydroxylate in Escherichia coli. First, they knocked out the FadD gene in E. coli and introduced the P450 enzyme gene into the bacteria to convert palmitic acid into omega-hydroxypalmitic acid. The results of the experiments demonstrate that the production of ω-hydroxypalmitic acid is increased in the FadD-deleted, P450 overexpressed mutants. The experimental results are shown in fig. 3 (a). [5]
Later, they overexpressed Fadl gene on the basis of FadD gene deletion and P450 expression, and found that the production of ω-hydroxypalmitic acid was greatly increased. The experimental results are shown in fig. 3 (B). Because LCFA accumulates in the periplasm through FadL membrane protein transport and is consumed in the cytoplasm through P450 enzyme, the diffusion of LCFA is driven and will not be participated in β-Oxidation. As a result, the yield of hydroxylation products increase. [5]
The P450 enzymes mentioned in the experiment are heme-containing monooxygenases. Although different CYP450 species require different electron transport systems, their catalytic reactions can generally be represented by the following general formula:
RH represents the substrate, which is catalyzed by P450 in the presence of molecular oxygen (O2) to form water and a hydroxylation product (ROH). The oxidation mechanism is shown in fig.4. Jin H’s paper shows that although palmitic acid and CYP153A were used as model in their experimental study, the method can also be applied to other LCFA or enzymes that use LCFA as a substrate. Therefore, referring to the above experimental results, in this part of the experiment, we will be able to obtain a high yield of hydroxylation products by overexpressing P450 BM3 and FadL genes in FadD-deficient E. coli cells which the laboratory has.
P450 BM3 is a particularly active member of the P450 monooxygenase family, which catalyzes the hydroxylation of ω-1, ω-2 and ω-3 positions of monounsaturated fatty acids, fatty alcohols and fatty amides with a chain length of C12 ~ C20. [3] [5] It not only conforms to the type of hydroxylase used in the literature, but also has stronger oxidation ability. The catalytic substrate is LCFA, and the expression content in Escherichia coli is high [3] [5], so we choose it as our oxidase.
After the completion of the plasmid design, we contacted the company to synthesize the fadL gene at the MCS I site of pCOLADuet-1, and linked it to the P450 gene by homologous recombination. At the same time, we extracted the plasmids which were successfully transferred into the cells and verified the vector part and P450 part by PCR. As shown in fig. 5.
Nucleic acid sequence:
Tips：
（1）The red sequences are T7 promoter.
（2）The green sequences are lactose operon.
（3）The blue part is fadL sequence.
（4）The purple part is P450 sequence.
Plasmid map:
Reference：
[1] Black, P. N. (1988). The fadL gene product of Escherichia coli is an outer membrane protein required for uptake of long-chain fatty acids and involved in sensitivity to bacteriophage T2. Journal of bacteriology, 170(6), 2850-2854. https://doi.org/10.1128/jb.170.6.2850-2854.1988
[2] Wang, X., Li, L., Zheng, Y., Zou, H., Cao, Y., Liu, H., … Xian, M. (2012). Biosynthesis of long chain hydroxyfatty acids from glucose by engineered Escherichia coli. Bioresource Technology, 114, 561–566. doi:10.1016/j.biortech.2012.02.119
[3] Brühlmann, F., Fourage, L., Ullmann, C., Haefliger, O. P., Jeckelmann, N., Dubois, C., & Wahler, D. (2014). Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid. Journal of Biotechnology, 184, 17–26. doi:10.1016/j.jbiotec.2014.05.002
[4] 刘星,孔建强.催化甾体羟基化的P450氧化酶BM3的蛋白质工程的研究进展[J].中国医药生物技术,2015,10(06):540-543.
[5] Bae Jin H; Park Beom Gi; Jung Eunok; Lee Pyung-Gang; Kim Byung-Gee. fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity[J].Applied Microbiology and BiotechnologyVolume 98, Issue 21. 2014. PP 8917-25
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