Production of fatty acid derivatives
一种脂肪酸衍生物、脂肪酸甲酯的技术，应用在脂肪酸衍生物的产生领域，能够解决增加脂肪酯成本和所需能量、植物油原料不是高效的能量来源、增加生物柴油生产成本和所需能量等问题
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Embodiment 1
[0267] Example 1 - Production of Escherichia coli MG1655ΔfadE
[0268] This example describes the construction of genetically engineered microorganisms in which the expression of fatty acid degrading enzymes is attenuated.
[0269] FadE was deleted from E. coli MG1655 (E. coli strain K) using the lambda Red (also known as Red-driven integration) system described in Datsenko et al., Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000) Gene, and the following changes were made at the same time.
[0270] Two primers used to generate the deletion:
[0271] Del-fad E-F
[0272] 5'-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATATTGATTCCGGGGATCCGTCGACC (SEQ ID NO: 1)
[0273] Del-fad E-R
[0274] 5'-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTTCCTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 2)
[0275] Del-fadE-F and Del-fadE-R primers were used to amplify kanamycin resistance (Km R )box. The PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 (as descr...
Embodiment 2
[0279] Example 2 - Production of Escherichia coli MG1655ΔfadEΔfhuA
[0280] This example describes the construction of genetically engineered microorganisms in which the expression of fatty acid degrading enzymes and outer membrane protein receptors has been attenuated.
[0281] The fhuA (also called tonA) gene of Escherichia coli MG1655 encoding the ferrochrome outer membrane transporter (GenBank accession number NP_414692) was deleted from Escherichia coli MG1655D1 of Example 1 using the λRed system described in Datsenko et al., supra. , but with the following changes.
[0282] Two primers used to generate the deletion:
[0283] Del-fhuA-F
[0284] 5'-ATCATTCTCGTTTACGTTATCATTCACTTTACATCAGAGATATACCAATGATTCCGGGGATCCGTCGACC (SEQ ID NO: 5);
[0285] Del-fhuA-R
[0286] 5'-GCACGGAAATCCGTGCCCCAAAAGAGAAATTAGAAACGGAAGGTTGCGGTTGTAGGCTGGAGCTGCTTC (SEQ ID NO: 6)
[0287] Del-fhuA-F and Del-fhuA-R primers were used to amplify Km from plasmid pKD13 by PCR R box. The obtained PCR p...
Embodiment 3
[0292] Example 3 - Production of Escherichia coli MG1655 ΔfadE, ΔfhuA, lacZ::'tesA fadD atfA1
[0293] This example describes the construction of a genetically engineered microorganism in which nucleotide sequences encoding thioesterase, acyl-CoA synthase and ester synthase are integrated into the chromosome of the microorganism.
[0294] The following nucleotide sequences: 'tesA, fadD and aftA1 were integrated into the lacZ locus of the chromosome of Escherichia coli MG1655ΔfadEΔfhuA strain (or DV2 strain, see Example 2). These sequences were integrated in the order 'tesA, then fadD, then aftA1.
[0295]'tesA is the nucleotide sequence comprising leaderless E. coli tesA (GenBank entry AAC73596, refseq accession number U00096.2). 'tesA encodes an E. coli thioesterase (EC 3.1.1.5, 3.1.2.-) in which the first 25 amino acids are deleted and alanine at position 26 is substituted with methionine. This methionine becomes the first amino acid of 'tesA. See Cho et al., J. Biol. Che...
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