Disruption of membrane transport systems
4.2. Introduction
The optimization of recombinant strains for the production of valuable compounds is a major goal of genetic engineering. A variety of rational approaches for engineering recombinant strains have been used for this purpose, including increasing the supply of metabolic precursors, deletion of negative regulators, overexpression of positive regulators, and removal of precursor-consuming and product-degrading pathways. The effect of genetic modifications based on rational engineering can be tested in a variety of strains to further improve the production of target compounds[95].
In the last decade, rational metabolic engineering has been frequently applied to increase microbial production of free fatty acids (FFAs) or their precursors[74, 86, 96]. Significant increases in FFA production have been achieved by overexpressing enzymes in the fatty acid (FA) synthesis pathway, including ACC, FabZ, and thioesterase[34, 38, 70]. Increased production of the FA precursor, malonyl-CoA, has been achieved by overexpression of accABCD, pgk, aceEF, and lpd and deletion of fumC and sucC, which resulted in a four-fold increase in the intracellular concentration of the metabolite[97]. Inactivation of FadD in the β-oxidation pathway has also been shown to improve FFA production[38]. Other strategies for increasing FFA production have involved overexpression or inactivation of regulators; for example, overexpression of FadR, a transcriptional regulator of FA synthesis, increased the mRNA and protein levels of FA synthesis pathway genes, resulting in a 7.5- fold increase in FFA production[72]. Likewise, deletion of FabR, a transcriptional regulator that represses expression of genes involved in unsaturated FA synthesis, yielded a 1.2-fold increase in FA production[70].
A prerequisite for the success of rational approaches for metabolic engineering is detailed knowledge of the metabolic pathway for production of the desired metabolite[98]. However, in some cases, a variety of cellular properties associated with production of the target product are poorly understood. In contrast, random approaches such as transposon mutagenesis do not require preliminary knowledge of the relevant metabolic pathways. In addition, random approaches to metabolic engineering have revealed previously unknown factors that contribute to increased metabolite production[99-102]. In a previous study, strains exhibiting increased FFA production were obtained by screening a library of random mutants; it was revealed that deletion of fadB (encoding enoyl-CoA hydratase) in a fadD deletion background strain resulted in an additional 1.2-fold increase in FFA production[103]. Further gains in titer, productivity, and yield may require the identification, using random approaches, of factors that improve FFA production via indirect mechanisms. Therefore, the aim of this study was to isolate E. coli strains exhibiting increased FFA production by screening of a library of random transposon mutants, and to identify and characterize the genes responsible for increased FFA production.
Table 4.2. Strains and plasmids used in this study
Strains and plasmids Genotype and description Reference
Strains
MG1655 E. coli K-12 F–λ–ilvG–rfb-50rph-1 [56]
MFDpir MG1655 RP4-2-Tc::[Mu1::aac(3)IV-∆aphA-∆nic35-
∆Mu2::zeo]∆dapA::(erm-pir) ∆recA [104]
SBF06 MG1655 with pBbB6c-‘tesA [105]
SBF22 MG1655 with ΔenvR::FRT and pBbB6c-‘tesA This study
SBF23 MG1655 with ΔgusC::FRT and pBbB6c-‘tesA This study
SBF24 MG1655 with ΔmdlA::FRT and pBbB6c-‘tesA This study
SBF25 MG1655 with ΔenvR::FRT, ΔgusC::FRT, ΔmdlA::FRT,
and pBbB6c-‘tesA This study
SBF26 MG1655 with pBbB6c-‘tesA and pBbA1k-gusC This study
SBF27 MG1655 with pBbB6c-‘tesA and pBbA1k-mdlA This study
SBF28 MG1655 with pBbB6c-‘tesA and pBbA1k-acrAB This study
SBF29 MG1655 with ΔenvR::FRT, ΔacrAB::FRT, and pBbB6c-
‘tesA This study
SBF30 MG1655 with ΔompF::FRT and pBbB6c-‘tesA This study
SBF31 MG1655 with Δaas::FRT and pBbB6c-‘tesA This study
SBF32 MG1655 with ΔfadL::FRT and pBbB6c-‘tesA This study
SBF33 SBF25 with ΔompF::FRT and pBbB6c-‘tesA This study
SBF34 SBF25 with Δaas::FRT and pBbB6c-‘tesA This study
SBF35 SBF25 with ΔfadL::FRT and pBbB6c-‘tesA This study
SBF36 SBF25 with ΔompF::FRT, Δaas::FRT, and pBbB6c-‘tesA This study SBF37 SBF25 with ΔompF::FRT, ΔfadL::FRT, and pBbB6c-
‘tesA This study
SBF38 SBF25 with Δaas::FRT, ΔfadL::FRT, and pBbB6c-‘tesA This study SBF39 SBF25 with ΔompF::FRT, Δaas::FRT, ΔfadL::FRT, and
pBbB6c-‘tesA This study
Plasmids
pBbB6c-‘tesA pBbB6c-gfp with Δgfp::‘tesA, CmR [105]
pFAB pBbE8a-fadR carrying PLR-tetA-rfp on AatII site [105]
pTNMod-OTc pMB1 ori, carrying RP4 oriT and Tn5 tnp, TetR [106]
pTNMod-R6K-KmR R6K ori, carrying RP4 oriT and Tn5 tnp, KmR This study
pBbA1k-gusC pBbB6c-rfp with Δrfp::gusC, KmR This study
pBbA1k-mdlA pBbB6c-rfp with Δrfp::mdlA, KmR This study
pBbA1k-acrAB pBbB6c-rfp with Δrfp::acrAB, KmR This study
Table 4.2. Primers used in this study.
Primers Sequence (5’-3’)
envR_del_FP GAACTGAATTTTCAGGACAGAATGTGAATTTACATGACACTTAATTCAT TGTGTAGGCTGGAGCTGCTTC
envR_del_RP ATTTTCTCACTCTGTGTCGAATATATTTATTTCCTGAATAATTAATCATG ATTCCGGGGATCCGTCGACC
envR_seq_FP GTTAAGCGAAGTTGACCCCGATCTC envR_seq_RP AAAGAAAAATACAGTTCGCTATCCT
gusC_del_FP CCGGAGATTTTTCTCTCCGGCGTTATTTTTTACTTCAGCATAAAGTCATA GTGTAGGCTGGAGCTGCTTC
gusC_del_RP ACTAATTAATATTCAATAAAAATAATCAGAACATCAAAGGTGCAACTATG ATTCCGGGGATCCGTCGACC
gusC_seq_FP GGCATGTTTGCGATGCCAGCTCTTA gusC_seq_RP CCGGCGTGCGAATTGAAGGGCTCAC
mdlA_del_FP TCTTTACCCATCGAATAAATATCCAGAATCAGGTCAGGACACAACGCGT GGTGTAGGCTGGAGCTGCTTC
mdlA_del_RP GCTTGAGAGTCGGCCACAGTTGGCTAAAACTACGCATCGACGGCCTCCT CATTCCGGGGATCCGTCGACC
mdlA_seq_FP AACGGCTGGAGGACGACGGTATCCT mdlA_seq_RP ACAGCAGCGACTGCGCGTAATGTAG
ompF_del_FP TTTTCGGCATTTAACAAAGAGGTGTGCTATTAGAACTGGTAAACGATACC GTGTAGGCTGGAGCTGCTTC
ompF_del_RP GACGGCAGTGGCAGGTGTCATAAAAAAAACCATGAGGGTAATAAATAAT GATTCCGGGGATCCGTCGACC
ompF_seq_FP TATAAGGAAATCATATAAATAGATT ompF_seq_RP TCATCTTTATAGACACCAATCCCGA
aas_del_FP TGTAATCTCCCTCCATTCGCTTTTACTGAATCAGAGCAAAGGGAGTTGG AGTGTAGGCTGGAGCTGCTTC
aas_del_RP TAACCGCTTTCATCCCCTTCGACCACAACGAAGTGTTAGTGTGCACTGA CCTGTCAAACATGAGAATTAA
aas_seq_FP TCGTCGTGCAGACCACAATC aas_seq_RP CCGACAAACGGCGCAAAAAG
fadL_del_FP TGTTACAGCACGTAACATAGTTTGTATAAAAATAAATCATTGAGGTTATG GTGTAGGCTGGAGCTGCTTC
fadL_del_RP CAGGTGACTTTATCCAGGCGAACGCGTTATCAGAACGCGTAGTTAAAGT TATTCCGGGGATCCGTCGACC
fadL_seq_FP GCTGCTCCAGTTGTTAATTC fadL_seq_RP AACGCTCATTTATTTAGAAC
R6K-Km_FP AAGGTACCAGATTGCAGCATTACACGTC
R6K-Km_RP CGAGCTCCTGAGATAGGTGCCTCACTG
Km_RP CGGTGCCCTGAATGAACTGC
R6K_FP CCCATGTCAGCCGTTAAGTGTTCCT
Km_seq_RP CGGTGCCCTGAATGAACTGC
R6K_seq_RP ATCAACAGGTTGAACTGCGGATCTT
Note: Underlined sequences indicate restriction enzyme sites.