Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

被引:91
作者
Lennen, Rebecca M. [1 ,2 ]
Politz, Mark G. [1 ]
Kruziki, Max A. [1 ]
Pfleger, Brian F. [1 ,2 ]
机构
[1] Univ Wisconsin Madison, Dept Chem & Biol Engn, Madison, WI 53715 USA
[2] Univ Wisconsin Madison, US DOE, Great Lakes Bioenergy Res Ctr, Madison, WI USA
关键词
ORGANIC-SOLVENT TOLERANCE; OUTER-MEMBRANE; MULTIDRUG-RESISTANCE; PUMP; EXPRESSION; OVERPRODUCTION; GENES; TOLC; MECHANISMS; BACTERIA;
D O I
10.1128/JB.01477-12
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB(+) strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts.
引用
收藏
页码:135 / 144
页数:10
相关论文
共 66 条
[1]   An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a [J].
Adebusuyi, Abigail A. ;
Foght, Julia M. .
BMC MICROBIOLOGY, 2011, 11
[2]  
ANDERSEN J, 1989, J BIOL CHEM, V264, P17961
[3]   Improvement of organic solvent tolerance level of Escherichia coli by overexpression of stress-responsive genes [J].
Aono, R .
EXTREMOPHILES, 1998, 2 (03) :239-248
[4]   Involvement of outer membrane protein TolC, a possible member of the mar-sox regulon, in maintenance and improvement of organic solvent tolerance of Escherichia coli K-12 [J].
Aono, R ;
Tsukagoshi, N ;
Yamamoto, M .
JOURNAL OF BACTERIOLOGY, 1998, 180 (04) :938-944
[5]   USE OF TRANSPOSON TNPHOA TO IDENTIFY GENES FOR CELL-ENVELOPE PROTEINS OF ESCHERICHIA-COLI REQUIRED FOR LONG-CHAIN FATTY-ACID TRANSPORT - THE PERIPLASMIC PROTEIN TSP POTENTIATES LONG-CHAIN FATTY-ACID TRANSPORT [J].
AZIZAN, A ;
BLACK, PN .
JOURNAL OF BACTERIOLOGY, 1994, 176 (21) :6653-6662
[6]   Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants:: the Keio collection [J].
Baba, Tomoya ;
Ara, Takeshi ;
Hasegawa, Miki ;
Takai, Yuki ;
Okumura, Yoshiko ;
Baba, Miki ;
Datsenko, Kirill A. ;
Tomita, Masaru ;
Wanner, Barry L. ;
Mori, Hirotada .
MOLECULAR SYSTEMS BIOLOGY, 2006, 2 (1) :2006.0008
[7]   CHARACTERIZATION OF FADL-SPECIFIC FATTY-ACID BINDING IN ESCHERICHIA-COLI [J].
BLACK, PN .
BIOCHIMICA ET BIOPHYSICA ACTA, 1990, 1046 (01) :97-105
[8]   TolC is involved in enterobactin efflux across the outer membrane of Escherichia coli [J].
Bleuel, C ;
Grosse, C ;
Taudte, N ;
Scherer, J ;
Wesenberg, D ;
Krauss, GJ ;
Nies, DH ;
Grass, G .
JOURNAL OF BACTERIOLOGY, 2005, 187 (19) :6701-6707
[9]   Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA [J].
Borges-Walmsley, MI ;
Beauchamp, J ;
Kelly, SM ;
Jumel, K ;
Candlish, D ;
Harding, SE ;
Price, NC ;
Walmsley, AR .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (15) :12903-12912
[10]  
Brown MH, 2001, J MOL MICROB BIOTECH, V3, P163