Alteration of Acyl-Acyl Carrier Protein Pools and Acetyl-CoA Carboxylase Expression in Escherichia coli by a Plant Medium-Chain Acyl-Acyl Carrier Protein Thioesterase

1995 ◽  
Vol 317 (1) ◽  
pp. 185-190 ◽  
Author(s):  
J. Ohlrogge ◽  
L. Savage ◽  
J. Jaworski ◽  
T. Voelker ◽  
D. Postbeittenmiller
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Medium Chain ◽  
Expression In Escherichia Coli ◽  
Chain Acyl

scholarly journals Overexpression of the Plant Medium-chain Acyl-carrier Protein Thioesterase BTE for the Overproduction of the nC14-surfactin Isoform with Promising MEOR Applications

2021 ◽  
Author(s):  
fangxiang hu ◽  
Weijie Cai ◽  
Junzhang Lin ◽  
Weidong Wang ◽  
Shuang Li
Keyword(s):  
Fatty Acid ◽  
Hydroxy Fatty Acid ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Medium Chain ◽  
Emulsification Index ◽  
Fatty Acid Chain ◽  
Umbellularia Californica ◽  
Chain Acyl ◽  
Surfactin Production

Abstract BackgroundSurfactin, a representative biosurfactant of popeptide mainly produced by Bacillus subtilis, consists of a cyclic heptapeptide linked to a β-hydroxy fatty acid chain. The functional activity of surfactin is closely related to the length and isomerism of the fatty acid chain. ResultsIn this study, the plant medium-chain acyl-carrier protein (ACP) thioesterase (BTE) from Umbellularia californica was overexpressed in a recombinant surfactin production strain based on B. subtilis 168. As a result, the surfactin yield after 24 h of cultivation improved by 23%, and the production rate increased from 0.112 to 0.177 g/L/h. The isoforms identified by RP-HPLC and GC-MS showed that the proportion of nC14-surfactin increased 6.4 times compared to the control strain. A comparison of further properties revealed that the product with more nC14-surfactin had higher surface activity and better performance in oil-washing. Finally, the product with more nC14-surfactin isoform had a higher hydrocarbon-emulsification index, and it increased the water-wettability of the oil-saturated silicate surface. ConclusionThe obtained results provide an original approach to modify the fatty acid chain of surfactin and further demonstrate the importance of the length and isomerism of the β-hydroxy fatty acid chain for the MEOR application of surfactin.

scholarly journals Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase

1994 ◽  
Vol 302 (3) ◽  
pp. 881-887 ◽  
Author(s):  
A Chapman-Smith ◽  
D L Turner ◽  
J E Cronan ◽  
T W Morris ◽  
J C Wallace
Keyword(s):  
Escherichia Coli ◽  
Exchange Chromatography ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
E Coli ◽  
Biotin Ligase ◽  
Domain Peptide

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

scholarly journals Isolation of cDNAs from Brassica napus encoding the biotin-binding and transcarboxylase domains of acetyl-CoA carboxylase: assignment of the domain structure in a full-length Arabidopsis thaliana genomic clone

1994 ◽  
Vol 301 (2) ◽  
pp. 599-605 ◽  
Author(s):  
K M Elborough ◽  
R Swinhoe ◽  
R Winz ◽  
J T Kroon ◽  
L Farnsworth ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Acyl Carrier Protein ◽  
Fatty Acid Synthetase ◽  
Genomic Clone ◽  
Full Length ◽  
Cdna Clones ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Biotin Binding

One independent and two overlapping rape cDNA clones have been isolated from a rape embryo library. We have shown that they encode a 2.3 kb and a 2.5 kb stretch of the full-length acetyl-CoA carboxylase (ACCase) cDNA, corresponding to the biotin-binding and transcarboxylase domains respectively. Using the cDNA in Northern-blot analysis we have shown that the mRNA for ACCase has a higher level of expression in rape seed than in rape leaf and has a full length of 7.5 kb. The level of expression during rape embryogenesis was compared with both oil deposition and expression of two fatty acid synthetase components enoyl-(acyl-carrier-protein) reductase and 3-oxoacyl-(acyl-carrier-protein) reductase. Levels of ACCase mRNA were shown to peak at 29 days after anthesis during embryonic development, similarly to enoyl-(acyl-carrier-protein) reductase and 3-oxoacyl-(acyl-carrier-protein) reductase mRNA. In addition, a full-length genomic clone (19 kb) of Arabidopsis ACCase has been isolated and partially sequenced. Analysis of the clone has allowed the first plant ACCase activity domains (biotin carboxylase-biotin binding-transcarboxylase) to be ordered and assigned. Southern-blot analysis using the Arabidopsis clone indicates that ACCase is a single-copy gene in Arabidopsis but is encoded by a small gene family in rape.

scholarly journals Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli

1998 ◽  
Vol 329 (3) ◽  
pp. 589-596 ◽  
Author(s):  
Pedro RECHE ◽  
Yi-Li LI ◽  
Christopher FULLER ◽  
Knut EICHHORN ◽  
N. Richard PERHAM
Keyword(s):  
Escherichia Coli ◽  
Lysine Residue ◽  
Sequence Motif ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Hairpin Loop ◽  
Acetyl Coa ◽  
E Coli

Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two β-sheets that make up the domain. A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E. coli. The biotinyl domain was readily released by cleavage with thrombin. Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes. No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro. The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated. It was also a poor substrate for lipoylation in vitro catalysed by the E. coli lipoyl protein ligase encoded by the lplA gene. The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes. The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E. coli but probably by a pathway different from that mediated by the lplA-dependent ligase. The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene

1987 ◽  
Vol 256 (1) ◽  
pp. 90-100 ◽  
Author(s):  
Phillip D. Beremand ◽  
David J. Hannapel ◽  
Daniel J. Guerra ◽  
David N. Kuhn ◽  
John B. Ohlrogge
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Cloning And Expression ◽  
Carrier Protein ◽  
Expression In Escherichia Coli ◽  
I Gene
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