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Multiple mechanisms contribute to increased neutral lipid accumulation in yeast producing recombinant variants of plant diacylglycerol acyltransferase 1.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2017 Oct 27; Vol. 292 (43), pp. 17819-17831. Date of Electronic Publication: 2017 Sep 12. - Publication Year :
- 2017
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Abstract
- The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn -1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield ( e.g. in canola-type Brassica napus ). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.<br /> (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Subjects :
- Amino Acid Substitution
Brassica napus enzymology
Mutation, Missense
Organisms, Genetically Modified genetics
Organisms, Genetically Modified metabolism
Recombinant Proteins biosynthesis
Recombinant Proteins genetics
Saccharomyces cerevisiae genetics
Brassica napus genetics
Diacylglycerol O-Acyltransferase biosynthesis
Diacylglycerol O-Acyltransferase genetics
Lipid Metabolism
Plant Proteins biosynthesis
Plant Proteins genetics
Saccharomyces cerevisiae enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 292
- Issue :
- 43
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 28900030
- Full Text :
- https://doi.org/10.1074/jbc.M117.811489