Journal of Oil Palm Research Vol. Special Issue  2008 July p.  118-133

Activity studies, gene characterization and manipulation of β-ketothiolase of oil palm (Elaeis guineensis Jacq.) mesocarp

Author(s): YIP Jin Teen* ; ABDUL MASANI Mat Yunus* ; AHMAD PARVEEZ Ghulam Kadir* ; SAMBANTHAMURTHI, Ravigadevi*

The enzyme β-ketothiolase plays a major role in iso­prenoid metab­o­lism as well as in poly­hy­drox­y­bu­tyrate (PHB) biosyn­the­sis. PHB is a mem­ber of the poly­hy­drox­yalka­noate (PHA) fam­ily of poly­mers with poten­tial as biodegrad­able replace­ments for the cur­rent petro­chem­i­cal plas­tics used. A cou­pled assay sys­tem for β-ketothiolase activ­ity in oil palm meso­carp crude extracts was designed and opti­mized. The high­est lev­els of β-ketothiolase spe­cific activ­ity were seen in oil palm fruits 8 to 11 weeks after anthe­sis (WAA) with ripe fruits show­ing lower activ­ity. A cDNA cod­ing for oil palm (Elaeis guineen­sis Jacq.) β-ketothiolase was iso­lated through RT-PCR and RACE tech­niques. The longest read­ing frame encoded a pro­tein of 415 amino acids with a pre­dicted rel­a­tive mol­e­c­u­lar weight of 43 217 Da, and con­sid­er­able sim­i­lar­i­ties to the gene/enzyme in other plant thi­o­lases and, to a lesser extent, prokary­otic thi­o­lases. There is no evi­dence for the pres­ence of a sig­nal pep­tide, sug­gest­ing that the β-ketothiolase cDNA encodes a cytoso­lic pro­tein.
Genomic DNA gel blot analy­sis sug­gested a small fam­ily of β-ketothiolase iso­genes. North­ern analy­sis revealed that β-ketothiolase mRNA tran­scripts are present in higher quan­ti­ties in the riper (13, 17, 20 WAA) than younger fruits at 6, 8 and 11 WAA, con­tra­dict­ing the bio­chem­i­cal activ­ity pro­file. This dis­crep­ancy may be caused by inter­fer­ing sub­stances in the oil palm crude extract such as lipids or com­pe­ti­tion for sub­strates by other enzymes. Other explana­tory fac­tors include geno­type depen­dency, thi­o­lase mRNA tran­scripts of the same size and post-translational mod­i­fi­ca­tion. The expres­sion pro­file obtained in the North­ern analy­sis is in agree­ment with that of sterol and carotenoid accu­mu­la­tion dur­ing fruit ripen­ing. Sequence analy­sis with bio­com­put­ing tools showed that β-ketothiolase cDNA is rel­a­tively lowly expressed in oil palm meso­carp through­out its devel­op­ment. The gene was used in an inter­ven­tion strat­egy to sub­sti­tute for bac­te­r­ial β-ketothiolase by redesign­ing the PHB trans­for­ma­tion vec­tor dri­ven by oil palm mesocarp-specific pro­moter (MSP1), for syn­the­siz­ing biodegrad­able plas­tics in oil palm. The new vec­tor, des­ig­nated pMS35, was later trans­formed into oil palm embryo­genic calli using the biolis­tics approach. Cur­rently, Basta-resistant embryo­genic calli have been obtained and are under­go­ing pro­lif­er­a­tion and regeneration.

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Author Information
* Malaysian Palm Oil Board, P. O. Box 10620. 50720 Kuala Lumpur, Malaysia.

Journal of Oil Palm Research Special Issue on Malaysia-MIT Biotechnology Partnership Programme: Volume 2 - Oil Palm Metabolic Engineering

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