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US 5,512,669 AGranted
Gene encoding bacterial acetoacetyl-COA reductase
AssigneeMassachusetts Institute of Technology
InventorsOliver P. Peoples, Anthony J. Sinskey
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AssigneeMassachusetts Institute of Technology
InventorsOliver P. Peoples, Anthony J. Sinskey
Estimating the term…
The present invention is a method for controlling biopolymer synthesis by determining the genetics and enzymology of polyhydroxybutyrate (PHB) biosynthesis at the molecular level. The purified enzymes and genes provide the means for developing new PHB-like bicpolymers having polyester backbones. Specific aims are to 1) control the chain length of the polymers produced in fermentation processes through genetic manipulation, 2) incorporate different monomers into the polymers to produce co-polymers with different physical properties, and 3) examine the physical/rheological properties of these new biopolymers in order to develop further design criteria at the molecular level. The method for engineering biopolymer synthesis includes: isolation and characterization of the genes for the enzymes in the synthetic pathway (beta-ketothiolase, acetoacetyl-CoA reductase and PHB synthetase); cloning of the genes in a vector(s); placement of the vector(s) under the control of regulated promoters; expression of the genes; determination of the function and use of other factors such as substrate specificity in polymer production and composition; and isolation and physical and chemical analysis of the resulting polymers.
An isolated DNA molecule encoding an acetoacetyl-CoA reductase having a molecular weight of approximately of 25,000 daltons, said DNA hybridizing to the sequence shown in FIG. 6 beginning at the ATG at nucleotide 37 to the TGA stop codon at nucleotide 760 when hybridized at 65.degree. C. for between sixteen and eighteen hours in 0.75M NaCl, 0.75M sodium citrate, 50 mM Na.sub.2 HPO.sub.4, 50 mM NaH.sub.2 PO.sub.4, 0.1% Ficoll, 0.1% polyvinylpyrrolidone and 0.1% BSA, 0.1% SDS, and 10 mM EDTA.
A DNA molecule encoding an acetoacetyl-CoA reductase having a molecular weight of approximately of 25,000 daltons, said DNA hybridizing to the sequence shown in FIG. 6 beginning at the ATG at nucleotide 37 to the TGA stop codon at nucleotide 760 when hybridized at 65.degree. C. for between sixteen and eighteen hours in 0.75M NaCl, 0.75M sodium citrate, 50 mM Na.sub.2 HPO.sub.4, 50 mM NaH.sub.2 PO.sub.4, 0.1% Ficoll, 0.1% polyvinylpyrrolidone and 0.1% BSA, 0.1% SDS, and 10 mM EDTA,
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