Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli

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• 作者：Hein ; Silke ; Sö ; hling ; Brigitte ; Gottschalk ; Gerhard ; Steinbü ; chel ; Alexander
• 关键词：Alcaligenes eutrophus ; Escherichia coli ; Clostridium kluyveri ; Biodegradable polyester ; Polyhydroxyalkanoic acid ; Poly(4-hydroxybutyric acid) ; 4-Hydroxybutyric acid metabolism ; PHA synthase ; 4-Hydroxybutyric acid-CoA transferase ; PHA ; polyhydroxyalkanoic
• 刊名：FEMS Microbiology Letters
• 出版年：1997
• 出版时间：August 15, 1997
• 年：1997
• 卷：153
• 期：2
• 页码：411-418
• 全文大小：587 K

文摘

The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C. kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS⁻ and pSK⁻, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80 % (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if γ-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or γ-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.