Silke Hein

Biochemical and molecular basis of microbial synthesis of polyhydroxyalkanoates in microorganisms.

Intensive research on the physiology, biochemistry, and molecular genetics of the metabolism of polyhydroxyalkanoates (PHA) during the last 15 years has revealed a dramatic increase of our knowledge on the biosynthesis of these polyesters in bacteria. This mainly very basic research has revealed several new, hitherto not described enzymes and pathways. In addition, many genes encoding the enzymes of these pathways and in particular the key enzyme of PHA biosynthesis, PHA synthase, were cloned and characterized at a molecular level. This knowledge was utilized to establish PHA biosynthesis in many prokaryotic and eukaryotic organisms, which were unable to synthesize PHAs, and to apply the methodology of metabolic engineering, thus opening new perspectives for the production of various PHAs by fermentation biotechnology or agriculture in economically feasible processes. This contribution summarizes the properties of PHA synthases and gives an overview on the genes for these enzymes and other enzymes of PHA biosynthesis that have been cloned and are available. It also summarizes our current knowledge on the regulation at the enzyme and gene level of PHA biosynthesis in bacteria.

Multiple evidence for widespread and general occurrence of type-III PHA synthases in cyanobacteria and molecular characterization of the PHA synthases from two thermophilic cyanobacteria: Chlorogloeopsis fritschii PCC 6912 and Synechococcus sp. strain MA19

Eleven different cyanobacteria were investigated with respect to their capabilities to synthesize poly-3-hydroxybutyrate [poly(3HB)] and the type of poly-beta-hydroxyalkanoic acid (PHA) synthase accounting for the synthesis of this polyester. Several methods, including (i) Southern blot analysis using a phaC-specific DNA probe, (ii) Western blot analysis using specific polyclonal anti-PhaE antibodies raised in this study against PhaE of Synechocystis sp. strain PCC 6803, (iii) generation and sequence analysis of PCR products using phaC-specific oligonucleotides as primers, and/or (iv) cloning and sequence analysis of PHA synthase structural genes, were used to provide evidence for the presence of a type-III PHA synthase in the following cyanobacteria: Synechococcus sp. strains MA19 and PCC 6715, Chlorogloeopsis fritschii PCC 6912, Anabaena cylindrica SAG 1403-2, Cyanothece sp. strains PCC 7424, PCC 8303 and PCC 8801, and Gloeocapsa sp. strain PCC 7428. The screening was compared with corresponding studies using crude protein extracts and genomic DNA of Synechocystis sp. strain PCC 6803, as a positive control, which is so far the only cyanobacterium for which molecular data of the PHA synthase genes are available. No evidence for the presence of a type-III PHA synthase could be obtained for only three of the eleven investigated cyanobacteria (Stanieria sp. strain PCC 7437, Cyanothece sp. strain PCC 8955 and Gloeothece sp. strain PCC 6501). The entire PHA synthase structural genes of the two thermophilic cyanobacteria Synechococcus sp. strain MA19 and Chlorogloeopsis fritschii PCC 6912, and in addition a central region of the phaC gene of Cyanothece sp. strain PCC 8303, were cloned, sequenced and also heterologously expressed in Escherichia coli.

Biosynthesis of polyesters in bacteria and recombinant organisms

Abstract Many bacteria are able to synthesize polyesters of hydroxyalkanoic acids (PHA), which occur as insoluble cytoplasmic inclusions in the cell and can contribute significantly to the cellular dry matter. More than 100 different hydroxyalkanoic acids have been detected as constituents in PHA. This article summarizes strategies and possibilities to obtain these biodegradable and thermoplastic/elastomeric polymers by fermentation of wild-type bacteria as well as of mutants and recombinant strains from renewable resources, waste substrates or special precursor substrates. In addition, in vitro biosynthesis of PHA employing the purified polymerizing enzyme (PHA synthase) is described. Throughout analysis and cloning of the bacterial PHA biosynthesis genes enabled scientists to also establish this pathway in non-PHA producing eukaryotic organisms such as yeast, plants and animals. This will allow new processes for cheap and economic production of PHA.

Cloning, nucleotide sequence and primary biochemical characterization of esterase EstA from Ralstonia eutropha CH34

A genomic library of Ralstonia eutropha CH34 was screened in Escherichia coli S17-1 for esterase activity by using α-naphthyl acetate and Fast Blue RR. A 1,711 bp DNA fragment was subcloned from an esterase-positive clone and sequenced. Esterase EstA was encoded by a 825-bp open reading frame and exhibited significant amino acid similarities with the enzymes involved in the meta-cleavage pathway. EstA is composed of 275 amino aicds with a predicted molecular mass of 30785 Da. The optimal pH for EstA was 7.0, and the enzyme retained more than 65% activity when incubated in buffers with pH 3.8–9.2 for 2 h. EstA was active at temperatures up to 80 °C and retained more than 77% activity after exposure to temperatures below 60 °C for 2 h.