Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold. Implications for nucleotide specificity.

The gene encoding a 4-hydroxybutyryl-Co A transferase has been isolated from bacteria and integrated into the genome of bacteria also expressing a polyhydroxyalkanoate synthase, to yield an improved production process for 4HB-containing polyhydroxyalkanoates using transgenic organisms, including both bacteria and plants. The new pathways provide means for producing 4HB containing PHAs from cheap carbon sources such as sugars and fatty acids, in high yields, which are stable. Useful strains are obtaining by screening strains having integrated into their genomes a gene encoding a 4HB-CoA transferase and/or PHA synthase, for polymer production. Processes for polymer production use recombinant systems that can utilize cheap substrates. Systems are provided which can utilize amino acid degradation pathways, α-ketoglutarate, or succinate as substrate.

Biological systems for manufacture of polyhydroxyalkanoate polymers containing 4-hydroxyacids

The nature of the last universal ancestor to all extent cellular organisms and the rooting of the universal tree of life are fundamental questions which can now be adressed by molecular evolutionists. Several scenarios have been proposed during the last years, based on the phylogenies of ribosomal RNA and of duplicated proteins, which suggest that the last universal ancestor was either an RNA progenote or an hyperthermophilic prokaryote. We discuss these hypotheses in the light of new data on the evolution of DNA metabolizing enzymes and of contradictions between different protein phylogenies. We conclude that the last universal ancestor was a member of the DNA world already containing several DNA polymerases and DNA topoisomerases. Furthermore, we criticize current data which suggest that the rooting of the universal tree of life is located in the eubacterial branch and we conclude that both rooting the universal tree and the nature of the last universal ancestor are still open questions.

The nature of the last universal ancestor and the root of the tree of life, still open questions

The gene encoding the NAD +-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M,) 49 295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-XaaXaa-Ala motif in the NADf-binding domain, which is more typical of the NADPf-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate sitedirected mutagenesis. The NAD +-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum catalyses the first step in the hydroxyglutarate pathway of glutamate fermentation 11, 21. This anaerobe is an abundant source of GDH, which amounts to approximately 15% of the total cell protein during growth with glutamate. The enzyme has been purified and extensively characterized (3 - 51 and peptides representing about 75% of the primary structure have been sequenced 161. In addition, C. symbiosum GDH has been crystallized in a form suitable for high-resolution X-ray analysis and the structure has been solved to 0.196 nm [7-lo]. The cloning of the gdh gene and the determination of its nucleotide sequence are important for deducing the complete amino acid sequence of the enzyme, for permitting a full interpretation of the molecular structure and for designing site-directed mutagenesis experiments. Conventional approaches for cloning the gdh gene of C. symbiosum

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1992.tb16912.x

The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.

The invention provides a non-naturally occurring microbial biocatalyst including a microbial organism having a 4-hydroxybutanoic acid (4-HB) biosynthetic pathway having at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, or α-ketoglutarate decarboxylase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce monomeric 4-hydroxybutanoic acid (4-HB). Also provided is a non-naturally occurring microbial biocatalyst including a microbial organism having 4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic pathways, the pathways include at least one exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase, succinyl-CoA synthetase, CoA-dependent succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA transferase, 4-butyrate kinase, phosphotransbutyrylase, α-ketoglutarate decarboxylase, aldehyde dehydrogenase, alcohol dehydrogenase or an aldehyde/alcohol dehydrogenase, wherein the exogenous nucleic acid is expressed in sufficient amounts to produce 1,4-butanediol (BDO). Additionally provided are methods for the production of 4-HB and BDO.

Compositions and methods for the biosynthesis of 1,4-butanediol and its precursors

The gene for the catabolic NAD-linked glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was cloned by selection of Escherichia coli for complementation of a biosynthetic defect. Cloned fragments containing the gene and the P. asaccharolyticus transcription and translation signals are very highly expressed in E. coli. The nucleotide sequence of the cloned gene was determined. It codes for a polypeptide of 421 amino acids, the sequence of which is similar to those of the NADP-accepting glutamate dehydrogenases. The sequence similarity of this protein to the mammalian glutamate dehydrogenases, which accept both NADP and NAD, is greater than its similarity to the bacterial NADP-specific dehydrogenases, suggesting that this NAD-specific bacterial glutamate dehydrogenase and the NADP-specific bacterial dehydrogenases diverged separately from the line leading to the dual-specificity mammalian glutamate dehydrogenases.

Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus.

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Selection, expression, and nucleotide sequencing of the glutamate dehydrogenase gene of Peptostreptococcus asaccharolyticus.