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All publicly accessible microbial genome databases were searched for the occurrence of genes encoding proteins homologous to the cyanophycin synthetase (CphA) of Synechocystis sp. strain PCC 6803 in order to reveal the capability of microorganisms not belonging to the cyanobacteria to synthesize cyanophycin. Among 65 genome sequences, genes homologous to cphA were found in Acinetobacter sp. strain ADP1 (encoding a protein homologous to CphA with 40% amino acid identity), Bordetella bronchiseptica strain RB50 (39%), Bordetella pertussis strain Tohama I (39%), Bordetella parapertussis strain 12822 (39%), Clostridium botulinum strain ATCC 3502 (39%), Desulfitobacterium hafniense strain DCB-2 (38%) and Nitrosomonas europaea strain ATCC 25978 (37%). The gene homologous to cphA from Acinetobacter sp. strain DSM 587 was amplified by PCR, ligated to the vector pBluescript SK(-) downstream of the lac promoter and introduced into Escherichia coli. The recombinant strain of E. coli expressed CphA activity at up to 1.2 U/mg protein and accumulated cyanophycin to up to 7.5% of the cellular dry matter, indicating that CphA of Acinetobacter sp. strain DSM 587 is functionally active. In Acinetobacter sp. strain DSM 587 itself, cyanophycin accumulated to up to 1.4% of the total protein under phosphate-limited conditions, and cyanophycin synthetase activity was detected, which indicated the function of cyanophycin as a storage compound in this strain.

Microorganisms are able to synthesize three different polyamides by enzymatic processes independently from ribosomal protein biosynthesis: poly(gamma-D-glutamic acid), poly(epsilon-L-lysine) and multi-L-arginyl-poly(L-aspartic acid) which is also referred to as cyanophycin. These polyamides, which occur mainly in Bacillus spp. (and only a few other eubacteria and the nematocysts of Cnidaria, in Streptomyces albulus or in cyanobacteria, respectively), have recently attracted considerable interest of the chemical industry and may be suitable for various applications. This review summarizes our current knowledge on the occurrence, biosynthesis, physiological functions, and biodegradation as well as on the properties and putative applications of these polyamides. Emphasis is placed on the enzymology of the polymerization and on the genes encoding the polymerizing enzymes, which have only recently become available for cyanophycin synthetases. Prospects for novel production processes. in particular for cyanophycin, are also presented.

The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase ( cphA ) and cyanophycinase ( cphB ), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-β- d -thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50°C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, l -arginine and l -aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, α-arginyl aspartic acid dipeptide, and poly-α,β- dl -aspartic acid as primers and also incorporated β-hydroxyaspartic acid instead of l -aspartic acid or l -canavanine instead of l -arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.

E2 (dihydrolipoamide acetyltransferase) and E3 (dihydrolipoamide dehydrogenase) of the Clostridium magnum acetoin dehydrogenase enzyme system were copurified in a three-step procedure from acetoin-grown cells. The denatured E2-E3 preparation comprised two polypeptides with M(r)s of 49,000 and 67,000, respectively. Microsequencing of both proteins revealed identical amino acid sequences. By use of oligonucleotide probes based on the N-terminal sequences of the alpha and beta subunits of E1 (acetoin dehydrogenase, thymine PPi dependent), which were purified recently (H. Lorenzl, F.B. Oppermann, B. Schmidt, and A. Steinbüchel, Antonie van Leeuwenhoek 63:219-225, 1993), and of E2-E3, structural genes acoA (encoding E1 alpha), acoB (encoding E1 beta), acoC (encoding E2), and acoL (encoding E3) were identified on a single ClaI restriction fragment and expressed in Escherichia coli. The nucleotide sequences of acoA (978 bp), acoB (999 bp), acoC (1,332 bp), and acoL (1,734 bp), as well as those of acoX (996 bp) and acoR (1,956 bp), were determined. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 35,532), E1 beta (M(r), 35,541), E2 (M(r), 48,149), and E3 (M(r), 61,255) exhibited striking similarities to the amino acid sequences of the corresponding components of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system and the Alcaligenes eutrophus acetoin-cleaving system, respectively. Significant homologies to the enzyme components of various 2-oxo acid dehydrogenase complexes were also found, indicating a close relationship between the two enzyme systems. As a result of the partial repetition of the 5' coding region of acoC into the corresponding part of acoL, the E3 component of the C. magnum acetoin dehydrogenase enzyme system contains an N-terminal lipoyl domain, which is unique among dihydrolipoamide dehydrogenases. We found strong similarities between the AcoR and AcoX sequences and the A. eutrophus acoR gene product, which is a regulatory protein required for expression of the A. eutrophus aco genes, and the A. eutrophus acoX gene product, which has an unknown function, respectively. The aco genes of C. magnum are probably organized in one single operon (acoABXCL); acoR maps upstream of this operon.

Cyanophycin [multi- l -arginyl-poly( l -aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae . The episomal vector systems pESC (with the galactose-inducible promoter GAL1 ) and pYEX-BX (with the copper ion-inducible promoter CUP1 ) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 ( cphA 6308 ) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.

By the use of Escherichia coli DH1 harboring cphA from Synechocystis sp. strain PCC6803, large-scale production of cyanophycin at 30- and 500-liter culture volumes was established. Transcription of cphA was controlled by the thermosensitive cI857 repressor, which enabled induction of cphA by a simple temperature shift in the culture fluid. Maximum cyanophycin cell content of up to 24% (wt/wt) of cellular dry matter was obtained by induction in the early exponential growth phase and cultivation of the cells in terrific broth complex medium. Synthesis of cyanophycin was found to be strongly dependent on the presence of complex components, and in mineral salts medium the cells synthesized and accumulated cyanophycin only if Casamino Acids were added. Cultivations were done at the 500-liter scale, allowing the provision of cell mass for the preparation of cyanophycin at the kilogram scale. Isolation of cyanophycin was achieved by a new acid extraction procedure which allowed large-scale purification of the polyamide from whole cells.

Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit with M(r) = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (beta-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in Escherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27-28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc((R)) and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of beta-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.

A 3878-bp genomic region from the cyanobacterium Synechocystis sp. strain PCC6308, amplified by inverse PCR, harbored the structural genes cphA (2625 bp) and cphB (819 bp) encoding cyanophycin synthetase and cyanophycinase, respectively. Both primary structures exhibited a high degree of similarity to the corresponding translational products from other cyanobacteria. Five regions were localized in the cyanophycin synthetase consensus sequence by their resemblance to conserved sites of ATP-dependent carboxylate-amine/thiol ligases and three substrate ligases. The functionality of cphA was proven by heterologous expression of active enzyme and synthesis of cyanophycin in Escherichia coli, which led to a maximum cyanophycin content of 26.6% (w/w) of cell dry mass. Furthermore, a modified radiometric enzyme assay for a more reliable and feasible measurement of cyanophycin synthetase activity was developed and applied to reveal the substrate specificity of the enzyme.

Use of oligonucleotide probes, which were deduced from the N-terminal sequences of the purified enzyme components, identified the structural genes for the alpha and beta subunits of E1 (acetoin:2,6-dichlorophenolindophenol oxidoreductase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehydrogenase) of the Pelobacter carbinolicus acetoin dehydrogenase enzyme system, which were designated acoA, acoB, acoC, and acoL, respectively. The nucleotide sequences of acoA (979 bp), acoB (1,014 bp), acoC (1,353 bp), and acoL (1,413 bp) as well as of acoS (933 bp), which encodes a protein with an M(r) of 34,421 exhibiting 64.7% amino acid identity to the Escherichia coli lipA gene product, were determined. These genes are clustered on a 6.1-kbp region. Heterologous expression of acoA, acoB, acoC, acoL, and acoS in E. coli was demonstrated. The amino acid sequences deduced from acoA, acoB, acoC, and acoL for E1 alpha (M(r), 34,854), E1 beta (M(r), 36,184), E2 (M(r), 47,281), and E3 (M(r), 49,394) exhibited striking similarities to the amino acid sequences of the components of the Alcaligenes eutrophus acetoin-cleaving system. Homologies of up to 48.7% amino acid identity to the primary structures of the enzyme components of various 2-oxo acid dehydrogenase complexes also were found. In addition, the respective genes of the 2-oxo acid dehydrogenase complexes and of the acetoin dehydrogenase enzyme system were organized very similarly, indicating a close relationship of the P. carbinolicus acetoin dehydrogenase enzyme system to 2-oxo acid dehydrogenase complexes.