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Scale-up of bialaphos production from a 3 l jar fermentor to a 300 kl fermentor was investigated. When the scale-up was based on power consumption (for agitation) per unit volume of the reactor, bialaphos production in the 300 kl fermentor was remarkably lower than that obtained with the 3 l jar fermentor. The reason for the unsuccessful scale-up was investigated by considering the major differences between the 3 l jar and the 300 kl fermentor. It was found that variation in the shear stress had no significant effect on bialaphos production. A special pressure-proof fermentor with pressure control system was used to study the effect of liquid pressure gradient in the 300 kl fermentor. When the cells in the special fermentor were subjected to cyclic variation in pressure (between 1,013 and 1,994 HPa) as though they are circulated between the top and the bottom of a 10 m liquid level in the 300 kl fermentor, bialaphos production was as low as that obtained in the 300 kl fermentor. The optimum DO concentration for bialaphos production in the 3 l fermentor was found to be 0.5 ppm and above this concentration, an inverse relationship between the DO concentration and bialaphos production was observed. The inhibition of bialaphos production at high pressure was therefore attributed to the increase in the DO concentration at high liquid pressures. When the scale-up was based on the DO concentration and the DO at the middle of the 300 kl fermentor was controlled at 0.5 ppm, bialaphos production increased to 85 % of that obtained with the 3 l jar fermentor. By taking the DO concentration gradient in the 300 kl fermentor into consideration, the DO at the bottom of the fermentor was maintained at 0.5 ppm. This resulted in a successful scale-up wherein bialaphos production increased to 96% of the maximum production in the 3 l jar fermentor.

We have developed a new series of R4L1 Gateway binary vectors (R4L1pGWB), which carry the bialaphos resistance gene (bar) or the UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as selection markers that confer BASTA® and tunicamycin resistance on plants respectively. R4L1pGWBs have an attR4-attL1-reporter and can accept an attL4-promoter-attR1 entry clone for easy construction of an attB4-promoter-attB1-reporter clone. The new R4L1pGWBs facilitate promoter:reporter analysis in pre-existing transgenic plants that are resistant to kanamycin or hygromycin.

We developed a simple and rapid method for the simultaneous determination of phosphorus-containing amino acid herbicides (glyphosate, glufosinate, bialaphos) and their major metabolites, aminomethylphosphonic acid (AMPA) and 3-methylphosphinicopropionic acid (MPPA), in human serum. Serum samples were filtrated through an ultrafiltration membrane to remove proteins. The filtrate was then washed with chloroform, and injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. Chromatographic separation was achieved on a hydrophilic interaction chromatography (HILIC) column. Determination of the target herbicides and metabolites was successfully carried out without derivatization or solid phase extraction (SPE) cartridge clean-up. The recoveries of these compounds, added to human serum at 0.2μg/mL, ranged from 94% to 108%, and the relative standard deviations (RSDs) were within 5.9%. The limits of detection (LODs) were 0.01μg/mL for MPPA, 0.02μg/mL for AMPA, 0.03μg/mL for both glyphosate and glufosinate, and 0.07μg/mL for bialaphos, respectively.

A sensitive gas chromatographic (GC)—ion-trap mass spectrometric (IT-MS) method has been developed to determine simultaneously the herbicides glyphosate, glufosinate and bialaphos and their major metabolises. A single-step derivatization is achieved at 80°C for 30 min with the reagent N-methyl-N-( tert. -butyldimethylsilyl)trifluoroacetamide in dimethylformamide, which introduces the tert. -butyldimethylsilyl group at active hydrogens and gives only a single peak for each compound. The derivatives of three herbicides, their metabolises and nineteen amino acids were simultaneously chromatographed and well separated in a single run on a DB-1 fused-silica capillary column. Each tert. -butyldimethylsilylated derivative produces an easily interpretable mass spectrum dominated by unique M - 15, M - 57, M - 85 and M - 159 fragment ions. The limits of detection were estimated to be 10–20 ng for glyphosate and glufosinate and their metabolises, and 500 ng for bialaphos, by GC-IT-MS. On the other hand, using GC analysis with flame ionization detection, glyphosate and glufosinate and their metabolises were detectable at levels of approximately 100 ng, but bialaphos could not be detected at a level of 5000 ng.

Bialaphos [a herbicide, 2-amino-4-(hydroxy)(methyl)-phospinoylbutyryl-alanyl-alanine] produced by Streptomyces hygroscopicus increased significantly as the oxygen supply decreased during both growth and production phases of batch culture. Under low oxygen partial pressure, there were decreases in sugar consumption by the cells, cell concentration and the activity of tricarboxylic acid cycle enzymes in the cells. On the other hand, the activity of glyoxylic acid cycle enzymes in the cells increased. These phenomena were more prominent in high producing than in low producing strains. From these results, it is suggested that in high bialaphos producing strains, low oxygen partial pressure suppresses the activity of tricarboxylic acid enzymes so that both acetyl-CoA and pyruvate (the substrates for bialaphos synthesis) are used mainly for secondary metabolism. This results in a high production of bialaphos.

Bialaphos foliar sprays were tested in eastern Quebec for control of red raspberry (Rubusidaeus L.), and the tolerance of planted black spruce (Piceamariana (Mill.) B.S.P.) seedlings was determined. Approximately 3 weeks after bialaphos was applied in July, August, or September, virtually all raspberry plants were killed. Efficiency of the June application, however, was much reduced. Spruce current-year foliage was more affected than older foliage. Foliar injury was more pronounced for bialaphos applications in June and July than for applications in August. First-year spruce survival was not affected by the treatments. Second-year evaluations revealed a significant quadratic relation of (i) month of application × bialaphos rate on raspberry density and (ii) bialaphos rate on raspberry height growth. Reduction in raspberry density and height growth was greater with the 2.0 and 2.5 kg active ingredient per hectare rates applied in August. As part of the evaluation of treatment efficiency, photosynthetically active radiation reaching spruce seedlings was measured using a portable integrating radiometer. Raspberry density and height growth were both negatively correlated with photosynthetically active radiation reaching spruce seedlings. A significant quadratic relation was found between bialaphos rate and 2nd-year spruce survival, which reached 95%, regardless of month and rate of application. A significant quadratic relation was found between spruce diameter growth increment and bialaphos rate. Except for calcium, 2nd-year spruce foliar mineral content was not affected by the treatments. Bialaphos treatments in August, with rates ranging from 1.0 to 2.0 kg active ingredient per hectare, had only slight or no adverse effects on spruce survival, foliar injury, and growth increment. Selective control of red raspberry in young black spruce plantations is a function of bialaphos rate and month of application.

The biosynthesis of bialaphos [a herbicide, 2-amino-4(hydroxy)(methyl)-phosphinoylbutyryl-alanylalanine] produced by Streptomyces hygroscopicus , proceeds through the degradation of glucose to phosphoenol pyruvate, the formation of a CP bond, acetic addition by acetyl-CoA and alanine addition. Therefore, bialaphos formation is considered to be closely related to glucose metabolism. Based on this hypothesis, sugar consumption and the activities of the tricarboxylic acid cycle enzymes were examined using a highly productive strain and a strain of the lower productivity. It was clear that the highly productive strain has a lower sugar consumption rate and lower yield of cells, compared with the lower productivity strain. The activities of the tricarboxylic acid cycle enzymes of the highly productive strain were lower than those of the lower productivity strain, while the activities of the glyoxylic acid cycle enzymes of the highly productive strain were higher. From these findings, it is suggested that the highly productive strain suppresses the flow from acetyl-CoA and pyruvate (as substrates of bialaphos) to the tricarboxylic acid cycle, and efficiently directs these substrates to the secondary metabolism by activation of the glyoxylic acid cycle, resulting in a high rate of bialaphos production in this strain.

A 64-year-old man with ethanol intoxication, ingested a bottle of Herbiace (100 ml, 32 w/v% of bialaphos, CAS #35597-43-4, Meiji Seika Kaisha, Tokyo, Japan). He had severe metabolic acidosis and was treated with infusions of sodium bicarbonate and furosemide, plus gastric lavage and enema. The metabolic acidosis improved 15 hours after treatment but nystagmus, apnea and convulsions were progressive.Although his sensorium was clear, spontaneous respirations were not observed for 64 hours. The electroencephalographic findings of atypical triphasic waves and slow waves suggest a unique response to bialaphos poisoning. His clinical course indicates that the management of apnea is critically important to recovery from bialaphos poisoning.

Bialaphos, l -2-amino-4-[(hydroxy) (methyl) phosphinoyl]butyryl- l -alanyl- l -alanine, induced a high level of sterility in rice, when applied

To study the relationship between citrate synthase and 2-phosphinomethylmalic acid (PMM) synthase, which catalyzes a very similar reaction comparable to citrate formation in the biosynthesis of a herbicide, bialaphos, citrate synthase was purified from the mycelium of Streptomyces hygroscopicus SF-1293, a bialaphos-producing organism. The overall purification was 440-fold with a yield of 4.4% from cell-free extract. Based on comparison with PMM synthase, it has been concluded that citrate synthase of S. hygroscopicus is quite different from PMM synthase in several aspects such as enzymatic properties, amino acid composition, N-terminal amino acid sequence, and stereo-chemical reaction mechanism.

A gene ( bar ) was identified adjacent to the hrdD sigma factor gene in Streptomyces coelicolor A3(2). The predicted bar product showed 32.2% and 30.4% identity to those of the pat and bar genes of the bialaphos (Bp) producers Streptomyces viridochromogenes and Streptomyces hygroscopicus , respectively; these genes encode phosphinothricin (PPT) N -acetyltransferases that function as enzymes in the Bp biosynthetic pathway and as resistance determinants. The S. coelicolor bar gene conferred high-level resistance to Bp when cloned in S. coelicolor on a high-copy-number vector. Enzymic assay showed that the S. coelicolor bar gene product inactivates PPT by transfer of acetyl groups from acetyl CoA. The S. coelicolor bar gene appears to be expressed from two promoters ( p 1 and p 2) and is divergently transcribed with respect to hrdD . The downstream ( barp2 ) transcript overlaps the hrdDp1 transcript and the upstream ( barp1 ) transcript overlaps both the hrdDp1 and hrdDp2 transcripts. Inactivation of hrdD did not prevent transcription from either bar promoter, indicating that σ hrdD is not essential for recognition of these sequences.

Bialaphos (BA) is a tripeptide produced by Streptomyces hygroscopicus SF-1293 and is characterized by the presence of a unique C-P-C bond. Through the biosynthetic studies on BA we have revealed the involvement of three enzymes catalyzing different C-P bond formation mechanisms. Two of them, phosphoenolpyruvate phosphomutase (PEP phosphomutase) and carboxyphosphonoenolpyruvate phosphonomutase (CPEP phosphonomutase), have been purified and characterized to catalyze the reactions of a similar type, i.e., intramolecular rearrangements of phosphate esters to form a C-P bond. On the other hand, the remaining one catalyzes P-methylation of phosphinic acid derivatives. In order to reveal these enzymatic properties in more detail, the corresponding genes were identified in the biosynthetic cluster of BA and then expressed in S. lividans. Introduction of PEP phosphomutase or CPEP phosphonomutase gene into S. lividans using pIJ680 resulted in efficient expression of the corresponding enzymes. On the other hand, the P-methylation enzyme was only expressed in S. lividans when the gene was under control of a thiostrepton-inducible strong promoter of pAK114.

One of the three C-P bond formation steps, defined as step 5 in the bialaphos (BA) biosynthetic pathway, was analyzed using a new BA non-producing mutant NP71. The mutant was derived from a BA producer by gene replacement of an unidentified region next to the gene responsible for the step 5 deficiency of the mutant NP213, obtained by conventional mutation procedures. Biochemical analysis of these two mutants indicated that NP71 was defective in the formation of carboxyphosphonoenolpyruvate (CPEP), while NP213 lacked the enzyme CPEP phosphonomutase, which catalyzed the intramolecular rearrangement of CPEP.

In bialaphos (a herbicide) fermentation by Streptomyces hygroscopicus , production of bialaphos is greatly reduced when the partial pressure of oxygen is increased. In order to determine the inhibitory effect of high oxygen partial pressure, the culture filtrate was analyzed revealing that large amounts of two possible biosynthetic intermediates of bialaphos were accumulated. These two compounds were identified to be C-P compounds having an amino group, one as a demethyl derivative of bialaphos (MP-102) and the other a demethyl derivative of AMPB [dealaninated bialaphos] (MP-101). It was also demonstrated that these demethyl derivatives were converted to bialaphos when they were added to the culture under low oxygen partial pressure, whereas the conversion was suppressed under high oxygen partial pressure. As a conclusion, methylation at the final step of the biosynthetic process of bialaphos did not proceed under high oxygen partial pressure, resulting in a low bialaphos concentration due to the accumulation of the demethyl derivatives.

Genetic studies suggest that either N-acetyldemethyl phosphinothricin (1, N-AcDMPT) or N-acetyldemethyl phosphinothricin tripeptide (2, N-AcDMPTT) is the substrate for the P-methylation reaction in the biosynthesis of phosphinothricin tripeptide (PTT), which is widely used as an herbicide. To study the mechanism for this unique P-methylation reaction catalyzed by the BcpD protein and the functions of the unusual nonribosomal peptide synthetases involved in PTT biosynthesis, this work reports the chemical syntheses of 1 and 2.

A 47-year-old Japanese woman undergoing maintenance hemodialysis (HD) was admitted to our hospital because of poisoning with the herbicide bialaphos. Respiratory arrest and loss of consciousness ensued rapidly, accompanied by convulsions and nystagmus. Treatment with HD and direct hemoperfusion, followed by HD alone, effectively removed bialaphos and its chief toxic metabolite (L-AMPB) from the circulation (bialaphos decreased from 0.33 to0.05 microg/ml and L-AMPB from 14 to 0.86 microg/ml). The glutamate concentration improved gradually after the removal of bialaphos and L-AMPB from plasma (plasma glutamate concentration: 250.4 nmol/l on day 5 to 120.6 nmol/l on day 26). Decreased glutamine concentration in cerebrospinal fluid was demonstrated for the first time as well as in plasma, indicating glutamine synthetase inhibition not only in plants but also in humans by bialaphos poisoning.

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml −1 , respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1–45, 0.3–32 and 0.1–50 μg ml −1 , respectively. The relative standard deviations ( n =5) were 1.7–3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.

A small plot experiment was carried out to investigate the effect of bialaphos, a microbially produced phytotoxin, on kalmia. Bialaphos was tested at five rates of application, including the control, and three dates of application, representing three phenological stages. Three weeks after bialaphos application in June and August, a complete defoliation of kalmia stems was observed. Second-year results revealed a significant quadratic effect of bialaphos rate on kalmia resurgence and a significant linear effect of bialaphos rate on kalmia height growth. Best control of kalmia resurgence (0 8/m²) and height growth (3.1 cm) was achieved with the highest application rate (2.5 kg a.i./ha). For both variables, the interaction between month of application and rate of application was not significant. The results obtained suggest that bialaphos could efficiently control kalmia on disturbed sites and thus prevent the establishment of an unproductive and persistent shrub seral stage. North. J. Appl. For. 8(4):147-149.

Bar gene conferring resistance to herbicide bialaphos (phmphinothricin) was cloned from Streptomyces hygroscopicus. Bar gene under 35S promoter of cauliflower mosaic virus was introduced in binary pBin19 vector and constructed plasmid was transferred into Agrcbacterium tumefaciens strain. Conditions of genetic transformation of cultivated buckwlwat and interspecific hybrid Fagopirum esculentum X F. tataricum were worked out. Buckwheat explants were inoculated by the strain with a plasmid carrying bar gene nearby NPTII gene. Molecular analysis of 8 regenereted plants that were selected for kanamycin resistance was performed. 5 plants gave positive signal by dol- and Southern hybridization using as a probe DNA fragment with bar gene that is an evidence of integration of bar gene into plant genome. Transformed plants grow and rooted at Basta concentrations in the medium, that totally inhibited nontransformed plants. Із Streptomyces hygroscopicus клонували bar-ген, який визначає стійкість до гербіціду біалафосу (фосфінотрицину). Ген під контролем 35S-промотора вірусу мозаїки цвітної капусти було введено у бінарний вектор рВin19 для трансформації рослин. Одержану рекомбінантну плазміду перенесено до шта­му 3850 Agrobacterium tumefaciens. Підібрано умови трансформації культурної гоечхи і міжвидового гібрида Fagopyrum esculentum х F. tatarium. Експланти гречки інокулювали агробактеріальним штамом, у якому bar-ген знаходиться на Тi плазміді поруч з NPTII геном. Здійснено молекулярно-біо­логічний аналіз восьми регенерантів, які пройшли канаміщновий відбір. П'ять рослин дали позитивну радіоавтогра­фічну відповідь при дот- і Саузерн-гібридизації з міченим зондом, який несе bar-ген, що свідчить про інтеграцію цього гена в геном рослин. Трансформовані рослини росли і утворю­вали коріння при концентрації гербіциди басти в середовищі, яке повністю пригнічувало нетрансформовані рослини, Из Streptomyces hygroscopicus клонировали bar-ген, определяю­щий устойчивость н гербициду биалафосу (фосфинотрицину). Ген под контролем 35S-промотора вируса мозаики цветной капусты был введен в бинарный вектор рВіп!9 для трансфор-мании растений. Полученная рекомбинантная плазмида перенесена в штамм 3850 Agrobacterium tumefaciens. Подобраны условия трансформации культурной гречихи и межвидового гибрида Fagopyrum esculentum X F. taiaricum. Экспланты гречи­хи инокулировали агробактериальным штаммом, в котором bar-ген находился на Тi плазмиде рядом с геном NPTII Проведен молекулярно-биологический анализ восьми регенераншов, прошедших канамициновый отбор. Пять растений дали положительный радиоавтографический ответ при дот- и Саузерн-гибридизации с меченым зондом, несущим bar-ген, что свидетельствует об интеграции этого гена в геном растений. Трансформированные растения росли и образовывали корни при концентрации гербицида басты в среде, полностью ингибирующей не трансформированные растения.

Publisher Summary This chapter provides an overview on the bialaphos (BA). Streptomyces hygroscopicus and Streptomyces viridochromogenes have the ability to convert intermediates of primary metabolism into BA, a tripeptide with a unique structure not identified anywhere else in nature. BA contains two L-alanine residues and an amino acid called phosphinothricin. The antimicrobial activity of BA results from its interactions with two different ubiquitous biological systems needed for peptide catabolism and nitrogen anabolism. PT is relatively nontoxic to most bacteria as amino acid uptake systems that are highly specific do not recognize it as glutamate. The biochemical studies that define the pathway of BA biosynthesis also provide a series of well characterized mutant strains defective in various conversions. Among a number of compounds related to BA in possessing reduced phosphate groups, 2-aminoethylphosphonic acid, fosfomycin, and FR33289 are biosynthesized from glucose via phosphoenolpyruvate. The possibility that the BA biosynthesis gene cluster might have been horizontally transferred between phylogenetically different organisms makes it impossible to determine the host in which the cluster is first assembled.

Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity profile of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411.

Summary The bialaphos resistance gene, bar, was used as a selectable marker to isolate the bialaphos production genes (bap) from the Streptomyces viridochromogenes genome. The S. viridochromogenes bar gene was cloned on overlapping restriction fragments using pIJ680 and pIJ702 in the bialaphos-sensitive host, S. lividans. Although the restriction endonuclease cleavage map of these fragments was not similar to the bap cluster of S. hygroscopicus, the presence and location of bar and four other bap genes as well as a gene required for the transcriptional activation of the cluster (brpA) was demonstrated by heterologous cloning experiments using a series of previously characterized bialaphos-nonproducing S. hygroscopicus mutants. Since recombination-deficient mutants of streptomycetes have not been isolated, restored function provided by cloned homologous DNA results from both recombination (marker rescue) and complementation in trans. In contrast to our previously reported homologous cloning experiments where we were able to define the position of mutant alleles by recombination, in these heterologous cloning experiments we observed little if any recombination between plasmid-cloned genes and the chromosome. As a result, this approach allowed us to define the location and orientation of functional genes using a genetic complementation test. The organization of the clustered S. viridochromogenes bap genes was indistinguishable from the corresponding S. hygroscopicus mutant alleles. The fact that the S. viridochromogenes transcriptional regulatory gene, brpA, functioned in S. hygroscopicus implied that some transcriptional regulatory signals may also be interchangeable. In these two Streptomyces species, which have considerable nucleotide sequence divergence, the complex biochemical and genetic organization of the bialaphos biosynthetic pathway is conserved.

Two oligopeptides containing phosphinothricin were accumulated in the culture of bialaphos (BA) producer Streptomyces hygroscopicus SF1293 when large amount of BA was added to the culture at idio phase. One of the oligopeptides was a new substance, BA dimer (phosphinothricyl-alanyl-alanyl-phosphinothricyl-alanyl-alan ine), and the other was a known substance called If (phosphinothricyl-alanyl-alanyl-phosphinothricin). Though BA none producing mutants which were blocked at the steps 1, 10 and 13, respectively in BA biosynthesis also converted BA to BA dimer and If, the mutants blocked at the step 11 (alanylation) could not curry out the conversion. Antibiotic activities of BA dimer and If were lower than 1/100 of BA.

Bialaphos resistant rice plants expressing a bar gene under the control of the maize ubiquitin promoter which were inoculated with mycelia of the sheath blight disease pathogen, Rhizoctonia solani, and subsequently treated with the herbicide were completely protected from symptomatic infection. Substantial suppression of sheath blight symptoms was also recorded when bialaphos was applied to transgenic plants which had been infected with R. solani two days before herbicide treatment. It may therefore be possible to design new agronomic strategies for the simultaneous control of weeds and fungal pathogens in fields of transgenic plants expressing a bar gene.

The nucleotide (nt) sequence of the Streptomyces hygroscopicus gene encoding carboxyphosphonoenolpyruvate (CPEP) synthase, that catalyzes a transesterification between phosphoenolpyruvate (PEP) and phosphonoformate (PF) in the bialaphos biosynthetic pathway, has been determined. The amino-acid sequence deduced from the nt sequence is similar to several eukaryotic 2-phospho-D-glycerate hydrolases (EC 4.2.1.11).

The nucleotide sequence of the Streptomyces hygroscopicus gene encoding P-methyltransferase, catalyzing the formation of a carbon-phosphorus bond, involved in bialaphos biosynthesis, has been determined. The amino-acid sequence deduced from the nt sequence, shows homology with those of magnesium-protoporphyrin IX monomethyl ester oxidative cyclase (Mg-ProtoMe cyclase) of Rhodobacter capsulatus and the enzyme catalyzing the methylation of the aldehyde carbon of phosphonoacetaldehyde in fosfomycin biosynthesis.