Use of long-chain fatty acid-CoA ligase (AMP-forming) from Pseudomonas fragi for the ''in vitro'' synthesis of natural penicillins

Five different naturally occurring penicillins containing as side chains hexanoic, trans-3-hexenoic, heptanoic, octanoic or trans-3-octenoic acids have been synthesized 'in vitro' by coupling long-chain fatty acid-CoA ligase (AMP-forming) (EC 6.2.1.3) from Pseudomonas fragi (LFCoA-L) with acyl-CoA: 6-aminopenicillanic acid acyltransferase (AT) from Penicillium chrysogenum. The quantity of penicillin produced was directly related with the carbon length of the side chain precursor tested, being maximal with octanoic acid. Fatty acids with a lower length than C5 were not recognized as substrates and nor were certain aromatic molecules.     

Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid

A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized. PA-CoA ligase was specifically induced by PAA when P. putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source. Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length). PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity. The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively. The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate. PA-CoA ligase was purified to homogeneity (513-fold). It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa. PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes. The enzyme is detected in P. putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.     

Alginate oligosaccharides as enhancers of penicillin production in cultures of penicillium chrysogenum

Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 mug/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. (c) 1997 John Wiley & Sons, Inc.     

Specificity of Penicillin Acylase of Fusarium and of Penicillium chrysogenum

Extracts containing penicillin acylase were obtained by shaking the mycelium of Fusarium avenaceum and of Penicillium chrysogenum in 0.2 M sodium acetate or sodium chloride solution. The optimum pH for conversion of penicillin V into 6-aminopenicillanic acid (6-APA) by the enzyme of Fusarium was about 7.5, and the reaction velocity was increased by a rise in temperature from 27 to 37 C. Penicillin G and penicillins with an aliphatic side chain were cleaved much less readily than was penicillin V. With the enzyme preparation obtained from a nonpenicillin-producing strain of P. chrysogenum, the reaction rate was higher at pH 8.5 than at pH 7.5 and pH 6.5. The acylase of P. chrysogenum hydrolyzes penicillin V more readily than penicillin G. In a series of aliphatic penicillins, the amount of 6-APA formed through the action of this enzyme increased with the number of carbon atoms of the side chain. Penicillins with a glutaryl or an adipyl group as side chain were unaffected by the enzyme of Fusarium and of Penicillium. No reaction was observed upon incubation of penicillin N (with a D-aminoadipyl side chain) or isopenicillin N (with an L-aminoadipyl side chain) with Fusarium and Penicillium extract. When the carboxy group of the side chain of these penicillins was esterified, formation of 6-APA was observed upon incubation with Penicillium extract, whereas no 6-APA or only very small amounts were obtained by acylase of Fusarium.     

Carbon catabolite regulation of phenylacetyl-CoA ligase from Pseudomonas putida

Phenylacetyl-CoA ligase (PA-CoA ligase) from P. putida U is a newly described enzyme involved in the aerobic catabolism of phenylacetic acid. The enzyme was specifically induced when P. putida was grown in a chemically defined medium containing phenylacetic acid as the sole carbon source. The induction of PA-CoA ligase was delayed by adding easily metabolizable carbon sources to the medium; the effect was more drastic in the presence of glucose. Glucose did not cause catabolic inactivation but rather catabolic repression, this effect being reversed by cAMP.     

Why did the Fleming strain fail in penicillin industry?

Penicillin, discovered 75 years ago by Sir Alexander Fleming in Penicillium notatum, laid the foundations of modern antibiotic chemotherapy. Early work was carried out on the original Fleming strain, but it was later replaced by overproducing strains of Penicillium chrysogenum, which became the industrial penicillin producers. We show how a C(1357)-->T (A394V) change in the gene encoding PahA in P. chrysogenum may help to explain the drawback of P. notatum. PahA is a cytochrome P450 enzyme involved in the catabolism of phenylacetic acid (PA; a precursor of penicillin G). We expressed the pahA gene from P. notatum in P. chrysogenum obtaining transformants able to metabolize PA (P. chrysogenum does not), and observing penicillin production levels about fivefold lower than that of the parental strain. Our data thus show that a loss of function in P. chrysogenum PahA is directly related to penicillin overproduction, and support the historic choice of P. chrysogenum as the industrial producer of penicillin.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

Aerobic catabolism of phenylacetic acid in Pseudomonas putida U: biochemical characterization of a specific phenylacetic acid transport system and formal demonstration that phenylacetyl-coenzyme A is a catabolic intermediate.

The phenylacetic acid transport system (PATS) of Pseudomonas putida U was studied after this bacterium was cultured in a chemically defined medium containing phenylacetic acid (PA) as the sole carbon source. Kinetic measurement was carried out, in vivo, at 30 degrees C in 50 mM phosphate buffer (pH 7.0). Under these conditions, the uptake rate was linear for at least 3 min and the value of Km was 13 microM. The PATS is an active transport system that is strongly inhibited by 2,4-dinitrophenol, 4-nitrophenol (100%), KCN (97%), 2-nitrophenol (90%), or NaN3 (80%) added at a 1 mM final concentration (each). Glucose or D-lactate (10 mM each) increases the PATS in starved cells (140%), whereas arsenate (20 mM), NaF, or N,N'-dicyclohexylcarbodiimide (1 mM) did not cause any effect. Furthermore, the PATS is insensitive to osmotic shock. These data strongly suggest that the energy for the PATS is derived only from an electron transport system which causes an energy-rich membrane state. The thiol-containing compounds mercaptoethanol, glutathione, and dithiothreitol have no significant effect on the PATS, whereas thiol-modifying reagents such as N-ethylmaleimide and iodoacetate strongly inhibit uptake (100 and 93%, respectively). Molecular analogs of PA with a substitution (i) on the ring or (ii) on the acetyl moiety or those containing (iii) a different ring but keeping the acetyl moiety constant inhibit uptake to different extents. None of the compounds tested significantly increase the PA uptake rate except adipic acid, which greatly stimulates it (163%). The PATS is induced by PA and also, gratuitously, by some phenyl derivatives containing an even number of carbon atoms on the aliphatic moiety (4-phenyl-butyric, 6-phenylhexanoic, and 8-phenyloctanoic acids). However, similar compounds with an odd number of carbon atoms (benzoic, 3-phenylpropionic, 5-phenylvaleric, 7-phenylheptanoic, and 9-phenylnonanoic acids) as well as many other PA derivatives do not induce the system, suggesting that the true inducer molecule is phenylacetyl-coenzyme A (PA-CoA). Furthermore, after P. putida U is cultured in the same medium containing other carbon sources (glucose or octanoic, benzoic, or 4-hydroxyphenylacetic acid) in the place of PA, the PATS and PA-CoA are not detected; neither the PATS nor PA-CoA is found in cases in which mutants (PA- and PCL-) lacking the enzyme which catalyzed the initial step of the PA degradation (phenylacetyl-CoA ligase) are used. PA-CoA has been extracted from bacteria and identified as a true PA catabolite by high-performance liquid chromatography and also enzymatically with pure acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum.     

Increased Penicillin Production in Penicillium chrysogenum Production Strains via Balanced Overexpression of Isopenicillin N Acyltransferase

Intense classical strain improvement has yielded industrial Penicillium chrysogenum strains that produce high titers of penicillin. These strains contain multiple copies of the penicillin biosynthesis cluster encoding the three key enzymes: δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS), isopenicillin N synthase (IPNS), and isopenicillin N acyltransferase (IAT). The phenylacetic acid coenzyme A (CoA) ligase (PCL) gene encoding the enzyme responsible for the activation of the side chain precursor phenylacetic acid is localized elsewhere in the genome in a single copy. Since the protein level of IAT already saturates at low cluster copy numbers, IAT might catalyze a limiting step in high-yielding strains. Here, we show that penicillin production in high-yielding strains can be further improved by the overexpression of IAT while at very high levels of IAT the precursor 6-aminopenicillic acid (6-APA) accumulates. Overproduction of PCL only marginally stimulates penicillin production. These data demonstrate that in high-yielding strains IAT is the limiting factor and that this limitation can be alleviated by a balanced overproduction of this enzyme.     

Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum

Homocitrate synthase in the first enzyme of the lysine biosynthetic pathway. It is feedback regulated by L-lysine. Lysine decreases the biosynthesis of penicillin (determined by the incorporation of [14C]valine into penicillin) by inhibiting and repressing homocitrate synthase, thereby depriving the cell of alpha-aminoadipic acid, a precursor of penicillin. Lysine feedback inhibited in vivo the biosynthesis and excretion of homocitrate by a lysine auxotroph, L2, blocked in the lysine pathway after homocitrate. Neither penicillin nor 6-aminopenicillanic acid exerted any effect at the homocitrate synthase level. The molecular mechanism of lysine feedback regulation in Penicillium chrysogenum involved both inhibition of homocitrate synthase activity and repression of its synthesis. In vitro studies indicated that L-lysine feedback inhibits and represses homocitrate synthase both in low- and high-penicillin-producing strains. Inhibition of homocitrate synthase activity by lysine was observed in cells in which protein synthesis was arrested with cycloheximide. Maximum homocitrate synthase activity in cultures of P. chrysogenum AS-P-78 was found at 48 h, coinciding with the phase of high rate of penicillin biosynthesis.     

Characterization of a phenylacetate-CoA ligase from Penicillium chrysogenum

Enzymatic activation of PAA (phenylacetic acid) to phenylacetyl-CoA is an important step in the biosynthesis of the beta-lactam antibiotic penicillin G by the fungus Penicillium chrysogenum. CoA esters of PAA and POA (phenoxyacetic acid) act as acyl donors in the exchange of the aminoadipyl side chain of isopenicillin N to produce penicillin G or penicillin V. The phl gene, encoding a PCL (phenylacetate-CoA ligase), was cloned in Escherichia coli as a maltose-binding protein fusion and the biochemical properties of the enzyme were characterized. The recombinant fusion protein converted PAA into phenylacetyl-CoA in an ATP- and magnesium-dependent reaction. PCL could also activate POA, but the catalytic efficiency of the enzyme was rather low with k(cat)/K(m) values of 0.23+/-0.06 and 7.8+/-1.2 mM(-1).s(-1) for PAA and POA respectively. Surprisingly, PCL was very efficient in catalysing the conversion of trans-cinnamic acids to the corresponding CoA thioesters [k(cat)/K(m)=(3.1+/-0.4)x10(2) mM(-1).s(-1) for trans-cinnamic acid]. Of all the substrates screened, medium-chain fatty acids, which also occur as the side chains of the natural penicillins F, DF, H and K, were the best substrates for PCL. The high preference for fatty acids could be explained by a homology model of PCL that was constructed on the basis of sequence similarity with the Japanese firefly luciferase. The results suggest that PCL has evolved from a fatty-acid-activating ancestral enzyme that may have been involved in the beta-oxidation of fatty acids.     

Penicillium chrysogenum Takes up the Penicillin G Precursor Phenylacetic Acid by Passive Diffusion

Penicillium chrysogenum utilizes phenylacetic acid as a side chain precursor in penicillin G biosynthesis. During industrial production of penicillin G, phenylacetic acid is fed in small amounts to the medium to avoid toxic side effects. Phenylacetic acid is taken up from the medium and intracellularly coupled to 6-aminopenicillanic acid. To enter the fungal cell, phenylacetic acid has to pass the plasma membrane. The process via which phenylacetic acid crosses the plasma membrane was studied in mycelia and liposomes. Uptake of phenylacetic acid by mycelium was nonsaturable, and the initial velocity increased logarithmically with decreasing external pH. Studies with liposomes demonstrated a rapid passive flux of the protonated species through liposomal membranes. These results indicate that phenylacetic acid passes the plasma membrane via passive diffusion of the protonated species. The rate of phenylacetic acid uptake at an external concentration of 3 mM is at least 200-fold higher than the penicillin production rate in the Panlabs P2 strain. In this strain, uptake of phenylacetic acid is not the rate-limiting step in penicillin G biosynthesis.     

Industrial production of beta-lactam antibiotics

The industrial production of beta-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the beta-lactam antibiotics, particularly penicillins and cephalosporins, represent the world's major biotechnology products with worldwide dosage form sales of approximately 15 billion US dollars or approximately 65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (approximately 80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.     

Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.     

alpha-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum

Intracellular amino acid pools in four Penicillium chrysogenum strains, which differed in their ability to produce penicillin, were determined under conditions supporting growth without penicillin production and under conditions supporting penicillin production. A significant correlation between the rate of penicillin production and the intracellular concentration of alpha-aminoadipate was observed, which was not shown with any other amino acid in the pool. In replacement cultivation, penicillin production was stimulated by alpha-aminoadipate, but not by valine or cysteine. Exogenously added alpha-aminoadipate (2 or 3 mM) maximally stimulated penicillin synthesis in two strains of different productivity. Under these conditions intracellular concentrations of alpha-aminoadipate were comparable in the two strains in spite of the higher rate of penicillin production in the more productive strain. Results suggest that the lower penicillin titre of strain Q 176 is due to at least two factors: (i) the intracellular concentration of alpha-aminoadipate is insufficient to allow saturation of any enzyme which is rate limiting in the conversion of alpha-aminoadipate to penicillin and (ii) the level of an enzyme, which is rate limiting in the conversion of alpha-aminoadipate to penicillin, is lower in Q 176 (relative to strain D6/1014/A). Results suggest that the intracellular concentration of alpha-aminoadipate in strain D6/1014/A is sufficiently high to allow saturation of the rate-limiting penicillin biosynthetic enzyme in that strain. The basis of further correlation of intracellular alpha-aminoadipate concentration and penicillin titre among strains D6/1014/A, P2, and 389/3, the three highest penicillin producers studied here, remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of δ-(L-α-aminoadipyl)-l-cysteinyl-d-valine and isopenicillin N biosynthesis in Penicillium chrysogenum by the α-aminoadipate pool size

The effect of changes in the intracellular concentration of alpha-aminoadipate on the formation of alpha-aminoadipyl-cysteinyl-valine (ACV) and isopenicillin N (IPN)--two intermediates of penicillin biosynthesis--by strains of Penicillium chrysogenum has been investigated by measuring the incorporation of radioactivity from (6-14C)-alpha-aminoadipate into cellular 14C-ACV and 14C-IPN. No ACV or IPN were found in any strain during cultivation on glucose, but were clearly detected in all three strains during growth on lactose, displaying increased formation in strains exhibiting increased penicillin productivity and increased intracellular alpha-aminoadipate pools. ACV and IPN formation was affected by subjected P. chrysogenum mycelia to either general amino acid control (by addition of amitrol) or by exogenous addition of 5 mM L-lysine. In all cases, the changes observed paralleled the changes in the intracellular alpha-aminoadipate pool. These results are consistent with the alpha-aminoadipate pool limiting the biosynthesis of ACV and IPN and hence penicillin biosynthesis in the present strains of P. chrysogenum.     

产黄青霉谷胱甘肽S-转移酶基因PcgstA的克隆与鉴定

从青霉素工业生产菌产黄青霉(Penicilliumchrysogenum)中首次克隆了一个谷胱甘肽S-转移酶(GST)基因,定名为PcgstA.该基因的开放阅读框长840bp,含有两个内含子,编码一个238氨基酸残基的蛋白质.其推断的氨基酸序列与一些已经鉴定的丝状真菌GST具有50%左右的序列一致性.PcgstA的完整编码区经RT-PCR扩增、验证,插入原核表达载体pET11a,转化大肠杆菌BL21(DE3)-RP菌株,表达得到重组PcGSTA蛋白.酶活测定证实,重组PcGSTA具有GST活性,其对底物CDNB(1-chloro-2,4-dinitrobenzene)的比活为(0.159±0.031)μmol/(min·mg).利用TaqMan探针法,对PcgstA的表达情况进行了比较.结果表明,在添加了侧链前体苯乙酸的青霉素生产培养基中,PcgstA的表达水平和在不含苯乙酸培养基中的表达相比明显下调,显示了该基因与苯乙酸代谢的关系.