Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.     

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.     

Homocitrate synthase from Penicillium chrysogenum. Localization, purification of the cytosolic isoenzyme, and sensitivity to lysine.

Subcellular fractionation of cell-free extracts obtained by nitrogen cavitation showed that Penicillium chrysogenum Q176 contains a cytosolic as well as a mitochondrial homocitrate synthase activity. The cytosolic isoenzyme was purified about 500-fold, and its kinetic and molecular properties were investigated. Native homocitrate synthase shows a molecular mass of 155 +/- 10 kDa as determined by gel filtration and a pH of 4.9 +/- 0.1 as determined by chromatofocusing. The kinetic behaviour towards 2-oxoglutarate is hyperbolic, with Km = 2.2 mM; with respect to acetyl-CoA the enzyme shows sigmoidal saturation kinetics, with [S]0.5 = 41 microM and h = 2.6. The enzyme was inhibited strongly by L-lysine (Ki = 8 +/- 2 microM; 50% inhibition by 53 microM at 6 mM-2-oxoglutarate), competitively with 2-oxoglutarate, in protamine sulphate-treated and desalted cell-free extracts and in partially purified preparations. The extent of this inhibition was strongly pH-dependent. Both isoenzymes are equally susceptible to inhibition by lysine. The same inhibition pattern is shown by the enzyme from strain D6/1014A, which is a better producer of penicillin than strain Q176.     

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.     

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q_p) and growth rate (µ) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q_p(µ) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed‐batch experiments, leads to errors in the prediction, because q_p is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes. In this paper a dynamic gene regulation model is presented, in which the specific rate of penicillin production is assumed to be a linear function of the amount of a rate‐limiting enzyme in the penicillin production pathway. Enzyme activity assays were performed and strongly indicated that isopenicillin‐N synthase (IPNS) was the main rate‐limiting enzyme for penicillin‐G biosynthesis in our strain. The developed gene regulation model predicts the expression of this rate limiting enzyme based on glucose repression, fast decay of the mRNA encoding for the enzyme as well as the decay of the enzyme itself. The gene regulation model was combined with a stoichiometric model and appeared to accurately describe the biomass and penicillin concentrations for both chemostat steady‐state as well as the dynamics during chemostat start‐up and fed‐batch cultivation. Biotechnol. Bioeng. 2010;106: 608–618. © 2010 Wiley Periodicals, Inc.     

胞外青霉素酰化酶产生菌的选育

利用纸片显色方法,从土壤甲诀速筛选出98株产胞外青霉素酰化酶的菌种,经复筛其中10株酶活力较高,经鉴定均属于巨大芽孢杆菌。经单株分离得46号菌,用这株菌进行了产酶条件的研究,在最适产酶条件下,酶话力比开始提高了3．6倍。在此基础上又进行了物理化学因素处理,得突变株UL-81,酶活力达720u／1 Ooml发酵液。对原株和突变株进行比较,发现UL-81菌落、细胞形态、诱导剂苯乙酸用量及添加时间等明显不同于原株。在500L罐发酵酶活达8 20u／1OOml发酵液,为开始酶活的16倍。     

Lysine biosynthesis in Penicillium chrysogenum is regulated by feedback inhibition of α-aminoadipate reductase

A partially purified preparation of alpha-aminoadipate reductase (EC 1.2.1.31) from Penicillium chrysogenum is competitively inhibited by lysine (Ki of 0.26 mM). Exogenous addition of 10 mM L-lysine to resting mycelia of P. chrysogenum increased the intracellular lysine pool concentration 2-fold, but decreased the incorporation of (6-14C)-alpha-aminoadipate into protein-bound lysine to a fifth. The distribution of radioactivity in the pathway metabolites alpha-aminoadipate, saccharopine and lysine was consistent with the assumption of a lysine sensitive enzyme step in vivo between alpha-aminoadipate and saccharopine. Hence lysine inhibition of alpha-aminoadipate reductase may be of physiologic importance.     

Microcalorimetric investigations of the metabolism of yeasts. Growth in chemostat cultures on glucose

Summary As part of a project on the production of penicillin, the penicillin production of two strains of Penicillium chrysogenum which have a different penicillin productivity was investigated in bubble column bioreactors and for comparison in stirred fermenters. The main interest of this study were the complicated interrelations between the stirrer speed, the stirrer type, the shear stress, the morphology of the mycelium and broth viscosity as well as the effect of the oxygen transfer behavior on antibiotic productivity.Stirred tank reactors with different turbine stirrers as well as with a draught tube and propeller were employed.The main variable investigated was the stirrer speed. At low stirrer speeds, gas dispersion is inadequate and the insufficient oxygen transfer rate is a limiting factor. At higher stirrer speeds, the oxygen supply of pulpy mycelia is improved and more cell mass is formed. This result is the same for both strains in all three reactors.If the oxygen partial pressure is near the lower cirtical value, a high percentage of the carbon source is converted into penicillin but the penicillin productivity is low due to a low percentage of penicillin producing cells. At oxygen partial pressures just above 8% saturation, the absolute penicillin productivity is maximal. At higher stirrer speeds and dissolved oxygen concentrations the penicillin production phase is shorter, cell growth is higher and a higher percentage of the carbon source is converted into CO₂.In reactors with a draught tube and propeller, a lower productivity is attained than in those with turbine stirrers.The behavior of the two strains is fairly similar. The higher producing strain, however, has a more distinct separation between its periods of growth and production than does the low producing one. At high stirrer speeds the increase in the cell growth rate is less significant and the substrate yield coefficients are higher for the high producing strain than for the low producing one.Symbols C Dissolved oxygen concentration (mg l^–1) - C^* C at saturation (mg l^–1) - k_La Volumetric mass transfer coefficient (h^–1) - OTR Oxygen transfer rate (mg l^–1 h^–1) - OUR Oxygen uptake rate (mg l^–1 h^–1) - rpm Impeller speed (min^–1) - X (Dry) biomass concentration (g kg^–1) - V_g Volumetric gas flow rate (Nl min^–1) - CMC Carboxymethyl cellulose     

Improved trehalose production from biodiesel waste using parent and osmotically sensitive mutant of Propionibacterium freudenreichii subsp. shermanii under aerobic conditions

Trehalose is an important nutraceutical of wide commercial interest in the food processing industry. Recently, crude glycerol was reported to be suitable for the production of trehalose using a food microbe, Propionibacterium freudenreichii subsp. shermanii, under static flask conditions. Similarly, enhanced trehalose yield was reported in an osmotically sensitive mutant of the same strain under anaerobic conditions. In the present study, an effort was made to achieve higher production of trehalose, propionic acid, and lactic acid using the parent and an osmotically sensitive mutant of P. freudenreichii subsp. shermanii under aeration conditions. Under aeration conditions (200 rpm in shake flasks and 30 % air saturation in a batch reactor), biomass was increased and approximately 98 % of crude glycerol was consumed. In the parent strain, a trehalose titre of 361 mg/l was achieved, whereas in the mutant strain a trehalose titre of 1.3 g/l was produced in shake flask conditions (200 rpm). In the mutant strain, propionic and lactic acid yields of 0.53 and 0.21 g/g of substrate were also achieved with crude glycerol. Similarly, in controlled batch reactor culturing conditions a final trehalose titre of approximately 1.56 g/l was achieved with the mutant strain using crude glycerol as the substrate. Enhanced production of trehalose using P. freudenreichii subsp. shermanii from waste under aeration conditions is reported here. Higher production of trehalose was not due to a higher yield of trehalose but to a higher final biomass concentration.     

Optimization of an Aspergillus niger glucose oxidase production process

The purpose of the present study was to ascertain the optimal concentration of dissolved oxygen in order to maximize the intracellular glucose oxidase formation in Aspergillus niger. Cultivations performed in a 3.5 l laboratory reactor showed that a dissolved oxygen concentration at 3% of saturation at a total pressure of 1.2 bar was optimal for maximizing intracellular glucose oxidase activity. Cultivations performed at higher dissolved oxygen concentrations did not produce as much glucose oxidase as those performed at 3%, although the formation rate was high. Experiments revealed that maximal intracellular glucose oxidase formation for the A. niger strain used, is accomplished by limiting the gluconic acid production rate by means of maintaining a low dissolved oxygen concentration. Several attempts to achieve higher intracellular glucose oxidase activity were also made by manipulating the glucose concentration at a 3% dissolved oxygen concentration. However, no enhancement in glucose oxidase activity was observed.     

Recurrent mutation and selection for increased penicillin titre in Aspergillus nidulans.

Recurrent mutation and selection has been used to increase penicillin titre in two closely related strains of Aspergillus nidulans. A selection programme was initiated from each of the two strains (programmes A and B) and continued through six cycles of mutation and selection. Near-ultraviolet light in conjuction with 8-methoxypsoralen was employed as the sole mutagen throughout programme A and ethyl methanesulphonate as the sole mutagen throughout programme B. Excluding the first cycle of A, where only 50 strains were assayed, the selection programmes were identical. In each programme, 100 survivors were assayed for penicillin titre after each mutagenic treatment and, on the basis of a single yield test, the best five strains were picked and carried forward to the next cycle. In both selection programmes, a near 300% increase in penicillin titre was achieved. This yield advance illustrates the effectiveness for strain development of experimental designs involving successive cycles of mutagenesis with a single-stage screen and the selection of the top few percent survivors in each cycle.     

Double mutation in photosystem II reaction centers and elevated CO2 grant thermotolerance to mesophilic cyanobacterium

Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43 °C but only at elevated CO(2) (1%). Under the same conditions, the Synechocystis control strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated Q(A)(-)→Q(B) electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO(2) concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant.     

Effect of penicillin precursors on antibiotic biosynthesis in various strains]

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.     

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation.     

Citric acid production by Candida strains under intracellular nitrogen limitation

A suitable strain and important factors influencing citric acid formation in yeasts were identified. Candida oleophila ATCC 20177 was chosen as the best citric acid producer from several Candida strains. Yields of 50 g/l citric acid were produced in shake flask and 80 g/l in fed-batch fermentations with 1.5 and 3 g/l NH(4)Cl under non-optimized conditions. Ammonium nitrogen was identified as the limiting substrate for citrate formation. Citric acid excretion begins a few hours after exhaustion of nitrogen in the medium. The importance of intracellular nitrogen limitation was clarified by elemental analysis of C. oleophila biomass. The nitrogen content of C. oleophila biomass decreased from 7.45% during the growth phase to 3.96% in the production phase. The biomass contained less carbon and more trace elements in the growth phase compared with the production phase. Relatively high intracellular NH(4)(+) concentration of about 1.2 mg/g biomass (~37.4 mM) was found during the production phase. The low intracellular nitrogen content and increase of intracellular NH(4)(+) concentration, possibly caused by proteolysis following extracellular nitrogen exhaustion, trigger citric acid production. Intracellular nitrogen limitation and the increase in intracellular NH(4)(+) concentration are the most important factors influencing citric acid formation in yeasts.     

Production of 6-APA using a recirculated packed bed batch reactor

Summary A recirculated packed bed batch reactor has been designed for the production of 6-aminopenicillanic acid. It was observed that the flow rate of penicillin G solution is a rate limiting step for its hydrolysis. Under the conditions used, the maximum rate of hydrolysis of penicillin G was observed at a flow rate of 3.0 L/min.     

Genetic and biochemical studies of mutants of Penicillium chrysogenum impaired in penicillin production.

Seventy-eight mutants of Penicillium chrysogenum strain NRRL 1951, that were impaired in penicillin production, were isolated following treatment with various mutagens. Twelve that yielded about 10% of their parental penicillin titre were studied in detail. Analyses of heterozygous diploids formed between them revealed the existence of at least five complementation groups with respect to penicillin production--V, W, X, Y and Z. Most mutants belonged to group Y. A biochemical investigation of the intracellular peptides in strains representing the five groups demonstrated the absence of the tripeptide alpha-aminoadipoylcysteinyl-valine from mutants of groups X, Y and Z. Extracts of mutants of groups W, Y and Z were able to catalyse a penicillin acyl-exchange reaction, a mutant of group V showed only a trace of activity and mutant from group X completely lacked this ability.     

Effect of dissolved oxygen level on ACV synthetase synthesis and activity during growth of Streptomyces clavuligerus

Summary The multi-subunit enzyme, -(L--aminoadipyl)-L-cysteinyl-D-valine (ACV) synthethase catalyses the first step in the biosynthetic pathway of the -lactam antibiotic, cephamycin C. In batch fermentations of Streptomyces clavuligerus, ACV synthetaase activity appeared during the rapid growth phase. Over the same period the dissolved oxygen (DO) content of the medium was depleted to zero and remained there for nearly 10 h. Maintainance of the DO at saturation throughout the fermentation did not change the maximum ACV synthetaase specific activity, but did reduce the in-vivo stability of the enzyme. Oxygen saturation lowered the maximum intracellular ACV levels to one-sixth of those accumulated in the fermentor with no oxygen control, due principally to an improvement in the conversion of ACV to the penicillin N intermediate. Increased oxygenation also improved ACV conversion to cephamycin C, which demostrated that the activity of both an early and a later enzymatic step in cephamycin biosynthesis was limiting antibiotic production under restricted oxygen conditions. The later step, catalysing the conversion of penicillin N to cephamycin C, showed the greatest sensitivity to the oxygen state of the culture. Offprint request to: D. W. S. Westlake