Industrial Production Of Penicillin Research Articles

Fault detection in automated production systems based on a long short-term memory autoencoder

Abstract In this paper, a hybrid model of regularized Long Short-Term Memory (LSTM) and autoencoder for fault detection in automated production systems is proposed. The presented LSTM autoencoder is used as a stochastic process model, which captures the normal behavior of a production system and allows to predict the probability distribution of sensor data. Discrepancies between the observed sensor data and the predicted probability density distribution are detected as potential faults. The approach combines the advantages of LSTMs and autoencoders: The correlations between individual sensor signals are exploited by an autoencoder, while the temporal dependencies are captured by LSTM neurons. A key challenge in training such a process model from historical data is to control the information passed through the latent space of the autoencoder. Different regularization methods are investigated for this purpose. Fault detection with the proposed LSTM autoencoder has been evaluated on the use case of an industrial penicillin production, achieving significantly improved results in comparison to the baseline LSTM.

Importación de penicilina y estreptomicina desde los Estados Unidos de Norteamérica entro los años 1946 y 1950 y su transporte mediante los buques de la Compañia Trasatlántica española

After the achievement of the industrial production of penicillin in the United States of America in 1944, there was a massive worldwide export of this drug. This note describes unknown data associated with the arrival of antibiotics in our country. The analysis of the bibliographic collection of the shipping company Pérez y Cía has allowed us to analyze in detail the importance of the Compañía Trasatlántica Española ships in importing penicillin and streptomycin from the US between 1946 and 1950. During the years 1946 to 1948, more than twenty pharmaceutical companies, both Spanish and North American, imported small quantities of these antibiotics. However, in the period between 1949 and 1950, the Spanish pharmaceutical company Pan-Química S.L. and the North American Interchange Commercial Corporation, a subsidiary of the Banco Exterior de España, cooperated to supply the country with large quantities of antibiotics, before the start-up of the two main national companies producing antibiotics, the Compañía Española de Penicilina y Antibióticos (CEPA) and Antibióticos S.A. in 1951.

The Contribution of Soviet Military Scientists to the Development of Industrial Technologies for the Production of the First Domestic Antibiotics (Penicillin and Streptomycin)

In the domestic literature, the industrial production of penicillin and streptomycin is commonly attributed to the representatives of civil institutions and enterprises. However, the key role of military scientists in solving this problem remains in the background. The purpose of this article is to summarize the available facts about the role of Soviet military scientists in creating a technology for obtaining the first antibiotics in the USSR. At the beginning of 1942, all the works, related to the penicillin in the Soviet Union, was headed by academician of AMS USSR Z. V. Ermolyeva. At the first stage of the research, the technology for obtaining penicillin was applied, based on the use of a surface method of growing cultures of the antibiotic-producing fungus in mattresses. But this method could not meet the country's needs for the drug. In early 1944, the Soviet government tasked the Research Institute of Epidemiology and Hygiene of the Red Army (NIIEG) in Kirov (now a branch of the Federal State Budgetary Institution «48th Central Research Institute» of the Ministry of Defense of the Russian Federation) to solve the case of mass production of penicillin and streptomycin. In 1944–1945 for the first time in the USSR military specialists from NIIEG developed a technology for the industrial production of penicillin by the deep method with aeration. The deep cultivation was carried out in cultivator devices designed by engineer-Lieutenant Colonel A. V. Krutyakov. During the research, a fungus strain (Penicillium chrysogenum 23248) was selected from a large number of studied strains, the use of which provided the highest yields of penicillin, and the most effective cultivation conditions were selected. The experience, obtained by the Soviet military specialists during the development of the technology for the deep production of penicillin, served in 1946-1947 as the basis for the creation of the method for obtaining domestic streptomycin from the domestic Streptomyces griseus. In 1947, for the first time in the world an employee of the NIIEG Lieutenant Colonel of medical service N. I. Nikolaev and civil doctors D. D. Fedorinov and V. I. Gorokhov used the NIIEG streptomycin successfully for the treatment of patients with pneumonic plague during the plague epidemic in Manchuria. In the late 1940s the technologies for the production of penicillin and streptomycin, as well as the relevant documentation, were transferred to civil healthcare institutions for the industrial development.

Penicillium chrysogenum, a Vintage Model with a Cutting-Edge Profile in Biotechnology.

The discovery of penicillin entailed a decisive breakthrough in medicine. No other medical advance has ever had the same impact in the clinical practise. The fungus Penicillium chrysogenum (reclassified as P. rubens) has been used for industrial production of penicillin ever since the forties of the past century; industrial biotechnology developed hand in hand with it, and currently P. chrysogenum is a thoroughly studied model for secondary metabolite production and regulation. In addition to its role as penicillin producer, recent synthetic biology advances have put P. chrysogenum on the path to become a cell factory for the production of metabolites with biotechnological interest. In this review, we tell the history of P. chrysogenum, from the discovery of penicillin and the first isolation of strains with high production capacity to the most recent research advances with the fungus. We will describe how classical strain improvement programs achieved the goal of increasing production and how the development of different molecular tools allowed further improvements. The discovery of the penicillin gene cluster, the origin of the penicillin genes, the regulation of penicillin production, and a compilation of other P. chrysogenum secondary metabolites will also be covered and updated in this work.

Biosynthetic process and strain improvement approaches for industrial penicillin production.

Penicillins and cephalosporins are the most important class of beta (β) lactam antibiotics, accounting for 65% total antibiotic market. Penicillins are produced by Penicillium rubens (popularly known as P. chrysogenum) were used to synthesize the active pharmaceutical intermediate (API), 6-aminopenicillinic acid (6-APA) employed in semisynthetic antibiotic production. The wild strains produce a negligible amount of penicillin (Pen). High antibiotic titre-producing P. chrysogenum strains are necessitating for industrial Pen production to meet global demand at lower prices. Classical strain improvement (CSI) approaches such as random mutagenesis, medium engineering, and fermentation are the cornerstones for high-titer Pen production. Since, Sir Alexander Fleming Discovery of Pen, great efforts are expanded to develop at a commercial scale antibiotics producing strains. Breakthroughs in genetic engineering, heterologous expression and CRISPR/Cas9 genome editing tools opened a new window for Pen production at a commercial scale to assure health crisis. The current state of knowledge, limitations of CSI and genetic engineering approaches to Pen production are discussed in this review.

Zhang Weishen: A Pioneer in the Development and Industrial Production of Penicillin in New China

The research and development of penicillin started with difficulty before 1949 and achieved certain results. In 1951, after the founding of the People's Republic of China, Zhang Weishen, as the only Chinese scientist who had been trained and worked in a penicillin research and development center in the United States for many years, overcame many difficulties and returned to China. In 1953, with the efforts of Zhang Weishen and his colleagues, China realized the industrialized production of penicillin, alleviating the urgent needs of the masses. Antibiotics has also become the first discipline to achieve major scientific and technological achievements after the founding of the New China. In the mid-1950s, the technical breakthrough in the localization of lactose substitutes marked the localization of the raw materials of the penicillin-producing culture medium, which paved the way for the industrialized production of penicillin with Chinese characteristics. Antibiotics have become one of the most widely used and affordable drugs for hundreds of millions of patients in China, and China has since ended the humiliating history of the "Sick Man of East Asia".

Real-time synchronization with expected distribution of synchronized index for on-line monitoring of uneven multiphase batch process

Because of the inconsistent batch duration, identifying the progress of an ongoing batch highly affects the performance of the on-line monitoring. In this paper, a real-time synchronization scheme is proposed based on relative proceeding rates, newly defined as the time difference between the trajectory of an ongoing batch and the nominal trajectory. As the relative proceeding rate indicates how early the point attains its desired value compared to the nominal trajectory, the synchronized index is obtained on-line by estimating the relative proceeding rate. Compared with the conventional methods that optimize the synchronized index with local constraints, the proposed method can estimate its distribution using the relative proceeding rate without applying local constraints. It can reduce synchronization errors and increase the accuracies of the state estimation and fault detection. The utility of the proposed method is verified via a case study of industrial penicillin production with a real-time unmeasurable variable.

Penicillium chrysogenum Fermentation and Analysis of Benzylpenicillin by Bioassay and HPLC.

Penicillium chrysogenum, recently re-identified as Penicillium rubens, is the microorganism used for the industrial production of penicillin. This filamentous fungus (mold) probably represents the best example of adaptation of a microorganism to industrial production conditions and therefore, it can be considered as a model organism for the study of primary and secondary metabolism under a highly stressful environment. In this regard, biosynthesis and production of benzylpenicillin can be used as an interesting phenotypic trait for those studies. In this chapter, we describe P. chrysogenum culture procedures for the production of benzylpenicillin and the process of antibiotic quantitation either by bioassay or by high-performance liquid chromatography (HPLC).

Characteristics of microbial community of soil subjected to industrial production of antibiotics.

Ecosystems worldwide are exposed to pollutants connected to the industrial production of pharmaceuticals. The objective of this study was to study the composition and characteristics of the soil microbial communities that had been exposed to long-term selection pressure caused by the industrial production of penicillin G. Soil samples from four sites among the penicillin G production plant were analysed using 16S rRNA profiling via Illumina MiSeq platform and were compared with the control samples from four sites outside the plant. Total metagenomic DNA from the impacted soil was also used for the preparation of E. coli T1R-based fosmid library which was consequently qualitatively tested for the presence of penicillin G acylase (PGA)-encoding genes using the method of sequence homology. Analyses of alpha diversity revealed that the long-term antibiotic presence in the soil significantly increased the microbial diversity and richness in terms of Shannon diversity index (p = 0.002) and Chao estimates (p = 0.004). Principal component analysis showed that the two types of communities (on-site and control) could be separated at the phylum, class and genus level. The on-site soil was enriched in Betaproteobacteria, Deltaproteobacteria, Gemmatimonadetes, Acidobacteria and Planctomycetia, while a significant decrease in Actinobacteria was observed. Metagenomic fosmid library revealed high hit rates in identifying PGAs (14 different genes identified) and confirmed the biotechnological potential of soils impacted by anthropogenic activity. This study offers new insights into the changes in microbial communities of soils exposed to anthropogenic activity as well as indicates that those soils may represent a hotspot for biotechnologically interesting targets.

The organization of industrial production of penicillin in the USSR

The article on the basis of analysis of declassified materials of the State Archive of the Russian Federation covers the history of the organization of industrial production of penicillin in the USSR in second half of the 1940s. The analysis of the documents demonstrated that existed huge gap between health needs of the antibiotic and actual quantity of its production by the end of 1945. This raised the question of organizing its large-scale production. The Soviet Government tried to solve the problem in the least expensive way i.e. through using available industrial areas for the development of penicillin production, refusing to buy ready-made penicillin plants abroad, maximal application of resources of international organizations. The problem of mastering advanced technologies of antibiotic production, as well as development of national equipment for penicillin plants was mainly solved through efforts of intelligence service activities. Due to organizational, technological, foreign policy causes, the development of new penicillin plants was delayed for a long time. The amounts of penicillin production by existing enterprises did not cover the needs of health care in the antibiotic.

Power input effects on degeneration in prolonged penicillin chemostat cultures: A systems analysis at flux, residual glucose, metabolite, and transcript levels.

In the present work, by performing chemostat experiments at 400 and 600 RPM, two typical power inputs representative of industrial penicillin fermentation (P/V, 1.00 kW/m3 in more remote zones and 3.83 kW/m3 in the vicinity of the impellers, respectively) were scaled-down to bench-scale bioreactors. It was found that at 400 RPM applied in prolonged glucose-limited chemostat cultures, the previously reported degeneration of penicillin production using an industrial Penicillium chrysogenum strain was virtually absent. To investigate this, the cellular response was studied at flux (stoichiometry), residual glucose, intracellular metabolite and transcript levels. At 600 RPM, 20% more cell lysis was observed and the increased degeneration of penicillin production was accompanied by a 22% larger ATP gap and an unexpected 20-fold decrease in the residual glucose concentration (Cs,out ). At the same time, the biomass specific glucose consumption rate (qs ) did not change but the intracellular glucose concentration was about sixfold higher, which indicates a change to a higher affinity glucose transporter at 600 RPM. In addition, power input differences cause differences in the diffusion rates of glucose and the calculated Batchelor diffusion length scale suggests the presence of a glucose diffusion layer at the glucose transporting parts of the hyphae, which was further substantiated by a simple proposed glucose diffusion-uptake model. By analysis of calculated mass action ratios (MARs) and energy consumption, it indicated that at 600 RPM glucose sensing and signal transduction in response to the low Cs,out appear to trigger a gluconeogenic type of metabolic flux rearrangement, a futile cycle through the pentose phosphate pathway (PPP) and a declining redox state of the cytosol. In support of the change in glucose transport and degeneration of penicillin production at 600 RPM, the transcript levels of the putative high-affinity glucose/hexose transporter genes Pc12g02880 and Pc06g01340 increased 3.5- and 3.3-fold, respectively, and those of the pcbC gene encoding isopenicillin N-synthetase (IPNS) were more than twofold lower in the time range of 100-200 hr of the chemostat cultures. Summarizing, changes at power input have unexpected effects on degeneration and glucose transport, and result in significant metabolic rearrangements. These findings are relevant for the industrial production of penicillin, and other fermentations with filamentous microorganisms.

A MAT1-2 wild-type strain from Penicillium chrysogenum: functional mating-type locus characterization, genome sequencing and mating with an industrial penicillin-producing strain.

In heterothallic ascomycetes, mating is controlled by two nonallelic idiomorphs that determine the ‘sex’ of the corresponding strains. We recently discovered mating-type loci and a sexual life cycle in the penicillin-producing fungus, Penicillium chrysogenum. All industrial penicillin production strains worldwide are derived from a MAT1-1 isolate. No MAT1-2 strain has been investigated in detail until now. Here, we provide the first functional analysis of a MAT1-2 locus from a wild-type strain. Similar to MAT1-1, the MAT1-2 locus has functions beyond sexual development. Unlike MAT1-1, the MAT1-2 locus affects germination and surface properties of conidiospores and controls light-dependent asexual sporulation. Mating of the MAT1-2 wild type with a MAT1-1 high penicillin producer generated sexual spores. We determined the genomic sequences of parental and progeny strains using next-generation sequencing and found evidence for genome-wide recombination. SNP calling showed that derived industrial strains had an uneven distribution of point mutations compared with the wild type. We found evidence for meiotic recombination in all chromosomes. Our results point to a strategy combining the use of mating-type genes, genetics, and next-generation sequencing to optimize conventional strain improvement methods.

Design of Robust Fault Detection Scheme for Penicillin Fermentation Process

Penicillin fermentation is a batch process used for the industrial production of penicillin. In this paper, a fault detection scheme is designed for this process for a reliable operation. Since practical systems are subjected to disturbances and noises, designed scheme might be unreliable. To cope with this problem, optimization is also considered in the design by identifying an optimized parity vector directly from the process data without going into the details of system model using subspace aided approach.

Prelude to rational scale-up of penicillin production: a scale-down study

Penicillin is one of the best known pharmaceuticals and is also an important member of the β-lactam antibiotics. Over the years, ambitious yields, titers, productivities, and low costs in the production of the β-lactam antibiotics have been stepwise realized through successive rounds of strain improvement and process optimization. Penicillium chrysogenum was proven to be an ideal cell factory for the production of penicillin, and successful approaches were exploited to elevate the production titer. However, the industrial production of penicillin faces the serious challenge that environmental gradients, which are caused by insufficient mixing and mass transfer limitations, exert a considerably negative impact on the ultimate productivity and yield. Scale-down studies regarding diverse environmental gradients have been carried out on bacteria, yeasts, and filamentous fungi as well as animal cells. In accordance, a variety of scale-down devices combined with fast sampling and quenching protocols have been established to acquire the true snapshots of the perturbed cellular conditions. The perturbed metabolome information stemming from scale-down studies contributed to the comprehension of the production process and the identification of improvement approaches. However, little is known about the influence of the flow field and the mechanisms of intracellular metabolism. Consequently, it is still rather difficult to realize a fully rational scale-up. In the future, developing a computer framework to simulate the flow field of the large-scale fermenters is highly recommended. Furthermore, a metabolically structured kinetic model directly related to the production of penicillin will be further coupled to the fluid flow dynamics. A mathematical model including the information from both computational fluid dynamics and chemical reaction dynamics will then be established for the prediction of detailed information over the entire period of the fermentation process and thereby for the optimization of penicillin production, and subsequently also benefiting other fermentation products.

Resolving phenylalanine metabolism sheds light on natural synthesis of penicillin G in Penicillium chrysogenum.

The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-(13)C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH(4))(2)SO(4) as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate.

Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum

Industrial penicillin production with the filamentous fungus Penicillium chrysogenum is based on an unprecedented effort in microbial strain improvement. To gain more insight into penicillin synthesis, we sequenced the 32.19 Mb genome of P. chrysogenum Wisconsin54-1255 and identified numerous genes responsible for key steps in penicillin production. DNA microarrays were used to compare the transcriptomes of the sequenced strain and a penicillinG high-producing strain, grown in the presence and absence of the side-chain precursor phenylacetic acid. Transcription of genes involved in biosynthesis of valine, cysteine and alpha-aminoadipic acid-precursors for penicillin biosynthesis-as well as of genes encoding microbody proteins, was increased in the high-producing strain. Some gene products were shown to be directly controlling beta-lactam output. Many key cellular transport processes involving penicillins and intermediates remain to be characterized at the molecular level. Genes predicted to encode transporters were strongly overrepresented among the genes transcriptionally upregulated under conditions that stimulate penicillinG production, illustrating potential for future genomics-driven metabolic engineering.

Improvement of penicillin yields in solid-state and submerged fermentation of Penicillium chrysogenum by amplification of the penicillin biosynthetic gene cluster

Penicillium chrysogenum low and high penicillin producing strains were transformed with a cosmid containing the whole penicillin biosynthetic gene cluster. The cosmid library was constructed in a newly developed cosmid vector, IztapaCos, which allows cloning and direct introduction of large DNA fragments in fungal recipients using phleomycin resistance as selection marker. The effect of increased gene dosage on penicillin production was evaluated both in submerged (SmF) and solid-state fermentation (SSF). Transformants from the low-producing strain Wis 54-1255, showed a 67.3 and 28.3% increased penicillin titer in SSF and SmF, respectively. In transformants from the high-producing strain P2-32 the increase was 92.9 and 158.4% respectively. Strain P2-32 already contains originally about 14 copies of the penicillin biosynthetic cluster, which shows that the strategy of increasing the gene dosage is still valid for high copy-number strains. The different behavior of the two strains in each type of culture is discussed, along with the practical implications for industrial penicillin production.

Functional characterization of the penicillin biosynthetic gene cluster of Penicillium chrysogenum Wisconsin54-1255

Industrial strain improvement via classical mutagenesis is a black box approach. In an attempt to learn from and understand the mutations introduced, we cloned and characterized the amplified region of industrial penicillin production strains. Upon amplification of this region Penicillium chrysogenum is capable of producing an increased amount of antibiotics, as was previously reported [Barredo, J.L., Diez, B., Alvarez, E., Martín, J.F., 1989a. Large amplification of a 35-kb DNA fragment carrying two penicillin biosynthetic genes in high yielding strains of Penicillium chrysogenum. Curr. Genet. 16, 453–459; Newbert, R.W., Barton, B., Greaves, P., Harper, J., Turner, G., 1997. Analysis of a commercially improved Penicillium chrysogenum strain series, involvement of recombinogenic regions in amplification and deletion of the penicillin gene cluster. J. Ind. Microbiol. 19, 18–27]. Bioinformatic analysis of the central 56.9 kb, present as six direct repeats in the strains analyzed in this study, predicted 15 Open Reading Frames (ORFs). Besides the three penicillin biosynthetic genes ( pcbAB, pcbC and penDE) only one ORF has an orthologue of known function in the database: the Saccharomyces cerevisiae gene ERG25. Surprisingly, many genes known to encode direct or indirect steps β-lactam biosynthesis like phenyl acetic acid CoA ligase and transporters are not present. Detailed analyses reveal a detectable transcript for most of the predicted ORFs under the conditions tested. We have studied the role of these in relation to penicillin production and amplification of the biosynthetic gene cluster. In contrast to what was expected, the genes encoding the three penicillin biosynthetic enzymes alone are sufficient to restore full β-lactam synthesis in a mutant lacking the complete region. Therefore, the role of the other 12 ORFs in this region seems irrelevant for penicillin biosynthesis.

Prediction of Key State Variables using Support Vector Machines in Bioprocesses

Support vector machines (SVM) are applied to the prediction of key state variables in bioprocesses, such as product concentration and biomass concentration, which commonly play an important role in bioprocess monitoring and control. A so-called rolling learning-prediction procedure is used to deal with the time variant property of the process, and to establish the training database for the SVM predictor, which is characterized with the rolling update of the training database. As an example, product concentration in industrial penicillin production is predicted, and a comparison is also made with three different artificial neural network architectures (FBNN, RBFN, and RNN). The test results indicate that a prediction accuracy of 1-3 % can be obtained for 4-40h ahead prediction using the SVM, which is better than the best of the three artificial neural networks (ANNs). Moreover, for noise-added training highly noisy data or small-sample learning, the SVM also clearly outper-forms FBNN, RBFN, and RNN.

Uptake of phenylacetic acid by two strains of Penicillium chrysogenum.

Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 microM), and in the high-yielding strain at low phenylacetic acid concentrations (2. 8-100 microM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin.