Physiological characterization of xylose metabolism in Aspergillus niger under oxygen-limited conditions

The physiology of Aspergillus niger was studied under different aeration conditions. Five different aeration rates were investigated in batch cultivations of A. niger grown on xylose. Biomass, intra- and extra-cellular metabolites profiles were determined and ten different enzyme activities in the central carbon metabolism were assessed. The focus was on organic acid production with a special interest in succinate production. The fermentations revealed that oxygen limitation significantly changes the physiology of the micro-organism. Changes in extra cellular metabolite profiles were observed, that is, there was a drastic increase in polyol production (erythritol, xylitol, glycerol, arabitol, and mannitol) and to a lesser extent in the production of reduced acids (malate and succinate). The intracellular metabolite profiles indicated changes in fluxes, since several primary metabolites, like the intermediates of the TCA cycle accumulated during oxygen limitation (on average three fold increase). Also the enzyme activities showed changes between the exponential growth phase and the oxygen limitation phase. In general, the oxygen availability has a significant impact on the physiology of this fungus causing dramatic alterations in the central carbon metabolism that should be taken into account in the design of A. niger as a succinate cell factory.     

基于CRISPR/Cas系统的黑曲霉基因组编辑技术

黑曲霉Aspergillus niger是有机酸与酶制剂的重要工业生产菌株,以极端环境耐受性、高生产经济性、强发酵鲁棒性与高食品安全性等优势成为不可多得的细胞工厂。合成生物学与系统生物学的快速发展,不仅拉开了全面揭示黑曲霉细胞工厂高效运转机制的序幕,而且为高效黑曲霉细胞工厂的创建优化提供了新技术体系。作为新一代的基因组编辑技术,基于CRISPR/Cas系统的基因组编辑技术为黑曲霉基因组定向改造与基因表达调控带来了革命性突破。本文重点综述该技术在黑曲霉中的最新进展及其在黑曲霉基因编辑与表达调控中的应用,并对其未来发展方向进行展望。     

黑曲霉作为细胞工厂:知识准备与技术基础

黑曲霉是一种重要工业微生物,在酶制剂、异源蛋白、有机酸等领域应用广泛。2007年黑曲霉基因组的公布将黑曲霉的研究引入后基因组时代,各种组学数据如雨后春笋般涌现,人们对黑曲霉高效生产机制的理解上升到系统、分子层次;与此同时,黑曲霉遗传操作系统也不断成熟,为系统地研究和改造黑曲霉、将黑曲霉打造成通用细胞工厂奠定了基础。     

Metabolic fate of glutamate and evaluation of flux through the 4-aminobutyrate (GABA) shunt in Aspergillus niger

Accumulation of GABA and a concurrent block in the Krebs cycle suggest a functional GABA bypass in the acidogenic Aspergillus niger. Apart from the demonstration of enzyme machinery required, a direct measurement of flux through this glutamate decarboxylation loop was attempted. The distribution of carbon from glucose and glutamate was studied using A. niger mycelia grown on different media. The uptake and incorporation of (14)C label into organic acids and amino acids was followed by paper chromatography. Flow of label from glucose into citrate, glutamate and GABA increased in cells harvested at later stages of acidogenic growth. Very little citrate was derived from glutamate while ten times more label reached GABA from labeled glutamate. Radioactivity from L-[U-(14)C]glutamate and not from L-[1-(14)C]glutamate was recovered in GABA. This demonstrated that alpha-decarboxylation of L-glutamate was the source of GABA. Unless grown on GABA, A. niger mycelia did not take up externally supplied GABA. A direct measure of GABA shunt flux was thus not feasible. Therefore a combination of metabolite balance technique and the kinetic approach was applied to evaluate flux from glutamate to succinate in normal and acidogenic A. niger. The flux relative to TCA cycle was estimated by using uptake rate for radiolabeled glutamate, rate of accumulation of certain metabolites and the reactions of GABA metabolism. The analysis indicated that GABA shunt is operative in A. niger and its operation is enhanced during acidogenic growth of the fungus. This is the first report of an estimation of the flux through GABA shunt in a fungus.     

黑曲霉组学研究进展

黑曲霉作为重要的工业发酵菌株,被广泛用于多种有机酸和工业用酶的生产。随着组学技术的日益发展和成熟,黑曲霉的基因组、转录组、蛋白质组、代谢组等组学数据不断增长,宣告着黑曲霉生物过程研究大数据时代的到来。从单一组学的数据分析、多组学的比较到以基因组代谢网络模型为中心的多组学整合研究,人们对黑曲霉高效生产机制的理解不断深入和系统,这为通过遗传改造和过程调控对菌株的生产性能进行理性的全局优化提供了可能。本文回顾和总结了近年来黑曲霉的组学研究进展,并提出黑曲霉组学研究未来的发展方向。     

Taxonomy and significance of black aspergilli

Members of Aspergillus section Nigri (formerly A. niger group) are distributed worldwide and are regarded as common food spoilage fungi. Some of them are widely used and studied for industrial purposes. They are common sources of extracellular enzymes and organic acids to be used in food processing and are also used in the production of traditional foods, especially in the Orient. Products produced by strains of Aspergillus niger hold the GRAS (Generally Recognised As Safe) status from the FDA. However some species in Aspergillus section Nigri can produce ochratoxin A, a nephrotoxic mycotoxin. In spite of their industrial importance, the taxonomy of black aspergilli ( Aspergillus section Nigri ) is not clear and many attempts have been made in order to find suitable taxonomic criteria. The aim of this paper is to provide an overview of the significance of black aspergilli focusing on all the approaches made in the taxonomy of this group of fungi. Some species, such as A. carbonarius and uniseriate species can be easily recognised. In the A. niger aggregate, although speciation at molecular level has been proposed, no morphological differences can be observed and species identification will therefore remain problematic. Phylogenetic analyses of ITS and 5.8S rDNA gene region of representative black Aspergillus species and a simple key to the most common species that can be easily distinguished by morphological criteria are also included.     

Bioleaching of spent fluid catalytic cracking catalyst using Aspergillus niger

The use of the fungus Aspergillus niger for the bioleaching of heavy metals from spent catalyst was investigated, with fluid catalytic cracking (FCC) catalyst as a model. Bioleaching was examined in batch cultures with the spent catalysts at various pulp densities (1-12%). Chemical leaching was also performed using mineral acids (sulphuric and nitric acids) and organic acids (citric, oxalic and gluconic acids), as well as a mixture of organic acids at the same concentrations as that biogenically produced. It was shown that bioleaching realised higher metal extraction than chemical leaching, with A. niger mobilizing Ni (9%), Fe (23%), Al (30%), V (36%) and Sb (64%) at 1% pulp density. Extraction efficiency generally decreased with increased pulp density. Compared with abiotic controls, bioleaching gave rise to higher metal extractions than leaching using fresh medium and cell-free spent medium. pH decreased during bioleaching, but remained relatively constant in both leaching using fresh medium and cell-free spent medium, thus indicating that the fungus played a role in effecting metal extraction from the spent catalyst.     

Physiology of Aspergillus niger in oxygen‐limited continuous cultures: Influence of aeration,carbon source concentration and dilution rate

In industrial production of enzymes using the filamentous fungus Aspergillus niger supply of sufficient oxygen is often a limitation, resulting in the formation of by‐products such as polyols. In order to identify the mechanisms behind formation of the different by‐products we studied the effect of low oxygen availability, at different carbon source concentrations and at different specific growth rates, on the metabolism of A. niger, using continuous cultures. The results show that there is an increase in the production of tricarboxylic acid (TCA) cycle intermediates at low oxygen concentrations. Indeed, at these conditions, a decrease in the mitochondrial respiratory chain activity leads to an accumulation of NADH and to a decreased ATP production which uncouples catabolism and anabolism, influences the intracellular pH and leads to production and excretion of organic acids. Moreover, mannitol is being produced in order to ensure reoxidation of NADH, and this is the main cellular response to balance the ratio NADH/NAD at low oxygen availability. Mannitol production is also coupled to low specific growth rate, which suggests a control of carbon catabolite repression on the mannitol pathway. The roles of two other polyols, erythritol and glycerol, were also investigated. Both compounds are known to accumulate intracellularly, at high osmotic pressure, in order to restore the osmotic balance, but we show that the efficiency of this system is affected by a leakage of polyols through the membrane. Biotechnol. Bioeng. Biotechnol. Bioeng. 2009;103: 956–965. © 2009 Wiley Periodicals, Inc.     

Coupling of microbial kinetics and oxygen transfer for analysis and optimization of gluconic acid production with Aspergillus niger

Gluconic acid fermentation has been widely used for the analysis of various aspects of kinetics and gas liquid transfer of oxygen. Most of these studies are, however, restricted to processes with bacteria. Mathematical models for industrially important productions with fungi have not been elaborated. In the experimental part of this work computer coupled fermentations of gluconic acid production with Aspergillus niger NRRL 3 have been performed. Knowledge of the stoichiometric relationship in the key reaction (glucose oxidase) provides an excellent opportunity for on-line estimation of glucose, biomass and product gluconate from oxygen uptake and carbon dioxide evolution rates. Starting then from experimental observations on the pH-depending oxygen kinetics of gluconic acid formation and influences of product concentrations on the growth of Aspergillus niger a mathematical framework is developed in which the kinetics of growth and production are coupled with gas liquid oxygen transfer. The model can be successfully applied to simulations of the experimental results of gluconic acid fermentations with cyclic addition of glucose. An important aspect in the coupling of transport and microbial reaction in this model is the incorporation of the influence of sugar and gluconate on the solubility of oxygen and k _La via changes of viscosities and molecular diffusivities. With the development of such a comprehensive model, it appears feasible to investigate the influence of various process conditions (sugar feeding, pressure, optimal pH profiles) and to study their possible impacts on the productivity of the overall process.     

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.     

Direct fermentation of potato starch to ethanol by cocultures of Aspergillus niger and Saccharomyces cerevisiae.

Direct fermentation of unhydrolyzed potato starch to ethanol by monocultures of an amylolytic fungus, Aspergillus niger, and cocultures of A. niger and Saccharomyces cerevisiae was investigated. Amylolytic activity, rate and amount of starch utilization, and ethanol yields increased several-fold in coculture versus the monoculture due to the synergistic metabolic interactions between the species. Optimal ethanol yields were obtained in the pH range 5 to 6 and amylolytic activity was obtained in the pH range 5 to 8. Ethanol yields were maximal when fermentations were conducted anaerobically. Increasing S. cerevisiae inoculum in the coculture from 4 to 12% gave a dramatic increase in the rate of ethanol production, and ethanol yields of greater than 96% of the theoretical maximum were obtained within 2 days of fermentation. These results indicate that simultaneous fermentation of starch to ethanol can be conducted efficiently by using cocultures of the amylolytic fungus A. niger and a nonamylolytic sugar fermenter, S. cerevisiae.     

Fumonisin and ochratoxin production in industrial Aspergillus niger strains

Aspergillus niger is perhaps the most important fungus used in biotechnology, and is also one of the most commonly encountered fungi contaminating foods and feedstuffs, and occurring in soil and indoor environments. Many of its industrial applications have been given GRAS status (generally regarded as safe). However, A. niger has the potential to produce two groups of potentially carcinogenic mycotoxins: fumonisins and ochratoxins. In this study all available industrial and many non-industrial strains of A. niger (180 strains) as well as 228 strains from 17 related black Aspergillus species were examined for mycotoxin production. None of the related 17 species of black Aspergilli produced fumonisins. Fumonisins (B(2), B(4), and B(6)) were detected in 81% of A. niger, and ochratoxin A in 17%, while 10% of the strains produced both mycotoxins. Among the industrial strains the same ratios were 83%, 33% and 26% respectively. Some of the most frequently used strains in industry NRRL 337, 3112 and 3122 produced both toxins and several strains used for citric acid production were among the best producers of fumonisins in pure agar culture. Most strains used for other biotechnological processes also produced fumonisins. Strains optimized through random mutagenesis usually maintained their mycotoxin production capability. Toxigenic strains were also able to produce the toxins on media suggested for citric acid production with most of the toxins found in the biomass, thereby questioning the use of the remaining biomass as animal feed. In conclusion it is recommended to use strains of A. niger with inactive or inactivated gene clusters for fumonisins and ochratoxins, or to choose isolates for biotechnological uses in related non-toxigenic species such as A. tubingensis, A. brasiliensis, A vadensis or A. acidus, which neither produce fumonisins nor ochratoxins.     

Hydrodynamics and mass transfer in Aspergillus niger fermentations in bubble column and loop bioreactors

The influence of Aspergillus niger broth rheology, bioreactor geometry, and superficial gas velocity on the volumetric liquid phase oxygen transfer coefficient (k(L)a(L)), riser gas holdup (epsilon(GR)), and circulating liquid velocity (u(LR)) was studied in a bubble column (BC) and two external-circulation-loop airlift (ECLAL) bioreactors. The results are compared to those of previous studies on homogeneous fluids and in particular with a recent study on non-Newtonian carboxymethylcellulose (CMC) solutions conducted in the same contactors used for the A. niger fermentations. As expected from the CMC-based studies, in the heterogeneous broths of A. niger epsilon(GR), k(L)a(L), and u(LR) decreased with increasing broth apparent viscosity; epsilon(GR) and k(L)a(L) decreased with increasing downcomer-to-riser cross-sectional area ratio, A(d)/A(r), whereas u(LR) increased with increasing A(d)/A(r). Gas holdup data in the airlift fermentations of A. niger were well predicted by the CMC-based correlation. However, the CMC-based correlations produced conservative estimations of k(L)a(L) and overestimates of u(LR) compared to the observed values in the A. niger broths.     

Fungal production of citric acid

Citric acid is the principal organic acid found in citrus fruits. To meet increasing demands it is produced from carbohydrate feedstock by fermentation with the fungus Aspergillus niger and the yeasts of Candida spp. Effect of various fermentation conditions and the biochemistry of citric acid formation by A. niger have been discussed. Commercially citric acid is produced by surface, submerged and solid state fermentation techniques. Recovery of pure acid from fermentation broth is done primarily by precipitation with lime and also by solvent extraction.     

Influence of dissolved oxygen concentration on intracellular pH for regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor

In this paper we report the regulation of Aspergillus niger growth rate during citric acid fermentation in a stirred tank bioreactor. For this, the influence of dissolved oxygen concentration in a medium on intracellular pH values and consequently on overall microbial metabolism was emphasized. Intracellular pH of mycelium grown under different concentrations of dissolved oxygen in the medium was determined. Sensitivity of proteins toward proton concentration is well recognized, therefore pH influences on the activities of key regulatory enzymes of Aspergillus niger were determined at pH values similar to those detected in the cells grown under lower dissolved oxygen concentrations. The results have shown significantly reduced specific activities of hexokinase, 6-phosphofructokinase and glucose-6-phosphate dehydrogenase in more acidic environment, while pyruvate kinase was found to be relatively insensitive towards higher proton concentration. As expected, due to the reduced specific activities of regulatory enzymes under more acidic conditions, overall metabolism should be hindered in the medium with lower dissolved oxygen concentration which was confirmed by detecting the reduced specific growth rates. From the studies, we conclude that dissolved oxygen concentration affects the intracellular pH and thus growth rate of Aspergillus niger during the fermentation process.