Citric Acid-producing Aspergillus Niger Research Articles

Genome-wide transcription landscape of citric acid producing Aspergillus niger in response to glucose gradient.

Aspergillus niger is the main industrial workhorse for global citric acid production. This fungus has complex sensing and signaling pathways to respond to environmental nutrient fluctuations. As the preferred primary carbon source, glucose also acts as a critical signal to trigger intracellular bioprocesses. Currently, however, there is still a knowledge gap in systems-level understanding of metabolic and cellular responses to this vital carbon source. In this study, we determined genome-wide transcriptional changes of citric acid-producing Aspergillus niger in response to external glucose gradient. It demonstrated that external glucose fluctuation led to transcriptional reprogramming of many genes encoding proteins involved in fundamental cellular process, including ribosomal biogenesis, carbon transport and catabolism, glucose sensing and signaling. The major glucose catabolism repressor creA maintained a stable expression independent of external glucose, while creB and creD showed significant downregulation and upregulation by the glucose increase. Notably, several high-affinity glucose transporters encoding genes, including mstA, were greatly upregulated when glucose was depleted, while the expression of low-affinity glucose transporter mstC was glucose-independent, which showed clear concordance with their protein levels detected by in situ fluorescence labeling assay. In addition, we also observed that the citric acid exporter cexA was observed to be transcriptionally regulated by glucose availability, which was correlated with extracellular citric acid secretion. These discoveries not only deepen our understanding of the transcriptional regulation of glucose but also shed new light on the adaptive evolutionary mechanism of citric acid production of A. niger.

Comprehensively dissecting the hub regulation of PkaC on high-productivity and pellet macromorphology in citric acid producing Aspergillus niger.

Summary Aspergillus niger, an important industrial workhorse for citric acid production, is characterized by polar hyphal growth with complex pelleted, clumped or dispersed macromorphologies in submerged culture. Although organic acid titres are dramatically impacted by these growth types, studies that assess productivity and macromorphological changes are limited. Herein, we functionally analysed the role of the protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) signalling cascade during fermentation by disrupting and conditionally expressing the pkaC gene. pkaC played multiple roles during hyphal, colony and conidiophore growth. By overexpressing pkaC, we could concomitantly modify hyphal growth at the pellet surface and improve citric acid titres up to 1.87‐fold. By quantitatively analysing hundreds of pellets during pilot fermentation experiments, we provide the first comprehensive correlation between A. niger pellet surface morphology and citric acid production. Finally, by intracellular metabolomics analysis and weighted gene coexpression network analysis (WGCNA) following titration of pkaC expression, we unveil the metabolomic and transcriptomic basis underpin hyperproductivity and pellet growth. Taken together, this study confirms pkaC as hub regulator linking submerged macromorphology and citric acid production and provides high‐priority genetic leads for future strain engineering programmes.

Isolation of citric acid-producing Aspergillus niger from soil and organic wastes

Introduction of new Aspergillus niger strains that are more productive than those currently in use is one of the important steps in promoting more effective commercial citric acid production. The present study was conducted to isolate and characterise indigenous A. niger from selected soil and organic wastes such as soil with buried bamboo, soil with bamboo on the surface, soil under cattle grazing, mango orchard soil, rotting plum fruit and rotting bread. Morphological identification of A. niger was based on the length and width of the conidiophores, vesicles, phialides, and spores. Citric acid-producing Aspergillus isolates were screened based on the citric acid production index. The present study found that the highest fungal spore counts (3.35±0.15 x 107 spores/g sample) were obtained from soil under cattle grazing, as were the highest A. niger counts (7.25±0.05 x 106 spores/g sample). The lowest total fungal counts came from rotting plum fruit (4.70±0.10 x 105 spores/g sample). A total of 14 isolates were collected, with five (NSA03, NSA06, NSA09, NSA12, and NSA14) showing morphological similarities with the reference isolate, A. niger Tiegh. All isolates were able to produce citric acid, but with varying efficiencies according to their citric acid production indices. The soil under cattle grazing area found to be the best site for sampling and isolation for citric acid-producing A. niger by using Czapek-Dox as a medium of isolation.

Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-ProducingAspergillus nigerWU-2223L

Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus niger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger

The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 °C, oxidative stress by exposure to 5mM paraquat or 1 mM t-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses.

Cloning and functional expression of the mitochondrial alternative oxidase gene ( aox1) of Aspergillus niger in Lactococcus lactis and its induction by oxidizing conditions

Lactococcus lactis is a widely used food bacterium mainly known for its fermentation metabolism. An important, and for long time overlooked, trait of this species is its ability to perform respiratory metabolism in the presence of heme and under aerobic conditions. There is no evidence however for the presence of an alternative respiration pathway and AOX activity. In this study, a cDNA fragment encoding the mitochondrial alternative oxidase, the enzyme responsible for alternative respiration, from a citric acid producing Aspergillus niger strain was cloned and expressed in L. lactis as a host strain. Expression of aox1 conferred on this organism cyanide-resistant and salicylhydroxamate-sensitive growth. Bioreactor cultures under fully aerobic conditions of the transformed L. lactis showed that the alternative respiratory pathway operates and improves significantly the microorganism's response to oxidizing stress conditions as it enhances biomass production, suppresses lactate formation, and leads to accumulation of large amounts of nisin.

Assessment of the homeostasis mechanisms in Aspergillus niger while in citric acid producing conditions

Mycelia of citric acid-producing Aspergillus niger WU-2223L show cyanide-insensitive respiration catalyzed by alternative oxidase. In this study, the constitutive expression of alternative oxidase gene (aox1) even at the stage of single-cell conidium in A. niger WU-2223L was found using the visual expression analysis system of aox1 with green fluorescent protein under microscopy observation.

Expression of alternative oxidase gene (aox1) at the stage of single-cell conidium in citric acid-producing Aspergillus niger

Mycelia of citric acid-producing Aspergillus niger WU-2223L show cyanide-insensitive respiration catalyzed by alternative oxidase. In this study, the constitutive expression of alternative oxidase gene (aox1) even at the stage of single-cell conidium in A. niger WU-2223L was found using the visual expression analysis system of aox1 with green fluorescent protein under microscopy observation.

Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger

In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc(1) to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.

Modeling the mechanisms of glucose transport through the cell membrane of Aspergillus niger in submerged citric acid fermentation processes

Data from batch fermentations of citric acid producing Aspergillus niger cultures in shake flasks, loop and stirred tank bioreactors, were used to construct diffusion models for the transport of glucose. It was found that the mediated diffusion model does not reflect the relationship between the observed uptake rate and glucose concentration, nor for the lack of sensitivity to citrate. This is due in part of the low value of K m in relation to the actual substrate concentration, which means that the carriers are saturated until the end of the process. The membrane barriers must be strongly inhibited under the standard production conditions. Instead, the simple diffusion model fits all the observed data and it explains the relationship between the specific uptake rate and the concentration of glucose, which should not exist under carrier-saturated conditions. This may account for the overproduction or organic acids under the specific process conditions. The simple nature of this mechanism also explains the similarity of the uptake relationships from different sources, despite the use of different growing conditions.

Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger

Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3–5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate.

Purification and properties of carnitine acetyltransferase from citric acid producing Aspergillus niger.

Carnitine acetyltransferase was purified from the citric acid producing A. niger mycelium with a protein band showing a relative molecular weight of 77,000 and a pH optimum of 7.3. The K(m) values for the purified enzyme for acetyl-CoA and for carnitine were 0.1 mM and 1 mM, respectively. Carnitine acetyltransferase was located both in the mitochondria and in the cytosol. Both mitochondrial and cytosolic enzyme were purified using ammonium sulfate precipitation, Mono Q and Superose 12 separation. Regarding the localization, except for maximum velocity, there were no differences observed in substrate specificity and inhibition. Inhibition of the enzyme with micromolar concentrations of Cu2+ could contribute to a greater citric acid biosynthesis. Carnitine acetyltransferase can be considered as an enzyme necessary for the transport of acetyl groups through mitochondrial membrane in both directions.

Morphological development of Aspergillus niger immobilized in Ca-alginate and K-carrageenan

The morphological development of citric acid producing Aspergillus niger immobilized in Ca-alginate and K-carrageenan was studied. The fungus normally developed a dense mycelium layer below and on the gel bead surfaces so that substrate and oxygen in this area had direct contact with mycelia. By this way mycelia are not only immobilized by entrapment but also in a “pellet-like” matter. Limitation of the nitrogen source induces a more interior mycelium growth, and outgrowing of free cells was minimized. In sucrose media no effect on the particle stability was observed whereas the application of potassium acetate as substrate caused the complete dissolving of the matrices.

Effect of aeration and agitation on the biosynthetic activity of diffusely growing Aspergillus niger.

A considerable lowering of aeration demands occurs during diffuse growth of citric acid-producing Aspergillus niger in a submerged cultivation. However, the diffuse culture poses stricter demands on the type of aeration and agitation. The impeller frequency affects considerably the morphology of the producer fungus and the accumulation of citric acid. The effect of impeller frequency on the distribution of air in the medium and on the amount of air fed into the diffuse culture is less important.

Phosphofructokinase as a regulatory enzyme in citric acid producing Aspergillus niger

Phosphofructokinase as a regulatory enzyme in citric acid producing Aspergillus niger