Reconstruction of a genome-scale metabolic model and in-silico flux analysis of Aspergillus tubingensis: a non-mycotoxinogenic citric acid-producing fungus.

BACKGROUND: Aspergillus tubingensis is a citric acid-producing fungus that can utilize sugars in hydrolysate of lignocellulosic biomass such as sugarcane bagasse and, unlike A. niger, does not produce mycotoxins. To date, no attempt has been made to model its metabolism at genome scale. RESULTS: Here, we utilized the whole-genome sequence (34.96 Mb length) and the measured biomass composition to reconstruct a genome-scale metabolic model (GSMM) of A. tubingensis DJU120 strain. The model, named iMK1652, consists of 1652 genes, 1657 metabolites and 2039 reactions distributed over four cellular compartments. The model has been extensively curated manually. This included removal of dead-end metabolites and generic reactions, addition of secondary metabolite pathways and several transporters. Several mycotoxin synthesis pathways were either absent or incomplete in the genome, providing a genomic basis for the non-toxinogenic nature of this species. The model was further refined based on the experimental phenotypic microarray (Biolog) data. The model closely captured DJU120 fermentative data on glucose, xylose, and phosphate consumption, as well as citric acid and biomass production, showing its applicability to capture citric acid fermentation of lignocellulosic biomass hydrolysate. CONCLUSIONS: The model offers a framework to conduct metabolic systems biology investigations and can act as a scaffold for integrative modelling of A. tubingensis.

Marine cyanobacterial biomass is an efficient feedstock for fungal bioprocesses.

BACKGROUND: Marine cyanobacteria offer many sustainability advantages, such as the ability to fix atmospheric CO2, very fast growth and no dependence on freshwater for culture. Cyanobacterial biomass is a rich source of sugars and proteins, two essential nutrients for culturing any heterotroph. However, no previous study has evaluated their application as a feedstock for fungal bioprocesses. RESULTS: In this work, we cultured the marine cyanobacterium Synechococcus sp. PCC 7002 in a 3-L externally illuminated bioreactor with working volume of 2 L with a biomass productivity of ~ 0.8 g L-1 day-1. Hydrolysis of the biomass with acids released proteins and hydrolyzed glycogen while hydrolysis of the biomass with base released only proteins but did not hydrolyze glycogen. Among the different acids tested, treatment with HNO3 led to the highest release of proteins and glucose. Cyanobacterial biomass hydrolysate (CBH) prepared in HNO3 was used as a medium to produce cellulase enzyme by the Penicillium funiculosum OAO3 strain while CBH prepared in HCl and treated with charcoal was used as a medium for citric acid by Aspergillus tubingensis. Approximately 50% higher titers of both products were obtained compared to traditional media. CONCLUSIONS: These results show that the hydrolysate of marine cyanobacteria is an effective source of nutrients/proteins for fungal bioprocesses.

Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate.

BACKGROUND: Citric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering. RESULTS: The novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase. CONCLUSIONS: In this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

Nutrient modulation based process engineering strategy for improved butanol production from Clostridium acetobutylicum.

The present study demonstrates a process engineering strategy to achieve high butanol titer and productivity from wild type Clostridium acetobutylicum MTCC 11274. In the first step, two different media were optimized with the objectives of maximizing the biomass and butanol productivity, respectively. In the next step, attributes of these two media compositions were integrated to design a two-stage fed-batch process which resulted in maximal butanol productivity of 0.55 g L-1 h-1 with titer of 13.1 g L-1 . Further, two-stage fed-batch process along with combinatorial use of magnesium limitation and calcium supplementation resulted in the highest butanol titer and productivity of 16.5 g L-1 and 0.59 g L-1 h-1 , respectively. Finally, integration of the process with gas stripping and modulation of feeding duration resulted in a cumulative butanol titer of 54.3 g L-1 and productivity of 0.58 g L-1 h-1 . The strategy opens up possibility of developing a viable butanol bioprocess. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2771, 2019.

Understanding regulation in substrate dependent modulation of growth and production of alcohols in Clostridium sporogenes NCIM 2918 through metabolic network reconstruction and flux balance analysis.

Flux Balance Analysis was performed for Clostridium sporogenes NCIM 2918 grown on sole glucose and glycerol or glucose-glycerol combinations at varied concentrations. During acidogenesis, glucose and glucose-glycerol combinations favored improved growth and butyric acid production. Glycerol fermentation was however marked by reduced growth and predominant ethanol synthesis. Further, with increase of glycerol fraction in glucose-glycerol blend, flux towards ethanol synthesis linearly increased with simultaneous decrease in butanol flux. Elevated ATP demand due to improved growth was satisfied by upregulated carbon flux towards butyric acid synthesis during both glucose and dual substrate fermentations. Possible repression of pyruvate carboxylase by glycerol resulting in downturn of carbon uptake flux towards TCA cycle through anaplerotic reaction may be responsible for reduced growth in glycerol fermentation. Ammonium acetate mediated induction of acetic acid utilization, during acidogenesis, led to excess acetyl-CoA generation and its subsequent metabolism to lesser reduced products, butyric acid or ethanol.

Substrate dependent modulation of butanol to ethanol ratio in non-acetone forming Clostridium sporogenes NCIM 2918.

Present study reports a non-acetone producing Clostridium sporogenes strain as a potential producer of liquid biofuels. Alcohol production was positively regulated by sorbitol and instant dry yeast as carbon and nitrogen sources respectively. Media optimization resulted in maximum butanol and ethanol titer (gL-1) of 12.1 and 7.9 respectively. Depending on the combination of carbon sources, the organism was found to manipulate its metabolism towards synthesis of either ethanol or butanol, thereby affecting the total alcohol titer. Among various dual substrate combinations, glucose-glycerol mixture in the ratio of 60:40 resulted in maximum butanol and ethanol titer (gL-1) of 11.9 and 12.1 respectively with total alcohol productivity of 0.59gL-1h-1. In the mixture, when pure glycerol was replaced with crude glycerol, butanol and ethanol titer (gL-1) of 11.2 and 11.7 was achieved. Hence, the strain shows immense potential for biofuels production using crude glycerol as cheap substrate.