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Studies on the production of fungal polyketides in Aspergillus nidulans using systems biology tools

ABSTRACT: Using genetic engineering tools available for the model organism Aspergillus nidulans, we constructed two recombinant strains; one expressing the model polyketide Penicillium griseofulvum 6-methylsalicylic acid (6-MSA) polyketide synthase gene, and one expressing the 6-MSA gene and overexpressing the native phosphoketolase (phk) for increasing the pool of polyketide precursor levels. The physiology of the recombinant strains and a reference wild type were characterized on glucose, xylose, glycerol and ethanol medium in controlled bioreactors. Glucose was found to be the preferable carbon source for 6-MSA production and 6-MSA titers up to 455 mg/L were achieved. Our findings indicate that overexpression of phk does not directly improve 6-MSA production on glucose but if the lower glycolysis is lowered, it is possible to obtain quite high conversion yields of sugar to 6-MSA. Systems biology tools were employed for in-depth analysis of the metabolic processes. Transcriptome analysis of 6-MSA producing strains on glucose and xylose in the presence and absence of phk overexpression combined with flux and physiology data enabled us to propose a model of phk/6msas interaction describing two different responses influencing 6-MSA production.

ORGANISM(S): Aspergillus niger Aspergillus nidulans Aspergillus oryzae

PROVIDER: GSE12859 | GEO | 2009/02/27

SECONDARY ACCESSION(S): PRJNA111093

REPOSITORIES: GEO

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Project description:Background: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of fuels. To release the major fermentable sugars such as glucose and xylose, pretreatment and enzymatic hydrolysis of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and eventually the production rate and yield. Acetate is one of the major inhibitors liberated from hemicelluloses during dilute acid pretreatment. An understanding of the toxic effects of acetate on the fermentation microorganism is critical to improving biofuel yields in the process. In addition, the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results: We have observed previously that some pretreatment inhibitors affect growth and performance in Zymomonas mobilis 8b differently when different sugars (e.g. glucose or xylose) are used as substrate. To investigate this phenomenon at the cellular level, microarray technology was used to investigate the acetate stress responses of Z. mobilis 8b when using single carbon sources of glucose or xylose, and mixed sugars of glucose and xylose. We designed a microarray based on the most up-to-date genome annotation for both coding sequences and intergenic regions. In the presence of acetate, 8b still can utilize all the glucose (though xylose utilization was inhibited) with similar ethanol yield although the growth, final biomass, and ethanol production rate were reduced. The presence of acetate caused genes related to biosynthesis, flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Our result indicates that Z. mobilis utilized different mechanism for xylose utilization compared to that of glucose, with even more dramatic results than those caused by treatment of the culture with the inhibitor acetate. Our study also suggests that redox imbalance caused by stressful conditions may trigger a metabolic reaction that leads to the accumulation of toxic intermediates such as xylitol, but Z. mobilis appears to be capable of managing its carbon and energy metabolism through the control of individual reactions to overcome the inhibition caused by stressful conditions. Several target gene candidates based on transcriptomic result have been selected for genetic manipulation and a TonB-dependent receptor knockout mutant was confirmed to have advantage on acetate tolerance. Conclusions: We have gained insights into the molecular responses of the model ethanologenic bacterium Z. mobilis to the inhibitor acetate when grown in different sugar sources. These insights will facilitate future metabolic modeling studies and help further strain metabolic engineering efforts for better xylose utilization and acetate tolerance. Two series of microarray studies using total RNA extracted from Zymomonas mobilis subsp mobilis 8b (an xylose-utilizing recombinant) were carried out to investigate the effect of carbon source and acetate on Z. mobilis. One study compared the acetate effect in either glucose or xylose at exponential phase and another study investigated the acetate effect in mixed sugar of glucose and xylose at three growth phases of exponential, transition, and stationary. Tthree biological replicates were used for each condition.

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Studies on the production of fungal polyketides in Aspergillus nidulans using systems biology tools

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