Xiang Zou

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end‐product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA‐producing Aureobasidium pullulans strain ZX‐10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high‐titer equivalent to 87.6 g/L of malic acid and high‐productivity of 0.61 g/L h in free‐cell fermentation in a stirred‐tank bioreactor. Fed‐batch fermentations with cells immobilized in a fibrous‐bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA‐900 anion‐exchange resins, achieving a ∼100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first‐order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113.

Adaptation and Transcriptome Analysis of Aureobasidium pullulans in Corncob Hydrolysate for Increased Inhibitor Tolerance to Malic Acid Production

Malic acid is a dicarboxylic acid widely used in the food industry, and is also a potential C4 platform chemical. Corncob is a low-cost renewable feedstock from agricultural industry. However, side-reaction products (furfural, 5-hydroxymethylfurfural (HMF), formic acid, and acetic acid) that severely hinder fermentation are formed during corncob pretreatment. The process for producing malic acid from a hydrolysate of corncob was investigated with a polymalic acid (PMA)-producing Aureobasidium pullulans strain. Under the optimal hydrolysate sugar concentration 110 g/L, A. pullulans was further adapted in an aerobic fibrous bed bioreactor (AFBB) by gradually increasing the sugar concentration of hydrolysate. After nine batches of fermentation, the production and productivity of malic acid reached 38.6 g/L and 0.4 g/L h, respectively, which was higher than that in the first batch (27.6 g/L and 0.29 g/L h, respectively). The adapted strain could grow under the stress of 0.5 g/L furfural, 3 g/L HMF, 2g/L acetic acid, and 0.5 g/L formic acid, whereas the wild type did not. Transcriptome analysis revealed that the differentially expressed genes were related to carbohydrate transport and metabolism, lipid transport and metabolism, signal transduction mechanism, redox metabolism, and energy production and conversion under 0.5 g/L furfural and 3 g/L HMF stress conditions. In total, 42 genes in the adapted strain were upregulated by 15-fold or more, and qRT-PCR also confirmed that the expression levels of key genes (i.e. SIR, GSS, CYS, and GSR) involved in sulfur assimilation pathway were upregulated by over 10-fold in adapted strain for cellular protection against oxidative stress.

Reconstruction of a genome-scale metabolic model and in silico analysis of the polymalic acid producer Aureobasidium pullulans CCTCC M2012223

Aureobasidium pullulans is a yeast-like fungus used for producing biopolymers e.g. polymalic acid (PMA) and pullulan. A high PMA producing strain, A. pullulans CCTCC M2012223, was isolated and sequenced in our previous study. To understand its metabolic performance, a genome-scale metabolic model, iZX637, consisting of 637 genes, 1347 reactions and 1133 metabolites, was reconstructed based on genome annotation and literature mining studies. The iZX637 model was validated by simulating cell growth, utilization of carbon and nitrogen sources, and gene essentiality analysis in A. pullulans. We further validated our model, designed a simulation program for the prediction of PMA production using experimental data, and further analyzed the carbon flux distribution and change with increasing PMA synthesis rates. Through the calculated flux distribution, NADPH- and NADH-dependent methylenetetrahydrofolate dehydrogenase (MTHFD) were found to be associated with the transfer of reducing equivalents from NADPH to NADH for supplementing NADH in the metabolic network. Furthermore, under the high PMA synthesis rate, a large amount of carbon flux was through pyruvate into malic acid via the reductive TCA cycle. Thus, pyruvate carboxylase, which can convert pyruvate to oxaloacetate with CO2 fixation, may also be an important target for PMA synthesis. These results illustrated that the model iZX637 was a powerful tool for optimization of A. pullulans metabolism and identification of targets for guiding metabolic engineering.

A novel rhodamine-based fluorescent pH probe for high-throughput screening of high-yield polymalic acid strains from random mutant libraries

Polymalic acid (PMA) is produced from the yeast-like fungus Auerobasidium pullulans, and is a water-soluble biopolymer with many useful properties for pharmaceutical applications. Its monomer, L-malic acid, is a potential C4 platform chemical and organic acid for food industry. Owing to the A. pullulans complex genetic background, random mutagenesis is still an effective tool for enhancing the production of PMA; however, ineffective screening methods for random mutant libraries still hamper this process. In this study, a high-throughput screening procedure was developed to solve this problem. A novel rhodamine-based fluorescent pH probe R was synthesized and characterized, and it showed a good quantitative relationship between fluorescent intensity and the acidic pH value (ranging from ∼2 to ∼5) of malic acid, the sole monomer of PMA, applied to evaluate the titer of PMA in fermentation broth. Based on this screening strategy, the mutant AH-21 was successfully selected from the atmospheric and room temperature plasma (ARTP) mutant libraries containing ∼1000 mutants. The titer of PMA was enhanced by 13.8% compared with that of wild type in a 3 L fermentor. Fed-batch fermentation in a 30 L fermentor achieved a high final PMA titer of 128.2 g L−1 (145.4 g L−1 of malic acid after hydrolysis) with the highest yield of 0.51 g g−1 from glucose. The high-throughput screening process established with this study represents a rapid and accurate approach to enhance the production of other similar organic acids from random mutant libraries.

Enhanced polymalic acid production from the glyoxylate shunt pathway under exogenous alcohol stress

Polymalic acid (PMA) is a water-soluble biopolymer produced by the yeast-like fungus Aureobasidium pullulans. In this study, the physiological response of A. pullulans against exogenous alcohols stress was investigated. Interestingly, ethanol stress was an effective inducer of enhanced PMA yield, although cell growth was slightly inhibited. The stress-responsive gene malate synthase (mls), which is involved in the glyoxylate shunt, was identified and was found to be regulated by exogenous ethanol stress. Therefore, an engineered strain, YJ-MLS, was constructed by overexpressing the endogenous mls gene, which increased the PMA titer by 16.2% compared with the wild-type strain. Following addition of 1% (v/v) of ethanol, a high PMA titer of 40.0 ± 0.38 g/L was obtained using batch fermentation with the mutant YJ-MLS in a 5-L fermentor, with a strongest PMA productivity of 0.56 g/L h. This study was the interesting report to show strengthening of the carbon metabolic flow from the glyoxylate shunt for PMA synthesis, and also provided a new sight for re-recognizing the regulatory behavior of alcohol stress in eukaryotic microbes.