Yanlin Liu

Production of pinoresinol diglucoside, pinoresinol monoglucoside, and pinoresinol by Phomopsis sp. XP-8 using mung bean and its major components.

Phomopsis sp. XP-8 is an endophytic fungus that has the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application for the biosynthesis of PDG independent of plants. When cultivated in mung bean medium, PDG production was significantly improved and pinoresinol monoglucoside (PMG) and pinoresinol (Pin) were also found in the culture medium. In this experiment, starch, protein, and polysaccharides were isolated from mung beans and separately used as the sole substrate in order to explore the mechanism of fermentation and identify the major substrates that attributed to the biotransformation of PDG, PMG, and Pin. The production of PDG, PMG, and Pin was monitored using high-performance liquid chromatography (HPLC) and confirmed using HPLC-MS. Activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) were analyzed and tracked during the cultivation. The reaction system contained the compounds isolated from mung bean in the designed amount. Accumulation of phenylalanine, cinnamic acid, p-coumaric acid, PDG, PMG, and Pin and the activities of PAL, C4H, and 4CL were measured during the bioconversion. PMG was found only when mung bean polysaccharide was analyzed, while production of PDG and Pin were found when both polysaccharide and starch were analyzed. After examining the monosaccharide composition of the mung bean polysaccharide and the effect of the different monosaccharides had on the production of PMG, PDG, and Pin, galactose in mung bean polysaccharide proved to be the major factor that stimulates the production of PMG.

Bioconversion of resveratrol using resting cells of non–genetically modified Alternaria sp.

Bioconversion of resveratrol is mainly achieved using plant cells and genetically modified microorganisms. We proposed a reaction system for resveratrol production using resting cells of a non–genetically modified strain, Alternaria sp. MG1, a resveratrol‐producing endophytic fungus isolated from the grape. Effects of phenylalanine concentration, inoculum size, resting time, bioconversion medium, cell age, and pH on resveratrol production in the bioconversion process were investigated and their levels were optimized. The resulting optimal bioconversion medium was 0.2 mM phosphate buffer (pH 6.5), 0.1 g/L MgSO4, 0.2 g/L CaSO4, and 4.66 mM phenylalanine. Resting cells obtained from cultures of liquid potato‐glucose medium after 4 days proved to be at the most suitable cell age for the bioconversion process with high resveratrol production and nonobvious cell growth. Highest resveratrol production (1.376 µg/L) was observed under the obtained optimal conditions of inoculum size, 12.16% (wet cell weight in 100 mL medium), and resting time, 21.3 H. The study provides a new way to produce resveratrol and establishes an essential reaction system for further study of the biosynthesis pathway of resveratrol in microorganisms, especially fungi.

Validation of reference genes for normalization of gene expression by qRT-PCR in a resveratrol-producing entophytic fungus ( Alternaria sp. MG1)

Alternaria sp. MG1, an endophytic fungus isolated from Vitis vinifera, can independently produce resveratrol, indicating that this species contains the key genes for resveratrol biosynthesis. Identification of these key genes is essential to understand the resveratrol biosynthesis pathway in this strain, which is currently unknown in microorganisms. qRT-PCR is an efficient and widely used method to identify the key genes related to unknown pathways at the level of gene expression. Verification of stable reference genes in this strain is essential for qRT-PCR data normalization, although results have been reported for other Alternaria sp. strains. In this study, nine candidate reference genes including TUBA, EF1, EF2, UBC, UFD, RPS5, RPS24, ACTB and 18S were evaluated for expression stability in a diverse set of six samples representing different growth periods. We compared cell culture conditions and an optimized condition for resveratrol production. The comparison of the results was performed using four statistical softwares. A combination of TUBA and EF1 was found to be suitable for normalization of Alternaria sp. MG1 in different developmental stages, and 18S was found to be the least stable. The reference genes verified in this study will facilitate further research to explore gene expression and molecular mechanisms as well as the improvement of secondary metabolite yields in Alternaria sp. MG1. To our knowledge, this is the first validation of reference genes in Alternaria with the capability to produce resveratrol. Additionally, these results provide useful guidelines for the selection of reference genes in other Alternaria species.

Comparison of pinoresinol diglucoside production by Phomopsis sp. XP-8 in different media and the characterisation and product profiles of the cultivation in mung bean.

BACKGROUND Phomopsis sp. XP-8 is an endophytic fungus with the ability to produce pinoresinol diglucoside (PDG) in vitro and thus has potential application in biosynthesis of PDG independent of plants. In order to enhance the production of PDG, 18 different natural materials were tested in solid-state cultivation of Phomopsis sp. XP-8. RESULTS Most of the tested natural materials promoted the production of PDG. A supplement derived from mung beans produced the highest PDG yield and better fungal growth than the other materials. Also, pinoresinol monoglucoside, pinoresinol and other substrates (phenylalanine, p-coumaric acid, cinnamic acid, caffeic acid, and ferulic acid) were obtained after fermentation on mung beans. Furthermore, PDG production was much higher when mung beans were incorporated into solid state agar versus a liquid medium. The highest pinoresinol diglucoside production (72.1 mg kg(-1) in fresh culture) was obtained in 9 days using a solid state culture of Phomopsis sp. XP-8 on a mung bean grain medium containing 100 g kg(-1) glucose. Mung bean water-soluble polysaccharide was identified as a major promoter of PDG production by Phomopsis sp. XP-8. CONCLUSION Mung bean, especially its water-soluble polysaccharide fraction, was an efficient natural material to promote PDG production by Phomopsis sp. XP-8.

Bioconversion of Pinoresinol Diglucoside and Pinoresinol from Substrates in the Phenylpropanoid Pathway by Resting Cells of Phomopsis sp.XP-8.

Pinoresinol diglucoside (PDG) and pinoresinol (Pin) are normally produced by plant cells via the phenylpropanoid pathway. This study reveals the existence of a related pathway in Phomopsis sp. XP-8, a PDG-producing fungal strain isolated from the bark of the Tu-chung tree (Eucommiaulmoides Oliv.). After addition of 0.15 g/L glucose to Phomopsis sp. XP-8, PDG and Pin formed when phenylalanine, tyrosine, leucine, cinnamic acid, and p-coumaric acid were used as the substrates respectively. No PDG formed in the absence of glucose, but Pin was detected after addition of all these substrates except leucine. In all systems in the presence of glucose, production of PDG and/or Pin and the accumulation of phenylalanine, cinnamic acid, or p-coumaric acid correlated directly with added substrate in a time- and substrate concentration- dependent manner. After analysis of products produced after addition of each substrate, the mass flow sequence for PDG and Pin biosynthesis was defined as: glucose to phenylalanine, phenylalanine to cinnamic acid, then to p-coumaric acid, and finally to Pin or PDG. During the bioconversion, the activities of four key enzymes in the phenylpropanoid pathway were also determined and correlated with accumulation of their corresponding products. PDG production by Phomopsis sp. exhibits greater efficiency and cost effectiveness than the currently-used plant-based system and will pave the way for large scale production of PDG and/or Pin for medical applications.

Lipopeptides from Bacillus subtilis have potential application in the winemaking process: inhibiting fungal and ochratoxin A contamination and enhancing esters and acids biosynthesis

Background and Aims Aspergillus carbonarius is considered the major cause of ochratoxin A (OTA) contamination in grape products, especially wine. The lipopeptides produced by Bacillus subtilis have been found to inhibit A. carbonarius contamination and OTA production in grapes. In order to assess the potential application of these lipopeptides during the winemaking process, they were tested alone or in combination with SO2 to evaluate their effect on fungal growth and OTA contamination. Methods and Results Lipopeptides at 200 and 400 mg/L were added alone or in combination with SO2 during the winemaking process using naturally contaminated grapes. Aspergillus carbonarius was also manually inoculated before fermentation to simulate grapes with a high fungal contamination and to investigate the inhibitory effect of lipopeptides on OTA-producing fungi. The lipopeptides inhibited significantly fungal contamination and OTA production, much stronger than SO2 against OTA-producing A. carbonarius, at an optimum level of 200 mg/L. Yeast growth and biosynthesis of esters and acids were also promoted by lipopeptides during winemaking, which led to an improvement of wine flavour. Conclusions Lipopeptides isolated from B. subtilis had a more potent inhibitory effect on fungal contamination and OTA accumulation than that of SO2 during winemaking. Moreover, lipopeptides had no negative effect on fermentation, wine composition and sensory attributes. Significance of the Study These findings highlight the interesting possibility for the application of lipopeptides to replace or reduce the amount of SO2 added during winemaking, especially when the inhibition of fungal contamination and OTA accumulation is considered.

Physiologically Shrinking the Solution Space of a Saccharomyces cerevisiae Genome-Scale Model Suggests the Role of the Metabolic Network in Shaping Gene Expression Noise.

Sampling the solution space of genome-scale models is generally conducted to determine the feasible region for metabolic flux distribution. Because the region for actual metabolic states resides only in a small fraction of the entire space, it is necessary to shrink the solution space to improve the predictive power of a model. A common strategy is to constrain models by integrating extra datasets such as high-throughput datasets and C13-labeled flux datasets. However, studies refining these approaches by performing a meta-analysis of massive experimental metabolic flux measurements, which are closely linked to cellular phenotypes, are limited. In the present study, experimentally identified metabolic flux data from 96 published reports were systematically reviewed. Several strong associations among metabolic flux phenotypes were observed. These phenotype-phenotype associations at the flux level were quantified and integrated into a Saccharomyces cerevisiae genome-scale model as extra physiological constraints. By sampling the shrunken solution space of the model, the metabolic flux fluctuation level, which is an intrinsic trait of metabolic reactions determined by the network, was estimated and utilized to explore its relationship to gene expression noise. Although no correlation was observed in all enzyme-coding genes, a relationship between metabolic flux fluctuation and expression noise of genes associated with enzyme-dosage sensitive reactions was detected, suggesting that the metabolic network plays a role in shaping gene expression noise. Such correlation was mainly attributed to the genes corresponding to non-essential reactions, rather than essential ones. This was at least partially, due to regulations underlying the flux phenotype-phenotype associations. Altogether, this study proposes a new approach in shrinking the solution space of a genome-scale model, of which sampling provides new insights into gene expression noise.

Verification of the phenylpropanoid pinoresinol biosynthetic pathway and its glycosides in Phomopsis sp. XP-8 using 13C stable isotope labeling and liquid chromatography coupled with time-of-flight mass spectrometry

Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (EucommiaulmoidesOliv), revealed the pinoresinol diglucoside (PDG) biosynthetic pathway after precursor feeding measurements and genomic annotation. To verify the pathway more accurately, [13C6]-labeled glucose and [13C6]-labeled phenylalanine were separately fed to the strain as sole substrates and [13C6]-labeled products were detected by ultra-high performance liquid chromatography-quantitative time of flight mass spectrometry. As results, [13C6]-labeled phenylalanine was found as [13C6]-cinnamylic acid and p-coumaric acid, and [13C12]-labeled pinoresinol revealed that the pinoresinol benzene ring came from phenylalanine via the phenylpropane pathway. [13C6]-Labeled cinnamylic acid and p-coumaric acid, [13C12]-labeled pinoresinol, [13C18]-labeled pinoresinol monoglucoside (PMG), and [13C18]-labeled PDG products were found when [13C6]-labeled glucose was used, demonstrating that the benzene ring and glucoside of PDG originated from glucose. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study verified the occurrence of the plant-like phenylalanine and lignan biosynthetic pathway in fungi. Importance Verify the phenylpropanoid-pinoresinol biosynthetic pathway and its glycosides in an endophytic fungi.

Fungal Spores Promote the Glycerol Production of Saccharomyces cerevisiae by Upregulating the Oxidative Balance Pathway

Fungal contamination is prevalent in grape berries and unavoidable during the winemaking process. In botrytized wine, Botrytis cinerea contamination of grape berries beneficially promotes the wine flavor, which is desirable especially with high glycerol content. To investigate the underlying mechanism, Aspergillus carbonarius and B. cinerea spores were separately cocultured with two different Saccharomyces cerevisiae strains in both grape juice and synthetic nutrient media. The results showed that both A. carbonarius and B. cinerea promoted glycerol accumulation and the consumption of sugars in the coculture systems but could not synthesize glycerol by themselves. The metabolites produced by fungal spores triggered these reactions. Reverse transcription-polymerase chain reaction analysis showed that the presence of A. carbonarius spores regulated the expression of GPP1 and GPD2, indicating that the reaction was triggered by regulating the oxidative balance pathway. The study revealed the beneficial impact of fungal contamination on wine quality by influencing yeast metabolism.