Jaslyn Lee

Metabolomic profiling of rhodosporidium toruloides grown on glycerol for carotenoid production during different growth phases

Carotenoid production from three strains of Rhodosporidium toruloides grown on glycerol was studied. A time-dependent metabolomics approach was used to understand its metabolism on glycerol and mechanism for carotenoid production in three strains during different growth phases (1, 4, 7, and 12 days). Strain CBS 5490 was the highest carotenoid producer (28.5 mg/L) and had a unique metabolic profile. In this strain, metabolites belonging to the TCA cycle and amino acids were produced in lower amounts, as compared to the other strains. On the other hand, it produced the highest amounts of carotenoid and fatty acid metabolites. This indicated that the lower production of the TCA cycle and amino acid metabolites promoted energy and metabolic flux toward the carotenoid and fatty acid synthesis metabolic pathways. This study shows that metabolomic profiling is a useful tool to gain insight into the metabolic pathways in the cell and to shed light on the different molecular mechanisms between strains.

Comparative Proteomics Profile of Lipid-Cumulating Oleaginous Yeast: An iTRAQ-Coupled 2-D LC-MS/MS Analysis

Accumulation of intracellular lipid in oleaginous yeast cells has been studied for providing an alternative supply for energy, biofuel. Numerous studies have been conducted on increasing lipid content in oleaginous yeasts. However, few explore the mechanism of the high lipid accumulation ability of oleaginous yeast strains at the proteomics level. In this study, a time-course comparative proteomics analysis was introduced to compare the non-oleaginous yeast Saccharomyces cerevisiae, with two oleaginous yeast strains, Cryptococcus albidus and Rhodosporidium toruloides at different lipid accumulation stages. Two dimensional LC-MS/MS approach has been applied for protein profiling together with isobaric tag for relative and absolute quantitation (iTRAQ) labelling method. 132 proteins were identified when three yeast strains were all at early lipid accumulation stage; 122 and 116 proteins were found respectively within cells of three strains collected at middle and late lipid accumulation stages. Significantly up-regulation or down-regulation of proteins were experienced among comparison. Essential proteins correlated to lipid synthesis and regulation were detected. Our approach provides valuable indication and better understanding for lipid accumulation mechanism from proteomics level and would further contribute to genetic engineering of oleaginous yeasts.

Comparative proteomic analysis of engineered Saccharomyces cerevisiae with enhanced free fatty acid accumulation.

The engineered Saccharomyces cerevisiae strain △faa1△faa4 [Acot5s] was demonstrated to accumulate more free fatty acids (FFA) previously. Here, comparative proteomic analysis was performed to get a global overview of metabolic regulation in the strain. Over 500 proteins were identified, and 82 of those proteins were found to change significantly in the engineered strains. Proteins involved in glycolysis, acetate metabolism, fatty acid synthesis, TCA cycle, glyoxylate cycle, the pentose phosphate pathway, respiration, transportation, and stress response were found to be upregulated in △faa1△faa4 [Acot5s] as compared to the wild type. On the other hand, proteins involved in glycerol, ethanol, ergosterol, and cell wall synthesis were downregulated. Taken together with our metabolite analysis, our results showed that the disruption of Faa1 and Faa4 and expression of Acot5s in the engineered strain △faa1△faa4 [Acot5s] not only relieved the feedback inhibition of fatty acyl-CoAs on fatty acid synthesis, but also caused a major metabolic rearrangement. The rearrangement redirected carbon flux toward the pathways which generate the essential substrates and cofactors for fatty acid synthesis, such as acetyl-CoA, ATP, and NADPH. Therefore, our results help shed light on the mechanism for the increased production of fatty acids in the engineered strains, which is useful in providing information for future studies in biofuel production.

Analysis of Soluble Microbial Products by Mass Spectrometry: Potential in Monitoring Bioprocesses of Wastewater Treatment

In biological wastewater treatment process, the analysis of metabolic compounds that are produced during the process is critical to monitor the performance of microorganisms. The soluble products present in the effluent directly affect the process efficiency and quality of the water after treatment, and it is also the major reason for fouling in membrane bioreactor. Currently, analytical methods are mainly restricted to the overall measurement of the total amount of polysaccharides, DNA and proteins without any specific identification of these compounds. Here we introduce an explorative mass spectrometry based strategy, for the analysis of soluble microbial products and other soluble impurities in the effluents of wastewater treatments using different digestion process. According to the results from this study, the two stage co digestion process indicated higher treatment efficiency compared with the single stage process, since fewer compounds were detected in the effluent. For the two-stage process, most of the fatty acids produced in the first stage of digestion by hydrolysis and acidogenesis, were digested in the second stage. The results also indicated that the digestion efficiency of the single stage process was lower than that of the separated two-stage process. This study is one of few exploration of analyzing and identifying unknown compounds using MS based technique from a metabolic analysis perspective. Our novel approach can be applied as an analytical platform to effectively monitor the biological processes and provides a different view point in wastewater treatment systems.

Analysis of Improved Nutritional Composition of Potential Functional Food (Okara) after Probiotic Solid-State Fermentation

Okara is a major agro-waste, generated as a byproduct from the soymilk and tofu industry. Since okara has a high nutritive value, reusing it as a substrate for solid state biofermentation is an economical and environmental friendly option. Rhizopus oligosporus and Lactobacillus plantarum were the probiotic FDA-approved food-grade cultures used in this study. The study revealed that biofermenting okara improves its nutritional composition. It was found that the metabolomic composition (by GC-MS analysis) and antioxidant activity (by DPPH test) improved after the microbial fermentations. Of the two, okara fermented with R. oligosporus showed better results. Further, the metabolites were traced back to their respective biosynthesis pathways, in order to understand the biochemical reactions being triggered during the fermentation processes. The findings of this entire work open up the possibility of employing fermented okara as a potential functional food for animal feed.

Dual Use of a Biopolymer From Durian (Durio zibethinus) Seed as a Nutrient Source and Stabilizer for Spray Dried Lactobacillus Plantarum

The search for natural and sustainable biopolymers is increasing. Biopolymers are used in applications, such as to encapsulate and stabilize probiotic bacteria. Currently, this process requires many steps, including the separation of bacteria from the culture media after fermentation, which is energy intensive. In this study, we developed a strategy to use a natural biopolymer from durian seed, durian seed gum (DSG), to address these issues. DSG was used to serve dual roles, firstly as a nutrient source, and secondly as an encapsulating agent. DSG was used in synergy with reconstituted skim milk (RSM), to be a complete nutrient source, for probiotic bacteria Lactobacillus plantarum (L. plantarum). Usually RSM requires supplementation, with costly yeast extract. DSG was characterized to be a suitable nutrient source as it contains polysaccharides, organic acids, amino acids and fatty acids. In the presence of DSG and RSM, the growth of L. plantarum increased from 8.73 log CFU/mL to 13.86 log CFU/mL, at the end of 72 h. In comparison, when grown with commercial gum arabic (GA) and RSM, growth of L. plantarum reached a lower 11.49 log CFU/mL at 72 h. Metabolomics revealed that several metabolites, including lactose, depleted after fermentation, in the DSG and RSM treatment group, as compared to GA and RSM treatment group. This suggested that glycolysis were up regulated. This correlated with the increased growth, lactic acid, malic acid and fatty acids production by L. plantarum when it was grown on DSG and RSM. Next, the entire culture of L. plantarum with DSG and RSM was taken for spray drying, without the need to separate the bacteria from the culture media. DSG and RSM was able to stabilize L. plantarum, to remain viable at 10 log CFU/g after storage for 10 weeks. On the other hand, the viability was 8.8 log CFU/g when L. plantarum was spray dried with GA and RSM. This study demonstrated an innovative process and low-cost strategy to produce spray dried probiotic powder. DSG could be a potential low cost and sustainable replacement for GA, to develop other functional foods.