Uncovering global lipid accumulation routes towards docosahexaenoic acid (DHA) production in Aurantiochytrium sp. SW1 using integrative proteomic analysis.

Aurantiochytrium sp., a marine thraustochytrid possesses a remarkable ability to produce lipid rich in polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA). Although gene regulation underlying lipid biosynthesis has been previously reported, proteomic analysis is still limited. In this study, high DHA accumulating strain Aurantiochytrium sp. SW1 has been used as a study model to elucidate the alteration in proteome profile under different cultivation phases i.e. growth, nitrogen-limitation and lipid accumulation. Of the total of 5146 identified proteins, 852 proteins were differentially expressed proteins (DEPs). The largest number of DEPs (488 proteins) was found to be uniquely expressed between lipid accumulating phase and growth phase. Interestingly, there were up-regulated proteins involved in glycolysis, glycerolipid, carotenoid and glutathione metabolism which were preferable metabolic routes towards lipid accumulation and DHA production as well as cellular oxidative defence. Integrated proteomic and transcriptomic data were also conducted to comprehend the gene and protein regulation underlying the lipid and DHA biosynthesis. A significant up-regulation of acetyl-CoA synthetase was observed which suggests alternative route of acetate metabolism for acetyl-CoA producer. This study presents the holistic routes underlying lipid accumulation and DHA production in Aurantiochytrium sp. SW1 and other relevant thraustochytrid.

Functional insight into Cordyceps militaris sugar transporters by structure modeling, network analysis and allosteric regulation.

Insights into the structures, functions and dynamics of Cordyceps militaris (C. militaris) sugar transporters are necessary for understanding their versatile metabolic capability for fungal growth. The sequence-function relationship study of 85 C. militaris sugar transporters showed that there is a gap between phylogenetic-based subfamily classification and their functions. Beyond protein sequences, structural modeling and principal component analysis of the structural ensemble revealed the different folds of the Car and Org subfamilies. Performing channel detection and network analysis found that the Alp and Hex subfamilies can be specifically distinguished from others by the betweenness of channel residues. Signature dynamics analysis further suggested that the Hex subfamily demonstrates different dynamics, with high flexibility at the H1 region in TM11. Furthermore, the H1 region as an allosteric site was examined by network parameter calculations that guided allosteric pathways between this region and the channel cavity. Together with gene expression data of C. militaris, e.g., Hex06741 in the Hex subfamily, it was promisingly expressed when sugar utilization was altered. This work demonstrates an in silico framework for investigating C. militaris sugar transporters as an example case study of the allosteric activity of the Hex subfamily and can facilitate sugar transporter engineering design that can further optimize the preferable sugar utilization and fermentation process of C. militaris.

Functional genomics and systems biology of Cordyceps species for biotechnological applications.

The use of Cordyceps species for the manufacture of natural products has been established; however, the tremendous advances observed in recent years in genetic engineering and molecular biology have revolutionized the optimization of Cordyceps as cell factories and drastically expanded the biotechnological potential of these fungi. Here, we present a review of systems and synthetic biology studies of Cordyceps and their implications for fungal biology and industrial applications. We summarize the current status of synthetic biology for enhancing targeted metabolites in Cordyceps species, such as cordycepin, adenosine, polysaccharide, and pentostatin. Progress in the systems and synthetic biology of Cordyceps provides a strategy for comprehensively comprehensive controlling efficient cell factories of natural bioproducts and novel synthetic biology toolbox for targeted engineering.

Revealing holistic metabolic responses associated with lipid and docosahexaenoic acid (DHA) production in Aurantiochytrium sp. SW1.

Aurantiochytrium sp. SW1, a marine thraustochytrid, has been regarded as a potential candidate as a docosahexaenoic acid (DHA) producer. Even though the genomics of Aurantiochytrium sp. are available, the metabolic responses at a systems level are largely unknown. Therefore, this study aimed to investigate the global metabolic responses to DHA production in Aurantiochytrium sp. through transcriptome and genome-scale network-driven analysis. Of a total of 13,505 genes, 2527 differentially expressed genes (DEGs) were identified in Aurantiochytrium sp., unravelling the transcriptional regulations behinds lipid and DHA accumulation. The highest number of DEG were found for pairwise comparison between growth phase and lipid accumulating phase where a total of 1435 genes were down-regulated with 869 genes being up-regulated. These uncovered several metabolic pathways that contributing in DHA and lipid accumulation including amino acid and acetate metabolism which involve in the generation of crucial precursors. Upon applying network-driven analysis, hydrogen sulphide was found as potential reporter metabolite that could be associated with the genes related to acetyl-CoA synthesis for DHA production. Our findings suggest that the transcriptional regulation of these pathways is a ubiquitous feature in response to specific cultivation phases during DHA overproduction in Aurantiochytrium sp. SW1.

Parallel screening and cheminformatics modeling of flavonoid activated aptasensors.

A parallel screening of 27 different flavonoids and chalcones was conducted using 6 artificial naringenin-activated riboswitches (M1, M2, M3, O, L and H). A quantitative structure-property relationship approach was applied to understand the physicochemical properties of the flavonoid structures resulting in specificity differences relied on the fluorescence intensity of a green fluorescent protein reporter. Robust models of riboswitches M1, M2 and O that had good predictive power were constructed with descriptors selected for their high correlation. Increased electronegativity and hydrophilicity of the flavonoids structures were identified as two properties that increased binding affinity to RNA riboswitches. Hydroxyl groups at the C-3' and C-4' positions of the flavonoid molecule were strictly required for ligand-activation with riboswitches M1 and M2. Riboswitches O and L preferred multi-hydroxylated flavones as ligands. Substitutions on the A ring of the flavonoid molecule were not important in the molecular recognition process. O-glycosylated derivatives were not recognized by any of the riboswitches, presumably due to steric hindrances. Despite the challenges of detecting RNA conformational change after ligand binding, the resulting models elucidate important physicochemical features in the ligands for conformational structural studies of artificial aptamer complexes and for design of ligands having higher binding specificity.

Whole genome analysis and elucidation of docosahexaenoic acid (DHA) biosynthetic pathway in Aurantiochytrium sp. SW1.

Aurantiochytrium sp., a fungoid marine protist that belongs to Stramenophila has proven its potential in the production of polyunsaturated fatty acids (PUFAs), especially docosahexaenoic acids (DHA). In this study, genomic characterisation of a potential producer for commercial production of DHA, Aurantiochytrium sp. SW1 has been carried out via whole genome sequencing analysis. The genome size of this strain is 60.89 Mb, with a total of 11,588 protein-coding genes. Among these, 9,127 genes could be functionally annotated into a total of 7,248 (62.5 %) from UniProt, 6,554 (56.6 %) from KEGG and 8,643 (74.6 %) genes from eggNOG protein database. The highest proportion of genes belongs to the protein family of metabolism were further assigned into 11 metabolic categories. The highest number of genes belonging to lipid metabolism (321 genes) followed by carbohydrate metabolism (290 genes), metabolism of cofactors and vitamins (197 genes) and amino acid metabolism (188 genes). Further analysis into the biosynthetic pathway for DHA showed evidence of all genes involved in PKS (polyketide synthase)-like PUFA synthase pathway and incomplete fatty acid synthase-elongase/desaturase pathway. Analysis of PUFA synthase showed the presence of up to ten tandem acyl carrier protein (ACP) domains which might have contributed to high DHA production in this organism. In addition, a hybrid system incorporating elements of FAS, Type I PKS and Type II PKS systems were found to be involved in the biosynthetic pathways of fatty acids in Aurantiochytrium sp. SW1. This study delivers an important reference for future research to enhance the lipid, especially DHA production in Aurantiochytrium sp, SW1 and establishment of this strain as an oleaginous thraustochytrid model.

Different Classes of Phytohormones Act Synergistically to Enhance the Growth, Lipid and DHA Biosynthetic Capacity of Aurantiochytrium sp. SW1.

In the present study, the impact of eight phytohormones from six different classes on the growth, lipid and docosahexaenoic acid (DHA) biosynthetic capacity of Aurantiochytrium sp. SW1 (SW1) was evaluated. Kinetin (KIN), jasmonic acid (JA) and gibberellic acid (GA) significantly enhanced the growth and DHA production of SW1 by 16%-28% and 66%-84% in comparison to the control, respectively. The synergistic effect of these three phytohormones, evaluated by the response surface methodology (RSM), showed that a combination of 3.6 mg/L GA, 2.0 mg/L KIN and 20.0 mg/L JA further increased the growth and DHA production of SW1 by 16% to 28% and 22% to 36%, respectively, in comparison to the individual supplementation. The synergistic effect of these phytohormones was also shown to be time-dependent, where feeding at 24 h of cultivation led to 15%, 26% and 35% further increments in the biomass, lipid and DHA production in comparison to that of 0 h, respectively. The determination of stress markers, antioxidant enzymes and key enzymes involved in fatty acid biosynthesis aided to elucidate the potential mechanism underlying the improvement of growth and DHA production by SW1 at various times of feeding. Supplementation with the phytohormones at 24 h exhibited the maximum impact on reducing the level of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as augmented the antioxidants (superoxide dismutase and catalase) and key metabolic enzymes involved in lipogenesis (malic, glucose-6-phosphate dehydrogenase and ATP-citrate lyase) in comparison to the control and other time points. This study signifies the potential application of phytohormones for improving the growth, lipid and DHA production in Aurantiochytrium spp.