Metabolic Regulation of Sugar Assimilation for Lipid Production in Aspergillus oryzae BCC7051 through Comparative Transcriptome Perspective.

Microbial lipid production with cost effectiveness is a prerequisite for the oleochemical sector. In this work, genome-wide transcriptional responses on the utilization of xylose and glucose in oleaginous Aspergillus oryzae were studied with relation to growth and lipid phenotypic traits. Comparative analysis of the active growth (t1) and lipid-accumulating (t2) stages showed that the C5 cultures efficiently consumed carbon sources for biomass and lipid production comparable to the C6 cultures. By pairwise comparison, 599 and 917 differentially expressed genes (DEGs) were identified in the t1 and t2 groups, respectively, in which the consensus DEGs were categorized into polysaccharide-degrading enzymes, membrane transports, and cellular processes. A discrimination in transcriptional responses of DEGs set was also found in various metabolic genes, mostly in carbohydrate, amino acid, lipid, cofactors, and vitamin metabolisms. Although central carbohydrate metabolism was shared among the C5 and C6 cultures, the metabolic functions in acetyl-CoA and NADPH generation, and biosynthesis of terpenoid backbone, fatty acid, sterol, and amino acids were allocated for leveraging biomass and lipid production through at least transcriptional control. This study revealed robust metabolic networks in the oleaginicity of A. oryzae governing glucose/xylose flux toward lipid biosynthesis that provides meaningful hints for further process developments of microbial lipid production using cellulosic sugar feedstocks.

Safety Assessment of a Nham Starter Culture Lactobacillus plantarum BCC9546 via Whole-genome Analysis.

The safety of microbial cultures utilized for consumption is vital for public health and should be thoroughly assessed. Although general aspects on the safety assessment of microbial cultures have been suggested, no methodological detail nor procedural guideline have been published. Herein, we propose a detailed protocol on microbial strain safety assessment via whole-genome sequence analysis. A starter culture employed in traditional fermented pork production, nham, namely Lactobacillus plantarum BCC9546, was used as an example. The strain's whole-genome was sequenced through several next-generation sequencing techniques. Incomplete plasmid information from the PacBio sequencing platform and shorter chromosome size from the hybrid Oxford Nanopore-Illumina platform were noted. The methods for 1) unambiguous species identification using 16S rRNA gene and average nucleotide identity, 2) determination of virulence factors and undesirable genes, 3) determination of antimicrobial resistance properties and their possibility of transfer, and 4) determination of antimicrobial drug production capability of the strain were provided in detail. Applicability of the search tools and limitations of databases were discussed. Finally, a procedural guideline for the safety assessment of microbial strains via whole-genome analysis was proposed.

Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum.

Protein production relies on time-consuming genetic engineering and in vivo expression, which is a bottleneck for functional studies in the postgenomic era. Cell-free protein synthesis (CFPS) overcomes the limitation of in vivo protein biosynthesis by processing in vitro transcription and translation of multiple genes to proteins within hours. We employed an automated CFPS to simultaneously synthesize proteins from 24 genes of the oomycete Pythium insidiosum (which causes the life-threatening disease pythiosis) and screen for a diagnostic and therapeutic target. CFPS successfully synthesized 18 proteins (∼75% success rate). One protein, namely, I06, was explicitly recognized by all pythiosis sera, but not control sera, tested. Py. insidiosum secreted a significant amount of I06. The protein architecture of I06 is compatible with the oligopeptide elicitor (OPEL) of the phylogenetically related plant-pathogenic oomycete Phytophthora parasitica The OPEL-like I06 protein of Py. insidiosum can stimulate host antibody responses, similar to the P. parasitica OPEL that triggers plant defense mechanisms. OPEL-like I06 homologs are present only in the oomycetes. Py. insidiosum contains two OPEL-like I06 homologs, but only one of the two homologs was expressed during hyphal growth. Twenty-nine homologs derived from 15 oomycetes can be phylogenetically divided into two groups. The OPEL-like genes might occur in the common ancestor, before independently undergoing gene gain and loss during the oomycete speciation. In conclusion, CFPS offers a fast in vitro protein synthesis. CFPS simultaneously generated multiple proteins of Py. insidiosum and facilitated the identification of the secretory OPEL-like I06 protein, a potential target for the development of a control measure against the pathogen.IMPORTANCE Technical limitations of conventional biotechnological methods (i.e., genetic engineering and protein synthesis) prevent extensive functional studies of the massive amounts of genetic information available today. We employed a cell-free protein synthesis system to rapidly and simultaneously generate multiple proteins from genetic codes of the oomycete Pythium insidiosum, which causes the life-threatening disease called pythiosis, in humans and animals worldwide. We aimed to screen for potential diagnostic and therapeutic protein targets of this pathogen. Eighteen proteins were synthesized. Of the 18 proteins, one was a secreted immunoreactive protein, called I06, that triggered host immunity and was recognized explicitly by all tested sera from pythiosis patients. It is one of the OPEL proteins; these proteins are present only in the unique group of microorganisms called oomycetes. Here, we demonstrated that cell-free protein synthesis was useful for the production of multiple proteins to facilitate functional studies and identify a potential target for diagnosis and treatment of pythiosis.

Systematic genome analysis of a novel arachidonic acid-producing strain uncovered unique metabolic traits in the production of acetyl-CoA-derived products in Mortierellale fungi.

The fungi in order Mortierellales are attractive producers for long-chain polyunsaturated fatty acids (PUFAs). Here, the genome sequencing and assembly of a novel strain of Mortierella sp. BCC40632 were done, yielding 65 contigs spanning of 49,964,116 total bases with predicted 12,149 protein-coding genes. We focused on the acetyl-CoA in relevant to its derived metabolic pathways for biosynthesis of macromolecules with biological functions, including PUFAs, eicosanoids and carotenoids. By comparative genome analysis between Mortierellales and Mucorales, the signature genetic characteristics of the arachidonic acid-producing strains, including Δ5-desaturase and GLELO-like elongase, were also identified in the strain BCC40632. Remarkably, this fungal strain contained only n-6 pathway of PUFA biosynthesis due to the absence of Δ15-desaturase or ω3-desaturase gene in contrast to other Mortierella species. Four putative enzyme sequences in the eicosanoid biosynthetic pathways were identified in the strain BCC40632 and others Mortierellale fungi, but were not detected in the Mucorales. Another unique metabolic trait of the Mortierellales was the inability in carotenoid synthesis as a result of the lack of phytoene synthase and phytoene desaturase genes. The findings provide a perspective in strain optimization for production of tailored-made products with industrial applications.

Genome Characterization of Oleaginous Aspergillus oryzae BCC7051: A Potential Fungal-Based Platform for Lipid Production.

The selected robust fungus, Aspergillus oryzae strain BCC7051 is of interest for biotechnological production of lipid-derived products due to its capability to accumulate high amount of intracellular lipids using various sugars and agro-industrial substrates. Here, we report the genome sequence of the oleaginous A. oryzae BCC7051. The obtained reads were de novo assembled into 25 scaffolds spanning of 38,550,958 bps with predicted 11,456 protein-coding genes. By synteny mapping, a large rearrangement was found in two scaffolds of A. oryzae BCC7051 as compared to the reference RIB40 strain. The genetic relationship between BCC7051 and other strains of A. oryzae in terms of aflatoxin production was investigated, indicating that the A. oryzae BCC7051 was categorized into group 2 nonaflatoxin-producing strain. Moreover, a comparative analysis of the structural genes focusing on the involvement in lipid metabolism among oleaginous yeast and fungi revealed the presence of multiple isoforms of metabolic enzymes responsible for fatty acid synthesis in BCC7051. The alternative routes of acetyl-CoA generation as oleaginous features and malate/citrate/pyruvate shuttle were also identified in this A. oryzae strain. The genome sequence generated in this work is a dedicated resource for expanding genome-wide study of microbial lipids at systems level, and developing the fungal-based platform for production of diversified lipids with commercial relevance.