QTLs for oil yield components in an elite oil palm (Elaeis guineensis) cross

Increased modern farming of superior types of the oil palm, Elaeis guineensis Jacq., which has naturally efficient oil biosynthesis, has made it the world’s foremost edible oil crop. Breeding improvement is, however, circumscribed by time and costs associated with the tree’s long reproductive cycle, large size and 10–15 years of field testing. Marker-assisted breeding has considerable potential for improving this crop. Towards this, quantitative trait loci (QTL) linked to oil yield component traits were mapped in a high-yield population. In total, 164 QTLs associated with 21 oil yield component traits were discovered, with cumulative QTL effects increasing in tandem with the number of QTL markers and matching the QT+ alleles for each trait. The QTLs confirmed all traits to be polygenic, with many genes of individual small effects on independent loci, but epistatic interactions are not ruled out. Furthermore, several QTLs maybe pleiotropic as suggested by QTL clustering of inter-related traits on almost all linkage groups. Certain regions of the chromosomes seem richer in the genes affecting a particular yield component trait and likely encompass pleiotropic, epistatic and heterotic effects. A large proportion of the identified additive effects from QTLs may actually arise from genic interactions between loci. Comparisons with previous mapping studies show that most of the QTLs were for similar traits and shared similar marker intervals on the same linkage groups. Practical applications for such QTLs in marker-assisted breeding will require seeking them out in different genetic backgrounds and environments

High density SNP and DArT-based genetic linkage maps of two closely related oil palm populations

Oil palm (Elaeis guineensis Jacq.) is an outbreeding perennial tree crop with long breeding cycles, typically 12 years. Molecular marker technologies can greatly improve the breeding efficiency of oil palm. This study reports the first use of the DArTseq platform to genotype two closely related self-pollinated oil palm populations, namely AA0768 and AA0769 with 48 and 58 progeny respectively. Genetic maps were constructed using the DArT and SNP markers generated in combination with anchor SSR markers. Both maps consisted of 16 major independent linkage groups (2n = 2× = 32) with 1399 and 1466 mapped markers for the AA0768 and AA0769 populations, respectively, including the morphological trait “shell-thickness” (Sh). The map lengths were 1873.7 and 1720.6 cM with an average marker density of 1.34 and 1.17 cM, respectively. The integrated map was 1803.1 cM long with 2066 mapped markers and average marker density of 0.87 cM. A total of 82% of the DArTseq marker sequence tags identified a single site in the published genome sequence, suggesting preferential targeting of gene-rich regions by DArTseq markers. Map integration of higher density focused around the Sh region identified closely linked markers to the Sh, with D.15322 marker 0.24 cM away from the morphological trait and 5071 bp from the transcriptional start of the published SHELL gene. Identification of the Sh marker demonstrates the robustness of using the DArTseq platform to generate high density genetic maps of oil palm with good genome coverage. Both genetic maps and integrated maps will be useful for quantitative trait loci analysis of important yield traits as well as potentially assisting the anchoring of genetic maps to genomic sequences

Systematic development of biomass overproducing Scheffersomyces stipitis for high-cell-density fermentations

The development of economically feasible bio-based process requires efficient cell factories capable of producing the desired product at high titer under high-cell-density fermentation. Herein we present a combinatorial approach based on systems metabolic engineering and metabolic evolution for the development of efficient biomass-producing strain. Systems metabolic engineering guided by flux balance analysis (FBA) was first employed to rationally design mutant strains of Scheffersomyces stipitis with high biomass yield. By experimentally implementing these mutations, the biomass yield was improved by 30% in GPD1, 25% in TKL1, 30% in CIT1, and 44% in ZWF1 overexpressed mutants compared to wild-type. These designed mutants were further fine-tuned through metabolic evolution resulting in the maximal biomass yield of 0.49 g-cdw/g-glucose, which matches well with predicted yield phenotype. The constructed mutants are beneficial for biotechnology applications dealing with high cell titer cultivations. This work demonstrates a solid confirmation of systems metabolic engineering in combination with metabolic evolution approach for efficient strain development, which could assist in rapid optimization of cell factory for an economically viable and sustainable bio-based process

Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform

Abstract Background Cordycepin (3′-deoxyadenosine) is an important bioactive compound in medical and healthcare markets. The drawbacks of commercial cordycepin production using Cordyceps spp. include long cultivation periods and low cordycepin yields. To overcome these limitations and meet the increasing market demand, the efficient production of cordycepin by the GRAS-status Aspergillus oryzae strain using a synthetic biology approach was developed in this study. Results An engineered strain of A. oryzae capable of cordycepin production was successfully constructed by overexpressing two metabolic genes (cns1 and cns2) involved in cordycepin biosynthesis under the control of constitutive promoters. Investigation of the flexibility of carbon utilization for cordycepin production by the engineered A. oryzae strain revealed that it was able to utilize C6-, C5-, and C12-sugars as carbon sources, with glucose being the best carbon source for cordycepin production. High cordycepin productivity (564.64 ± 9.59 mg/L/d) was acquired by optimizing the submerged fermentation conditions. Conclusions This study demonstrates a powerful production platform for bioactive cordycepin production by A. oryzae using a synthetic biology approach. An efficient and cost-effective fermentation process for cordycepin production using an engineered strain was established, offering a powerful alternative source for further upscaling

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\ua0bps 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

Additional file 1 of Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform

Supplementary Material 1: Fig. S1: The purine nucleotide pathway towards cordycepin biosynthesis in fungi [1, 2]. APRT, adenine phosphoribosyltransferase; AMPD, AMP deaminase; ADK, adenosine kinase; NT5E, 5’-nucleotidase; ADA, adenosine deaminase; PNP, purine nucleoside phosphorylase; PDE, phosphodiesterases; CNS1, oxidoreductase/dehydrogenase; CNS2, metal-dependent phosphohydrolase; CNS3 (ATPPRT), ATP phosphoribosyl transferase. The straight and dashed lines show validated and predicted pathways of cordycepin, respectively. Question mark indicates those reactions are unknown. Fig. S2: Schematic map of pAoCordy plasmid and integration event of expression cassette into A. oryzae genome based on homologous recombination mechanism. The flanking regions corresponding to the PyrG targeted locus are represented by dark-gray boxes (PyrG-LF and PyrG-RF). The dotted arrows indicate the positions of primer pairs used for determination of the integration event in engineered strain. The expected PCR fragment corresponding to the 5’- and 3’-regions of the targeted pyrG integration into the genome are shown. Fig. S3: Genetic and phenotypic stability of the AoCordy-T1 transformant. The spore of 1st-, 3rd- and 5th-subculturing transformants were inoculated and grown in the SM medium for 48 h. The genomic DNA of the transformant was subjected to PCR analysis for verifying its genetic stability (A). Lane M indicates a 1-kb DNA marker. Lanes 1, 3, and 5 show the amplified products of the cns1 expression cassette. Lanes 2, 4, and 6 show the amplified products of the cns2 expression cassette. The phenotypic stability in cordycepin production of the transformant was analyzed by HPLC-UV (B

Additional file 1: Figure S1. of The codon-optimized Δ6-desaturase gene of Pythium sp. as an empowering tool for engineering n3/n6 polyunsaturated fatty acid biosynthesis

Comparison of deduced amino acid sequences between PyDes6 of Pythium sp. BCC53698 and other ∆6-desaturases, including P. infestans (PinDES6), P. splendens (PspDES6), N. oculata (NoDES6), M. alpina (MaDES6), M. rouxii (MrDES6) and Borage officinalis (BoDES6). The conserved histidine boxes and cytochrome b 5 heme-binding motif are underlined and boxed, respectively. Figure S2. Phylogenetic relationships between PyDes6 (Pythium sp. BCC53698) and ∆6-desaturase genes of other organisms. Bootstrap values from 1000 replicates test are shown at individual nodes. Abbreviations of other desaturase genes in individual organisms: PciDES6, P. citrophthora; PsoDES6, P.sojae; PinDES6, P. infestans; PirDES6, P. irregular; PspDES6, P. splendens; AlDES6, Albugo laibachii; NoDES6, N. oculata; EsDES6, Ectocarpus siliculosus; TpDES6, Thalassiosira pseudonana; PtDES6, P. tricornutum; MaDES6, M. alpina; RsDES6, Rhizopus stolonifer; McDES6, Mucor circinelloides; MrDES6, M. rouxii; MpDES6, Marchantia polymorpha; PpDES6, Physcomitrella patens; BoDES6, B. officinalis. Figure S3. Growth of the recombinant yeast strains carrying the empty vector (pYES2), PyDes6 and MPyDes6 genes. All strains were cultivated in SD medium containing 20 g/l of raffinose for 96 h. Cell growth is represented in terms of dry cell weight (grey bar) and cell density at OD600 (upward diagonals bar). (DOCX 1439 kb