3.1.1.2 Feed Processing and Handling DL2 Final Report

This milestone report is the deliverable for our Feed Processing and Handling project. It includes results of wet biomass feedstock analysis, slurry pumping information, fungal processing to produce a lignin-rich biorefinery residue and two subcontracted efforts to quantify the amount of wet biomass feedstocks currently available within the corn processing and paper processing industries

The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

<p>Abstract</p> <p>Background</p> <p><it>Dunaliella salina </it>Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because <it>D. salina </it>can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of <it>D. salina </it>have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of <it>D. salina </it>and compares them with those of the model green algae <it>Chlamydomonas reinhardtii </it>and <it>Volvox carteri</it>.</p> <p>Results</p> <p>The <it>D. salina </it>organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the <it>D. salina </it>plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the <it>D. salina </it>ptDNA.</p> <p>Conclusions</p> <p>These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of <it>D. salina </it>and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the <it>D. salina </it>organelle DNA sequences will aid in the development of a viable plastid transformation system for this model alga, and they will complement the forthcoming <it>D. salina </it>nuclear genome sequence, placing <it>D. salina </it>in a group of a select few photosynthetic eukaryotes for which complete genome sequences from all three genetic compartments are available.</p

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND:Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS:The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4&nbsp;g/L. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism

Regulation of Yeast-to-Hyphae Transition in <i>Yarrowia lipolytica</i>

The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae We confirmed that the Y. lipolyticamsn2 (Ylmsn2) ortholog is required for hyphal growth and found that overexpression of Ylmsn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G1-to-S phase progression. We found that overexpression of either the Ylmbp1 or Ylswi6 homologs decreased hyphal growth and that deletion of either Ylmbp1 or Ylswi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Ylchk1 and Ylnik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Ylssk2, Ylpbs2, and Ylhog1 Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways.IMPORTANCE Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in Yarrowia lipolytica We confirmed that the transcription factor Ylmsn2 is required for the transition to hyphal growth and found that signaling by the histidine kinases Ylchk1 and Ylnik1 as well as the MAP kinases of the HOG pathway (Ylssk2, Ylpbs2, and Ylhog1) regulates the transition to hyphal growth. These results suggest that Y. lipolytica transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response

A comparative genomics study of 23 Aspergillus species from section Flavi

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.Peer reviewe

Investigation of inter- and intraspecies variation through genome sequencing of Aspergillus section Nigri

Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter-and intraspecies genomic variation. We further predicted 17,903 carbohydrateactive enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.Peer reviewe

Review of the algal biology program within the National Alliance for Advanced Biofuels and Bioproducts

In 2010,when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortiumbegan, littlewas known about themolecular basis of algal biomass or oil production. Very fewalgal genome sequenceswere available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy\u27s Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played bymetabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels

Identification of Genes Associated with Morphology in Aspergillus niger by Using Suppression Subtractive Hybridization

The morphology of citric acid production strains of Aspergillus niger is sensitive to a variety of factors, including the concentration of manganese (Mn(2+)). Upon increasing the Mn(2+) concentration in A. niger (ATCC 11414) cultures to 14 ppb or higher, the morphology switches from pelleted to filamentous, accompanied by a rapid decline in citric acid production. The molecular mechanisms through which Mn(2+) exerts effects on morphology and citric acid production in A. niger cultures have not been well defined, but our use of suppression subtractive hybridization has identified 22 genes responsive to Mn(2+). Fifteen genes were differentially expressed when A. niger was grown in media containing 1,000 ppb of Mn(2+) (filamentous form), and seven genes were expressed in 10 ppb of Mn(2+) (pelleted form). Of the 15 filament-associated genes, seven are novel and eight share 47 to 100% identity with genes from other organisms. Five of the pellet-associated genes are novel, and the other two genes encode a pepsin-type protease and polyubiquitin. All 10 genes with deduced functions are either involved in amino acid metabolism-protein catabolism or cell regulatory processes. Northern blot analysis showed that the transcripts of all 22 genes were rapidly enhanced or suppressed by Mn(2+). Steady-state mRNA levels of six selected filament-associated genes remained high during 5 days of culture in a filamentous state and remained low under pelleted growth conditions. The opposite behavior was observed for four selected pellet-associated genes. The full-length cDNA of the filament-associated clone, Brsa-25, was isolated. Antisense expression of Brsa-25 permitted pelleted growth and increased citrate production at concentrations of Mn(2+) that were higher than the parent strain could tolerate. These results suggest the involvement of the newly isolated genes in the regulation of A. niger morphology

Intrahypothalamic Pituitary Adenylate Cyclase-activating Polypeptide Regulates Energy Balance via Site-specific Actions on Feeding and Metabolism

Numerous studies have demonstrated that both the hypothalamic paraventricular nuclei (PVN) and ventromedial nuclei (VMN) regulate energy homeostasis through behavioral and metabolic mechanisms. Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are abundantly expressed in these nuclei suggesting PACAP may be critical for the regulation of feeding behavior and body weight. To characterize the unique behavioral and physiological responses attributed to select hypothalamic cell groups, PACAP was site-specifically injected into the PVN or VMN. Overall food intake was significantly reduced by PACAP at both sites, however meal pattern analysis revealed only injections into the PVN produced significant reductions in meal size, duration, and total time spent eating. PACAP-mediated hypophagia in both the PVN and VMN was abolished by PAC1R antagonism, whereas pretreatment with a VPACR antagonist had no effect. PACAP injections into the VMN produced unique changes in metabolic parameters, including significant increases in core body temperature and spontaneous locomotor activity that was PAC1R dependent whereas, PVN injections of PACAP had no effect. Finally, PACAP-containing afferents were identified using the neuronal tracer, cholera toxin subunit B (CTB), injected unilaterally into the PVN or VMN. CTB signal from PVN injections was co-localized with PACAP mRNA in the medial anterior bed nucleus of the stria terminalis (BNST), VMN, and lateral parabrachial nucleus (LPB). Whereas CTB signal from VMN injections was highly co-localized with PACAP mRNA in the medial amygdala (MeA) and LPB. These brain regions are known to influence energy homeostasis perhaps, in part, through PACAP projections to the PVN and VMN