Reconstruction And Application Of Scheffersomyces Stipitis Genome-scale Metabolic Model

Along with the increasing stress on the shortage of oil reserves and the negativeecological impacts of greenhouse gas emissions, a popular research topic is to get an highefficient cellulosic ethanol producer. As a naturally occurring cellulose-fermenting yeast,Scheffersomyces stipitis has its unique metabolic traits-fermenting different types of biomasssubstrates with high yield of ethanol and little byproducts. However, many problems remainfor ethanol production in S. stipitis, such as relatively low sugar uptake rate, the tough controlof dissolved oxygen and the low tolerance of ethanol. Thus, a systematic understanding of itsphysiological features and metabolic capacities is great need. A genome-scale metabolicmodel (GSMM) of S. stipitis was reconstructed based on genome annotation. A series ofmodeling and analysis (dry experiment) were conducted by the use of constraint-basedanalysis model. A preliminary study on the Transcription Factors in S. stipitis was performedbased on comparative genomics. The main results were described as follows:(1) This GSMM reconstruction of S. stipitis was based on the published genomesequence as well as data extracted from public biochemical databases and literature. Theavailability of protein sequences of S. stipitis enables us to carry out the genome annotationby BLAST and KAAS. A draft model with detailed gene-protein-reaction (GPR) associationswas obtained with Matlab programs. The draft model was curated with the biochemicalinformation from databases and literature. The resulting genome-scale metabolic modeliTL885comprises885metabolic genes,1240reactions,870metabolites, and three cellularcompartments (cytosol, mitochondrion and extracellular). Gene coverage of iTL885is15.2%.About92%reactions in this model are with gene associations.(2) The capabilities of different carbon sources utilization were investigated from theperspective of sugar transport, metabolic pathway, cell growth phenotype and essential genes.By the combination of information from the sequenced genome, transcriptional expressionand experimental data,36putative sugar transporters were reannotated which gives us a sightinto the molecular basis of sugar transport. A map illustrating the central carbohydratemetabolism with corresponding gene annotation was provided. Cell growth and ethanolsynthesis on xylose, glucose and rhamnose was modeled with FBA. It was found that onxylose, glucose can produce ethanol but not rhamnose. In the model iTL885,130genes(14.7%of the total genes) were predicted to be essential on a minimal medium with glucoseas carbon source.(3) With iTL885the rate-limiting steps for ethanol production were identified byconstraints-based COBRA algorithms. Robustness analysis of oxygen demonstrated themaximum ethanol production (4.72mmol/gDCW/h) could be achieved only under therelatively low oxygen condition (1.15mmol/gDCW/h). FBA pointed out the impacts of oxygenation on the flux distribution of central metabolism and flux to ethanol formationreactions (PDC, ALD, ADH) was increased about10-fold with the decreased oxygen level.The investigation of xylose utilization from the perspectives of sugar transport andmetabolism partly accounts for the high efficient bioconversion of xylose in S. stipitis. For theoverproduction of ethanol from xylose, three candidate knockout targets (GLY, ALA2, GDH3)were identified and the effects of20common amino acids addition to cell growth and ethanolsynthesis were simulated. Among the different amino acids addition, glutamate addition wasmost effective, leading to the ethanol production rate increased by27.7%compared to thecontrol.(4) Eighty-seven high-confidence Transcription Factors were predicted in S. stipitisbased on comparative genomics and database information, which partly belong to the344TFspool downloaded from Fungal Transcription Factors Database (FTFD). Compared with theTFs regulating core metabolism in Saccharomyces cerevisiae, it was found that someregulatory proteins are fairly well conserved among S. stipitis and S. cerevisiae such asproteins involved in the pathway for oxygen regulation and glucose sensing. Ethanol toleranceand glycolytic regulator seems unique to S. cerevisiae, indicating S. cerevisiae has evolved tosurvive in high-sugar environments. While S. cerevisiae represses respiration in the presenceof glucose, S. stipitis induces fermentative activities in response the oxygen limitation,differences in the glucose and oxygen regulatory pathways would be expected.