Genomic and transcriptomic analysis of Hansenula polymorpha on carbon sources and sulfur metabolism

Abstract

In the post-genomic era, metabolic reconstruction and subsequent in silico modeling has become a useful implement to understand and exploit biological processes at systems level. As the availability of complete genome of many microorganisms has been increasing, efforts are ongoing to make sense of this mass of information in terms of the gene products encoded and their interactions in the growth, development and survival under various conditions, and its regulation by systematic approaches. The thermotolerant methylotrophic yeast Hansenula polymorpha has been used as a model organism for basic research in peoxisomal function and biogenesis and also successfully applied to the industrial production of heterologous proteins. Particularly for application purposes, H. polymorpha has several advantages over Saccharomyces cerevisiae, including non-immunogenic glycosylation, stable and strong expression system, and capability of dense growth on simple media. Recently this thermotolerant yeast has drawn attraction as a promising organism for development of biofuel-producing host due to its ability to ferment pentose sugar, such as xylose, derived from plant biomass at high temperature up to $48^\circ C$. Information from the complete genome sequence will allow extensive exploration of genome-wide analysis for understanding of cellular physiology and characteristics, which can subsequently be used further development of useful systems to supplement the strong platforms that exist for H. polymorpha. The genome of H. polymorpha, approximately 8.9 Mb in total size, is organized as six chromosomes ranging in size from 0.9 to 1.8 Mb. The whole genome sequencing of H. polymorpha DL1-L was carried out by analysis of shotgun and fosmid libaray clones, and subsequent assembly of 9 collections of big stretches of the genome. Sequence variation among three representative H. polymorpha strains, DL-1, CBS4732, and NCYC495, which show considerable differences in physiology and metabolic regulation, was analyzed at the genome level by comparative genomic hybridization analysis using the H. polymorpha whole-genome cDNA microarray to obtain information on parallel the changes in DNA copy number and expression levels of thousands of genes in the same sample. The H. polymorpha cDNA microarray was also used for transcriptome analysis of H. polymorpha to obtain comprehensive information on gene function and regulatory networks involved in the metabolism of major nutrient elements carbon and sulfur. First, the temporal change of gene expression accompanying the carbon source shift from glucose to methanol was observed because the majority of H. polymorpha processes generally utilize methanol as substrate and inducer for heterologous protein production. The expression profiles showed that several genes involved in glyoxylate cycle and pentose-phosphate pathway were significantly induced, whereas quite a few genes encoding the enzymes of the tricarboxylic acid (TCA) cycle were decreased in the cells cultivated on methanol. From this result, one of highly methanol-induced genes whose expression was up-regulated more than 10-fold, was selected and validated as a novel promoter for application to heterologous production. H. polymorpha is one of naturally pentose-fermenting yeasts at higher temperature so that this yeast as a promising host system to produce biofuels from renewable raw materials. The expression profiles of two carbon source xylose and glucose showed the induction of several genes involved in xylose utilization and pentose-phosphate pathway, while repression of quite a few genes associated with the glycolysis in the cells cultivated on xylose. Since glutathione (GSH), a sulfur containing compound, participates in the detoxification of various products of methanol oxidation, H. polymorpha has been a good model system to study the metabolism and functions of GSH in the response to stress. Based on H. polymorpha genome information, the putative sulfur metabolic pathway was reconstructed and unique biosynthetic pathway of sulfur-containing amino acids was discovered. Also systematic functional analysis of a master transcriptional regulator, MET4p, was carried out to obtain comprehensive information on the HpMet4p-mediated regulatory networks in association with the Cd detoxification and sulfur regulation. In conclusion, the whole genome sequencing and global transcriptional profiling study allow us to reconstruct the regulatory network of major metabolic pathways, carbon and sulfur pathways, in H. polymorpha. The comprehensive information obtained in this study on the genome organization and metabolic regulatory networks of H. polymorpha will be useful for the rational design of engineered yeast strains with artificial genetic circuits to develop improved host strains for the production of fuels, chemicals, food ingredients as well as pharmaceutical proteins.