Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway.

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.

Synthetic hybrids of six yeast species.

Allopolyploidy generates diversity by increasing the number of copies and sources of chromosomes. Many of the best-known evolutionary radiations, crops, and industrial organisms are ancient or recent allopolyploids. Allopolyploidy promotes differentiation and facilitates adaptation to new environments, but the tools to test its limits are lacking. Here we develop an iterative method of Hybrid Production (iHyPr) to combine the genomes of multiple budding yeast species, generating Saccharomyces allopolyploids of at least six species. When making synthetic hybrids, chromosomal instability and cell size increase dramatically as additional copies of the genome are added. The six-species hybrids initially grow slowly, but they rapidly regain fitness and adapt, even as they retain traits from multiple species. These new synthetic yeast hybrids and the iHyPr method have potential applications for the study of polyploidy, genome stability, chromosome segregation, and bioenergy.

Comparative genomics provides new insights into the diversity, physiology, and sexuality of the only industrially exploited tremellomycete: Phaffia rhodozyma.

BACKGROUND: The class Tremellomycete (Agaricomycotina) encompasses more than 380 fungi. Although there are a few edible Tremella spp., the only species with current biotechnological use is the astaxanthin-producing yeast Phaffia rhodozyma (Cystofilobasidiales). Besides astaxanthin, a carotenoid pigment with potent antioxidant activity and great value for aquaculture and pharmaceutical industries, P. rhodozyma possesses multiple exceptional traits of fundamental and applied interest. The aim of this study was to obtain, and analyze two new genome sequences of representative strains from the northern (CBS 7918T, the type strain) and southern hemispheres (CRUB 1149) and compre them to a previously published genome sequence (strain CBS 6938). Photoprotection and antioxidant related genes, as well as genes involved in sexual reproduction were analyzed. RESULTS: Both genomes had ca. 19 Mb and 6000 protein coding genes, similar to CBS 6938. Compared to other fungal genomes P. rhodozyma strains and other Cystofilobasidiales have the highest number of intron-containing genes and highest number of introns per gene. The Patagonian strain showed 4.4 % of nucleotide sequence divergence compared to the European strains which differed from each other by only 0.073 %. All known genes related to the synthesis of astaxanthin were annotated. A hitherto unknown gene cluster potentially responsible for photoprotection (mycosporines) was found in the newly sequenced P. rhodozyma strains but was absent in the non-mycosporinogenic strain CBS 6938. A broad battery of enzymes that act as scavengers of free radical oxygen species were detected, including catalases and superoxide dismutases (SODs). Additionally, genes involved in sexual reproduction were found and annotated. CONCLUSIONS: A draft genome sequence of the type strain of P. rhodozyma is now available, and comparison with that of the Patagonian population suggests the latter deserves to be assigned to a distinct variety. An unexpected genetic trait regarding high occurrence of introns in P. rhodozyma and other Cystofilobasidiales was revealed. New genomic insights into fungal homothallism were also provided. The genetic basis of several additional photoprotective and antioxidant strategies were described, indicating that P. rhodozyma is one of the fungi most well-equipped to cope with environmental oxidative stress, a factor that has probably contributed to shaping its genome.