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Alleviating classB vitamin requirement for fast growth of Saccharomyces cerevisiae in oxic conditions

ABSTRACT: Saccharomyces cerevisiae is bradytroph for class B vitamins, it means that yeast cells exhibit slower growth in the absence of an external source of these metabolites. Alleviating these nutritional requirements for optimal growth performance would represent a valuable phenotypic characteristic for industrial strains since this would result in cheaper processes that would also be less susceptible to contaminations. In the present study, suboptimal growth of S. cerevisiae in absence of either pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and biotin were corrected by single or double gene overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and the Cyberlindnera fabianii BIO1, respectively. Several strategies were attempted to improve growth of S. cerevisiae CEN.PK113-7D in absence of thiamine, revealing that overexpression of ScTHI4 and ScTHI4/ScTHI5 improved growth up to 83% of the maximum specific growth rate of the reference CEN.PK113-7D in vitamins containing medium. By combining overexpression of solely seven S. cerevisiae genes and the heterologous CfBIO1, the strain IMX2816 was fast-growing at a maximum specific growth rate of 0.33 ± 0.01 h-1 in medium devoid of all vitamins. Secondly, this strain exhibited physiological performances in oxic glucose-limited chemostat cultures at a dilution rate of 0.1 h-1 in absence of vitamins similar to that of the reference strain CEN.PK113-7D grown in fully supplemented medium. These physiological similarities were further emphasized by the limited differences observed in comparative transcriptome analysis from the chemostat culture grown cells that were essentially affecting genes of the class B vitamins biosynthetic pathways. This work paves the way towards construction of the first fast growing vitamin-independent S. cerevisiae strain.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE225473 | GEO | 2023/02/21

REPOSITORIES: GEO

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Project description:Saccharomyces cerevisiae is bradytroph for class B vitamins, it means that yeast cells exhibit slower growth in the absence of an external source of these metabolites. Alleviating these nutritional requirements for optimal growth performance would represent a valuable phenotypic characteristic for industrial strains since this would result in cheaper processes that would also be less susceptible to contaminations. In the present study, suboptimal growth of S. cerevisiae in absence of either pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and biotin were corrected by single or double gene overexpression of native FMS1, ABZ1/ABZ2, SNZ1/SNO1, INO1 and the Cyberlindnera fabianii BIO1, respectively. Several strategies were attempted to improve growth of S. cerevisiae CEN.PK113-7D in absence of thiamine, revealing that overexpression of THI4 and THI4/THI5 was able to improve growth up to 83% of the maximum specific growth rate of the reference CEN.PK113-7D in medium including all vitamins. Although the initial aim of this study was to combine all identified mutations in a single strain, the engineered strain IMX2210 only harboured genes to correct biotin, pABA, pantothenate and inositol bradotrophies. Firstly, this strain was fast-growing at a maximum specific growth rate of 0.28 ± 0.01 h-1 in medium devoid of all vitamins. Secondly, this strain exhibited physiological variables in aerobic glucose limited chemostat cultures at a dilution rate of 0.1 h-1 in absence of vitamins similar to that of the reference strain CEN.PK113-7D grown in the same conditions but in a fully supplemented complete medium. These physiological similarities were further emphasized by the limited differences observed in comparative transcriptome analysis from the chemostat culture grown cells that were essentially affecting genes of the class B vitamins biosynthetic pathways. This work paves the way towards construction of the first fast growing vitamin-independent S. cerevisiae strain.

Project description:The needs for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. In this work we establish proof-of-concept that whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,873 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being non-silent (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at www.sysbio.se/cenpk. Keywords: Two strains and two different carbon sources

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Alleviating classB vitamin requirement for fast growth of Saccharomyces cerevisiae in oxic conditions

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