Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

Abstract

Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h^-1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h^-1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h^-1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h^-1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast.IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are insufficient to sustain fast growth and product formation. Consequently, this expensive vitamin is routinely added to baker's yeast cultures. In this study, laboratory evolution in biotin-free growth medium yielded new strains that grew as fast in the absence of biotin as in its presence. By analyzing the DNA sequences of evolved biotin-independent strains, mutations were identified that contributed to this ability. This work demonstrates full biotin independence of an industrially relevant yeast and identifies mutations whose introduction into other yeast strains may reduce or eliminate their biotin requirements.

Keywords: Saccharomyces cerevisiae; adaptive laboratory evolution; biotin; prototrophy; reverse metabolic engineering; vitamin biosynthesis; whole-genome sequencing.

FIG 1

Biotin biosynthesis in S. cerevisiae. BIO genes encode the following enzymes: Bio1, pimeloyl-CoA synthetase; Bio6, KAPA synthetase; Bio3, DAPA aminotransferase; Bio4, dethiobiotin synthase; Bio2, biotin synthase; SAM, S-adenosylmethionine; and PLP, pyridoxal phosphate. The protein names in parentheses indicate the corresponding bacterial enzymes. In S. cerevisiae, biotin and its precursor, KAPA, can be imported via the proton symporter Vht1 and the KAPA permease Bio5, respectively.

FIG 2

Laboratory evolution of S. cerevisiae CEN.PK113-7D cells in a sequential batch reactor (SBR) for improved growth in biotin-free synthetic medium. Shown are off gas CO₂ (percent) profiles during an SBR experiment in which automated empty-refill cycles were based on the CO₂ concentration in the off gas, leaving ca. 5% of the culture as an inoculum for each subsequent batch cycle (28). CO₂ production in the initial cycle reflects depletion of biotin pools in the inoculum. Specific growth rates (μ) were calculated from the exponential increase of the off gas CO₂ concentration in each cycle. Graphs representing the increase of dilution rates in accelerostats over time are depicted in Fig. S2 in the supplemental material.

FIG 3

mRNA levels of BIO genes in strains evolved for biotin prototrophy. (A) Transcript levels in cultures grown on SMD with biotin. Shown are transcript levels of BIO1, BIO2, BIO3, BIO4, and BIO6 in the parent strain, CEN.PK113-7D (hatched bars), and in the evolved strains IMS0478 (white bars), IMS0480 (gray bars), IMS0481 (black bars), and IMS0496 (cross-hatched bars) relative to ACT1 expression levels. (B) Transcript levels in cultures grown on SMD without biotin. Shown are transcript levels of BIO1, BIO2, BIO3, BIO4, and BIO6 in the evolved strains IMS0478 (white bars), IMS0480 (gray bars), IMS0481 (black bars), and IMS0496 (cross-hatched bars) relative to ACT1 expression levels. All qPCR experiments were carried out on duplicate cultures, with analytical triplicates for each culture. Relative expression levels were determined according to the ΔΔC_T method (45). The error bars represent the SEM of duplicate analyses.

FIG 4

Chromosomal copy number variations in yeast strains evolved for full biotin prototrophy. Strains IMS0478, IMS0480, and IMS0481 were evolved in accelerostats, while strain IMS0496 was evolved in a sequential batch reactor. Copy numbers of chromosomes and chromosomal regions were calculated from sequence data with the Magnolya algorithm (67). The results for the parental strain, S. cerevisiae CEN.PK113-7D, and for the evolved strains are indicated in blue and red, respectively. Individual chromosomes, indicated by Roman numerals, are separated by dashed lines.

FIG 5

Karyotyping and chromosomal localization of BIO1 in evolved biotin-prototrophic yeast strains. (A) Pulsed-field gel electrophoresis (PFGE) of the chromosomes of evolved biotin-prototrophic strains IMS0478, IMS0480, IMS0481, and IMS0496 and the parent strain, S. cerevisiae CEN.PK113-7D. Chromosome numbers and sizes (kilobases) were obtained using S. cerevisiae S288C as a reference strain. (B) Southern blot of the PFGE gel. Hybridization with BIO1 probe revealed copies of BIO1 on multiple chromosomes in the evolved strains.