Vitamin requirements and biosynthesis in Saccharomyces cerevisiae

Abstract

Chemically defined media for yeast cultivation (CDMY) were developed to support fast growth, experimental reproducibility, and quantitative analysis of growth rates and biomass yields. In addition to mineral salts and a carbon substrate, popular CDMYs contain seven to nine B-group vitamins, which are either enzyme cofactors or precursors for their synthesis. Despite the widespread use of CDMY in fundamental and applied yeast research, the relation of their design and composition to the actual vitamin requirements of yeasts has not been subjected to critical review since their first development in the 1940s. Vitamins are formally defined as essential organic molecules that cannot be synthesized by an organism. In yeast physiology, use of the term "vitamin" is primarily based on essentiality for humans, but the genome of the Saccharomyces cerevisiae reference strain S288C harbours most of the structural genes required for synthesis of the vitamins included in popular CDMY. Here, we review the biochemistry and genetics of the biosynthesis of these compounds by S. cerevisiae and, based on a comparative genomics analysis, assess the diversity within the Saccharomyces genus with respect to vitamin prototrophy.

Keywords: Saccharomyces cerevisiae; fermentation; growth requirements; synthetic media; vitamin biosynthesis.

Figure 1

PLP and TDP de novo synthesis pathway in Saccharomyces cerevisiae. D‐glyceraldehyde 3‐phosphate, L‐glutamine, and keto‐D‐ribose 5‐phosphate are converted to PLP by the catalytic activity of the SNO1, 2,3 and SNZ1, 2,3 gene products . Gcn4 acts as positive regulator of de novo PLP biosynthesis, whereas Bas1 acts as an inhibitor. Gcn4 is inhibited by amino acids and activated under amino‐acid starvation. Bas1 instead is upregulated in the presence of glycine. PN, PM, and PL are imported by Tpn1. PN is converted at the expense of ATP to PNP by Bud16 whereupon Pdx3, produces PLP and hydrogen peroxide in an oxygen‐dependent reaction. Similarly, PLP can be formed starting from PM in two steps by action of Bud16 and Pdx3, with PMP as intermediate. Moreover, PL can also be converted to PLP by action of Bud16. PLP is used as cofactor or converted to HMP‐P by one of the four homologous enzymes Thi5, Thi11, Thi12, and Thi13, under consumption of L‐histidine. HMP‐P is the intermediate for the formation of the pyrimidyl moiety of thiamine (shown in cyan). Thi20 and Thi21 further phosphorylate HMP‐P to HMP‐PP. The thiazole moiety (shown in yellow) is synthesized by activity of Thi4 in a suicide mechanism, leading to HET‐P. HMP‐PP and HET‐P are merged by the gene product of THI6 to TMP. The following reaction catalyzed by an acid phosphatase (EC number 3.1.3.2) yields thiamine. Thiamine can be taken up with the aid of the transporter Thi10. Finally, thiamine is converted to its biologically active form TDP under consumption of ATP by Thi80. Pdc2, Thi2, and Thi3 are responsible for the upregulation of transcription of THI5/11/12/13, THI20/21, THI6 and THI4. Alcohol and methyl substitutions on the pyridoxine pyrimide ring are shown in magenta and purple, respectively. Metabolites, proteins, and positive regulators are shown in bold, blue, and green, respectively. ATP, adenosine triphosphate; HET‐P, 5‐(2‐hyroxylethyl)‐4‐methylthiazole phosphate; HMP‐P, 4‐amino‐2‐methyl‐5‐pyrimidine phosphate; HMP‐PP, 4‐amino‐2‐methyl‐5‐pyrimidine diphosphate; PL, pyridoxal; PLP, pyridoxal‐5'‐phosphate; PM, pyridoxamine; PMP, pyridoxamine‐5'‐phosphate; PN, pyridoxine; PNP, pyridoxine‐5'‐phosphate; TDP, thiamine diphosphate; TMP, thiamine phosphate [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Biotin de novo biosynthesis pathway in Saccharomyces cerevisiae. Biotin is composed of an ureido and a tetrahydrothiophene ring (shown in cyan) fused to a valeric acid chain (shown in yellow). The five final steps of de novo biotin synthesis are carried out by Bio1, Bio6, Bio3, Bio4, and Bio2. Origin of pimelic acid remains elusive in S. cerevisiae (indicated by question mark(?)). Pimeloyl‐CoA formed by Bio1 is converted via 8‐amino‐7‐oxonanoate (KAPA) to 7,8‐diaminopelargonate (DAPA) by Bio6 and Bio3. DAPA is subsequently converted by Bio4 to dethiobiotin and finally to biotin by Bio2. The intermediate KAPA and biotin can be imported via the membrane transporters Bio5 and Vht1, respectively. In the absence of biotin, the regulator Vhr1 upregulates expression of genes encoding the transporters Vht1 and Bio5 as well as Bio2. In iron and amino acid rich conditions the transcriptional regulator genes AFT1 and GCN4 are transcriptionally repressed, which under iron and amino‐acid scarce conditions would not activate transcription of BIO3, BIO4, and BIO2 and relieve BIO5 expression. Metabolites, proteins, positive, and negative regulators are shown in bold, blue, green, and red, respectively [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 3

Pantothenate de novo synthesis pathway in Saccharomyces cerevisiae and transcription profiles of pantothenate biosynthetic genes under different growth conditions. Pantothenate can be imported by the proton symporter Fen2 or synthesized de novo by condensation of pantoate (shown in cyan) and β‐alanine (shown in yellow) in an ATP‐dependent reaction catalyzed by Pan6. Pantoate is formed in a two‐step pathway from 2‐keto‐isovalerate catalyzed by Ecm31 and Pan5 with 2‐dehydropantoate as intermediate. β‐alanine is formed starting from spermine by the enzymes Fms1 and Ald2‐3 via 3‐aminopropanal. ATP, adenosine triphosphate; NADP⁺, nicotinamide adenine dinucleotide phosphate [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 4

Heatmap showing mRNA levels for pantothenate biosynthetic genes measured under 70 different conditions in chemostat cultures. Each row shows a gene involved in de novo pantothenate biosynthesis, whereas each column represents one condition. Data are derived from (Knijnenburg et al., 2007; Knijnenburg et al., 2009), and code for generating this plot is available at https://gitlab.tudelft.nl/rortizmerino/sacch_vitamins [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

pABA and myo‐inositol de novo synthesis pathways in Saccharomyces cerevisiae. (a) The genes ABZ1 and ABZ2 code for a two‐step pathway producing pABA from chorismate via the intermediate 4‐amino‐4‐deoxychorismate. Chorismate is synthesized from erythrose‐4‐phosphate and phosphoenolpyruvate via the shikimate pathway. In addition to being precursor for pABA biosynthesis, chorismate also serves as precursor for tryptophan, phenylalanine and tyrosine biosynthesis. (b) myo‐Inositol is formed from glucose‐6‐phosphate via Ino1 yielding L‐myo‐inositol‐1‐phosphate, which is in a second step converted to myo‐inositol by Inm1 or Inm2. INO2, INO4 genes encode INO1 transcriptional activators while OPI1 encodes the antagonist regulator of the gene encoding the initial step of inositol synthesis. Metabolites, proteins, positive regulators, and positive regulators are shown in bold, blue, green, and red, respectively. pABA, para‐Aminobenzoic acid [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 6

Nicotinic acid de novo synthesis and salvage pathway in Saccharomyces cerevisiae. NAD⁺ is de novo synthesized from L‐tryptophan in nine catalytic steps involving the Bna enzyme family and enzymes Nma1, Nma2, and Qns1. Nicotinic acid can be imported into the cell via Tna1 and enters the NAD synthesis pathway as NaMN by catalytic activity of Npt1. Similarly, NaR can be salvaged by catalytic activity of Nrk1 to form NaMN. NaR can be also converted to nicotinic acid by Urh1 and Pnp1. Nrk1 also converts NR into NMN subsequently converted to NAD⁺ by Nma1 and Nma2. NR is imported by activity of Nrt1 transporter and might be used by Pnp1 or Urh1 to form nicotinamide. Alternatively, nicotinamide can be synthesized via Sir2 from NAD⁺. Pnc1 uses nicotinamide to form nicotinic acid. The regulators Hst1 (with aid of Rfm1 and Sum1) and Mac1 repress the expression of genes encoding Bna enzymes upon binding to NAD⁺ and nicotinic acid. Metabolites, proteins and negative regulators are shown in bold, blue and red respectively. NAD⁺, nicotinamide adenine dinucleotide; NaMN, nicotinic acid mononucleotide; NAR, nicotinamide riboside; NMN, nicotinamide nucleotide; NR, nicotinamide riboside [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 7

Occurrence of vitamin biosynthesis annotated genes in Saccharomyces species. A homology search was conducted using HMMER v3 (Eddy, 2011) with Saccharomyces cerevisiae S288C proteins as queries (left side row names) against a database of annotated proteins from the Saccharomyces species listed in the column headers. For BIO1 andBIO6, S. cerevisiae K7 proteins were used as queries (indicated with *) because S288C is known to lack such proteins. Available genome annotations from species in the monophyletic Saccharomyces clade (formely known as sensu stricto; Table 3) were used to build a protein sequence database. Besides S. cerevisiae S288C and CEN.PK113‐7D, sequences in the database belong to type strains. This database was then searched for sequence homologs using the queries listed on the left‐hand side. Queries are grouped and labelled on the right‐hand side and depending on the biosynthetic pathway they are involved in. Boxes are coloured depending on the number of hits (e‐value > 1e‐5, percentage of alignment > 75%) obtained by each query on each strain. The colour code is shown at the bottom. Hits from queries belonging to the same biosynthetic pathway were ranked according to lowest e‐value then highest percentage of alignment and best hits were uniquely assigned to each query (i.e., a sequence considered as best hit is never used more than once and best hits with a count >1 are all identical). This last step accounts for the presence of paralogs and the high level of similarity between proteins in the same pathway, especially in the pyridoxine and thiamine pathways (see Thi5 and Thi20 for instance). Code for this search is available in https://gitlab.tudelft.nl/rortizmerino/sacch_vitamins and sequences are deposited under BioProject accession PRJNA578688 as indicated in Table 3 [Colour figure can be viewed at http://wileyonlinelibrary.com]