An alternative, arginase-independent pathway for arginine metabolism in Kluyveromyces lactis involves guanidinobutyrase as a key enzyme

Abstract

Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologues of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of the deletion mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and (13)C-(15)N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic (13)C(15)N-enrichment in γ-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1Δ mutant of a guanidinobutyrase (EC.3.5.3.7), a key enzyme in a new pathway for arginine degradation. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi.

Fig. 1

Overview of the key reactions in eukaryotic arginine metabolism. Thick lines indicate ureohydrolase reactions. EC 1.2.1.3: aldehyde dehydrogenase:, EC 3.5.3.11: agmatinase, EC 4.1.1.19: arginine decarboxylase, EC 4.3.2.1: argininosuccinate lyase, EC 6.3.4.5: argininosuccinate synthase, EC.1.14.13.39: nitric oxide synthase, EC 1.4.3.10: putrescine oxidase, EC 2.1.3.3: ornithine carbamoyltransferase, EC 3.5.3.1: arginase, EC 4.1.1.17: ornithine decarboxylase, EC 2.6.1.13: ornithine aminotransferase, EC 1.5.1.2: pyrroline-5-carboxylate reductase, EC 1.5.99.8: proline dehydrogenase, EC 1.5.1.12: l-Δ¹-pyrroline-5-carboxylate dehydrogenase.

Fig. 2

Phenotypic analysis of K. lactis GG1632 (KlCAR1, KlCAR2, KlPRO3), IMK432 (Klcar1Δ), IMK433 (Klcar2Δ), IMK434 (Klpro3Δ) and S. cerevisiae IMZ312 (CAR1, CAR2, PRO3), IMZ310 (car1Δ KlCAR1↑), IMZ311 (car2Δ KlCAR2↑), IMK342 (pro3Δ KlPRO3↑), IMK435 (car1Δ), IMK436 (car2Δ), IMK445 (pro3Δ) for growth on various nitrogen sources. The strains were plated on synthetic medium with glucose and either ammonium, arginine, ornithine or proline. The plates were incubated at 30°C and scored after 72 h.

Fig. 3

Adaptive growth profile of the strain IMK432 (Klcar1Δ Arg⁺) in sequential shake flask cultivations. The strain was grown in four successive shake flaks with chemically defined medium with glucose and arginine as nitrogen source. A single colony isolate from the last shake flask was selected, and named IMS0367.

Fig. 4

Growth kinetics of K. lactis GG1632 (left) and IMS0367 Klcar1Δ (right).A. Optical density at 660 nm (blue) and extracellular concentration of arginine (green), glucose (red), urea (purple) and ammonia (orange) were followed in aerobic batch cultures on glucose synthetic medium with arginine as nitrogen source.B and C. The total Carbon (B) and Nitrogen (C) pools were determined by HPLC and off-gas measurements taken during the exponential growth phase (15 h for GG1632 and 52 h for IMS0367) based on standard biomass composition estimation (Supplemental information) and are shown as a percentage of the initial pool.

Fig. 5

Enrichment of intracellular metabolites and flux distribution of K. lactis GG1632 (KlCAR1) and K. lactis IMS0367 (Klcar1Δ Arg⁺) grown on ¹³C₆¹⁵N₄ arginine. Enrichment profiles are shown next to the corresponding compounds in plots with light blue background for strain GG1632 and green for the IMS0367 strains. Red lines refer to the ¹⁵N species only. The fluxes are indicated in boxes (expressed in μmol gdw⁻¹ min⁻¹), fluxes indicated in blue correspond to strain GG1632 (KlCAR1 Arg⁺) and those indicated in green to IMS0367 (Klcar1Δ Arg⁺).

Fig. 6

Typical growth profile of K. lactis and S. cerevisiae strains on guanidinobutyrate: The K. lactis strains GG1632 (KlCAR1) (•), IMS0367 (Klcar1Δ) (▼), IMK560 (Klcar1ΔKlgbu1Δ) (▪) and the S. cerevisiae strain IME215 (TDH3_pr::KlGBU1::CYC1_ter) (♦) were grown in shake flasks on glucose synthetic medium containing guanidinobutyrate as sole nitrogen source.

Fig. 7

Phylogenetic tree of Saccharomycotina yeast species and analysis of the distribution of K. lactis KlGBU1/KLLA0F27995g and KLLA0F04235g orthologues within this subphylum. The tree is based on Kurtzman (2003) and Dujon (2010). K. lactis orthologous amino acid sequences were identified by Blastp or tBlastn in genomics data of each individual species mentioned. The branch length is arbitrary. The reported species are divided in several groups: The subphylum Saccharomycetaceae (in blue) includes the following clades: 1 – Saccharomyces sensu stricto, 2 – Kazachstania, 3 – Naumovozyma, 4 – Nakaseomyces, 5 – Vanderwaltozyma, 6 – Zygosaccharomyces, 7 – Lachancea, 8 – Kluyveromyces, 9 – Eremothecium, 10 – Torulaspora, 11 – Ogataea, The CGT group (in green) includes the clades: 12 – Debaryomyces, 13 – Clavispora, 14 – Candida I, 15 – Candida II; The Dipodascaceae and related families (*) (in orange) includes: 16 – Komagataella, 17 – Yarrowia. Label accession indicates the specific amino acid sequence accession number and E-value indicates the significance of the comparison, the significance threshold was set at 1.0E-70. The K. lactis sequences used as query for the Blast analysis were indicated in a box.

Fig. 8

Updated arginine catabolism in K. lactis. Thick lines indicate the new guanidinobutyrase pathway linking arginine to succinate. The grey lines represent the reactions absent in K. lactis but present in other eukaryotes (as shown in Fig. 1). Reactions: EC 3.5.3.1: arginase, EC 4.1.1.17, ornithine decarboxylase, EC 2.6.1.13: ornithine aminotransferase, EC 1.5.1.2: pyrroline-5-carboxylate reductase, EC 1.5.99.8: proline dehydrogenase, EC 1.5.1.12: 1-pyrroline-5-carboxylate dehydrogenase, EC 2.6.1.: aminotransferase, EC 4.1.1.75: 2-oxo acid decarboxylase, EC 1.2.1.54: gamma-guanidinobutyraldehyde dehydrogenase, EC 3.5.3.7: guanidinobutyrate, EC 2.6.1.19: GABA transaminase, EC 1.2.1.16: succinate-semialdehyde dehydrogenase.