Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal Nitrogen scavenging from amino acids and peptides in the model alga Chlamydomonas reinhardtii. The role of extracellular L-amino oxidase Victoria Calatrava a , Erik F.Y. Hom b , Ángel Llamas a , Emilio Fernández a , Aurora Galván a, ⁎ a Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, Spain b Department of Biology, University of Mississippi, University, MS, USA ARTICLE INFO Keywords: Algae Chlamydomonas Extracellular L-amino acid oxidase LAAO LAO1 Amino acids Peptides Nitrogen mineralization ABSTRACT Phytoplankton live under constantly changing environments in which concentrations of inorganic nitrogen can be limiting but organic nitrogen sources (urea, amino acids or peptides) are available. Understanding how algae, as primary producers, assimilate organic nitrogen is of eco-physiological importance although such studies are rare. Given genetic tractability, the green microalga Chlamydomonas reinhardtii is an excellent model system for elucidating amino acid assimilation by algal species. This alga can extracellularly deaminate most amino acids using a L-Amino acid Oxidase (LAO1), which generates ammonium that can then be taken up as a source of nitrogen. In this work, we have used lao1 mutant strains to investigate the impact of the absence of this enzyme on C. reinhardtii growth using amino acids and di-/tri-peptides as sole nitrogen sources. Our results show that LAO1 enzyme is crucial for growth on most proteinogenic amino acids and peptides by this alga. We present findings from an analysis of algal genomes that reveal a new evolutionary branch for algal L-amino acid oxidase genes (ALAAO) that includes Rhodophyta, Alveolata, Heterokonta, Haptophyta, and Dinophyta species. Interestingly, C. reinhardtii appears to be the only green algal that contains an ALAAO homolog, and we present a hypothesis about the possible origins of ALAAO genes in algae based on a comparative analysis of currently available and assembled algal genomes. 1. Introduction Inorganic nitrogen is often the most abundant form of nitrogen in marine and freshwater systems, with concentrations that can regularly change spatially or temporarily reach low levels, while organic nitrogen sources like urea and free amino acids are often at higher concentra- tions (reviewed by [1]). In many aquatic systems, terrestrial leaking and runoff are important inputs of organic nitrogen. Sewage and other anthropogenic activities lead to significant infusions of organic nitrogen into rivers that are eventually transported to estuarine and coastal waters. These organic inputs into water systems have dramatically in- creased over the last century due to industrial growth and global warming, and have great impact on the biodiversity of associated ecosystems [1–4]. Although bacteria have long been considered to be the dominant consumers of organic nitrogen, many phytoplankton species have been shown to also use organic nitrogen as evidenced during algal blooms [1,3,5,6]. Thus, primary producers may compete with other heterotrophic organisms like bacteria for organic nitrogen, which should be factored into models of ecosystem balance. The green microalga Chlamydomonas reinhardtii has served as a model organism for physiological studies for more than fifty years [7] and nitrogen metabolism in this alga has been one of the most ex- tensively studied of any alga [8]. This alga preferentially assimilates inorganic nitrogen over other sources [9]. Under inorganic nitrogen deficiency, C. reinhardtii can grow on organic nitrogen sources such as urea, purines and amino acids. L-arginine is the only reported amino acid that is imported by a high affinity transporter in C. reinhardtii [10]. Once intracellular, L-arginine is deaminated by an arginine deiminase (ADI), producing ammonium is incorporated into carbon skeletons by the GS-GOGAT cycle [11]. C. reinhardtii also bears a periplasmic L- amino acid oxidase encoded by the gene LAO1 that deaminates a wide range of L-amino acids according to the reaction: + + → + + + L‐amino acid H O O keto acid NH HO 2 2 4 2 2 The resulting ammonium is then imported into the cell by ammo- nium transporters (AMTs); the α-keto acids are not further metabolized and remain extracellular [12]. The LAO1 protein is highly expressed during nitrogen starvation but is repressed in the presence of https://doi.org/10.1016/j.algal.2018.101395 Received 9 October 2018; Received in revised form 19 December 2018; Accepted 20 December 2018 ⁎ Corresponding author. E-mail address: bb1gacea@uco.es (A. Galván). Algal Research 38 (2019) 101395 2211-9264/ © 2019 Elsevier B.V. All rights reserved. T