Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal A synthetic cdo/csad taurine pathway in the green unicellular alga Chlamydomonas reinhardtii Rahul Tevatia a,b , Sophie Payne a , James Allen c , Derrick White a , Thomas E. Clemente d,e , Heriberto Cerutti a,e , Yaşar Demirel b , Paul Blum a,f, ⁎ a School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, NE 68588, USA b Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA c Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA d Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE 68588, USA e Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA f Department of Microbiology and Toxicology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA ARTICLE INFO Keywords: Microalgae Taurine Chloroplast transformation Biotechnology Synthetic biology ABSTRACT Synthetic taurine is an important animal feed supplement critical for fish and cat nutrition. However, there is a demand for natural and renewable sources of this nutraceutical additive. The objective of this study was to increase the production of taurine using genetically engineered Chlamydomonas reinhardtii modified to have the cysteine dioxygenase/cysteine sulfinic acid decarboxylase (CDO/CSAD) taurine synthesis pathway from animals. Because unicellular green microalgae produce taurine at low levels, methods to increase taurine content may qualify the use of these microbes as a feed supplement. Here, increased microalgal taurine content was ac- complished using a synthetic approach based on addition of the CDO/CSAD taurine biosynthetic pathway from animals into the chloroplasts of Chlamydomonas reinhardtii. To optimize synthesis, genes were codon optimized and transformed as either separate genes (cdo::csad) or as a fusion protein (cdo-csad). Genotyping confirmed the identity of stable homoplasmic cdo::sad and cdo-csad transformants while HPLC-MS was used to identify pathway intermediates and the end product taurine. While the cdo-csad fusion transformants produced only the first taurine intermediate, cysteine sulfinic acid (CSA), the cdo::csad transformants produced CSA, hypotaurine, and taurine indicating successful synthetic expression of the animal taurine pathway in microalgae. Unsuccessful expression of the fusion construct explained the lack of end product formation and resulted from premature transcript cleavage as indicated by Northern and Western blot analysis. Furthermore, culture supplementation with the pathway precursors, cysteine and methionine, improved production of CDO/CSAD pathway inter- mediates. These results support the feasibility of using engineered microalgae coupled with culture supple- mentation to meet taurine requirements for use as an animal feed supplement. 1. Introduction Taurine (2-aminoethanesulfonic acid), has essential biological roles as an antioxidant, osmoregulator, and membrane stabilizer, along with important though poorly understood roles in cardiovascular function and the development of muscle and nervous systems [1,2]. Because taurine is an essential nutrient for developing fish larvae among other animal species, it has become a prominent nutritional supplement in the animal feed industry. However, commercial taurine is produced only by chemical synthesis leaving room for novel production methods that better meet the needs of the food industry. Animals produce high levels of taurine, while plants and fungi generally lack it [1–4]. Recently we reported that microalgae have the capacity for taurine synthesis [5] though they produce only low levels. As such, the en- gineering of microalgae that produce adequate amounts of taurine would provide an economically feasible method to enable their use as an animal feed supplement. In fish aquaculture, microalgae can be used as a taurine supplement and reduce or eliminate the use of fishmeal from wild caught fish meal as a taurine additive. This depends however on whether the cultivated fish are freshwater or marine species. For example, Spriluna planetis was found to improve growth of common carp (Cyprinus carpio)[6], and a combination of Spriluna planetis, Chlorella vulgaris, and Azolla pinnata in combination could fully replace fishmeal [7]. Furthermore, https://doi.org/10.1016/j.algal.2019.101491 Received 9 November 2018; Received in revised form 3 April 2019; Accepted 3 April 2019 ⁎ Corresponding author. E-mail address: pblum1@unl.edu (P. Blum). Algal Research 40 (2019) 101491 Available online 12 April 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved. T