A synthetic cdo/csad taurine pathway in the green unicellular alga Chlamydomonas reinhardtii

Rahul Tevatia, Sophie Payne, James Allen, Derrick White, Thomas E. Clemente, Heriberto D Cerutti, Yaşar Demirel, Paul H Blum

Research output: Contribution to journalArticle

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 accomplished 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 intermediates. These results support the feasibility of using engineered microalgae coupled with culture supplementation to meet taurine requirements for use as an animal feed supplement.

Original languageEnglish (US)
Article number101491
JournalAlgal Research
Volume40
DOIs
StatePublished - Jun 1 2019

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taurine
Chlamydomonas reinhardtii
Chlorophyta
cysteine
feed supplements
microalgae
acids
animals
synthesis
codons
Northern blotting
functional foods
genotyping
biochemical pathways
methionine
Western blotting
genes
chloroplasts
cats
nutrition

Keywords

  • Biotechnology
  • Chloroplast transformation
  • Microalgae
  • Synthetic biology
  • Taurine

ASJC Scopus subject areas

  • Agronomy and Crop Science

Cite this

A synthetic cdo/csad taurine pathway in the green unicellular alga Chlamydomonas reinhardtii. / Tevatia, Rahul; Payne, Sophie; Allen, James; White, Derrick; Clemente, Thomas E.; Cerutti, Heriberto D; Demirel, Yaşar; Blum, Paul H.

In: Algal Research, Vol. 40, 101491, 01.06.2019.

Research output: Contribution to journalArticle

Tevatia, Rahul ; Payne, Sophie ; Allen, James ; White, Derrick ; Clemente, Thomas E. ; Cerutti, Heriberto D ; Demirel, Yaşar ; Blum, Paul H. / A synthetic cdo/csad taurine pathway in the green unicellular alga Chlamydomonas reinhardtii. In: Algal Research. 2019 ; Vol. 40.
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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 accomplished 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 intermediates. These results support the feasibility of using engineered microalgae coupled with culture supplementation to meet taurine requirements for use as an animal feed supplement.",
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