Artificial microRNA-mediated knockdown of pyruvate formate lyase (PFL1) provides evidence for an active 3-hydroxybutyrate production pathway in the green alga Chlamydomonas reinhardtii

Steven J. Burgess, Gregory Tredwell, Attila Molnar, Jacob G. Bundy, Peter J. Nixon

Research output: Contribution to journalArticlepeer-review

Abstract

Artificial microRNA technology was investigated as a means of down regulating metabolic pathways in the green alga Chlamydomonas reinhardtii, targeting pyruvate formate lyase (PFL1), which catalyzes the conversion of pyruvate to acetyl-CoA and formate during anoxic conditions. Two transformants with an 80-90% reduction in target protein and mRNA levels were identified. Nuclear magnetic resonance spectroscopy confirmed a substantial decrease in the production of formate in the knockdown lines during dark anoxic conditions and a re-routing of metabolism leading to enhanced production of ethanol and lactate. Under microaerobic conditions in the light, induced by sulphur-deprivation, knock-down of PFLI resulted in reduced formate and ethanol production, increased net consumption of acetate and the excretion of lactate but no increase in the production of hydrogen. In addition the production of 3-hydroxybutyrate was identified in knock-down line cultures during the transition between microaerobic and anoxic conditions. Overall our results indicate that microRNA knock-down is a useful tool to manipulate anaerobic metabolism in C. reinhardtii.

Original languageEnglish
Pages (from-to)57-66
Number of pages10
JournalJournal of Biotechnology
Volume162
Issue number1
Early online date8 Jun 2012
DOIs
Publication statusPublished - 30 Nov 2012

Keywords

  • Chlamydomonas
  • Pyruvate formate lyase
  • Artificial microRNA
  • Hydrogen production
  • 3-Hydroxybutyrate
  • HYDROGEN-PRODUCTION
  • FERMENTATIVE METABOLISM
  • NUCLEAR TRANSFORMATION
  • GENE-EXPRESSION
  • H-2 PRODUCTION
  • DEHYDROGENASE
  • EVOLUTION
  • ELECTRON
  • LIGHT
  • MITOCHONDRIA

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