Heterologous Gene Expression System Using the Cold-Inducible CnAFP Promoter in Chlamydomonas reinhardtii

Abstract

To increase the availability of microalgae as producers of valuable compounds, it is necessary to develop novel systems for gene expression regulation. Among the diverse expression systems available in microalgae, none are designed to induce expression by low temperature. In this study, we explored a cold-inducible system using the antifreeze protein (AFP) promoter from a polar diatom, Chaetoceros neogracile . A vector containing the CnAFP promoter ( pCnAFP ) was generated to regulate nuclear gene expression, and reporter genes ( Gaussia luciferase ( GLuc ) and mVenus fluorescent protein ( mVenus )) were successfully expressed in the model microalga, Chlamydomonas reinhardtii . In particular, under the control of pCnAFP , the expression of these genes was increased at low temperature, unlike pAR1 , a promoter that is widely used for gene expression in C. reinhardtii . Promoter truncation assays showed that cold inducibility was still present even when pCnAFP was shortened to 600 bp, indicating the presence of a low-temperature response element between –600 and –477 bp. Our results show the availability of new heterologous gene expression systems with cold-inducible promoters and the possibility to find novel low-temperature response factors in microalgae. Through further improvement, this cold-inducible promoter could be used to develop more efficient expression tools.

Enhanced Recombinant Protein Production Under Special Environmental Stress

“…Moreover, this promoter, in combination with a capB 5 UTR regulatory element and the transcription terminator of B. subtilis cold-inducible bkd operon caused a higher mRNA stability and thus a further significant increase in protein expression, which has been verified by heterologous production of β-galactosidase from Pseudoalteromonas haloplanktis TAE79A, xylanase from B. subtilis and α-glucosidase from S. cerevisiae (Janiyani and Ray, 2002). A new cold-inducible expression vector with the CnAFP promoter from the polar diatom Chaetoceros neogracile was constructed and a promising transcriptional regulatory element responsive to low temperature was developed (Kim et al, 2020). Similar to cold induction, thermally induced gene expression was achieved through pL and/or pR phage lambda promoters regulated by the thermolabile cI857 repressor (Valdez-Cruz et al, 2010).…”