Five year maintenance of the inducible expression of anthranilate synthase in Catharanthus roseus hairy roots

Abstract

Transgenic hairy root cultures have the potential to be an industrial production platform for a variety of chemicals. This report demonstrates the long-term stability of a transgenic Catharanthus roseus hairy root line containing the inducible expression of a feedback-insensitive anthranilate synthase (AS). After 5 years in liquid culture, the presence of the inserted AS gene was confirmed by genomic PCR. The inducible expression of AS was confirmed by enzyme assay and by changes in terpenoid indole alkaloid concentrations. This report also demonstrates that it may take as long as 2 years for the metabolite profile to stabilize.

Vanillin production in metabolically engineered Beta vulgaris hairy roots through heterologous expression of Pseudomonas fluorescens HCHL gene

“…1b), was confirmed by PCR in both the Bv 42 and Bv18HR clones following five years of cultivation which confirmed stability of root clones. This corroborates earlier finding the long term stability of the concerning anthranilate synthase gene expression in Catharanthus roseus hairy roots (Peebles et al, 2008). Growth kinetic analysis revealed that both the HR clones attained their maximum growth potentials on the 45 days of culture in full strength MS media.…”

“…It is pertinent to mention that the stable expression of the HCHL transgenes along with the vanillin synthesizing potential of both the HR clones of Bv after five year of their establishment (Rahman et al, 2009) emphasized the significance of the present study, in view of the fact that long term expression of transgenes remained, by and large unpredictable as found in cases of Cinchona ledgeriana and Catharanathus roseus hairy roots (Peebles et al, 2008). Undoubtedly the presently observed stabilization of the transgene after prolong culture period comply with the essentials prerequisite for the proper functioning and utilization of the targeted gene activity in terms of vanillin synthesis.…”

“…In the present paper we are reporting successful production of vanillin from the aforesaid B. vulgaris hairy root clones (Rahman et al, 2009) that reached up to the level of 0.1% of vanillin, which is quite significant being comparable to that of the natural vanillin from Vanilla planifolia pods. The present findings indeed facilitated addressing two major concerns: (i) does the HCHL transgene expression in hairy roots really need an intermediate time gap for stabilization of its proper function through long term cultivation as elucidated earlier (Peebles et al, 2008); and (ii) does the availability of precursor and/or other chemical parameters resuscitate the proper function of the HCHL gene?…”

Still stable after 11 years: A Catharanthus roseus Hairy root line maintains inducible expression of anthranilate synthase

“…Datura stramonium hairy roots showed stable alkaloid production for 5 years . C. roseus hairy roots have been reported to maintain the stability of an inducible GFP gene for 4.5 years and an inducible anthranilate synthase (AS) gene for 5 years . This article further demonstrates that C. roseus hairy root lines can maintain inducible expression of a gene for 11 years.…”

“…The cultures were then frozen at −80°C until they were lyophilized. Tryptophan (Sigma Aldrich), tryptamine (Sigma Aldrich), ajmalicine (AvaChem Scientific), serpentine (AvaChem Scientific), catharanthine (Qventas), hörhammericine (gift from Dr. Jacqueline V. Shanks at Iowa State University), lochnericine (gift from Dr. Jacqueline V. Shanks at Iowa State University) and tabersonine (AvaChem Scientific) were extracted and quantified by a previously reported HPLC based protocol …”