Optimization of recombinant expression and purification of tomato cutin synthase (CUS1) in Nicotiana benthamiana

November 26, 2025

Polyesters dominate synthetic polymer production, but their environmental impact drives the search for bio-based alternatives. Tomato cutin synthase 1 (CUS1), an enzyme that synthesizes cutin, a natural polyester, offers promising potential for sustainable polyester production. This work focused on the recombinant production and preliminary characterization of CUS1. A Design of Experiments (DoE) approach incorporating four parameters (optical density, harvest time, 5′ untranslated region (UTR), and N- and C-terminal signal peptides) was applied to optimize transient expression in Nicotiana benthamiana using the pTRA vector system. The pTRA-CHS-SSE-CUS1 construct was predicted to reach the highest accumulation at 3 days post infiltration (18.76 mg kg-1). Substitution of the CHS 5’UTR with GGR increased purity 2.5-fold but reduced CUS1 yield by 32%. The protein extraction process efficiency was low (~5%), and the quantification method showed substantial variability in CUS1 quantification. CUS1 exhibited previously unreported esterase activity toward p-nitrophenyl decanoate, with an optimum at pH 8.0. DoE blanching optimization, for host-cell protein removal, evaluated by CUS1 concentration, activity, and total soluble protein, identified optimal conditions at 47.4 °C for 14 min at pH 5.53 and 20 mS cm⁻¹, yielding a 2.24-fold increase in purity but a 31% loss of activity. IMAC purification provided a 25-fold purification factor but only 10–20% final purity. There was evidence of heterogeneity due to modified, truncated, and degraded forms.

Authors in the community:

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  A

  André Afonso dos Santos Redondo

  ist199578

Supervisors of this institution:

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  M

  Maria Ângela Cabral Garcia Taipa Meneses de Oliveira

  ist13278

industrial-biotechnology - Industrial Biotechnology

eng - English

Instituto Superior Técnico