Engineering Chlamydomonas reinhardtii as a microalgal cell factory for recombinant human serum albumin production.

Abstract

Human serum albumin (HSA) is an important biopharmaceutical protein, but its recombinant production is still limited by low productivity and high manufacturing cost in conventional expression systems. Here, we engineered Chlamydomonas as a microalgal cell factory for recombinant HSA (rHSA) production. Expression cassette optimization and sequential nuclear transformation were employed to enhance transgene expression, leading to the identification of a high-producing transformant, eCrHSA-3. To further improve production performance, a three-stage pH-controlled fed-batch strategy was developed and successfully implemented in a 5 L bioreactor. Under optimized conditions, biomass reached 48.9 g/L after 10 days of cultivation, with rHSA accumulating to 10.0 mg/L. The identity of the purified recombinant protein was verified by mass spectrometry. Circular dichroism (CD) spectroscopy and 8-anilino-1-naphthalenesulfonic acid (ANS)-binding fluorescence assays revealed similar secondary structure and surface-hydrophobicity features to those of commercially purified human serum albumin, suggesting similarities in their structural characteristics. This study integrates genetic and process engineering for microalgal recombinant protein production and provides a laboratory-scale proof-of-concept for high-density rHSA biosynthesis, highlighting the potential of C. reinhardtii as an alternative host for recombinant protein production.