Heterologous expression of L-lactate dehydrogenase in Chlorella pyrenoidosa: A beginning for poly(L-lactic acid) biosynthesis from CO2 by eukaryotic microalgae.

Lactic acid is a monomer of poly(lactic acid), and the use of sugar-based raw materials in conventional lactic acid production poses a risk to food security. CO2 is an abundant carbon resource, and microalgae are crucial for the development of third-generation bio-manufacturing through CO2 utilization. In this study, the transformation efficiency of Chlorella pyrenoidosa (C. pyrenoidosa) was investigated during Agrobacterium-mediated transformation, and an efficient transformation system was established. L-lactate dehydrogenases (LLDHs) from Bos taurus, Lactobacillus plantarum, and Bacillus coagulans were successfully integrated into C. pyrenoidosa, and the expression of the target genes was confirmed through RT-PCR and enzyme activity assays. The engineered strains expressing BCLLDH, LPLLDH, and BTLLDH produced L-lactic acid titers of 76.64 mg/L, 153.18 mg/L, and 98.21 mg/L, respectively, and their transgenic stability was verified. Homology modeling of the three LLDHs was performed, and molecular docking with pyruvate was conducted to investigate the interaction mechanism. Moreover, binding energy and interaction network analyses supported the obtained enzyme activity results. Ultimately, L-lactic acid biosynthesis was achieved in C. pyrenoidosa, offering a potential pathway for eukaryotic microalgae to synthesize poly(L-lactic acid) based on the generated precursor.