Lactic acid has significant applications in food and beverage manufacturing (Rawoof et al., 2021), and it naturally occurs in two optical isomers: D-lactic acid and L-lactic acid (Tong et al., 2024). L-lactic acid is metabolizable by the human body, which supports its broader application in food preservation compared with D-lactic acid (Wang et al., 2022). Lactic acid can be polymerized into poly(lactic acid) (PLA) (Muller et al., 2017), which is the most widely used biopolymer in the food packaging industry. And the annual production of PLA has reached 140,000 t (Swetha et al., 2023). In addition, PLA has been designated as Generally Recognized As Safe (GRAS) by the United States Food and Drug Administration (FDA), ensuring its safety for all food packaging applications (Conn et al., 1995, Swetha et al., 2023). And PLA is typically synthesized via either the ring-opening polymerization of lactide or the direct polymerization of lactic acid (Lajus et al., 2020, Mehta et al., 2005). Currently, commercial lactic acid production primarily relies on sugar-derived feedstocks, such as sugarcane or cornstarch (Singh et al., 2022). However, the use of sugar-based raw materials has raised concerns regarding food security. Consequently, it is necessary to develop PLA production processes that utilize non-edible feedstocks (Tan et al., 2022). Under mild fermentation conditions, biological processes can produce high yields of optically pure lactic acid from a wide range of substrates (Kong et al., 2019). Recent studies have demonstrated lactic acid biosynthesis in Saccharomyces cerevisiae (Zhu et al., 2022), Corynebacterium glutamicum (Tsuge et al., 2019), and Pichia pastoris (Wu et al., 2025) through the expression of heterologous genes, and these microorganisms primarily utilize glucose and methanol as substrates. Carbon dioxide is an abundant carbon resource. Since the onset of the Industrial Revolution, atmospheric CO2 concentration has increased by over 40 % (from 270 to 400 ppm), and global emissions currently amount to approximately 30 Gt per year. This has disrupted the natural carbon cycle and contributed to atmospheric accumulation (Moreira and Pires, 2016). In the context of third-generation bio-manufacturing, CO2 utilization presents a promising approach for simultaneously enhancing food security and promoting environmental sustainability (Liu et al., 2020). Therefore, microalgae can be directly cultured to produce the required lactic acid for further PLA biosynthesis from CO2.

Eukaryotic microalgae, which are globally distributed, exhibit exceptional photosynthetic capabilities by utilizing CO2 and light energy (Xie et al., 2023). And these organisms are responsible for fixing approximately 40 % or more of global primary production (Field et al., 1998). As sustainable “natural green factories”, eukaryotic microalgae have been employed in the biosynthesis of various high-value bioproducts (Forján et al., 2015). Furthermore, Agrobacterium-mediated transformation has proven to be an effective system for constructing genetically modified organisms (GMOs) through the integration of large DNA fragments into host genomes, which is stable, cost-effective, and broadly applicable across host species (Simon et al., 2015). And it has been successfully applied in Chlamydomonas reinhardtii (Tran and Kaldenhoff, 2020), Chlorella vulgaris (Yedahalli et al., 2018), and Dunaliella tertiolecta (Norzagaray-Valenzuela et al., 2018). Available Agrobacterium strains for algae transformation include Agrobacterium tumefaciens (A. tumefaciens) LBA4404 (Norashikin et al., 2018, Norzagaray-Valenzuela et al., 2018), A. tumefaciens EHA105 (Becker et al., 2021, Liang et al., 2023) and A. tumefaciens GV3101 (Sobieh et al., 2022). In the genetic transformation of Chlamydiae reinhard mediated by A. tumefaciens, the presence of acetosyringone increased transformation efficiency by approximately 45-fold compared with conditions lacking phenolic compounds (Mosey et al., 2021). Notably, explants pretreatment, co-culture duration and agrobacterium density also influence T-DNA delivery and integration efficiency (Ziemienowicz, 2014).

This study explored the heterologous expression of L-lactate dehydrogenases (LLDHs) in Chlorella pyrenoidosa (C. pyrenoidosa), focusing on LLDHs derived from different sources. By optimizing parameters in the Agrobacterium-mediated transformation process, a stable genetic transformation system was established, enabling the direct biosynthesis of L-lactic acid from CO2 in C. pyrenoidosa. In addition, homology modeling was performed for the three LLDHs, followed by molecular docking with pyruvate to elucidate the interaction mechanisms. This work provides a foundational step toward the biosynthesis of poly(L-lactic acid) from CO2 by eukaryotic microalgae.