Currently, global supply chains face numerous challenges from unstable factors, with the recycling and high-cost conversion of waste oils being particularly prominent. Taking China as an example, a significant amount of waste cooking oil (WCO) is generated daily, yet nearly half of it remains uncollected through legal channels and unused effectively ^[1]. Chongqing, a major hub for China's catering industry and the birthplace of hotpot culture, generates substantial amounts of used cooking oil (UCO), including waste cooking oil and hotpot oil. Despite strengthened municipal oversight and recycling efforts, issues like illegal collection and potential food safety risks persist. This not only wastes resources but also poses food safety and environmental hazards. Meanwhile, regions like the EU are promoting biofuel adoption through policies such as mandating a gradual increase in sustainable energy in aviation fuel to 70% by 2050 starting in 2025, providing clear direction for waste oil resource utilization^[1]. However, anti-dumping tariffs and technical costs still constrain its large-scale development. Against this backdrop, converting waste oils into biodiesel has become a vital pathway for advancing the circular economy and supporting energy transition.

Figure 1 The production of waste oil

Biodiesel can be produced through transesterification of triglycerides derived from renewable vegetable oils, animal fats, or microbial oils using short-chain alcohols (methanol and ethanol). Although alkali catalysis is widely recognized for its high reaction rates and biodiesel yields, it requires excess methanol or ethanol and high temperatures. On the other hand, acidic oils containing free fatty acids require acid catalysts for neutralization during transesterification^[2]. Catalysis by these alkalis or acids leads to environmental pollution and equipment corrosion, thus failing to qualify as a green production process.

In contrast, an enzyme protein called lipase in living organisms can efficiently perform both esterification and transesterification simultaneously under mild, environmentally friendly conditions with easily separable byproducts. Thus, the bioenzymatic method represents a green production alternative to chemical synthesis^[3], inspiring us to employ synthetic biology to construct an efficient microbial cell factory that optimizes this bioenzymatic process and promotes waste oil recovery.

Figure 2 Synthesis of biodiesel through biological enzymatic method catalyzed by lipase

We selected Pichia pastoris as the chassis organism because it is a safe model organism with a clear genetic background that facilitates genetic modification. Additionally, P. pastoris exhibits high protein expression levels and supports high-density fermentation, demonstrating advantages for large-scale industrial production of lipase. Since the lipase production level in wild-type fungi is insufficient, we decided to screen and optimize the lipase source, promoter and signal peptide, so as to improve the expression level of lipase in P. pastoris^[4].

Different microbial lipases exhibit variations in activity, reaction time, and thermostability, which impact the efficiency of biodiesel conversion. Through literature review, we identified three lipases with potential advantages: CALB, GGL and LipZ01, originating from Candida antarctica, Galactomyces geotrichum Y05 and oil-contaminated soil, respectively. Among these, CALB is one of the most extensively studied and applied lipases^[5]; GGL exhibits thermotolerance and organic solvent resistance, making it an excellent industrial biocatalyst^[6]; while LipZ01 features high thermal stability, high activity, and high conversion rates^[7].

For the promoters regulating lipase transcription and based on the widely used AOX1 methanol-inducible promoter in P. pastoris, we selected three additional inducible promoters—AOX713, FDH1 and FLD1^[8-11]. These promoters, previously reported in studies, demonstrate excellent application potential and were chosen to enhance lipase expression levels. Finally, building upon this foundation, we attempted to replace the original signal peptide with 0030α-factor, SWP1-α-factor, and KRE1-α-factor signal peptides which have been successfully validated in research cases, to enhance lipase secretion efficiency^[12-13].

Lipase from microorganisms can catalyze the conversion of oils and fats into biodiesel, which is of great significance for the recycling and utilization of waste oils in daily life. We selected P. pastoris as the chassis organism for lipase expression. However, lipases from different microorganisms exhibit specificity, making it necessary to modify and optimize them through genetic engineering to enhance the compatibility of exogenous high-performance lipases in P. pastoris, thereby improving their thermal stability and expression levels to meet the demands of industrial production. In this study, we used protein expression level and enzymatic activity as indicators to screen and optimize three aspects: lipase source, promoter, and signal peptide (Figure 3).

1. We selected three lipases—CALB, GGL, and LipZ01—originating from Candida antarctica, Galactomyces geotrichum Y05, and oil-contaminated soil, respectively. These were cloned into the P. pastoris expression vector pPICZαA to identify the optimal lipase.

2. The AOX1 promoter in the plasmid was replaced with AOX713, FDH, and FLD1 promoters to screen for the promoter most suitable for lipase expression with the highest strength. Then, the α-factor signal peptide in the plasmid was replaced with 0030α-factor, SWP1-α-factor, and KRE1-α-factor signal peptides to obtain the signal peptide most conducive to promoting lipase secretion.

3. The optimal combinations of lipase, promoter, and signal peptide were reassembled to construct an optimized expression plasmid, which was transformed into P. pastoris. Further optimizations were conducted regarding pH and temperature conditions to enhance the expression level and catalytic efficiency of the lipase.

Figure 3 The design concept of the project.
(A) Technical route, (B) DBTL cycles.

This study will provide valuable insights and a robust microbial platform for the industrial production of biodiesel from waste oils, contributing to sustainable energy development and environmental protection.

1.Optimize Lipase Expression: Design an optimal combination of lipase gene, promoter, and signal peptide for high-yield lipase production.

2.Develop a High-Efficiency Strain: Obtain a recombinant P. pastoris strain capable of highly expressing and secreting the target lipase.

3.Improve Catalytic Process: Establish a reaction process that markedly improves the catalytic efficiency and stability of the lipase.

4.Enable Biodiesel Production from Waste Oils: Establish a sustainable microbial platform for converting waste oils into biodiesel, promoting environmental sustainability.

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[3] Stergiou P Y, Foukis A, Filippou M, et al. Advances in lipase-catalyzed esterification reactions [J]. Biotechnology Advances, 2013, 31(8): 1846-1859.

[4] Matsumoto T, Takahashi S, Kaieda M, et al. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production [J]. Applied Microbiology and Biotechnology, 2001, 57(4): 515-520.

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[6] Yan J Y, Yang H K, Xu L, et al. Gene cloning, overexpression and characterization of a novel organic solvent tolerant and thermostable lipase from Galactomyces geotrichum Y05 [J]. Journal of Molecular Catalysis B-Enzymatic, 2007, 49(1-4): 28-35.

[7] Zheng J H, Liu C N, Liu L G, et al. Characterisation of a thermo-alkali-stable lipase from oil-contaminated soil using a metagenomic approach [J]. Systematic and Applied Microbiology, 2013, 36(3): 197-204.

[8] Vogl T, Glieder A. Regulation of Pichia pastoris promoters and its consequences for protein production [J]. New Biotechnology, 2013, 30(4): 385-404.

[9] Liu Q, Li Y H, Tao L F, et al. Rational design and characterization of enhanced alcohol-inducible synthetic promoters in Pichia pastoris [J]. Applied and Environmental Microbiology, 2025, 91(1): 11.

[10] Komeda T, Yurimoto H, Kato N, et al. Cis-acting elements sufficient for induction of FDH1 expression by formate in the methylotrophic yeast Candida boidinii [J]. Molecular Genetics and Genomics, 2003, 270(3): 273-280.

[11] Ahmad M, Hirz M, Pichler H, et al. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production [J]. Applied Microbiology and Biotechnology, 2014, 98(12): 5301-5317.

[12] Lv X Q, Zhang Y T, Wang L R, et al. Expression and antimicrobial activity of the recombinant bovine lactoferricin in Pichia pastoris [J]. Synthetic and Systems Biotechnology, 2024, 9(1): 26-32.

[13] Richard Zahrl,Wien (AT);Oezge Ata Aykol,Wien (AT);Diethard Mattanovich,Wien (AT);Brigitte Gasser,Wien (AT)signal peptides for increased protein secretion_patent.US20240141363A1. 02 May 2024