For the Contribution, we completed the experimental characterization of the previous parts （BBa_K2935068、BBa_K925000、BBa_K346085 and BBa_K2117000） about efficient production or accumulation in Yarrowia lipolytica. These investigations included the effect of IDH2 knockdown on lipid accumulation, verification of delta 12 desaturase function from Synechocystis sp. PCC 6803, introduction of the strong promoter PT7 and measurement of the expression intensity of the TEF promoter under different conditions, and these data were added to the corresponding BioBricks. All of these may be helpful to other teams and we hope it will make some contribution to the iGEM community.

BioBricks	Codes in the lab	Contribution from us
BBa_K2935068	IDH2 Gene	Charactering the effect of the IDH2 gene for lipid synthesis and growth in Y.lipolytica.
BBa_K925000	Delta-12 desaturase	Charactering the leakage ability of the Ptet and the transcription level of T7 RNAP
BBa_K346085	Ptet-RBS-T7polymerase	Introducing it into Y.lipolytica and transcribing gene expression
BBa_K2117000	Constitutive TEF1 promoter	Charactering its function on growth and AMPs function

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Isocitrate dehydrogenase (IDH) is a key enzyme in the TCA cycle, which can provide reducing power for cell growth, and participate in algal energy metabolism and anabolism[1]. IDH2 is the gene encoded in Y.lipolytica. It was registered in 2019. In order to characterize the effect of the IDH2 gene in lipid synthesis and growth, we constructed a knockout strain po1f ΔylIDH2 using homologous recombination. The YNB medium have been chosen for testing Results showed that the strain po1f ΔylIDH2 can effectively increase the accumulation of biomass and fatty acid content. Among them, the strain po1f ΔylIDH2 biomass and fatty acid content increased by 5.63% and 10.10%, respectively. These results provide references for future iGEM teams to improve biomass and lipid accumulation in Y.lipolytica.

Fig. 1. The Growth and lipid synthesis of strain po1f ΔylIDH2 under different culture conditions. A. Changes of growth curves in two fermentation mediums. B. Changes of fatty acid content in the two fermentation mediums at 120h

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Delta 12 Desaturase (Δ-12 desaturase), a trans-membrane enzyme, can introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (C18:1, Δ9). This converts the substrate to linoleic acid (C18:2 Δ9,12), which is further used as a substrate to produce EPA in the alternating action of desaturases and elongases[2]. Δ-12 desaturase from Synechocystis sp PCC 6803 (SyElo9) was registered in 2012. Here, we verified that SyElo9 had functional activity in Y.lipolytica. First, we knocked out the endogenous Δ-12 desaturase (encoded by the Fad2 gene) in Y. lipolytica polf to facilitate the examination of the extension activity for SyElo9. The gas chromatography results showed that overexpression of SyElo9 in strain polf ΔylFad2 can convert oleic acid to linoleic acid (Fig 2). Further, the introduction of SyElo9 into the Y. lipolytica polf genome and fermentation revealed a significant increase in the proportion of linoleic acid, up to 59.895% (Fig 2). These results provide references for future iGEM team to select suitable sources of Δ-12 desaturase.

Fig 2. The fatty acid distribution in various strains

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T7 RNA polymerase (RNAP) from λ prophage can specifically recognize the T7 promoter (PT7) and achieve high gene expression at a transcriptional rate 8-fold higher than that of Escherichia coli RNAP. This expression system has been successfully introduced into a variety of yeast hosts and has driven the CRISPR-Cas9 system to enable gene editing. However, the above systems are expressed using constitutive promoters and do not allow for precise regulation[3].

Based on this, we introduced the component Ptet-RBS-T7polymerase into Y. lipolytica and tested its regulatory function. To ensure that this element is functional, we added nuclear localization sequences (BBa_K4343135) to both terminals of the T7 RNAP and integrated a green fluorescent protein expression cassette regulated by the PT7 in the genome (polf-T7RNAP-T7GFP or polf-T7GFP). The strain polf-T7RNAP-GFP exhibited brighter fluorescence when the anhydrote tracycline (aTC) inducer was added, with a more than 5-fold increase in unit fluorescence intensity after induction (Fig 3A). At the same time, the system has no significant growth burden on the host (Fig 3B). This shows that the optimised BBa_K346085 (BBa_K4343135) can work in lipolytic yeast and provides a reference for future iGEM teams.

Fig. 3. A. The GFP expression in the polf-T7GFP and polf-T7RNAP-T7GFP. B. The growth of Y. lipolytica and polf-T7RNAP-GFP.

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The translation elongation factor-1α (TEF1) promoter is a strong constitutive promoter. Native for the oleaginous yeast Y.lipolytica, which was first registered in 2016. In order to determine the expression intensity of PTEF1 under different mediums (YPD, YP2D4, YEA, YNBDL), we integrated the expression cassette of enhanced green fluorescent protein (eGFP) into the Y.lipolytica. Next, we performed shake flask fermentations under different mediums and tested the fluorescence intensity at different time periods. The experimental result showed that the growth curves differed significantly under different medium, the expression intensity of PTEF1 is also affected (Fig 4). This demonstrates that PTEF1 does not efficiently express the target gene in all states. These results provide references for future iGEM team to select suitable promoter according to the culture medium.

Fig. 4. Changes in eGFP expression intensity mediated by the TEP promoter under different medium.

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[1] Morgunov, I. G., Solodovnikova, N. Y., Sharyshev, A. A., Kamzolova, S. V., & Finogenova, T. V. (2004). Regulation of NAD+-Dependent isocitrate dehydrogenase in the citrate producing yeast Yarrowia lipolytica. Biochemistry (Moscow), 69(12), 1391-1398.

[2] Gombos, Z., Wada, H., Varkonyi, Z., Los, D. A., & Murata, N. (1996). Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity. Biochimica et Biophysica Acta (BBA)-Lipids and Lipid Metabolism, 1299(1), 117-123.

[3] Morse, N. J., Wagner, J. M., Reed, K. B., Gopal, M. R., Lauffer, L. H., & Alper, H. S. (2018). T7 polymerase expression of guide RNAs in vivo allows exportable CRISPR-Cas9 editing in multiple yeast hosts. ACS synthetic biology, 7(4), 1075-1084.