Contribution
For the Contribution, we supplemented the experimental characteristics of the part elements in Yarrowia lipolytica and Escherichia coli (BBa_K3753000, BBa_K2533034, BBa_K4180002). These investigations included the identification of heterologous phenylacetaldehyde synthase, cofactor equilibrium and verification of lipid accumulation-related gene Snf1, 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_K3753000 | PAAS-Petunia | Identification of the capacity of to Y.lipolytica produce 2-PE in lipolytic yeast |
| BBa_K2533034 | RBS-mdh | Identification of the mdh gene that supplies NADH in Escherichia coli and demulcent Y.lipolytica to enhance production |
| BBa_K4180002 | snf1Δ2-306aa N-truncated | Charactering the effect of the SNF1 gene for lipid synthesis and growth in Y.lipolytica. |
Microbially produced 2‑Phenylethanol (2-PE) is mainly obtained by two routes, containing the de novo pathway from glucose and bioconversion from L-phenylalanine by the Ehrlich pathway. Compared to the multi-step pathway, the Ehrlich pathway provides more efficient synthesis of 2-PE In the Ehrlich pathway, the generation of L-phenylalanine by oxidative decarboxylation to phenylethylaldehyde is a key step in the high yield of 2-PE(Farhi et al. 2010) (Fig. 1). Petunia hybrida hybrid phenylacetaldehyde synthase (PAAS) was first registered in 2021, which had been shown to have functional activity in Saccharomyces cerevisiae BY4741. We introduced this gene into the genome of Y. lipolytica polf and tested it in shake flask fermentation with 4 g/L of L-phe. The experimental result showed that overexpression of PhPAAS (BBa_K3753000) does not affect the normal growth of Y. lipolytica. Simultaneously, there was a significant increase in the yield of 2-PE, reaching 957.35 mg/L (Fig. 1). These results provide references for future iGEM team to select suitable sources of PAAS.
Fig. 1. A. The key synthetic steps of the Ehrlich pathway. B. Overexpression of PhPAAS enhances 2-PE yield.
Malate dehydrogenase (MDH) is one of the key enzymes in biological metabolism, which can catalyze the reversible conversion between malate and oxaloacetate. Based on different coenzyme specificities, MDHs are classified into NAD-dependent and NADP-independent. RBS-mdh was first registered in 2021. With the overexpression of mdh, Shewanella could produce NADH more efficiently, which brings more electricity to be produced. To verify whether this gene could be used in other microorganisms to provide sufficient NADH for enhanced production, we first introduced the part in E. coli BL21(DE3). It was found that overexpression of this gene was effective in increasing the yield of various hard-to-express proteins, including heterologous toxic protein (GDH, BBa_K2239014) and endogenous membrane proteins (Oxa1 family membrane protein insertase (YidC, BBa_K4297069), F-ATPase subunit b (Ecb, BBa_K4297070), and cytosine transporter protein (CodB, BBa_K4297071) (Fig. 2A).
Further, phenylethylaldehyde reduction is a NADH-dependent reduction reaction. Therefore, we hope to be able to offer additional cofactors to enhance the 2-PE yield to the maximum. To ensure that the gene can be expressed in Y. lipolytica, we replaced the promoter and terminator elements of the original part and constructed TEF-mdh-CYC1t (BBa_K4297066). this part was introduced into the genome of Y. lipolytica polf and tested it in shake flask fermentation with 4 g/L of L-phe. The results showed that NADH supplementation was effective in increasing the yield of 2-PE up to 1.08 g/L (Fig. 2B). These results provide effective guide for future iGEM team to improve the production by NADH supplementation.
Fig 2. A. Overexpression of MDH enhances the yield of various recombinant proteins in E.coli. B. Overexpression of MDH enhances the 2-PE yield in Y.lipolytica.
Sucrose non-fermenting (Snf1) protein kinase, the yeast ortholog of mammalian AMP-activated protein kinase, is the key component of the glucose repression pathway and the energy sensor in yeast. When the ratio of adenosine monophosphate (AMP) to adenosine triphosphate (ATP) is high, it increased catabolic processes to obtain more ATP and decreases anabolic processes to reduce energy consumption. When truncated from the N-terminal amino acid 2 to amino acid 306, the kinase structure of SNF1 can be deleted and the protein inactivated(Liu et al. 2020). It was first registered in 2022. In order to characterize the effect of the IDH2 (BBa_K4297072) gene in lipid synthesis and growth, we constructed a knockout strain po1f ΔylSNF1 using homologous recombination and tested for fermentation in YNB mediums. The experimental results show that the strain po1f ΔylSNF1 can effectively increase the accumulation of biomass and fatty acid content in YNB medium, up to 83.77% and 159.68% at 120h, respectively (Fig.3). These results provide references for future iGEM teams to improve biomass and lipid accumulation in Y.lipolytica.
Fig. 3. The growth and lipid synthesis of strain po1f ΔylSNF1 in YNB medium. A. Changes of growth curves in YNB medium. B. Changes of fatty acid content in YNB medium at 120h.
Farhi M, Lavie O, Masci T, Hendel-Rahmanim K, Weiss D, Abeliovich H, Vainstein A (2010) Identification of rose phenylacetaldehyde synthase by functional complementation in yeast. Plant Mol Biol 72(3):235-245.
Farhi M, Lavie O, Masci T, Hendel-Rahmanim K, Weiss D, Abeliovich H, Vainstein A (2010) Identification of rose phenylacetaldehyde synthase by functional complementation in yeast. Plant Mol Biol 72(3):235-245.
Liu XY, Yu XJ, Wang ZP, Xia J, Yan YB, Hu L, Wang XY, Xu JX, He AY, Zhao PS (2020) Enhanced erythritol production by a Snf1-deficient Yarrowia lipolytica strain under nitrogen-enriched fermentation condition. Food Bioprod Process 119:306-316.