Under extreme conditions (such as temperatures of 39°C), researchers observed trisomy of chromosome III in yeast cells. To elucidate the underlying mechanism, we introduced synthetic chromosome III into haploid yeast cells. Through synthetic chromosome rearrangement and modification by LoxP-mediated evolution （SCRaMbLE） screening, we successfully identified a series of genes enhancing stress resistance. Utilizing mass spectrometry analysis, we discovered a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This yeast trehalose synthesis pathway has not been previously reported in the literature.

We obtained a yeast strain containing the artificially synthesised chromosome III from Professor Wu. Through a narrow-down screening strategy, five key genes were identified using PCR tag technology. By means of stepwise assembly, the recombinant plasmid pRS413-trehalose, containing all five target genes, was ultimately obtained.

At the same time, we also introduced a yeast surface display system and successfully displayed antifreeze proteins outside the cells.

Number	Name	Short Description
BBa_25YHZVNV	pRS413	A yeast centromeric vector containing the HIS3 marker and a multiple cloning site (MCS) derived from pBLUESCRIPT II.
BBa_258AOSKU	Downstream of the trehalose pathway	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This is miscellaneous downstream of the trehalose pathway.
BBa_2521MLHD	Essential Component for Trehalose Production 1	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. It represents the first identified component involved in trehalose synthesis.
BBa_25SU4YOW	The miscellaneous between key components 1 and 2 in trehalose production	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This sequence lies between key components 1 and 2 and is classified as miscellaneous.
BBa_25MBKAJ6	Essential Component for Trehalose Production 2	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. It represents the second component confirmed to participate in trehalose synthesis.
BBa_251HXQC6	The miscellaneous between key components 2 and 3 in trehalose production	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This sequence lies between key components 2 and 3 and is classified as miscellaneous.
BBa_25XUM0RM	Essential Component for Trehalose Production 3	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. It represents the third component confirmed to participate in trehalose synthesis.
BBa_25504V39	The miscellaneous between key components 3 and 4 in trehalose production	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This sequence lies between key components 3 and 4 and is classified as miscellaneous.
BBa_25O12L2L	Essential Component for Trehalose Production 4	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. It represents the fourth component confirmed to participate in trehalose synthesis.
BBa_25UKBYRR	The miscellaneous between key components 4 and 5 in trehalose production	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This sequence lies between key components 4 and 5 and is classified as miscellaneous.
BBa_25Z80NNB	Essential Component for Trehalose Production 5	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. It represents the fifth component confirmed to participate in trehalose synthesis.
BBa_25UNJIBC	Upstream of the trehalose metabolic pathway	Through SCRaMbLE screening and mass spectrometry analysis, we identified a gene cluster comprising five key coding regions corresponding to the yeast endogenous trehalose synthesis pathway. This is miscellaneous upstream of the trehalose pathway.
BBa_25WZNWWE	NEW11-10	It is an coding zone.By substituting a helical segment from the 4MTJ structure with one from 6A8K, a novel antifreeze protein was constructed
BBa_255P1F01	NEW11-9	It is an coding zone.By substituting a helical segment from the 4MTJ structure with one from 4NU2, a novel antifreeze protein was constructed
BBa_25M7R0TL	NEW11-B	It is an coding zone.A new antifreeze protein obtained by replacing a segment of the original protein's helix with another De novo helix.
BBa_25Q2MI51	NEW11-A	It is an coding zone.A new antifreeze protein obtained by replacing a segment of the original protein's helix with another De novo helix.
BBa_254GP0FQ	pYD1 Linearisation vector	It is a plasmid_backbone.The carrier for the yeast surface display system carries the GAL1 promoter, enabling expression of the target gene upon galactose induction. It also carries a tryptophan expression gene, allowing the yeast to survive on tryptophan-deficient nutrient medium
BBa_25337LH2	Superfolding GFP	It is an coding zone.Emits fluorescence under microscopic observation.
BBa_25ILR6EM	tMATα	It is Terminator，Transcription termination site on the pYD1 plasmid.
BBa_25VKZD81	GS linker	It is an coding zone.Linking Aga2 to the target protein enables surface anchoring of the target protein.
BBa_25E804IN	Aga2	It is an coding zone.Under galactose induction and regulation by the GAL1 promoter, expression of the Aga2 protein binds to Aga1 on the yeast cell surface, anchoring the protein to the yeast cell surface.
BBa_25P03NBK	4MTJ	It is an coding zone.Encode an antifreeze protein derived from Clostridium butyricum.
BBa_25G5LZEK	6A8K	It is an coding zone.Encode an antifreeze protein derived from Fragilariopsis cylindrus.
BBa_25LH8M07	4NU2	It is an coding zone.Encode an antifreeze protein derived from Flavobacterium frigoris PS1.
BBa_25O3AS58	3WP9	It is an coding zone.Encode an antifreeze protein derived from Colwellia sp. SLW05.
BBa_25F6MECA	3P4G	It is an coding zone.Encode an antifreeze protein derived from Marinomonas primoryensis
BBa_25EWFA1M	2PNE	It is an coding zone.Encode an antifreeze protein derived from Hypogastrura harveyi.
BBa_259VUFV8	1M8N	It is an coding zone.Encode an antifreeze protein derived from Choristoneura fumiferana.
BBa_25WJJ9KP	3ULT	It is an coding zone.Encode an antifreeze protein derived from Lolium perenne.
BBa_25TJJ1PD	1UCS	It is an coding zone.Encode an antifreeze protein derived from Lycodichthys dearborni.
BBa_25PX1D04	2ZIB	It is an coding zone.Encoding an antifreeze protein derived from Brachyopsis segaliensis
BBa_25CE36NZ	4KE2	It is an coding zone.Encoding an antifreeze protein derived from the American flounder.
BBa_25KNQ0KV	De novo6	It is an coding zone.De novo synthesis of an antifreeze protein based on inverse folding optimisation design.
BBa_251FZYUC	De novo5	It is an coding zone.De novo synthesis of an antifreeze protein based on inverse folding optimisation design.
BBa_25UHWY3N	De novo4	It is an coding zone.De novo synthesis of an antifreeze protein based on inverse folding optimisation design.
BBa_25DFCXRO	De novo3	It is an coding zone.De novo synthesis of an antifreeze protein based on inverse folding optimisation design.
BBa_25LOZE72	De novo2	It is an coding zone.De novo synthesis of an antifreeze protein based on inverse folding optimisation design.
BBa_25JXF50Q	De novo1	It is an coding zone.De novo synthesis of an antifreeze protein based on inverse folding optimisation design.
BBa_25J7GQ4H	4NU2-IF	It is an coding zone.Novel antifreeze proteins based on inverse folding design.
BBa_25E03GWV	3WP9-IF	It is an coding zone.Novel antifreeze proteins based on inverse folding design.
BBa_25FINPKW	6A8K-IF	It is an coding zone.Novel antifreeze proteins based on inverse folding design.
BBa_25668EJ5	Rigid linker	It is an coding zone.Fusing domains from different frost-resistant proteins into a novel frost-resistant protein.
BBa_251KX4GR	RD3N-G	It is an coding zone.Fusing domains from different frost-resistant proteins into a novel frost-resistant protein.
BBa_25UBI5DR	pET-28a Linearisation vector	It is a plasmid_backbone.Carrying a lactose-operon system and T7 promoter, with IPTG added to induce expression of the target gene, whilst also carrying kanamycin resistance to screen for successful transfer of the target gene.
BBa_25E66014	MUT10	It is an coding zone.By introducing targeted mutations into the gene sequence of the original protein（6A8K）, new proteins with varying levels of frost resistance can be obtained.
BBa_25YKZOE3	MUT9	It is an coding zone.By introducing targeted mutations into the gene sequence of the original protein（4NU2）, new proteins with varying levels of frost resistance can be obtained.
BBa_25GSNX92	MUT8	It is an coding zone.By introducing targeted mutations into the gene sequence of the original protein（3WP9）, new proteins with varying levels of frost resistance can be obtained.
BBa_25G76SCK	MUT4	It is an coding zone.By introducing targeted mutations into the gene sequence of the original protein（3ULT）, new proteins with varying levels of frost resistance can be obtained.
BBa_257VFWBC	wpy4	It is an coding zone.Frost-resistant proteins with varying freeze tolerance obtained through structural domain rearrangement.
BBa_25P61588	wpy3	It is an coding zone.Frost-resistant proteins with varying freeze tolerance obtained through structural domain rearrangement.
BBa_25DJMWLT	wpy2	It is an coding zone.Frost-resistant proteins with varying freeze tolerance obtained through structural domain rearrangement.
BBa_25R74RPR	wpy1	It is an coding zone.Frost-resistant proteins with varying freeze tolerance obtained through structural domain rearrangement.

Number	Name	Short Description
BBa_25LK9WHI	Endogenous Trehalose Pathway in Yeast	Gene clusters on chromosome 3 identified through mass spectrometry and SCRaMbLE screening.
BBa_255UOOXM	pGAL1-Aga2-GS linker-NEW11-10-tMATα	This is a gene expression module. Under galactose induction, transcription commences from the pGAL1 promoter, expressing a fusion protein comprising Aga2 and NEW11-10. Within this fusion protein, Aga2 forms disulphide bonds with Aga1 on the surface of EBY100, anchoring it to the yeast cell surface. This enables the NEW11-10 protein to be displayed on the yeast cell surface, forming a new layer of antifreeze proteins.
BBa_25JB37GY	pGAL1-Aga2-GS linker-NEW11-9-tMATα	This is a gene expression module.Under galactose induction, transcription commences from the pGAL1 promoter, expressing a fusion protein comprising Aga2 and NEW11-9. Within this fusion protein, Aga2 forms disulphide bonds with Aga1 on the surface of EBY100, anchoring it to the yeast cell surface. This enables the NEW11-9 protein to be displayed on the yeast cell surface, forming a new layer of antifreeze proteins.
BBa_25COQ0NM	pGAL1-Aga2-GS linker-NEW11-B-tMATα	This is a gene expression module.Under galactose induction, transcription commences from the pGAL1 promoter, expressing a fusion protein comprising Aga2 and NEW11-B. Within this fusion protein, Aga2 forms disulphide bonds with Aga1 on the surface of EBY100, anchoring it to the yeast cell surface. This enables the NEW11-B protein to be displayed on the yeast cell surface, forming a new layer of antifreeze proteins.
BBa_25QYHXD3	pGAL1-Aga2-GS linker-NEW11-A-tMATα	This is a gene expression module.Under galactose induction, transcription commences from the pGAL1 promoter, expressing a fusion protein comprising Aga2 and NEW11-A. Within this fusion protein, Aga2 forms disulphide bonds with Aga1 on the surface of EBY100, anchoring it to the yeast cell surface. This enables the NEW11-A protein to be displayed on the yeast cell surface, forming a new layer of antifreeze proteins.
BBa_259ZX5TT	pGAL1-Aga2-GS linker-Superfolding GFP-tMATα	This is a gene expression module. Under galactose induction, transcription commences at the pGAL1 promoter, expressing a downstream fusion protein comprising Aga2 and Superfolding GFP. The Aga2 protein forms disulphide bonds with the Aga1 protein on the surface of EBY100 yeast cells, thereby displaying the Superfolding GFP protein on the yeast cell surface. Observation under a microscope reveals fluorescence on the cell surface.
BBa_251433F2	pGAL1-Aga2-GS linker-MUT4-tMATα	This is a gene expression module. Building upon the surface display of the native protein, MUT4 from the site-directed mutagenesis design was selected for surface display. Upon galactose induction, the pGAL1 promoter is activated, expressing a fusion protein of Aga2 and MUT4. Aga2 forms disulphide bonds with Aga1 on the surface of EBY100 yeast cells, anchoring the fusion protein to the yeast cell surface. This process displays MUT4 on the yeast cell surface, forming a layer of antifreeze proteins.
BBa_25FHHEP0	pGAL1-Aga2-GS linker-4MTJ-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 4MTJ. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 4MTJ protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25S5YLV9	pGAL1-Aga2-GS linker-6A8K-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 6A8K. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 6A8K protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25INSP64	pGAL1-Aga2-GS linker-4NU2-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 4NU2. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 4NU2 protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25O40TB7	pGAL1-Aga2-GS linker-3P4G-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 3P4G. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 3P4G protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25RO6WMC	pGAL1-Aga2-GS linker-3WP9-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 3WP9. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 3WP9 protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25P9IJJJ	pGAL1-Aga2-GS linker-2PNE-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 2PNE. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 2PNE protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25IZTU85	pGAL1-Aga2-GS linker-1M8N-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 1M8N. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 1M8N protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25606GOP	pGAL1-Aga2-GS linker-3ULT-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 3ULT. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 3ULT protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25V9YYJ1	pGAL1-Aga2-GS linker-1UCS-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 1UCS. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 1UCS protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.
BBa_25ZDIXV8	pGAL1-Aga2-GS linker-2ZIB-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter activates expression of a fusion protein comprising Aga2 and 2ZIB. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast cell surface, anchoring the fusion protein to the cell membrane. This process exposes 2ZIB on the cell surface, forming a layer of antifreeze proteins on the yeast cell surface.
BBa_25YPV182	pGAL1-Aga2-GS linker-4KE2-tMATα	This is a gene expression module.Under galactose induction, the pGAL1 promoter initiates expression of a fusion protein comprising Aga2 and 4KE2. The Aga2 protein forms disulphide bonds with the Aga1 protein on the yeast surface, thereby displaying the 4KE2 protein on the yeast cell surface. This process establishes a protective layer of antifreeze proteins.