Combinatorial Evolution of Enzymes and Synthetic pathways Using

Transcription

1 Supplementary data Combinatorial Evolution of Enzymes and Synthetic pathways Using One-Step PCR Peng Jin,, Zhen Kang,,, *, Junli Zhang,, Linpei Zhang,, Guocheng Du,, and Jian Chen, The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu , China The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi , China * To whom correspondence should be addressed. Tel: ; Fax: ; zkang@jiangnan.edu.cn 1

2 Contents Supplementary Figure S1. Optimization of the DNA ligases for improving..3 the efficiency of RECODE. Supplementary Figure S2. Effects of Mg 2+ concentration on the RECODE..4 reaction. Supplementary Figure S3. Optimizing the cycles of RECODE PCR. Supplementary Figure S4. The DNA products of the variants were generated by one-step and two-step of PCR systems of RECODE. Supplementary Figure S5. DNA sequence analysis of insertion and deletion in..7 gfp gene by RECODE. Supplementary Figure S6. Alignment of the nucleic acid sequences of the..8 ALA synthetic pathway variants by RECODE. Supplementary Table S1. List of primers used in this study. Supplementary Table S2. Phenotype analysis of the mutant library of galactosidase and esterase. Supplementary methods 12 2

3 Supplementary Figures Figure S1. Comparison and screening DNA ligases for the RECODE reaction system. Lane 1, Taq DNA ligase (NEB); lane 2, 9 N DNA ligase (NEB); lane 3, Ampligase thermostable DNA Ligase (Epicentre); M, DNA marker. 3

4 Figure S2. Effects of Mg 2+ concentration on two-step RECODE reaction. (a) The RECODE buffers containing various Mg 2+ concentrations ( mm) were used for the preparation of single-stranded mutant library in the 1 st step PCR reaction, the corresponding product amounts of the 2 nd step PCR were analyzed in agarose gel electrophoresis (indicated by the arrow). (b) The amplification capability of DNA polymerase in RECODE buffers containing various Mg 2+ concentrations ( mm) was tested to amplify the DNA fragment (1500 base pairs) as the conventional PCR operation. Lane 1, 10 mm; lane 2, 7.5 mm; lane 3, 5 mm; lane 4, 3 mm; lane 5, 1.5 mm; M, DNA marker. 4

5 Figure S3. Optimization of PCR thermal cycles in the 1 st step PCR. Based on the optimized ligase and Mg 2+ in RECODE buffers, PCR thermal cycling were performed with 1-25 cycles, respectively. The corresponding mutant products of the 2 nd step PCR were indicated by the arrow. Lane 1, 25 cycles; lane 2, 15 cycles; lane 3, 5 cycles; lane 4, 1 cycle. 5

6 Figure S4. The final mutant products generated by different RECODE reaction systems. Lane 1, one-step PCR reaction system; Lane 2, Two-step PCR reaction system. 6

7 Figure S5. DNA sequence analysis of insertion and deletion in the gfp gene by RECODE. Red bases represent the inserted nucleotides and the red arrows indicate the insertion sites. - denotes the deleted nucleotides and the green arrow indicates the deletion site. The wild-type sequence is shown at the top. The size of the nucleotides inserted (+) or deleted ( ) are specified. The efficiencies of insertion and deletion were calculated according to DNA sequencing of the randomly picked 216 clones from insertion library and 132 clones from deletion library, respectively. 7

8 Figure S6. Alignment of the nucleic acid sequences of the ALA synthetic pathway variants by RECODE. Combinatorial engineering were denoted and distributed on the pathway enzymes (Red in the background) and the regulatory elements (RBS, yellow in the background; Promoter, green in background). The wild-type sequence is shown at the top. 8

9 Supplementary Tables Table S1. Primers used in this study Name Sequence (5 3 ) Primers of lethal mutations of lacz-estc23 genes RECODE/LacZ-P1F GTGACTGGGAAAACCCTGGCTAAACCCAACTTAATCGCCTTGC AGCA RECODE/Est-P2F GTGCTTTATCTGCACGGTGGCGGCTAAGTTATCGGCTCGATCAA CACGC RECODE/Est-P3F CGATATGGAAGGAGTCGGCGATTCGTGAAGACGAAGGCGGCTG TCG LZE-UAP CATCACAGTCTTGCTAAAGCGCAAGCGTTGGCCGATTCATTAAT GCAGCTG LZE-DAP CGACAAAATTGGAGCGTTCATCCGCGCCAACGCCGAGTAAATC ACTAGTCTGAATCGGCC LZE-F CATCACAGTCTTGCTAAAGCGCAAG LZE-RP GGCCGATTCAGACTAGTGAT Primers of rpos promoter evolution RECODE/rpoSp-F TTCCACCGTTGCTGTTGCGTNNNNNNNNNNNNNNNNNTATTCT GAGTCTTCGGGTGAAC RECODE/rpoSp4-F CATAACGACACAATGCTGGTNNNNNNNNNNNNNNAAGTTAAG GCGGGGCAAAAAATAGC RECODE/rpoSp3-F TAGCACCGGAACCAGTTCAANNNNNNNNNNNNNNNNAATTCG TTACAAGGGGAAATCCG RECODE/rpoSp21-F(D AGCGATAAATCGGCGGAACNNNNNNNNNNNNNNNNTGNTCCG AP) TCAAGGGATCACGGGTAGGAGCCACCTTATGGGTAAGGGAGAA GAACT RpoSp-UAP CACTATAGGGCGAATTGGAGCTCCATACGCGCTGAACGTTGGTC AG rposp-f CACTATAGGGCGAATTGGAGCT rposp-rp AGTTCTTCTCCCTTACCCAT PBBR2-gfp-F ATGGGTAAGGGAGAAGAACTTTTCAC PBBR2-gfp-R CCGGAATTCTTATTTGTATAGTTCATCCATG Primers of hyaluronidase evolution RECODE/LHyal-P1F TCTCCGAAAGTTTCCATVNNVNNVNNNNNGATGSGVNNVNNTT TTCACCGAAAGGGTTG RECODE/LHyal-P2F ATCACATCACCGARATTGNNNVNNCTCVNNVNNVNNCTCTCTC CAGSTTWTTTCCGCGT RECODE/LHyal-P3F GTCGGAGGGACGNNNVNNVNNTKGTTAVNNTTTRRCCYCGATG AAAACAACAAATGGAA RECODE/LHyal-P4F GTCAAAYTCRCCAAMKGATCTVNNVNNVNNNTGMTGNTTNAT TTAAACGCTGAAGTCAG RECODE/LHyal-P5F AAGGCTATGGAGATNACVNNRRCTGGGAAVNNVNNVNNVNNC CGGATCATACGTCCGCA 9

10 10 RECODE/LHyal-P6F CATAAAGTGCTGGAAAAMNATVNNVNNVNNVNNVNNVNNNCA TTANTGGGCCCTGACGT RECODE/LHyal-P7F ACGCTTCACCASTACKDSNTTRACGGCMRWNCCKCARMTRDG AGCACATACCTGGACGC RECODE/LHyal-P8F GCACCAAAGATSTTTCGNVVNVVNNTVNNVNNGGTTTTNTTWS GCTTGACAAACTGGGT RECODE/LHyal-P9F AGCCGAATCCAGATTATTGGCTGVNNVNNVNNVNNVNNTCGTT AGTAGGGMVTACGGTC RECODE/LHyal-P10F GAGTGTACGCACAMTGCNCCAAMVNNVNNNCAVNNVNNVNN CAGAGTCGTTWCTACAAG LHyal-UAP GAGGCTGAAGCTTACGTAGAATTCCACCACCACCACCACCACA TGAAAGAGATCGCGGTGACAATAG LHyal-DAP TGTAGTCAGCGATGCAAATGTTGAAGCGTGCAAAAAGTAAGCG GCCGCGAATTAATTCGC LHyal-F GAGGCTGAAGCTTACGTAGAATTC LHyal-RP GCGAATTAATTCGCGGCCGC Primers of engineering of ALA pathway RECODE/hemL-P1F (heml-uap) AACTTTAATAAGGAGGGATCCATAAAAGGRRRDDDDTATATGAG TAAGTCTGAAAATCT RECODE/hemL-P2F CGTGGTTTAAGCTTTGGTNCANCANNCVNNVNNVNNNTGNAAA TGGCGCAACTGGTGA RECODE/hemL-P3F CGCCTGGCCCGTGGTTTTNCCNNTVNNVNNANNNNTNTTAAAT TTGAAGGGTGTTACC RECODE/hemL-P4F ACTTATAATGATCTGGCTNCTVTAVNNVNNNCANNTVNGCAATA CCCGCAAGAGATTGC RECODE/hemL-P5F GGTGGTCGTCGTGATGTAATGNATVNNVNNVNNVNNNCGNGTC CGGTCTATCAGGCGG RECODE/hemA-P6F (hema-uap) TATGACTGTGATGATATTAGDDRRRRRDDDDDATGNNCVNNVN GCTTTTAGCGCTCGGTA RECODE/hemA-P7F AATGACGCCGTCAGCCACNTGNTGVNNVNNVNNNGCNGTCTG GATTCACTGGTGCTGGG RECODE/hemA-P8F AGCGCCGTCTCCGTCGCGNTTNCCVNNNNTVNNVNNGCCCGCC AAATCTTTGAATCGCT RECODE/hemA-P9F ATTATCGCCAACCGAACCNGCVAGVNNVNNVAANCCCTGGCGG ATGAGGTAGGCGCCG RECODE/hemA-P10F GCATTAAAAAGCCGTCGTNACVNGVNNNTGVNNVNNGTGGATA TCGCCGTACCGCGCG RECODE/hemF-P11F (hemf-uap) CCGCATAATCGAAATTAATANNNNNNNNTATAGGGGAATTGTGA GCGGATAAC RECODE/hemF-P12F GTATATTAGTTAAGTATAAGRRRRRRDDDDDDATGAAACCCGAC GCACACCAGG RECODE/hemF-P13F CATCGCCCGGAACTTGCCNGGVNNVNNNNCGAGGCGATGGGC GTTTCACTG RECODE/hemF-P14F TTCTATGGTTTTGAAGAAGATNCTNNTNNNNNGNATCGCACCG

11 CCCGTGACCTGTGCC heml-f TGTTTAACTTTAATAAGGAGGGATCC heml-dap CACCATCGATGCTGCACGTCGGGTGTTTGCGAAGTTGTAATATG ACTGTGATGATATTAG heml-rp CTAATATCATCACAGTCATA hema-dap ACGCCTGAATATTCTGCGCGACAGCCTCGGGCTGGAGTAACCG CATAATCGAAATTAATA hema-rp TATTAATTTCGATTATGCGG hemf-dap GGCGTTAAGTGAGTTTATTAAGGTCAGGGATTGGGTGTAAGCAC TTCGTGGCCGAGCTCG hemf-rp CGAGCTCGGCCACGAAGTGC PRSFDUET-F GCACTTCGTGGCCGAGCTCGAGTCTGGTAAAGAAACCGCTGC PRSFDUET-R CTCCTTATTAAAGTTAAACAAAATTATTTCTACAG Primers of insertion and deletion of elements RECODE/gfp-I196 CTTGGCCAACACTTGTCACTACTCTTACTACATGCCACTGGTAT GGTGTTCAATGCTTTTCAAGATACC RECODE/gfp-I601 CCTGTCCTTTTACCAGACAACCATTACTACTGGCACCTGTCCAC ACAATCTGCCCTTTCGAAAGA RECODE/gfp-D607 CTGTCCTTTTACCAGACAACCATTACCTGTCCTCTGCCCTTTCG AAAGATCCCAACGAA puc19-f GGCGTAATCATGGTCATAGCTGTTTCC puc19-r TCGTGACTGGGAAAACCCTGGCG gfp-uap GCTATGACCATGATTACGCCATGGGTAAGGGAGAAGAACTTTTC AC gfp-dap TGGATGAACTATACAAATAACTGGCCGTCGTTTTACAACGTCGT GACTGGGAAAACCCTG gfp-ap CAGGGTTTTCCCAGTCACGACGTTG Table S2. Phenotype analysis of the mutant library of galactosidase and esterase a Blue a Bule b White b White c White-hole d Bule-hole e Mutation sites / / Two-steps RECODE f NF NF One-step RECODE NF NF 346 clones from Two-steps RECODE library and 73 clones from One-step RECODE library were analyzed and evaluated the recombination efficiency and quality of the libraries by DNA sequencing. a :Blue phenotypic clone with galactosidase activity; b : white phenotypic clone c : white phenotypic clone with esterase hydrolysis clear zone; d : Blue and clear zone phenotypic clone with galactosidase and esterase activity (parents); e : the order of mutation sites in lacz-estc23 fragment (first site in lacz, second and third sites in estc23, : No introduced 11

12 mutation; : introduced mutation ).; f NF: no found phenotypic clone. Supplementary methods Screening and Identification of the Ligase in the RECODE Reaction System To optimize the use of ligases, three thermostable ligases and their corresponding modified buffers for the RECODE system are listed below: Taq DNA ligase (NEB) with the reaction buffer (20 mm Tris-HCl, 25 mm KAc, 1.5 mm Mg 2+, 1 mm NAD, 2 mm dntps, 0.1% Triton X-100, ph 7.6); 9 N DNA ligase (NEB) with the reaction buffer (10 mm Tris-HCl, 0.6 mm ATP, 1.5 mm Mg 2+, 2.5 mm DTT, 2 mm dntps, 0.1% Triton X-100, ph 7.6) and Ampligase thermostable DNA ligase (Epicentre) with the reaction buffer (20 mm Tris-HCl, 25 mm KCl, 1.5 mm Mg 2+, 0.5 mm NAD, 2 mm dntps, 0.1% Triton X-100, ph 8.3). Other components of the RECODE system included 0.1 µm of each phosphorylated oligonucleotides and anchor primers (UAP and phosphorylated DAP), template DNA (0.01 pmol), and 1 U Phusion DNA polymerase (NEB). The following PCR conditions were performed: 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, 2 min at 72 C and 3 min at 45 or 66 C; and a hold period at 4 C. The 1 st step of RECODE PCR products with three ligases were purified after the digestion of the plasmid templates. The double-strand products were then generated with an antisense primer in the new PCR system: 24 µl of the purified products, 1 µl of the antisense primer, and 25 µl of 2 Super pfu PCR MasterMix (Hangzhou Biosci Co., Ltd, China.). The PCR conditions were performed: 2 min at 94 C; then 3 cycles of 30 s at 94 C, 30 s at 50 C and 90 s at 72 C; 5 min at 72 C, and a hold period at 4 C. All the RECODE products were analyzed by agarose gel electrophoresis. The RECODE reaction with Ampligase thermostable DNA ligase generated small amounts of mutant products under the above reaction conditions test, whereas target products were invisibility using the other two ligases. Consequently, Ampligase thermostable DNA ligase was used in this study. Optimization of the Mg 2+ Concentration of the RECODE Reaction System Following the optimization of the ligase and its buffer, we set out to further optimize 12

13 the system by testing the Mg 2+ concentration between 1.5 and 10 mm in the RECODE reaction system. The RECODE reaction mixture was 0.1 µm of each phosphorylated oligonucleotides and anchor primers (UAP and phosphorylated DAP), template DNA (0.01 pmol), 1 U Phusion DNA polymerase, 5 U Ampligase thermostable DNA ligase and the buffer (20 mm Tris-HCl, 25 mm KCl, 0.5 mm NAD, 2 mm dntps, 0.1% Triton X-100, ph 8.3), with the final Mg 2+ concentration in RECODE varied over five concentrations (1.5, 3, 5, 7.5, and 10 mm). The PCR conditions were used: 2 min at 94 C; then 25 cycles of 30 s at 94 C, 30 s at 50 C, 2 min at 72 C and 3 min at 66 C; and a hold period at 4 C. The 1 st step of RECODE PCR products under different Mg 2+ concentrations were purified after the digestion of the plasmid templates. The double-strand products were then generated with an antisense primer in the new PCR system: 24 µl of the purified products, 1 µl of antisense primer, and 25 µl of 2 Super pfu PCR MasterMix (Hangzhou Biosci Co., Ltd, China.). The PCR conditions used were: 2 min at 94 C; then 3 cycles of 30 s at 94 C, 30 s at 50 C and 90 s at 72 C; 5 min at 72 C and a hold period at 4 C. Agarose gel electrophoresis was used to analyze the effects of the Mg 2+ concentrations on product yield using RECODE. As showed in Supplementary Figure S2a, maximum amount of target products was generated when the final Mg 2+ concentration was 5 mm. Based on the above research, the optimal 1 buffer for RECODE were confirmed as the follow: 20 mm Tris-HCl, 25 mm KCl, 0.5 mm NAD, 5 mm Mg 2+, 2 mm dntps, 0.1% Triton X-100, ph 8.3. Optimization of the PCR Thermal Cycles of the RECODE On the basis of the optimized Ampligase thermostable DNA ligase and the RECODE buffer (20 mm Tris-HCl, 25 mm KCl, 5 mm Mg 2+, 0.5 mm NAD, 2 mm dntps, 0.1% TritonX-100, ph 8.3), the number of thermal cycles of the RECODE PCR was optimized by varying the cycle number between 1 and 25. The RECODE reaction mixture was 0.1 µm of each phosphorylated oligonucleotide and anchor primers (FAP and DAP), template DNA (0.01 pmol), 1 U Phusion DNA polymerase, 5 U Ampligase 13

14 thermostable DNA ligase, and the buffer (20 mm Tris-HCl, 25 mm KCl, 5 mm Mg 2+, 0.5 mm NAD, 2 mm dntps, 0.1% Triton X-100, ph 8.3). The PCR conditions used were: 2 min at 94 C; then 25 cycles of 30 s at 94 C, 30 s at 50 C, 2 min at 72 C and 3 min at 66 C; and a hold period at 4 C. The 1 st step of PCR products of RECODE with various cycles were purified after the digestion of the plasmid templates. The double-strand products were then generated with an antisense primer in the new PCR system: 24 µl of the purified products, 1 µl of the antisense primer and 2 Super pfu PCR MasterMix 25 µl (Hangzhou Biosci Co., Ltd, China.). The PCR conditions used were: 2 min at 94 C; then 3 cycles of 30 s at 94 C, 30 s at 50 C and 90 s at 72 C; 5 min at 72 C, and a hold period at 4 C. Agarose gel electrophoresis was used to analyze the effects of thermal cycles on product yield using RECODE. As showed in Supplementary Figure S3, the target products were clearly generated when 25 thermal cycles were used in RECODE, whereas negligible amounts of the products were observed when the number of cycles was lower. Insertions and Deletions of DNA Bases by RECODE In order to show RECODE s excellent ability of editing DNA such as targeted deletion and insertion bases, the targeted edit for green fluorescent protein gfp were designed (primers in Supplementary Table S1) by deleting 6 nucleotides (from 607 to 612 site) and inserting 12 nt (at 196 site) and 9 nt (at 601 site), respectively (Supplementary Figure S5). For generating the insertion mutant library, primers RECODE/gfp-I196, RECODE/gfp-I601 and gfp-dap were phosphorylated in 50 µl reactions containing 300 pmol of a primer mixture, 1 T4 DNA ligase buffer (NEB), and 8 U polynucleotide kinase (NEB). The reaction was for 30 min at 37 C. The polynucleotide kinase was subsequently heat inactivated for 10 min at 75 C. The RECODE reaction was carried out in 50 µl system containing 0.1 µm of each phosphorylated mutant primers and gfp-uap, 0.2 µm gfp-ap, 0.01 pmol DNA template, 1 U Phusion DNA polymerase (NEB), 5 U Ampligase thermostable DNA 14

15 ligase (Epicentre) and 1 optimized RECODE reaction buffer (20 mm Tris-HCl, 25 mm KCl, 0.5 mm NAD, 2 mm dntps, 5 mm Mg 2+, 0.1% Triton X-100, ph 8.3). The one-step reaction system was performed with the following PCR conditions: 2 min at 94 C; 25 cycles of 30 s at 94 C, 30 s at 50 C, 2 min at 72 C, and 3 min at 66 C; and a hold period at 4 C. The insertion mutant library was constructed by assembling the purified PCR products into the puc19 vector which amplified with primers puc19-f and puc19-r. Also, the deletion mutation of gfp at site was performed with one mutagenic primer RECODE/gfp-D607 as the above manipulation. 216 and 132 colonies of deletion and insertion libraries were sequenced for assaying the proportions of mutation, respectively. As showed in Supplementary Figure S5, the proportion of simultaneously inserting12 nt (at 196 site) and 9 nt (at 601 site) was up to 44%, and that of targeted deletion one site (6 nt) was up to 98%. These suggest that the RECODE method also has high efficiency on targeted insertion and deletion of DNA. 15