Luận án tiến sĩ: Development of inducer-free expression vectors for Bacillus subtilis based on Pgrac promoters

TRAN THI MINH DINH DEVELOPMENT OF INDUCER-FREE EXPRESSION VECTORS FOR Bacillus subtilis BASED ON Pgrac PROMOTERS PhD THESIS OF MICROBIOLOGY HO CHI MINH CITY — 2022 TRAN THI MINH DINH DEVELOPMENT OF INDUCER-FREE EXPRESSION VECTORS FOR Bacillus subtilis BASED ON Pgrac PROMOTERS Speciality: Microbiology Code: 62420107 Reviewer |: Prof. Le Huyen Ai Thuy Reviewer 2: Assoc. Ngo Thi Hoa Reviewer 3: Dr. Nguyen Thi Thanh Thao Independent reviewer 1: Dr.

Tran Thi Hai Yen Independent reviewer 2: Dr. Nguyen Thi Thanh Thao SUPERVISOR 1. Nguyen Duc Hoang 2. Wolfgang Schumann HO CHI MINH CITY — 2022 DECLARATION OF AUTHORSHIP I hereby declare that the PhD thesis in Microbiology with the topic "Development of inducer-free expression vectors for Bacillus subtilis based on Pgrac promoters" is my own scientific work under the guidance of Assoc.

Nguyen Duc Hoang and Prof. The research results of the thesis are completely honest, accurate, and do not overlap with the published works. PhD student Tran Thi Minh Dinh ACKNOWLEDGEMENT First and foremost, I would like to express my sincere thanks and gratitude to Associate Professor Nguyen Duc Hoang. His wholehearted support and continuous advice went through the process of completion of my thesis.

His encouragement and comments had significantly enriched and improved my work. Without his motivation and instructions, the thesis would have been impossible to be done effectively. I would like to express my deep gratitude to Professor Wolgang Schumann and Associate Professor Phan Thi Phuong Trang for their generous support, patient guidance, enthusiastic encouragement, valuable and constructive suggestions, and valuable critiques of this research work. I heartily appreciate Huynh Thi Kim Phuong, Dang Thi Kim Ngan, Phan Thi Thu Hanh, Nguyen Thi My Linh, and Nguyen Thi My Ngoan for their valuable assistance and for making the warm atmosphere in the Lab.

I am also grateful to all of the CBBers for their help and encouragement throughout this work. My special thanks approve to my parents for their endless love, care, and have the most effective assistance and motivation for my whole life. I also would like to thank to my brother and sisters for their support and care for me all the time. I do not know how to express my thanks to my husband, Do Thanh Hung.

He always persevered through late-night lab and study sessions. He understands my goals and helps me every step of the way. His understanding and love mean the world to me. I could not finish my thesis without him.

I would like to say a special thank to my little angel, Do Tue Minh. She joined all my study sessions and my labwork during my pregnancy. I also appreciate for her lovely patience during her infancy and toddlerhood. Last but not least, I am really grateful for financial and the whole support from Center of Bioscience and Biotechnology.

il CONTENTS DECLARATION OF AUTHORSHIP. 223111113 119 11911 1 ng ngư1 ACKNOWLEDGEMENTT.-- 9H Hà HH HH ng ng il CONTENTS 0.-- HH HH TH HH HH nh Hư Vii LIST OF TABLES 1177. Vii LIST OF FIGURES 17.-- 0 HH ng TH TH TH HH nh 1 1. Rationale of the Study.

Objective of the SfUỈY. TH TH HH ng 2 3. Research SUJ€CfS.- -- - c1 111v TH TH ng HH HH ng 3 4.-- G- L1 SH HH HH HH HH 3 hN. Scientific and practical significance of the theS1S.- SH HH TH HH HH Hư 5 1.

Bacillus subtilis as cell factories for recombinant protein production. SH HH HH kh 5 1. subtilis in recombinant protein producfion.3, Advances in improving B. subtilis cellular perfOrIMAanC€S.

Expression systems for B. Characteristics ofa promoter Of B. Characteristics of an expression vector for B. Inducible expression SVSÍ©HHS.

Ăn HH ng vệ 21 1. Inducer-free €XDT€SSỈOH SVSÍCTH(S. BgaB reporter DYOf€ITI. Hypothesis of the Study.

MATERIALS AND METHODOLOGY. Bacterial strains, plasmids, oligonucleotides, antibiotics, and media. BACtCrIAL St GINS nan ốốốố. HH TH HH HT TH như 51 QDS, MCdiIQ anh h.

Enzymes, biochemicals, chemicals, and KI(S. SH HH TH 52 2. DNA and protein ÏQ(Ï@LF. Biochemicals and Chemicals.

- - 6 2< 111191211911 1 91H HH HH Hệ 54 2. PCR and Colony PCR. RECOMDINANE StVAINS €H€T(fÏOH. Growth and collection Of SAMPLES.

BgaB activity m€aSUT€TT€TIE.- 5 0 1011930 91199119 11 vn HH nệt 59 2. Inducer-free replicative VeCtOr CONSÍTHCÍÏOH. Construction of integrative expression vectors allowing insertion at the ,1/4058199/10NhhOC. Construction of integrative expression vectors allowing insertion at the VACA LOCUS SE nh.

RESULTS AND DISCUSSION. Inducer-free replicative EXPpressSiON VECTOTS. Inducer-free plasmid based on PgrdCÔÏ. The inducer-free plasmids based on strong promoters, Pgrac57 and PRTACLOO voecccccccsccccesssceessssecseneeeessneeesseeeeeneesseeeeseseeesseseeessaueeeneaaeeseseeseeaseseneaaes 73 3.

Influence of differrent lengths of lacI deletion on the production of BgaB %ẮẦ. Inducer-free expression plasmid with the Pgrac212 promof†er. Discussion of inducer-free expression pÏASIH(ÌS. Inducer-free integrative EXPresSSiON V€CẨOTS.

Inducer-free integrative vector based on PgracO]. Vectors with strong promoters Pgrac57, Pgracl00, Pgrac212 for high production levels of the bgaB repOrter QeCNE. Increasing the production of reporter protein by introducing two expression cassettes into PB. Discussion of inducer-free integrative CExXPTeSSION VECTOTS.

CONCLUSION AND SUGGESTION. 113 LIST OF AUTHOR'S PUBLICA TIONS. - HH gi, 114 REFERENCES. Án ng TH TH HH HH HH nhe 115 APPENDIX 07.

Maps of vectors generated in this SEUd|y.- 5 SH ng ey a A2. Electrophoresis of the colony PCR of inducer-free integrative expression VECCOTS TT. Sequencing of inducer-free pÏaSIT1đS. Sequencing of inducer-free integrative V€CfOFS.

The schematic map showed the primer sites and PCR product sizes for checking the integration of the expression CaSSffC. Electrophoresis of the colony PCR for integrating checking at the amyE A7. Electrophoresis of the colony PCR for integrating checking at both the amyE and 220 on". Growth of some B.

subtilis recombinant strains habouring IPTG-inducible or inducer-free expression vectors with PgracO1] promOf€T. Variance analysis of data using Stagraphics plus 3.«--<+5 r vi ABBREVIA TIONS Abbreviations Full words bp base pair Cas CRISPR-associated CoSE Controllable Stabilizing Elements CRISPR Clustered Regularly Interspaced Short Palindromic Repeats DMSO DiMethyl SulfOxide DNA DeoxyriboNucleic Acid dNTP deoxyriboNucleotide TriPhosphate EGFP Enhanced Green Fluorescent Protein GFP Green Fluorescent Protein IPTG IsoPropyl B-D-1-ThioGalactopyranoside LB Luria-Bertani MCS Multiple Cloning Sites MUG 4-MethylUmbelliferyl-8-D-Glucuronide min minute ori origin of replication PCR Polymerase Chain Reaction RBS Ribosome Binding Site RFP Red Fluorescent Protein RNA RiboNucleic Acid SDS Sodium Dodecyl Sulfate SDS-PAGE Plectrophovea Sulfate PolyAcrylamide Gel sfGFP superfolder Green Fluorescent Protein X-gal 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside Vil LIST OF TABLES Table 1. Some inducible promoters for B. Sequences of some Pgrac promoters.

Bacterial strains used 1n this Work. Plasmids used in this Study. Oligonucleotides used 1n this WOTK. Antibiotic solutions used in this Work.

Components of PCR for cÏOn1ng. Components of colony PCT.- ---- 5 5 + vn ng ng ng trên 55 Table 2. Components of the restriction cleavage with KpnI and Sac]l. Components of ligation reaction.

Components of the restriction cleavage with BamHI and Aaill. Components of the restriction cleavage with BamHI and Kpnl. Components of the restriction cleavage with SnaBI and Sacl. Components of the restriction cleavage with BamHI and EcoRl.

Components of the restriction cleavage with BglII and EcoRI. Components of the restriction cleavage with BamHI and Spel. Components of the restriction cleavage with BamHI and Sacl. ODeoo value of B.

subtilis recombinant sfTa1nS. Repression of different expression vectors 1n E. Inducer-free replicative expression plasmids with different lengths of lacI Table 3. BgaB production of B.

subtilis recombinant strains carrying plasmids with different lengths of lacI deπfIOT. --- c5 1113111311119 1111 111v ng rey 81 Table 3. Inducer-free integration vectors carrying Pgrac57, Pgracl00, or Pgrac212 promoters allowing integration at the amyE locus in this study. Inducer-free integration vectors carrying PgracO1, Pgrac57, Pgrac100, or Pgrac212 promoters allowing integration at the /acA locus constructed in this study Table 3.

Percentage of BgaB in comparison with total intracellular proteins in B. subtilis Wte grants 20. 104 Vili LIST OF FIGURES Fig. Colony morphology and scanning electron microscopic image of B.

The schematic model of the organization - segregation cycle in B. The schematic plasmid map showing the main component of typical EXPTESSION VECCOTS 2. Location of some well-characterized loci for ectopic integration on B. SUbtilis circular FENOME.

Q2 HH TH HH 20 Fig. The Pgrac01 promoter and RBS sequence in the pNDH33 plasmid. Secondary structures of the 5’-steMm-lOOPS .cc:cccescesseeeseeeeteeeeeeeseeeees 24 Fig. Schematic map of the pHTO1 plasimid.- -- 5 «<< £+s£<se+se+sessxs 27 Fig.

Control of ColEl repÏ1CafIOII. --- -- - < + +3 + VE*kkseieeeeeeereere 28 Fig. P43 promoter S€QU€TCG. G2 0111311119111 911191111 HH ng ng 29 Fig.

The cry3Aa promoter sequence and some of its derivatives. The schematic representation of repession and expression mechanism of the target gene ON PHT V€CfOTS 00. eee eeesccesseseeeceseecesceceseeeeseesaeceeeeeeeseaecesaeeeeeesaes 37 Fig. DNA and protein ladder used in this sfUdyy.

The schematic diagram shows the sites of oligonucleotides used for checking the double crossover at both amyE in B. subtilis recombinant strains. Schematic diagram of the construction of inducer-free expression vectors harboring the Pgrac100 promoter and the DgaB gene. Schematic diagram of the construction of inducer-free expression vectors harboring the Pgrac212 promoter and the DgaB øene.- -- 5 «<< <+sx+se+sxs 63 Fig.

Schematic diagram of the construction of inducer-free expression vectors integrating into the AMYE ÏOCUS. -- -- 5c 111191018910 19 111911 ng ky 64 Fig. Schematic diagram of the construction of inducer-free expression vectors integrating into the [ACA ÏOCUS.- s5 6+ 1E E919 211 9101 911101 hi HH 66 Fig. BgaB activity of B.

subtilis recombinant strains carrying the Pgrac01 PLOMOLEL 00111777. SDS-PAGE revealed BgaB synthesis of Bs/pHT1655. subtilis recombinant strains on LB-agar-Xøgal. BgaB activities of E.

coli strains carrying the PgrzcOÖ]. Gel electrophoresis of the colony PCR product of pHT1658 on agarose 2% 2111810150300). Gel electrophoresis on 2% agarose of PCR product for cloning of PHT 1656. BgaB activities of B.

subtilis recombinant strains carrying inducer-free plasmids with the Pgrac01, Pgrac57, or Pgrac100 promoters. SDS-PAGE showing BgaB production of B. subtilis recombinant strains carrying inducer-free plasmids with PgracO1, Pgrac57, or Pgrac100. Gel electrophoresis on agarose 2% of colony PCR of pHT2079 with the product size Of V29).

Gel electrophoresis on 2% agarose of PCR products for cloning of 2:0. BgaB protein of B. subtilis carrying inducer-free plasmids with PgracO1, Pgrac57, OF PgraclOO PT 43. Gel electrophoresis on 2% agarose of colony PCR of pHT2080 with the product size Of 587 Dp.- sọ TT Hà Hà HH HH gà 85 Fig.

BgaB activities of B. subtilis carrying plasmids with Pgrac212. BgaB protein in B. subtilis carrying plasmids with Pgrac212.

The supposed DNA-loop formed by binding of tetrameric LacR on the TWO LAC OPECTALOTS Tai. Cloning result of PHT 2170 0. Background expression of plasmids carrying Pgrac01 promoter in E. Confirmation of double crossover of the expression cassette in B.

BgaB synthesis of integrative strains carrying Psrac01 in comparison with other strains carrying replicative plasmids with the same or different promoters Fig. BgaB production of recombinant B. subtilis strains habouring expression cassettes at the GME LOCUS 0. Confirmation of the insertion of the expression cassette at both the amyE and the lacA loci by a double crossover €V€TI.

cv ersep 103 Fig. BgaB synthesis of B. subtilis recombinant strains carrying two inserted expression Cassettes On the GENOME .- <1 13011991 E991 91 1v ng ng nếp 105 XI SUMMARY Thank to its numerous favourable properties, B. subtilis is an ideal cellular factory for producing heterologous proteins.

The inducer-free expression vectors in B. subtilis gain preference because of the low costs and absence of toxicity of the inducers. However, the majority of the expression vectors in B. subtilis - Escherichia coli shuttle vectors, so the background expression can interfere the efficiency of cloning steps in E.

Therefore, generation of expression vectors that could produce the heterologous proteins in the absence of inducers at a high level in B. subtilis while they still repress leaky expression in E. coli is a demand in heterologous protein production.