Stable Expression and Secretion of Polyhydroxybutyrate Depolymerase of Paucimonas lemoignei in Escherichia coli

다국어 초록 (Multilingual Abstract)

An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase
(PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of
PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form
of PhaZ1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing
PhaZ1 and its own N-terminal signal peptide (PrePhaZ1) enabled the secretion of active PhaZ1 into the
extracellular medium. However, the PrePhaZ1 construct was harmful to the host cell and resulted in atypical
growth and instability of the plasmid during the cultivation. In contrast, INPNC-PhaZ1 and INPNCPrePhaZ1
fusion constructs did not affect growth of host cells. INPNC-PhaZ1 was successfully displayed
on the cell surface with its fusion form, but did not retain PhaZ1 activity. In the case of INPNC-PrePhaZ1,
the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active
PhaZ1 was consequently released into the culture medium. The amount of PhaZ1 derived from E. coli
(INPNC-PrePhaZ1) was almost twice as great as that directly expressed from E. coli (PrePhaZ1), and was
predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently
secreted into the extracellular medium when cultivated at 37°C.

참고문헌 (Reference)

1 Jung, H.C., "Surface display of Zymomonas mobilis levansucrase by using the ice-nucleation protein of Pseudomonas syringae" 16 : 576-580, 1998

2 Hannig, G, "Strategies for optimizing heterologous protein expression in Escherichia coli" 16 : 54-60, 1998

3 Jana, S, "Strategies for efficient production of heterologous proteins in Escherichia coli" 67 : 289-298, 2005

4 Wu, C.M, "Signal peptide of eosinophil cationic protein is toxic to cells lacking signal peptide peptidase" 322 : 585-592, 2004

5 Choi, J.H, "Secretory and extracellular production of recombinant proteins using Escherichia coli" 64 : 625-635, 2004

6 Mergulh, F.J.M., "Recombinant protein secretion in Escherichia coli" 23 : 177-202, 2005

7 Braaz, R., "Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis" 209 : 237-241, 2002

8 Schober, U., "Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei" 66 : 1385-1392, 2000

9 Zinn, M., "Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate" 53 : 5-21, 2001

10 Mergulhao, F.J.M., "Medium and copy number effects on the secretion of human proinsulin in Escherichia coli using the universal stress promoters uspA and uspB" 61 : 495-501, 2003

11 Humphreys, D.P., "High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: Importance of codon usage at the 5'end of the coding sequence" 20 : 252-264, 2000

12 Denefle, P., "Heterologous protein export in Escherichia coli: Influence of bacterial signal peptides on the export of human interleukin 1" 85 : 499-510, 1989

13 Yim, S.K, "Functional expression of mammalian NADPH-cytochrome p450 oxidoreductase on the cell surface of Escherichia coli" 49 : 292-298, 2006

14 Stinson, M.W, "Extracellular enzyme secretion by Pseudomonas lemoignei" 119 : 152-161, 1974

15 Bassi, A.S., "Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the icenucleation protein of Pseudomonas syringae" 16 : 557-563, 2000

16 Jung, H.C., "Expression of carboxymethylcellulase on the surface of Escherichia coli using Pseudomonas syringae ice nucleation protein" 22 : 348-354, 1998

17 Pfeiffer, T., "Effects of signal peptide exchange on hiv-1 glycoprotein expression and viral infectivity in mammalian cells" 580 : 3775-3778, 2006

18 Beena, K., "Effect of signal peptide on the stability and folding kinetics of maltose binding protein" 43 : 3608-3619, 2004

19 Chung, S.H., "Effect of levulinic acid on the production of poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) by Ralstonia eutropha KHB-8862" 39 : 79-82, 2001

20 Samuelson, P., "Display of proteins on bacteria" 96 : 129-154, 2002

21 Sei, K., "Design of PCR primers and a gene probe for extensive detection of poly(3-hydroxybutyrate)(PHB)-degrading bacteria possessing fibronectin type III linker type-PHB depolymerases. Appl" 53 : 801-806, 2001

22 Tokiwa, Y, "Degradation of microbial polyesters. Biotechnol" 26 : 1181-1189, 2004

23 Jiang, Y., "Cloning and expression of the polyhydroxyalkanoate depolymerase gene from Pseudomonas putida, and characterization of the gene product" 26 : 1585-1588, 2004

24 Jendrossek, D., "Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product" 218 : 701-710, 1993

25 Lee, S.Y., "Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals" 65 : 363-368, 1999

26 Kim, D.Y., "Characterization of an extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas alcaligenes LB-19" 3 : 291-296, 2002

27 Kwak, Y.D., "Cell surface display of human immunodeficiency virus type 1 gp120 on Escherichia coli by using ice nucleation protein" 6 : 499-503, 1999

28 Shokri, A., "Cell and process design for targeting of recombinant protein into the culture medium of Escherichia coli" 60 : 654-664, 2003

29 Do Young Kim, "Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates" 한국미생물학회 45 (45): 87-97, 2007

30 Kim, D.Y, "Biodegradation of microbial and synthetic polyesters by fungi" 61 : 300-308, 2003

31 Jendrossek, D., "Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system" 177 : 596-607, 1995

32 Jung, H.C., "Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities" 26 : 177-184, 2003

33 Kang, S.M., "Bacterial cell surface display for epitope mapping of hepatitis c virus core antigen" 226 : 347-353, 2003

34 Handrick, R., "A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids" 276 : 36215-36224, 2001