PHA synthase 활성에 의한 polyhydroxybutyrate(PHB)의 분자량 및 분자량 분포의 in vivo 제어

심상준, 조영상, 최광진, 김영대, A. J. Sinskey*

한국과학기술연구원 화공연구부

PHA Synthase Activity controls the molecular weight and polydispersity of Polyhydroxybutyrate (PHB) in vivo

Sang Jun Sim, Young Sang Cho, Guang Jin Choi, Young Dae Kim, and Anthony J. Sinskey*

Division of Chemical Engineering, Korea Institute of Science and Technology, Korea

*Department of Biology, Massachusetts Institute of Technology, MA 02139 USA

Introduction

Although considerable attention has been devoted to the fernentation of genetically engineered E. coli PHA producers studies have focused predominantly on the PHA yield and production rate of the cells. Such information is of preeminent commercial importance especially because production costs of PHAs currently exceed those of comparable conventional or biodegradable plastics1,2. However, they offer no insight to the final nature or properties of the polymer. The average moleclar weight and moleclar weight distribution are perhaps the most important properties for the commercial suitability of a polymer product yet only a few researchers1 have characterized the size of PHAs produced in recombinant E. coli strains.

In this study, the average molecular weight and molecular weight distribution of polyesters isolated from recombinant E. coli constructs were examined by multi-angle light scattering. Investigations focused on a plasmid-encoded native A. eutrophus operon and a synthetic operon encoding the PHA bioynthetic enzymes. Since previous studies have demonstrated that expression of the native A. eutrophus operon in E. coli provides little synthase activity3,4, the synthetic operon was designed to yield high levels of in vivo PHA synthase. This was achieved by placing a strong inducible promenter and an E. coli ribosome binding site (RBS) in front of the synthase gene. Fermentation of E. coli strains prossessing the two constructs and characterization of both the molecular weight and molecular weight distribution of the isolated polyesters has suggested a role for PHA synthase activity in controlling the molecular weight of PHAs produced in vivo in a host organism5.

Results

Native Operon Evaluation. E. coli DH5□/pAeT41 was utilized to establish a reference point from which to compare future genetically engineered E. coli PHB producers. Plasmid pAeT41 contains a 5 kb genetic fragment encoding the native, unmodified PHA biosynthetic operon from A. eutrophus inserted into the vector pUC18. As with all other strains in this study, a 13 L batch fermentation was performed in LB medium containing 20 g/L glucose to provide material for molecular weight determination, enzyme assays, and fermentation parameters for a range of time points during the fermentation period. polymer was extracted from lyophilized cells in refluxing chloroform, precipitated with hexane, and the weight average molecular weight (Mw) of the isolated polymer was characterized using gel permeation chromatography (GPC) in line with multi-angle light scattering.

Synthetic Operon Construction and Evaluation. Expression of the native A. eutrophus operon in E. coli has consistently yielded low levels of synthase activity. High levels of synthase activity have been obtained with constructs expressing only the gene encoding the synthase upon replacement of the native A. eutrophus synthaes RBS with an E. coli RBS. This strategy was therefore used to construct a synthetic operon designed to yield increased synthase activity relative observed in DH5□/pAeT41. Plasmid pSP2 was constructed in the medium copy number vector pTRCN and contains a strong inducible trc promoter, a gene encoding PHA synthase with an optimal E. coli RBS, and genes encoding the thiolace and reductase. The presence of a plamid-borne lacIq gene allows the transcription of all three PHA biosynthetic genes in this synthetic operon to be tightly controlled by the amount of isopropyl □-D-thiogalactoside (IPTG) added to the culture medium.

Comparison of Enzyme Activities in DH5□/pAeT41 and DH5□/pSP2. With the decrease in polymer molecular weight in DH5□/pSP2 induced with 0.4 mM IPTG (Figure 1A), there is a concurrent increase in PHB concintration. This indicates that the dicrease in molecular weight is not due to polymer degradation during the fermentation. Enzyme activities of the thiolase, reductase, and synthase were therefore monitored to determine if a difference in enzyme activities coincided with the observde differences in Mw of polymer isolated from strains DH5□/pAeT41 and DH5□/pSP2 (0.4 mM IPTG). Despite localization of the A. eutrophus PHA biosynthetic operon on the high copy plamid pUC18, no soluble PHA synthase activity was detected during the entire DH5□/pAeT41 fermentation (Figure 2A).

The presence of polymer in cell lysates often results in localization of synthase activity in the insoluble portion of the lysate. An additional assay was thus performed for all fermentation samples by measuring the combined soluble and insoluble fractions in the crude lysate prior to centrifugation. DH5□/pAeT41 lysates assayed in this way still contained no detectable synthase activity (Figure 2B).  Synthas activity was predominently insoluble during the fermentation of DH5□/pSP2 (0.4 mM IPTG) yielding a maximum specific activity of 1.4 U/mg in the combined soluble and insoluble fractions at 13.3 h (Figure 2B).  Soluble synthase levels in DH5□/pSP were 0.11 U/mg at 18.3 h (Figure 2A). Assays for thiolase activity in DH5□/pAeT41 yielded a maximum of 2.5 U/mg at 14.6 h whereas thiolase activity in DH5□/pSP2 approached 3.6 U/mg at 11.5 h (Figure 2C). Reductase activity was only observed at 18.7 h at a level of 0.33 U/mg during the fermentation of DH5□/pAeT41. No other detectable reductase activity was observed in the DH5□/pAeT41 fermentation or during the entire fermentation of DH5□/pSP2.

References.

1. Marchessault, R. H. 1996. Tender Morsels for Bacteria: Recent Developments in Microbial Polyesters. Trends Poylm. Sci. 4:163-168

2. Poirer, Y.;Nawrath, C.;Somerville, C. 1995. Production of Polyhydroxyalkanoates, a Family of Biodegradable Plastics and Elastomers, in Bacteria and Plants. Bio/technolgy 13:142-150

3. Peoples, O. P.; sinskey, A. J. 1989. Poly-□-hydroxybutyrate (PHB) Biosynthesis in Alcaligenes eutrophus H16: Identification and Characterization of the PHB Polymerase Gene (phbC). J. Biol. Chem. 264:15298-15303.

4. Gerngross, T. U.; Snell, K. D.; Peoples, O. P.; Sinskey, A. J.; Csuhai, E.; Masamune, S.; Stubbe, J. 1994. Overexpression and Purification of the Soluble Polyhydroxyalkanoate Synthase from Alcaligenes eutrophus: Evidence for a Required Posttranscriptional Modification for Catalytic Activity. Biochemistry. 33:9311-9320.

5. Sang Jun Sim, S. Hogan, K. D. Snell, A. J. Sinskey, C. K. Rha, and J. Stubbe "PHA synthase activity controls the molecular weight and polydispersity of Polyhydroxybutyrate in vivo", Nature Biotechnology, 15(1), 63-67 (1997) USA

Figure 1 (A) Weight average molecular weight of polymer isolated from fermentations of strains E. coli DH5□/pAeT41 (△), DH5□/pSP2 with 0.4 mM IPTG induction (O), and DH5□/pSP2 with 5.0 mM IPTG induction (◆).

(B) Polydispersity

Figure 2. (A) Synthase activity in soluble lysate fractions of strains E. coli DH5□/pAeT41 (△), DH5□/pSP2 with 0.4 mM IPTG induction (O), and DH5□/pSP2 with 5.0 mM IPTG induction (◆). (B) Combined soluble and insoluble PHA synthase activity (C) Thiolase activity