변이주와 형질전환균주를 이용한 Alcaligenes eutrophus의 poly-□-hydroxybutyrate 생합성 조절기작의 규명

정 영 미, 이 용 현

경북대학교 자연과학대학 유전공학과

Regulation mechanism of poly-□-hydroxybutyrate biosynthesis of Alcaligenes eutrophus using PHB negative mutant and transformants

Young-Mi Jeoung and Yong-Hyun Lee

Department of Genetic Engineering, Colleage of Natural Sciences

Kyungpook National University, Taegu 702-701, Korea

INTRODUCTION

Poly-□-hydroxybutyrate (PHB) is an intracellular energy storage material accumulated by many microorganisms under the abnormal growth conditions such as limitation of nitrogen, phosphate, and oxygen.  The PHB biosynthetic pathway and its regulation mechanism have been studied extensively in Alcaligenes eutrophus.  The biosynthesis of PHB from intermediatry metabolite acetyl-CoA is accomplished by three enzymes: □-ketothiolase, acetoacetyl-CoA reductase, and PHB sybthase, which are the gene products of phbA, phbB, and phbC, respectively.

In our previous works, PHB accumulation increased significantly in transformants of A. eutrophus harbouring cloned phbCAB and phbC genes.  Especially, transformant harbouring phbC gene showed highly increased HV mol fraction in P(3HB-3HV) accumulation and these results indicate that the PHB synthase is the most critical enzyme for PHB biosynthesis [Park et al., 1995c].

In this work, the regulatory mechanism of PHB biosynthesis was investigated in the respect of cultural and metabolic characteristics to investigate using PHB (-) mutant Alcaligenes eutrophus DSM 541 damaged at phbC gene, transformants of PHB negative A. eutrophus harbouring cloned PHB biosynthesis genes, phbC and phbCAB genes, respectively, and applied for the accumulation of PHB and P(3HB-3HV).

MATERIALS AND METHODS

Strains : The parent strain Alcaligenes eutrophus H16 (ATCC 17699), mutant strain A. eutrophus (DSM 541), and transformants of PHB(-) A. eutrophus MAR3 and MAR5 harbouring the recombinant plasmids containing phbC and phbCAB genes, respectively, were used.  [Park et al., 1995b; Park et al., 1995c]

Media and cultivation :  The cultivation was carried out in the minimal medium composed of 20 g l-1 of fructose and 2.0  g l-1 of NH4Cl and cultivated at 30℃ and pH 7.0, using a shaking flask.

Determination of PHB and P(3HB-3HV) : PHB and P(3HB-3HV) concentration was determined by the modified method of Braunegg et al. using gas chromatography, with PHB and P(3HB-3HV) powder of A. eutrophus (Sigma Co.) as a standard.

Measurement of enzyme acitivies : The cell extract was prepared after the disruption of suspended cells in 50 mM phosphate buffer (pH 7.0) by ultrasonication at 4℃.  The enzymatic activities in the cell extract were determined by absorbance analysis.

Analytical method : The concentrations of metabolic intermediates were determined by high-pressure liquid chromatography.  Pyruvate conentration was determined by enzymatic method.

RESULTS AND DISCUSSION

1. Cell growth and accumulation of metabolic intermediate of PHB(-) mutant A. eutrophus

To examine the characteristics of cell growth and the accumulation of metabolic intermediates during cultivation, total cell mass, residual cell mass, and concentrations of accumulated intermediates were measured for the mutant and parent strains during cultivation in minimal medium.  In the mutant, total cell growth was notably decreased because of no accumulation of PHB due to the low activity of PHB synthase. However, residual cell growth was not much different from the parent strain.  Also, metabolic intemediates were excreted only in the mutant and among of them pyruvate was excreted significantly as shown in Fig. 1.  The specific activity of pyruvate dehydrogenase was remarkably decreased compared to that of the parent strain.  The excretion seems to be the low availability of CoASH and the product inhibition of pyruvate dehydrogenase by accumulated acetyl-CoA due to the blockage of PHB biosynthesis.

Fig. 1. Comparison of pyruvate excretion (A) and PHB accumulation of mutant and parent strains (B).

□: mutant strain A. eutrophus DSM 541,  □: parent strain A. eutrophus H16

open symbols: concentration of pyruvate and PHB,  closed symbols: NH4Cl concentration

2. Effect of cloned phbC and phbCAB genes on  pyruvate excretion of PHB(-) mutant

To investigate the effects of cloned phbC and phbCAB genes on pyruvate excretion and PHB accumulation, the concentrations of excreted pyruvate and accumulated PHB were measured for transformants MAR3 and MAR5 harbouring phbC and phbCAB genes and the mutant in the minimal medium.   In transformant MAR3 harbouring phbC gene, the pyruvate excretion was notably decreased and PHB accumulation was lower than that of the parent strain.  Meanwhile, in transformant MAR5 harbouring phbCAB gene, pyruvate excretion did not occurred and PHB accumulation was proceeded similar to that of the parent strain.  The specific activities of PDH were highly increased in two transformants compared to that of the mutant strain.  These results indicate that the PHB biosynthesis can be controlled by the activity of pyruvate dehydrogenase.

3. PHB accumlation of transformant of PHB (-) A. eutrophus harbouring phbC and phbCAB genes

To analyze the effects on PHB accumulation by transformation of phbC, phbCAB genes, the transformant MAR3 and MAR5 were cultivated in the minimal medium using two-stage cultivation and total cell mass, concentration and content of PHB, and PHB yield per fructose were measured.  In transformant MAR3 harbouring phbC gene, the content and yield of PHB were similar to those of the parent strain, however, total cell mass and PHB concentration were significantly decreased compared to those of the parent strain.  Meanwhile, transformant MAR5 harbouring phbCAB gene showed the similar patterns of cell growth and PHB accumulation compared to those of the parent strain.

4. P(3HB-3HV) accumulation of transformant of PHB (-) A. eutrophus harbouring phbC and phbCAB genes

To investigate the effects of cloned phbC and phbCAB genes on P(3HB-3HV) accumulation, the transformant MAR3 harbouring phbC gene and MAR5 harbouring phbCAB gene were cultivated in the minimal medium containing 1%(W/V) propionate using two-stage cultivation and total cell mass, concentration and content of P(3HB-3HV) and 3HV mol fraction were measured. In transformant MAR5 harbouring phbCAB gene, total cell growth and P(3HB-3HV) accumulation was not much different from those of the parent strain.  Meanwhile, in transformant MAR3 harbouring phbC gene, total cell growth, accumulation of P(3HB-3HV) were significantly decreased compared to that of the parent strain. Especially, in transformant of PHB(-) A. eutrophus MAR5, the mol fraction of 3HV was remarkably increased compared to that of the transformant MAR5 and parent strain.

5. Relationship between activty regulation of PHB biosynthetic enzymes and the biosynthesis of PHB and P(3HB-3HV)

Table 1 shows the specific activities of three PHB biosynthetic enzymes for the mutant strain, two transformants, and the parent strain with the characteristics of PHB and P(3HB-3HV) accumulation to investigate the relationship between the activity regulation of PHB biosynthetic enzymes and the biosynthesis of PHB and P(3HB-3HV) after 48 h cultivation.

Table 1. Relative activity of PHB biosynthetic enzymes and accumulation of PHB and  P(3HB-3HV).

		Parent strain (H16)	PHB negative mutant (DSM 541)	Transformant harbouring phbC gene (MAR3)	Transformant harbouring phbCAB (MAR5)
Relative specific activity (phbA: phbB: phbC)		7.1: 3.6: 7.6	2.2: 1.3: 0.001	3.5: 1.8: 7.6	6.8: 3.3: 7.0
PHB	Concentration (g/L)	4.23	ND	3.12	4.21
	Accumulation rate (g/□. h)	0.313	ND	0.22	0.315
P(3HB-3HV)	Concentration (g/L)	4.13	ND	3.05	4.15
	Accumulation rate (g/□. h)	0.303	ND	0.211	0.31
	HV fraction (mol %)	43	ND	56	42

(ND : not detected)

This results indicate that the relative lower specific activities of □-ketothiolase, acetoacetyl-CoA reductase than PHB synthase affect on the accumulation rate of PHB and P(3HB-3HV), and the higher specific activity of PHB synthase did not affect on the accunulation rate of P(3HB-3HV) but affect on the determination of 3HV mol fraction.

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