Poly(4-hydroxybutyrate) (P4HB) production in recombinant Escherichia coli: P4HB synthesis is uncoupled with cell growth

Abstract

Background: Poly(4-hydroxybutyrate) (P4HB), belonging to the family of bacterial polyhydroxyalkanoates (PHAs), is a strong, flexible and absorbable material which has a large variety of medical applications like tissue engineering and drug delivery. For efficient production of P4HB recombinant Escherichia coli has been employed. It was previously found that the P4HB synthesis is co-related with the cell growth. In this study, we aimed to investigate the physiology of P4HB synthesis, and to reduce the total production cost by using cheap and widely available xylose as the growth substrate and sodium 4-hydroxybutyrate (Na-4HB) as the precursor for P4HB synthesis.

Results: Six different E. coli strains which are able to utilize xylose as carbon source were compared for their ability to accumulate P4HB. E. coli JM109 was found to be the best strain regarding the specific growth rate and the P4HB content. The effect of growth conditions such as temperature and physiological stage of Na-4HB addition on P4HB synthesis was also studied in E. coli JM109 recombinant in batch culture. Under the tested conditions, a cellular P4HB content in the range of 58 to 70% (w w(-1)) and P4HB concentrations in the range of 2.76 to 4.33 g L(-1) were obtained with a conversion yield (Y(P4HB/Na-4HB)) of 92% w w(-1) in single stage batch cultures. Interestingly, three phases were identified during P4HB production: the "growth phase", in which the cells grew exponentially, the "accumulation phase", in which the exponential cell growth stopped while P4HB was accumulated exponentially, and the "stagnation phase", in which the P4HB accumulation stopped and the total biomass remained constant.

Conclusions: P4HB synthesis was found to be separated from the cell growth, i.e. P4HB synthesis mainly took place after the end of the exponential cell growth. High conversion rate and P4HB contents from xylose and precursor were achieved here by simple batch culture, which was only possible previously through fed-batch high cell density cultures with glucose.

Figure 1

Comparison of P4HB accumulation in six recombinant E. coli strains. Cultures were grown in shake flasks at 37°C in modified E2 minimal medium containing either glucose or xylose (10 g L^-1). Error bars represent the standard deviations from four independent measurements. There is a significant difference from t-test in the P4HB accumulation between the strains growing on glucose and on xylose with t(5) value of 3.71 and p < 0.01.

Figure 2

Influence of temperature on the growth and P4HB accumulation of E. coli JM109 (pKSSE5.3). The cells were grown in modified E2 minimal medium supplemented with 10 g L^-1 xylose, 4 g L ^-1 Na-4HB, 1 g L^-1 NZ-amines, 100 μg mL^-1 ampicillin and 0.015 g L^-1 thiamine. Four different temperatures were tested: 30°C (♦), 32°C (■), 34°C (▲) and 37°C (●). Error bars represent the deviations from two independent measurements.

Figure 3

Influence of the physiological stage of 4HB addition on P4HB synthesis. E. coli JM109 (pKSSE5.3) were grown in a 1 L bioreactor at 32°C on modified E2 medium supplemented with 10 g L^-1 xylose, 4 g L ^-1 Na-4HB, 1 g L^-1 NZ-amines, 100 μg mL^-1 ampicillin and 0.015 g L^-1 thiamine. The black arrows represent the addition of the Na-4HB precursor. Panel A: addition of Na-4HB at different growth stages. I: Addition of Na-4HB at the beginning of the culture; II: Addition of Na-4HB at the end of the exponential growth phase; III: Combination of addition of Na-4HB at the beginning and at the end of exponential growth phase. Panel B: Time courses of P4HB content and concentration presented in log-scale. Panel C: P4HB productivity. The P4HB accumulation rate for the described conditions is obtained from three independent cultivations.

Figure 4

P4HB production in batch culture in 1 L bioreactors.E. coli JM109 (pKSSE5.3) were grown in modified E2 medium with 10 g L^-1 xylose as the carbon source at 32°C with an agitation of 500 rpm. The substrate consumption and product formation were followed with time. Error bars represent measurement errors of the same sample in triplicates.

Figure 5

Hypothetic metabolic pathway of P4HB synthesis from Na-4HB in recombinant E. coli. Blue color represents growth phase, purple color represents P4HB synthesis phase.