Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1

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Abstract

Growth conditions for mass production of luciferase-bacterial magnetic particles (BMPs) by a recombinant Magnetospirillum magneticum AMB-1 were investigated in a pH-regulated fed-batch culture system. Enrichment of growth medium with L-cysteine, yeast extract and polypeptone enhanced both bacterial growth and BMP production. The presence of L-cysteine in the medium was useful for induction of cell growth. Strict anaerobic conditions led to a prolonged lag phase and limited the final cell density. Trace oxygen enhanced cell growth with increasing BMP production. As iron sources, ferrous sulfate and ferric gallate dramatically enhanced BMP yield as compared with ferric quinate, an iron chelate conventionally used. The optimized conditions increased cell density to 0.59 ± 0.03 g cell dry weight/liter and BMP production to 14.8 ± 0.5 mg dry weight/liter in fed-batch culture for four days.

Introduction

Magnetic bacteria have been isolated from fresh and marine sediments and are known to produce magnetic particles (Fe3O4) [1]. Current researches of magnetic bacteria focus on two aspects. One illustrates the biomineralization process during synthesis of magnetic particles [2], [3]. The other investigates practical applications of this intriguing nano-magnetic particles. Bacterial magnetic particles (BMPs) are small in size (50–100 nm), disperse very well because they are covered with a stable lipid membrane and can be separated simply using a magnet [4]. Antibodies and DNA immobilized on BMPs have been used for highly sensitive immunoassays and mRNA recovery [4], [5], [6]. Genetic analysis of BMPs synthesis has resulted in the isolation of the magA gene from Magnetospirillum magneticum AMB-1, which encodes an iron transport membrane protein on BMPs [7]. Functional proteins have been displayed on BMPs using MagA as an anchor protein through gene fusion [8]. ProteinA displayed on BMPs by fusion with MagA protein has also been used in immunoassays [9], [10]. These applications emphasize the need to enhance BMP production for biotechnological applications.

Isolation and cultivation of magnetic bacteria has been proven to be difficult. However, several strains, including Magnetospirillum magneticum AMB-1, have been successfully cultured in chemically defined media [11], [12]. Although the majority of isolated strains are obligately microaerophilic, AMB-1 is a facultative anaerobe and capable of growing aerobically [12]. Poor information about natural habitats and difficulty mimicking them in the laboratory limit efficient cell growth and BMP production. Our previous works have provided important information regarding this aspect [13], [14].

In this paper, growth media, iron sources and atmospheric oxygen concentrations have been investigated for efficient production of luciferase-BMP complexes by a recombinant Magnetospirillum magneticum AMB-1.

Section snippets

Bacterial strain and plasmid

A recombinant Magnetospirillum magneticum AMB-1 transformed by a plasmid pKML was used throughout this research. A plasmid pKML is derived from pRK415 (Tcr, lacZ, mob+) by cloning luc downstream of the C-terminal hydrophilic domain of magA[8]. The plasmid pRK415 is a broad-host range vector for Gram-negative bacteria, and stably maintained in Magnetospirillum magneticum AMB-1 [15].

Media and culture conditions

M. magneticum AMB-1 was cultured in 40 ml of magnetic spirillum growth medium (MSGM) containing the following

Luciferase-BMP production in MSGM enriched with polypeptone, yeast extract and L-cysteine

Our previous work shows that fed-batch cultivation with MSGM needs at least 6–7 days to reach a final cell density of about 0.35 g dry weight producing 6 mg BMPs per liter culture [14]. When MSGM enriched with polypeptone, yeast extract and L-cysteine was employed for BMP production, the lag phase was reduced from 48–60 h to within 24 h (Fig. 1). The final cell density increased to 0.5 g dry weight/liter with BMP production increasing to 7.4 mg/liter (Table 1). This medium was efficient for

Discussion

MSGM enriched with L-cysteine, yeast extract and polypeptone could enhance BMP productivity. Addition of yeast extract had no effect on BMP production and polypeptone only improved the final cell density and therefore slightly improved BMP production, whereas L-cysteine induced cell growth leading to a short lag phase and higher BMP productivity. The reason for enhancement of BMP production by L-cysteine is unknown, because it is not associated with lower redox potentials in the presence of

Acknowledgements

This work was partially supported by Grant-in Aid for Scientific Research on Priority Areas (A) no. 10145102, from the Ministry of Education, Science, Sports and Culture and the Proposal-Based New Industry Creative Type Technology R & D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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