High-Level Heterologous Production of d-Cycloserine by Escherichia coliApplied and Environmental Microbiology

About

Authors
Takanori Kumagai, Tomoki Ozawa, Momoko Tanimoto, Masafumi Noda, Yasuyuki Matoba, Masanori Sugiyama
Year
2015
DOI
10.1128/AEM.02187-15
Subject
Biotechnology / Food Science / Ecology / Applied Microbiology and Biotechnology

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Text

High-Level Heterologous Production of D-Cycloserine by

Escherichia coli

Takanori Kumagai,a Tomoki Ozawa,b Momoko Tanimoto,b Masafumi Noda,a Yasuyuki Matoba,a Masanori Sugiyamaa

Department of Molecular Microbiology and Biotechnology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japana; Department of

Pharmaceutical Sciences, Hiroshima University, Hiroshima, Japanb

Previously, we successfully cloned a D-cycloserine (D-CS) biosynthetic gene cluster consisting of 10 open reading frames (designated dcsA to dcsJ) from D-CS-producing Streptomyces lavendulae ATCC 11924. In this study, we put four D-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) in tandem under the control of the T7 promoter in an Escherichia coli host. SDS-PAGE analysis demonstrated that the 4 gene products were simultaneously expressed in host cells. When L-serine and hydroxyurea (HU), the precursors of D-CS, were incubated together with the E. coli resting cell suspension, the cells produced significant amounts of

D-CS (350 20M). To increase the productivity of D-CS, the dcsJ gene, which might be responsible for the D-CS excretion, was connected downstream of the four genes. The E. coli resting cells harboring the five genes produced D-CS at 660 31M. The dcsD gene product, DcsD, formsO-ureido-L-serine fromO-acetyl-L-serine (OAS) and HU, which are intermediates in D-CS biosynthesis. DcsD also catalyzes the formation of L-cysteine fromOAS and H2S. To repress the side catalytic activity of DcsD, the E. coli chromosomal cysJ and cysK genes, encoding the sulfite reductase  subunit and OAS sulfhydrylase, respectively, were disrupted. When resting cells of the double-knockout mutant harboring the four D-CS biosynthetic genes, together with dcsJ, were incubated with L-serine and HU, the D-CS production was 980 57M, which is comparable to that of D-CS-producing S. lavendulae ATCC 11924 (930 36M).

The cyclic structural analog of the amino acid D-cycloserine(D-CS) (Fig. 1) is a broad-spectrum antibiotic produced by

Streptomyces lavendulae and Streptomyces garyphalus (1). The antibiotic inhibits both alanine racemase and D-alanyl–D-alanine ligase, which are necessary for the biosynthesis of peptidoglycan in the bacterial cell wall (2, 3). Rifampin and isoniazid have been clinically used for the treatment of tuberculosis caused by infection with Mycobacterium tuberculosis (4). However, M. tuberculosis that is resistant to these drugs has recently occurred. Presently,

D-CS is clinically used as a second-line-of-defense drug against these antibiotic-resistant M. tuberculosis strains (4). In this connection, it has been shown that M. smegmatis overproducing alanine racemase is resistant to D-CS (5, 6). Recently, D-CS has been shown to function as a partial agonist for the N-methyl-D-aspartate receptor. As a result, the application of D-CS for the treatment of some psychological dysfunctions has been extensively studied (7–9).

Our group has successfully cloned a D-CS biosynthetic gene cluster from the chromosomal DNA of D-CS-producing S. lavendulaeATCC 11924, which is composed of 10 open reading frames, designated dcsA to dcsJ (10). The functions of dcsI and dcsJ had previously been analyzed using the corresponding genes cloned from other D-CS-producing strains, i.e., S. lavendulae ATCC 25233 (11) and S. garyphalus (CSH) 5-12 (12), demonstrating that both gene products are responsible for self-resistance in the D-CS producer. Gene disruption and recombinant protein analyses have demonstrated that the revised D-CS biosynthetic pathway is as follows. L-Serine is O-acetylated by DcsE to generate O-acetylL-serine (OAS) (10, 13). The resultant OAS reacts with hydroxyurea (HU) to yield O-ureido-L-serine by use of DcsD, which is a pyridoxal phosphate-dependent enzyme (14). O-Ureido-Lserine is racemized by DcsC (14, 15), followed by cyclization with

DcsG, which is a member of the ATP-grasp fold family of proteins (10, 14) (Fig. 1).

We have previously hypothesized that L-arginine, as a precursor in the D-CS biosynthetic pathway, must be hydroxylated by nitric oxide synthase (NOS) expressed in D-CS-producing S. lavendulae (10). However, we have corrected the hypothesis as follows: DcsA as a heme protein, but not as an NOS protein, contributes to the formation ofN-hydroxy-L-arginine (16). As shown in

Fig. 1, HU is generated by the hydrolysis of N-hydroxy-L-arginine with DcsB (10).

In recent years, the heterologous expression of secondary metabolic pathways using a surrogate host, such asEscherichia coli, has emerged as an effective way of producing natural products. However, practical antibiotics have not yet been successfully produced using E. coli as a host. Our goal is to realize high production of

D-CS by expressing its biosynthetic genes (dcsA to -dcsE and dcsG) in E. coli as a host cell. In this study, we tried to introduce the four

D-CS biosynthetic genes (dcsC, dcsD, dcsE, and dcsG) into E. coli cells to express these gene products and to construct a D-CS production system by incubating resting cells with precursors of

D-CS. We show that coexpression of the four D-CS biosynthetic genes and a self-resistance gene, dcsJ, which encodes a putative

D-CS efflux protein from D-CS-producing S. lavendulae, in combination with metabolic engineering of the E. coli host, is effective for the high production of the antibiotic.

Received 4 July 2015 Accepted 1 September 2015

Accepted manuscript posted online 4 September 2015

Citation Kumagai T, Ozawa T, Tanimoto M, Noda M, Matoba Y, Sugiyama M. 2015.

High-level heterologous production of D-cycloserine by Escherichia coli. Appl

Environ Microbiol 81:7881–7887. doi:10.1128/AEM.02187-15.

Editor:M. A. Elliot

Address correspondence to Masanori Sugiyama, sugi@hiroshima-u.ac.jp.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

November 2015 Volume 81 Number 22 aem.asm.org 7881Applied and Environmental Microbiology o n

O ctober 21, 2015 by CM