Genetic Engineering of Corynebacteria

Masato Ikeda; Seiki Takeno

doi:10.1128/9781555816827.ch16

Chapter 16 : Genetic Engineering of Corynebacteria

Authors: Masato Ikeda, Seiki Takeno

Content Type: Reference

Publication Year: 2010

Category: Applied and Industrial Microbiology

Book DOI: 10.1128/9781555816827

Chapter DOI: 10.1128/9781555816827.ch16

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Abstract:

This chapter describes the technology and provides strategies for molecular strain improvement using current genetic engineering tools, global analysis techniques, and genome-based engineering approaches, with particular emphasis on the industrially important Corynebacterium glutamicum. The transposon (Tn) mutagenesis experiments described in this chapter were performed using special Tn delivery vectors containing insertion sequences (ISs). As a tool for the mutagenesis of C. glutamicum ATCC 13032, Tn vector pAT6100 has been constructed. In C. glutamicum, comparative genomic analysis between two C. glutamicum strains, R and ATCC 13032, revealed that 11 strain-specific islands are scattered in the genomes. Such strain-specific islands are thought to be composed of dispensable genes acquired by horizontal gene transfer. Determining the whole genome sequence of C. glutamicum is aimed at gaining sufficient information to manipulate the metabolism or physiology on a global scale, eventually integrating the information for development of more efficient production strains. This chapter discusses strain improvement strategies, and genome-based strain reconstruction. Exhaustive studies have been directed to metabolic engineering of C. glutamicum for lysine production, resulting in a large body of literature. Comprehensive screening of secretion signal sequences has been conducted in C. glutamicum R by using bioinformatic analysis and a high-throughput secretion assay, which identified a total of 108 candidate signal sequences that could secrete heterologous α-amylase in the organism. The two examples of lysine and arg+cit production described in this chapter will be a paradigm for future strain development in the fermentation industry.

Citation: Ikeda M, Takeno S. 2010. Genetic Engineering of Corynebacteria, p 225-237. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch16

Key Concept Ranking

Basic Amino Acids: 0.7577955
Genetic Elements: 0.5128887
Amino Acids: 0.47721872
Bacterial Proteins: 0.4746503
Chemicals: 0.45239395
Horizontal Gene Transfer: 0.42106524

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Genetic Engineering of Corynebacteria

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/content/10.1128/9781555816827.ch16.ch16fig01

FIGURE 1

Cre/loxP-mediated deletion of the C. glutamicum genome. Boxes A and B are short segments of the C. glutamicum genome. These PCR-amplified segments are integrated into the genome by homologous recombination using two separate vectors. After integration, the Cre-containing plasmid is introduced into the recombinant cell in order to excise the target region. Kmr, kanamycin resistance gene; Spr, spectinomycin resistance gene.

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FIGURE 1

/content/10.1128/9781555816827.ch16.ch16fig02

FIGURE 2

Random segment deletion of the C. glutamicum genome. Two IS31831-based transposon vectors are serially introduced into the C. glutamicum cell, randomly integrating two loxP sites into the genome, followed by Cre-mediated recombination. Cmr, chloramphenicol resistance gene; L-IR, left inverted repeat; R-IR, right inverted repeat.

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FIGURE 2

/content/10.1128/9781555816827.ch16.ch16fig03

FIGURE 3

Methodology to create a minimally mutated strain. Useful mutations relevant to amino acid production are indicated (stars), together with unnecessary mutations (×).

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10.1128/9781555816827/f0231-01.gif

FIGURE 3

Methodology to create a minimally mutated strain. Useful mutations relevant to amino acid production are indicated (stars), together with unnecessary mutations (×).

/content/10.1128/9781555816827.ch16.ch16fig04

FIGURE 4

Fermentation kinetics of the newly developed strain RBid at 38°C in 5-liter jar fermentor cultivation. For comparison, the profiles of the best classical producer, A-27, which was cultured under its optimal 30°C conditions, are shown as controls.

, arg+cit of strain RBid; •, growth of strain RBid;

, arg+cit of strain A-27;

, growth of strain A-27.

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FIGURE 4

/content/10.1128/9781555816827.ch16.ch16fig05

FIGURE 5

Schematic diagram of the creation of new strain RBid. Useful mutations identified in classical producers I-30, α-27, and D-77 are indicated (stars), together with unnecessary mutations (×).

10.1128/9781555816827/f0232-02_thmb.gif

10.1128/9781555816827/f0232-02.gif

FIGURE 5

Schematic diagram of the creation of new strain RBid. Useful mutations identified in classical producers I-30, α-27, and D-77 are indicated (stars), together with unnecessary mutations (×).

References

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