Synthetic biology tools for engineering Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is a promising organism for the industrial production of amino acids, fuels, and various value-added chemicals. From the whole genome sequence release, C. glutamicum has been valuable in the field of industrial microbiology and biotechnology. Continuous discovery of genetic manipulations and regulation mechanisms has developed C. glutamicum as a synthetic biology platform chassis. This review summarized diverse genomic manipulation technologies and gene expression tools for static, dynamic, and multiplex control at transcription and translation levels. Moreover, we discussed the current challenges and applicable tools to C. glutamicum for future advancements.

Keywords: Corynebacterium glutamicum; Dynamic control; Gene expression regulation; Genome editing; Multiplex control; Static control.

Graphical abstract

Fig. 1

Genome editing and gene regulation tools associated with C. glutamicum: (A) Improved allelic exchange methods with different selection strategies and expression of heterologous recombination proteins, and CRISPR-associated systems for genome editing; (B) Gene regulation tools through static, dynamic, and multiplex controls at transcriptional and translational levels.

Fig. 2

Selection methods of allelic exchange in C. glutamicum: (A) sacB, a counter-selection marker; (B) rpsL, a counter-selection marker; (C) upp, a counter-selection marker; (D) Cre/loxP system; (E) I-Sce-based system. Each arrow represents its recombination event followed by the selection marker.

Fig. 3

Optimizations of CRISPR-associated systems in C. glutamicum: (A) The optimization for CRISPR/Cpf1 system was done by different PAM sequences and lengths of crRNA. The optimal PAM sequence was 5′-NYTV-3′ and length of crRNA, 21 bp; (B) For CRISPR/Cas9 system, lowering Cas9 expression level was optimized through an inducible promoter and origin change; (C) For base editor, large sgRNA array was designed for better genome-targeting scope and the combination of CBE and ABE for better base transition capability.

Fig. 4

Static gene regulation tools and their applications in C. glutamicum: (A) Development of C. glutamicum promoters. Native and synthetic promoter sets with various strengths were found based on the conserved sequences; (B) RBS libraries design from Shine-Dalgarno sequence of C. glutamicum.

Fig. 5

Dynamic gene regulation tools and their applications in C. glutamicum: (A) ʟ-Isoleucine- and ʟ-serine-responsive transcription activation; (B) Lrp-based biosensor for adaptive laboratory evolution of ʟ-valine producing strain; (C) TF-based biosensors for screening mutant strains or plasmid libraries; (D) ʟ-Lysine-OFF riboswitch. Inhibited gltA expression in the presence of lysine increased metabolic flux towards the lysine production; (E) ʟ-Lysine-ON riboswitch. lysE is upregulated when intracellular lysine concentration increases, facilitating the lysine export through the membrane.

Fig. 6

Multiplex gene regulation tools and their applications in C. glutamicum: (A) CRISPRi-based multiple gene repression. Simultaneous knockdown of target genes improved product yields; (B) sRNA-based gene repression. Downregulation of pyk, ldhA, and odhA led to the enhanced glutamate production; (C) asRNA-mediated gene regulation. HA production increased within attenuation of fba.