Identification, repair and characterization of a benzyl alcohol-inducible promoter for recombinant proteins overexpression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial organism for the production of a variety of biological commodities. We discovered a promoter encoded by the gene NCgl2319 in C. glutamicum, which could be induced by benzyl alcohol, could be used as an efficient tunable expression system. In initial attempts, this promoter failed to function in a recombinant expression system. This was remedied by extending the original genetic context of the promoter, generating a new version Pcat-B. The Pcat-B transcription initiation site, its critical active regions, and its effect of inducers were fully characterized resulting in tunable expression. This approach proved to be very efficient in producing a pharmaceutical protein, N-terminal pro-brain natriuretic peptide (NT-proBNP). Production of approximately 440.43 mg/L NT-proBNP was achieved with the Pcat-B expression system demonstrating its application for controllable pharmaceutical protein production in C. glutamicum.

Glycerol transporter 1 (Gt1) and zinc-regulated transporter 1 (Zrt1) function in different modes for zinc homeostasis in Komagataella phaffii (Pichia pastoris).

OBJECTIVES: To identify the zinc transport function of the membrane proteins Gt1 and Zrt1 in Komagataella phaffii (Pichia pastoris) and study their regulatory mode. RESULTS: Two membrane proteins that might have zinc transport function were found in K. phaffii. GT1 was known to encode a glycerol transporter belonging to the Major Facilitator Superfamily. ZRT1 was predicted to resemble the zinc transporter gene in Saccharomyces cerevisiae. Consistent with the prediction, protein plasma-membrane localizations were confirmed by ultracentrifugation and confocal microscopy. Their zinc binding abilities were identified by ITC in vitro, and the impaired zinc uptake activity caused by their deficiencies was confirmed by zinc fluorescence quantification in vivo. Furthermore, zinc excess could turn the two channels off, while zinc deficiency induced their expressions. Gt1 could only function to maintain zinc homeostasis in glycerol, while the block of Gt1 function might lead to Zrt1 upregulation in glucose. CONCLUSIONS: The zinc transport capabilities of Gt1 and Zrt1 were identified in vivo and in vitro. Their regulatory mode to maintain zinc homeostasis in K. phaffii is a new inspiration.

Identification and validation of appropriate reference genes for qRT-PCR analysis in Corynebacterium glutamicum.

Real-time quantitative PCR (qRT-PCR) is a fast and efficient technology for detecting gene expression levels in the study of the Corynebacterium glutamicum protein expression system, but it requires normalization to ensure the reliability of the results obtained. We selected 13 genes from the commonly used housekeeping genes and from transcriptome data as candidate reference genes. The Ct values of the 13 genes were obtained by qRT-PCR at different fermentation stages and under three stress conditions (temperature, acid and salt). The expression stability of the reference genes was evaluated by geNorm and NormFinder software. For the study of different growth stages, the most appropriate reference genes are Ncgl2772 and leua, which encode acetyl-CoA carboxylase beta subunit and 2-isopropylmalate synthase, separately. For the study of different stress factors, the optimal minimum number of reference genes is 3, with Ncgl2772, gyrb encoding DNA gyrase B and siga encoding RNA polymerase sigma factor A as the most suitable combination. Additionally, clpx and clpc, encoding ClpX and ClpC protease subunits, were used to validate the candidate reference genes. The identification of new reference genes makes qRT-PCR more convenient, and using these genes for normalization can improve the accuracy and reliability of the measurements of target gene expression levels obtained by qRT-PCR for C. glutamicum.

Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.

BACKGROUND: Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. RESULTS: In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. CONCLUSIONS: In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.