Dataset Information

Bicistronic expression strategy for high-level expression of recombinant proteins in Corynebacterium glutamicum.

ABSTRACT: Directly using the promoter associated with 5'-untranslated region of a high-protein-abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5' coding sequence of the source gene and an embedded Shine-Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5' untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP-BEP4, was obtained from a heterologous sequence source, leading to a 2.24-fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single-chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.

SUBMITTER: Liu X

PROVIDER: S-EPMC6999366 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Bicistronic expression strategy for high-level expression of recombinant proteins in <i>Corynebacterium glutamicum</i>.

Liu Xiuxia X Zhao Zihao Z Zhang Wei W Sun Yang Y Yang Yankun Y Bai Zhonghu Z

Engineering in life sciences 20170814 10

Directly using the promoter associated with 5'-untranslated region of a high-protein-abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5' coding sequence of the source gene and an embedded Shine-Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide ...[more]

PMID: 32624739

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Project description:In a manner similar to ubiquitin, the prokaryotic ubiquitin-like protein (Pup) has been shown to target proteins for degradation via the proteasome in mycobacteria. However, not all actinobacteria possessing the Pup protein also harbor a proteasome, suggesting fates for pupylated proteins other than degradation via a proteasome or degradation at all. In the present study we set out to study pupylation in the proteasome-lacking non-pathogenic model microorganism and biotechnological workhorse Corynebacterium glutamicum. A defined pup deletion mutant of C. glutamicum ATCC 13032 grew as the control indicating that pupylation seems to be dispensable under the conditions tested. By expression of homologous Pup carrying a poly-histidine tag in C. glutamicum ATCC 13032 we purified the first pupylome of a microorganism lacking a proteasome. Multidimensional Protein Identification Technology (MudPIT) unraveled 54 proteins being pupylated in this organism. Similar to mycobacteria, the majority of pupylated proteins in C. glutamicum can be classified as enzymes of the metabolism or as involved in translation. These results help to elucidate the common target pathways of pupylation in bacteria. Sample 1: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done. Sample 2-4: For growth in CGXII minimal media, a preculture 1 was grown in 5 ml BHI medium inoculated with a single colony from a fresh BHI agar plate and incubated at 170 rpm for 8 hours. Then 500 M-BM-5l of preculture 1 were used to inoculate preculture 2 in 100 ml shake flasks with 20 ml CGXII minimal medium containing 4 % (w/v) glucose and incubated over night (140 rpm , 30 M-BM-0C ). Subsequent main cultures (50 ml CGXII medium with 4 % glucose) were inoculated with cells from preculture 2 to an OD600 of about 0.4. For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done for each growth medium. Sample 5-6: For growth experiments in complex BHI medium, 20 ml brain heart infusion (BHI) medium (Difco Laboratories, Detroit, USA) were inoculated with a single colony from a fresh BHI agar plate and incubated at 140 rpm as overnight cultures. Subsequent main cultures (50 ml BHI medium) were inoculated with cells from overnight cultures to an optical density at 600 nm (OD600) of about 1. For DNA microarray analysis, cells were harvested in the exponential growth phase at an OD600 of 4 to 5. After extraction of total RNA 25M-BM-5g of total RNA from C. glutamicum Delta-pup and C. glutamicum WT were compared in two-color microarray analysis. Overall three biological replicates were done.

Dataset Information

Bicistronic expression strategy for high-level expression of recombinant proteins in Corynebacterium glutamicum.

Publications

Bicistronic expression strategy for high-level expression of recombinant proteins in <i>Corynebacterium glutamicum</i>.

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