Identification and application of a growth-regulated promoter for improving l-valine production in Corynebacterium glutamicum

Abstract

BackgroundPromoters are commonly used to regulate the expression of specific target genes or operons. Although a series of promoters have been developed in Corynebacterium glutamicum, more precise and unique expression patterns are needed that the current selection of promoters cannot produce. RNA-Seq technology is a powerful tool for helping us to screen out promoters with expected transcriptional strengths.ResultsThe promoter PCP_2836 of an aldehyde dehydrogenase coding gene from Corynebacterium glutamicum CP was identified via RNA-seq and RT-PCR as a growth-regulated promoter. Comparing with the strong constitutive promoter Ptuf, the transcriptional strength of PCP_2836 showed a significant decrease that from about 75 to 8% in the stationary phase. By replacing the native promoters of the aceE and gltA genes with PCP_2836 in the C. glutamicum ATCC 13032-derived l-valine-producing strain AN02, the relative transcriptional levels of the aceE and gltA genes decreased from 1.2 and 1.1 to 0.35 and 0.3, and the activity of their translation products decreased to 43% and 35%, respectively. After 28 h flask fermentation, the final cell density of the obtained strains, GRaceE and GRgltA, exhibited a 7–10% decrease. However, l-valine production increased by 23.9% and 27.3%, and the yield of substrate to product increased 43.8% and 62.5%, respectively. In addition, in the stationary phase, the intracellular citrate levels in GRaceE and GRgltA decreased to 27.0% and 33.6% of AN02, and their intracellular oxaloacetate levels increased to 2.7 and 3.0 times that of AN02, respectively.ConclusionsThe PCP_2836 promoter displayed a significant difference on its transcriptional strength in different cell growth phases. With using PCP_2836 to replace the native promoters of aceE and gltA genes, both the transcriptional levels of the aceE and gltA genes and the activity of their translation products demonstrated a significant decrease in the stationary phase. Thus, the availability of pyruvate was significantly increased for the synthesis of l-valine without any apparent irreversible negative impacts on cell growth. Use of this promoter can enhance the selectivity and control of gene expression and could serve as a useful research tool for metabolic engineering. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1031-7) contains supplementary material, which is available to authorized users.

Physiology and Transcriptional Analysis of (p)ppGpp-Related Regulatory Effects in Corynebacterium glutamicum

“…A regulatory influence of (p)ppGpp on the pyruvate concentration could directly contribute to the optimization of the cellular resource distribution with respect to given conditions and thus to the regulation of growth behavior. The entry of pyruvate into the TCA cycle through the conversion to acetyl-CoA has already been directly associated with the regulation of growth rate for C. glutamicum in this context (Buchholz et al, 2013; Ma et al, 2018).…”

Development of a Hyperosmotic Stress Inducible Gene Expression System by Engineering the MtrA/MtrB-Dependent NCgl1418 Promoter in Corynebacterium glutamicum

“…under different culture conditions and bioinformatics analysis of specific recognition sequences of transcriptional regulators are the main strategies for the identification of new promoters (Dahl et al, 2013;Liu et al, 2015;Kim et al, 2016;Ma et al, 2018). However, the strength and induction activity of natural promoters are often not sufficient for practical applications.…”

“…For example, based on the LysR-type transcriptional regulator (LTTR) LysG and the lysE promoter with the LysG-binding site, a sensor suitable for intracellular lysine detection was developed ( Binder et al, 2012 ). Two growth-regulated promoters (P cg3141 and P CP 2836 ) had been identified ( Kim et al, 2016 ; Ma et al, 2018 ). The P cg3141 promoter was verified with the production of glutathione S-transferase as a model protein, and the P CP_2836 promoter was used for improving the production of valine and 5-aminolevulinic acid ( Kim et al, 2016 ; Ma et al, 2018 ; Zhang et al, 2020 ).…”

“…Two growth-regulated promoters (P cg3141 and P CP 2836 ) had been identified ( Kim et al, 2016 ; Ma et al, 2018 ). The P cg3141 promoter was verified with the production of glutathione S-transferase as a model protein, and the P CP_2836 promoter was used for improving the production of valine and 5-aminolevulinic acid ( Kim et al, 2016 ; Ma et al, 2018 ; Zhang et al, 2020 ).…”

Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum

“…Therefore, a reduction rather than inactivation of CS activity was preferable. Moreover, regulation of the gltA gene, which is responsible for 95% of CS activity in C. glutamicum [42], was widely applied to improve the biosynthesis of pyruvate-derived products [34,[43][44][45]. However, the impact of reducing CS activity on acetoin production is still unclear.…”