Double promoter expression systems for recombinant protein production by industrial microorganisms

Ozturk, Sibel
Ergun, Burcu Gunduz
Çalık, Pınar
Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the change-over from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more effective and adaptive processes to meet the increasing demand of r-protein industry.


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Intracellular phenomena such as promoter strength, mRNA secondary structure, translation efficiency and codon preference, 5′-untranslated region processing, and protein turnover, have impacts directly on the expression of heterologous genes. Design of multi-promoter expression systems with constituent strong promoters and engineered promoter variants is a novel metabolic engineering strategy for increasing the promoter strength further, and tuning the expression for recombinant protein (r-protein) productio...
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Demir, Irem; Çalık, Pınar (Springer Science and Business Media LLC, 2020-10-01)
Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants P(ADH2-Cat8-L2)and P(mAOX1)and with the naturally occurring promoter P(GAP)to enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were e...
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The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (P-ADH2) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Ca...
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In this study, the aim is to develop an effective feeding strategy for therapeutically important protein, recombinant human growth hormone (rhGH) production by Bacillus subtilis scoC knockout (scoC-) strain carrying pMK4::pre(subC)::hGH plasmid. In the first part of the research program, laboratory scale air filtered shake bioreactor experiments were conducted to optimize the production conditions. Thereafter, semi-batch production processes at pilot scale bioreactor system were performed by designing expon...
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Citation Formats
S. Ozturk, B. G. Ergun, and P. Çalık, “Double promoter expression systems for recombinant protein production by industrial microorganisms,” APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, pp. 7459–7475, 2017, Accessed: 00, 2020. [Online]. Available: