Realization of Robust and Precise Regulation of Gene Expression by Multiple Sigma Recognizable Artificial Promoters

Abstract

Precise regulation of gene expression is fundamental for tailor-made gene circuit design in synthetic biology. Current strategies for this type of development are mainly based on directed evolution beginning with a native promoter template. The performances of engineered promoters are usually limited by the growth phase because only one promoter is recognized by one type of sigma factor (σ). Here, we constructed multiple-σ recognizable artificial hybrid promoters (AHPs) composed of tandems of dual and triple natural minimal promoters (NMPs). These NMPs, which use σ A , σ H and σ W , had stable functions in different growth phases. The functions of these NMPs resulted from an effect called transcription compensation, in which AHPs sequentially use one type of σ in the corresponding growth phase. The strength of the AHPs was influenced by the combinatorial order of each NMP and the length of the spacers between the NMPs. More importantly, the output of the precise regulation was achieved by equipping AHPs with synthetic ribosome binding sites and by redesigning them for induced systems. This strategy might offer promising applications to rationally design robust synthetic promoters in diverse chassis to spur the construction of more complex gene circuits, which will further the development of synthetic biology.

Data-Driven and in Silico-Assisted Design of Broad Host-Range Minimal Intrinsic Terminators Adapted for Bacteria

“…These novel synthetic devices and systems largely rely on standardized biological elements and parts. A plethora of genetic parts in bacteria (especially Escherichia coli and Bacillus subtilis), such as synthetic promoters, toehold switches, and riboswitches, have been designed with repertoires to regulate gene expression at the transcription, − posttranscription, and translation levels. − …”

Importance of the 5′ regulatory region to bacterial synthetic biology applications

“…Hybrid promoters commonly comprise a mix of two or more different promoters (Fig. 2) (Zhang et al ., 2012; Jiao et al ., 2017; Chen et al ., 2018b; Han et al ., 2020). For example, the widely used inducible tac promoter is composed of the naturally occurring trp promoter and the synthetic lac UV5 promoters (Boer et al ., 1983).…”

“…Rational design efforts are increasingly applied, especially with the advancement of deep learning methods. However, accurately predicting the promoter output is difficult to achieve as promoter strength varies depending on distinct features, such as the associated σ‐factor (Wang et al ., 2019a ), associated transcription factors (Ishihama et al ., 2016 ), UP elements (Lee et al ., 2012 ), competition for binding sites (Ishihama et al ., 2016 ; Han et al ., 2020 ), the sequence to which holoenzymes bind (Kinney et al ., 2010 ), and the spacer length and sequence between the motifs (Nair and Kulkarni, 1994 ; Jensen and Hammer, 1998 ; Han et al ., 2019 ). The distinct features can be a target for promoter engineering approaches; however, it is not always clear how these different features factor into promoter strength.…”

“…Experimental design for multi‐input systems, such as genetic circuits and metabolic pathways, is still mostly based on time‐consuming trial‐and‐error procedures. Because individual parts are removed from their natural or tested environment and placed in a new genetic context, combining numerous parts frequently produces unexpected consequences, which may be troublesome even when dealing with standardized DNA parts in model hosts (Zong et al ., 2018 ; Han et al ., 2020 ). When assembling pathways, some genetic elements behave relatively stable, while other parts vary depending on the genetic context (Kosuri et al ., 2013 ; Mutalik et al ., 2013b ).…”

Development of a Glycerol-Inducible Expression System for High-Yield Heterologous Protein Production in Bacillus subtilis

“…Efficient and precise expression of a gene of interest (GOI) has been a longstanding and essential research area for the construction of biological systems required for enzyme production, natural product synthesis, and synthetic biology ( 1 – 3 ). The GOI is expressed in either constitutive or inducible systems ( 4 – 6 ). Constitutive systems are unable to modulate the strength and timing of the GOI expression, making it difficult to rationally allocate limited resources between cell growth and product synthesis.…”