Contemporary Tools for Regulating Gene Expression in Bacteria

Abstract

Insights from novel mechanistic paradigms in gene expression control have led to the development of new gene expression systems for bioproduction, control, and sensing applications. Coupled with a greater understanding of synthetic burden and modern creative biodesign approaches, contemporary bacterial gene expression tools and systems are emerging that permit fine-tuning of expression, enabling greater predictability and maximisation of specific productivity, while minimising deleterious effects upon cell viability. These advances have been achieved by using a plethora of regulatory tools, operating at all levels of the so-called 'central dogma' of molecular biology. In this review, we discuss these gene regulation tools in the context of their design, prototyping, integration into expression systems, and biotechnological application. From Overexpression to Precise Regulation: Engineering Gene Expression Control Historically, researchers have relied on a relatively small number of mechanisms to regulate heterologous gene expression [1]. These traditional systems have focussed on massive overexpression of genes in an effort to maximise product yield (see Glossary). Often systems developed for overexpression are adapted ad hoc for integration into genetic circuits. While suited for the rapid accumulation of high levels of protein, these tools are often insufficient for developing the more precise systems required for many modern applications in sensing, computation and production. Recent years have seen improvements in the functionality of gene regulation tools, enhancing utility for dynamic, tuneable regulation. With any bioproduction effort, there is often an important tradeoff between yield, biomass accumulation, and titre that must be considered (Figure 1A). This balance is important for an array of biotechnological applications to ensure process viability and stability. As our ability to engineer more complex systems increases, the importance of harmonising gene expression with the metabolic capacity of the host organism, or chassis, by reducing the burden associated with the engineered function only increases. In the case of recombinant protein production, aberrant protein synthesis can lead to an overload in cellular capacity, such as synthesis or secretion. Both resource limitations, such as limited amino acid and ribosome availability [2], and symptoms of cellular capacity overload, such as impaired protein folding and secretion capacity [3-5], can impact cellular viability. Resource demand and overload also present a challenge to metabolic engineering efforts where substrate, intermediate, and product concentration can all have deleterious effects on cell viability [6]. This issue is exacerbated when central metabolites and/or cofactors are consumed, creating further competition for cellular resources.

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…As our knowledge on metabolic engineering continues to increase, we have learned that balanced expression levels and feedback mechanisms often result in higher yields and healthier cell populations 39 . To reach these desired expression levels, predictable, robust, and tuneable genetic constructs are a must and can only be built from well-characterized, reliable, and context-independent biological parts as demonstrated by Nielsen et al 40 .…”

“…In contrast to the relatively large set of activators, the number of (characterized) repressors for P. putida is low and limits the construction of complex genetic feedback mechanisms for SynBio applications (Table 2 and Supplementary Table 1 ). In some applications, the lack of a suitable repressor can be substituted by CRISPR interference (CRISPRi) technology 39 . This technology enables the simultaneous, tunable, and transient repression of multiple genes by expression of gene-specific sgRNAs and a single nuclease-deficient Cas9 (dCas9) and has been successfully applied in P. putida 49 , M. smegmatis 50 , K. pneumoniae 51 , B. subtilis 51 , and L. lactis 52 with up to 98% repression.…”

“…Riboswitches and RNA thermometers are often used in SynBio to tune expression levels in response to their cognate ligand 69 . For E. coli , genome-wide riboswitch identification, extensive characterization, optimization, and standardization of 5′ sequences have resulted in riboswitches with reliable functioning 39 , 68 . In contrast, these parts are not yet exploited as regulatory SynBio tools for non-model hosts.…”

“…Moreover, the first riboswitches and RNA thermometers have only recently been characterized in vivo in non-traditional bacteria 70 – 73 , except for B. subtilis for which riboswitch research is more mature 69 . The trans -acting counterparts of riboswitches are called translation riboregulators or antisense RNAs and can be used for multiplexed genome-wide knock-down of gene expression 39 , but their utilization as biological parts in non-traditional hosts is still limited. This might change in the near future as a synthetic sRNA toolbox for P. putida has recently been developed 74 .…”

“…RNA-based regulatory parts allow an additional level of gene expression regulation complementary to the classical biological parts such as promoters and RBSs 39 , 68 . In general, RNA-based parts do not depend on additional proteins or translation.…”