There is an increasing demand for the production of novel small molecules and biomaterials including drugs, chemicals, biofuels and biopolymers. To efficiently produce these molecules and to reduce the production cost of existing compounds in engineered microbial cells, a major challenge is to regulate the expression of a large number of genes to optimize the production of a biosynthesis pathway. The introduction of genes into production hosts is typically achieved using plasmids previously assembled and propagated in E. coli. Synthetic biology can make a significant contribution to speed up this process by using standardized plasmids where all the components are synthesized with standard formats to facilitate easy exchange and testing [3, 30].
Here we present a synthetic biology framework for genetic engineering of C. glutamicum, a microorganism of industrial interest for which synthetic biology tools have not been developed yet. In the design described herein, all the plasmid components are flanked by unique restriction sites to enable a standardized replacement of genetic elements. In most cases, the parts and pathway genes used in our laboratory are synthetic and designed so as to avoid NdeI, EcoRI, XbaI, SpeI, NheI and AvrII sites, although natural sequences lacking these sites and PCR amplified to add the flanking restriction sites are used as well. Short regulatory sequences, like promoters and RBSs, can be simply added by using overlapping oligonucleotides. Codon usage of heterologous genes can be optimized to facilitate its expression in C. glutamicum. The whole process of gene design, including restriction site generation/removal and codon optimization, can be achieved in one step using free web based programs like Optimizer [31, 32].
Several formats for the construction of synthetic plasmids and for the assembly of parts have been proposed [30, 33–36]. The most accepted method in the synthetic biology community was created by Knight and co-workers . They proposed the BioBrick standard for the assembly of biological parts, where all parts are flanked by a standard set of restriction sites to allow joining and combination with further parts. The plasmids described here can be adapted to fit the BioBrick assembly method by replacing the set of restriction sites, although some limitations have been described for this format as well .
Optimal levels of enzyme to maximize production from a biosynthetic pathway can be achieved as a result of fine tuning gene expression by, for example, modulating transcription or translation. For this purpose, a toolbox with a collection of promoters and RBSs capable of providing different levels of gene expression is desirable. The pTGR system may serve as a standardized test to evaluate the strength of promoter sequences. To validate this application, three promoters were tested using the eGFP as a reporter gene. Synthetic DNA fragments containing the E. coli tac promoter, and the sod and cspB promoter from C. glutamicum were inserted using the standard format. Three levels of fluorescence intensity were obtained: the tac promoter provided the strongest signal, the sod promoter the lowest amount of fluorescence and the cspB an intermediate intensity. The result is not surprising since, (i) other groups have used E. coli lac derived promoters like tac and trc to over-express genes in C. glutamicum[15, 39, 40], and (ii) the sequence of the −10 box of the tac promoter is identical to that of the consensus promoter of C. glutamicum. Interestingly, this promoter may provide another instance for fine tuning gene expression by using different concentrations of the IPTG inducer . Although a GFP based approach to characterize promoters has been previously described by Knoppova and co-workers , the vector described does not possess the versatility of the pTGR platform for testing multiple regulatory sequences.
Initiation is the rate limiting step of translation. Provided that there are not secondary structures between the RBS and the coding sequence, it was shown that RBSs can be used as a regulatory part since they affect translation initiation and, therefore, gene expression [42, 43]. Moreover, a method was recently described for automatic design of artificial RBSs to control gene expression, expanding the potential of these sequences to be used in genetic circuits . In this way, the pTGR may also be used to test RBSs modulator effect on gene expression. To validate this application, three different RBSs were tested using eGFP as a reporter gene under the tac promoter. The sequences provided variable amounts of fluorescence intensity, validating the use of the system to populate a collection of these gene-expression regulatory sequences.
The pTGR provides a rapid means to create constructions for the expression of multiple genes. The design allows the assembly of constructs -operons or gene clusters- in as many cloning steps as genes are assembled by: (i) inserting all the genes to be expressed into a pTGR vector with the desired regulatory sequences and (ii) the sequential transfer of the genes with corresponding regulator sequences to a vector containing another gene(s) to extend an operon or gene cluster as illustrated in Figure 1B.
It is tempting to expect that the output of promoters and RBSs in single gene expression experiments from probe vectors may anticipate the performance of these parts in a more complex context like operons or multiple gene clusters. For example, the level of expression of both proteins in the experiments shown in Figure 4 is similar to that expected from the individual expression of each protein (Figure 3). This would facilitate the classification of regulatory parts to accurately regulate gene expression in pathways requiring multiple proteins. However, the output of many parts may be context dependant. Thus, obtaining the optimal balance for all the proteins, specially for pathways containing a high number of genes, may require multiple tests to find the appropriate regulatory sequences. Moreover, in most cases the optimal level of expression of a given protein in a pathway is unknown, and combinatorial constructions using a variety of regulatory sequences are necessary to find the right combination [45–47]. The pTGR platform may contribute to speed up these tests by facilitating the rapid assembly of combinatorial constructs and exchange of parts involved in gene expression regulation.
Other features of the pTGR platform may provide additional levels of regulation. For example, the copy number of a gene cluster or an operon may be regulated by using origins of replication from medium or low copy number plasmids. This can be easily achieved in one cloning step using the KpnI and PstI sites flanking the replication origin. Alternatively, a sequence for the insertion of the genes into the chromosome may be inserted into this place.
In principle, the platform described here for C. glutamicum may be extended to other microorganisms by replacing its origin of replication by an appropriate counterpart from, for example, Bacillus and Streptomyces. Such experiments are in progress in our laboratory to validate the use of the pTGR system in other microorganisms of industrial interests.