Proof of Concept | TeamFreiburg:chAMBER

chAMBER is a system that aims to improve biosynthesis through compartmentalisation and modulation via non-canonical amino acids (ncAA). Our assumption was that, by bringing key enzymes of a pathway physically close together in a compartment distinct from the cytoplasm, we would obtain a higher yield of the compound due to lower build-up of toxic by-products, faster reaction rates and less interference from other reacting molecules in the cytoplasm.

To obtain a proof of principle, we cloned the pathway for the production of Indigo and Indirubin, whereby two of the enzymes were cloned with and without localisation tags that would bring them into the compartment. We then tested indigo production under different concentrations of the inducer of the expression of the genes in the pathway and found that compartmentalisation had a positive effect on the production level, thus proving the initial assumption. We repeated the experiments a total of three times. For a more in-depth description and interpretation of our experiments, please read our results page. A simplified representative graph is shown in fig. 1, displaying the results of the third repetition of the experiments.
Bioproduction of Indigo and Indirubin in bacteria with and without microcompartments.

Fig. 1: Bioproduction of Indigo and Indirubin in bacteria with and without microcompartments. Measurement of production of Indigo/Indirubin by bacteria transformed with the pathway enzymes and, either an empty pTrc99A backbone (grey), or the pHT1spysnpT2T3, which encodes genes for the wiffleball proteins (green). A: Overview of the plasmids transformed into each sample. B: Indigo production over time, measured via fluorescence (ex: 612 nm, em: 670 nm). C: Indirubin production over time, quantified via its absorbance at 540 nm.

The samples shown are E. coli BL21 transformed with two plasmids: Every sample is transformed with a plasmid which encodes enzymes from the Indigo/Indirubin pathway tagged with SnoopCatcher and SpyCatcher and spectinomycin resistance (pCDFDuet-1). The second plasmid varies; either it is an empty pTrc99a backbone or the pHT1 backbone containing the sequence for the bacterial microcompartment (BMC) or wiffleball and an ampicillin resistance. As shown on the graph, the presence of wiffleballs, while keeping all other conditions the same, positively affects the production of Indigo (A) and Indirubin (B). The samples expressing the wiffleball compartmentalisation protein in grey show a higher absorption than those samples only expressing the synthesis enzymes along an empty backbone in green. We, therefore, conclude that a higher amount of the product of interest is produced. We could show this effect in 2 out of 3 repetitions of this experiment.

However, chAMBER does not only comprise compartmentalisation, but is also meant as a tool to use the broad spectrum of ncAA and their applications in order to make a system more modulable or more robust. In an experiment, we expressed the complete chAMBER system in E. coli BL21, which includes the synthesis enzymes, the wiffleballs with an amber stop codon mutation, a plasmid encoding a tRNA synthetase and the supply of the respective ncAA into the medium. A graph with representative results is shown in fig. 2. Bioproduction of Indigo and Indirubin in bacteria with microcompartments and with or without ncAA incorporation.

Fig. 2: Bioproduction of Indigo and Indirubin in bacteria with microcompartments and with or without ncAA incorporation. Measurement of production of Indigo/Indirubin by bacteria transformed with the pathway enzymes and pHT1spysnpT2T3, which encodes genes for the wiffleball proteins (BMC). Either no additional plasmid was transformed (grey), or an additional plasmid was transformed encoding the pAzF tRNA synthetase (blue). A: Overview of plasmids transformed in each sample. B: Indigo production over time, measured via fluorescence (ex: 612 nm, em: 670 nm). B: Indirubin production over time, quantified via its absorbance at 540 nm.

In fig. 2, the grey bars represent the amount of Indigo (A) and Indirubin (B) of E. coli BL21 which express the synthesis enzymes and wiffleballs (grey), or a variation in which the T1 BMC has a mutation leading to an amber stop codon and therefore to early termination of translation. Additionally, a plasmid encoding a tRNA synthetase (pAzFS) is transformed. Through amber stop codon suppression technology, the respective ncAA pAzF is integrated in the T1 BMC, restoring suppression.

Comparing the samples with mutation-less wiffleballs (grey) with those that incorporate the ncAA into the T1 BMC protein (blue), the amount of Indigo and Indirubin that is synthesised is similar. Unfortunately, this experiment lacks appropriate controls. Furthermore, by measuring the absorption of Indigo and Indirubin in the medium we do not gain structural information about the actual process, therefore we can only speculate whether the compartments are successfully synthesised and assembled. A hint that this is likely to be the case give us the western blots we performed, in which we could show that BMCs are successfully synthesised and assembled using the complete chAMBER system with amber suppression technology. The successful BMC assembly shown in the western blot and the steady production of Indigo and Indirubin suggest that the chAMBER system may be functional and lead to successful biosynthesis.

To conclude, we could show that our compartmentalisation system truly has an increasing effect on the production of compounds via biosynthesis pathways in E. coli. We could also show exciting preliminary data about the usage of ncAA with our compartmentalisation system, showing that BMCs can form using amber stop codon suppression, and biosynthesis is likely to be successful.