Modelling hCDKL5 Heterologous Expression in Bacteria

Abstract

hCDKL5 refers to the human cyclin-dependent kinase like 5 that is primarily expressed in the brain. Mutations in its coding sequence are often causative of hCDKL5 deficiency disorder, a devastating neurodevelopmental disorder currently lacking a cure. The large-scale recombinant production of hCDKL5 is desirable to boost the translation of preclinical therapeutic approaches into the clinic. However, this is hampered by the intrinsically disordered nature of almost two-thirds of the hCDKL5 sequence, making this region more susceptible to proteolytic attack, and the observed toxicity when the enzyme is accumulated in the cytoplasm of eukaryotic host cells. The bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) is the only prokaryotic host in which the full-length production of hCDKL5 has been demonstrated. To date, a system-level understanding of the metabolic burden imposed by hCDKL5 production is missing, although it would be crucial for upscaling of the production process. Here, we combined experimental data on protein production and nutrients assimilation with metabolic modelling to infer the global consequences of hCDKL5 production in PhTAC125 and to identify potential overproduction targets. Our analyses showed a remarkable accuracy of the model in simulating the recombinant strain phenotype and also identified priority targets for optimised protein production.

Keywords: CDKL5; genome-scale metabolic modelling; protein production.

Figure 1

(A) Growth curves of WT and hCDKL5 strains, as experimentally determined. (B) Comparison between the model-predicted and measured growth rates in the wild-type strain. (C) Comparison between the measured hCDKL5 production rate in the recombinant strain and the one predicted by the model. (D) Production enveloper for hCDKL5.

Figure 2

Difference in amino acid composition between the PhTAC125 proteome and CDKL5.

Figure 3

The effect of putative nutrients to be added to GG medium on the CDKL5 production flux. The y-axis indicates the log fold change of the CDKL5 production flux with respect to the recomb model grown on GG medium.

Figure 4

Pathways including reactions identified as potential overexpression targets by the FSEOF algorithm for (A) the Entner–Doudoroff pathway and (B) glutamate catabolism. Only pathways including 2 or more reactions are shown in (C).