Dataset Information

From Sea to Therapy: Development and analytical control of recombinant human CDKL5 production in the marine bacterium Pseudoalteromonas haloplanktis TAC125

ABSTRACT: Marine bacteria are increasingly explored as alternative microbial platforms for the production of high-value biopharmaceuticals, owing to their distinctive physiological and biochemical features. In this study, we focus on the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125), an unconventional host that has proven capable of producing soluble and active human Cyclin-Dependent Kinase-Like 5 (hCDKL5). This serine/threonine kinase is essential for neuronal development as its deficiency causes CDKL5 Deficiency Disorder, a severe and currently untreatable neurodevelopmental disease. Recombinant production of hCDKL5 represents a necessary prerequisite for the development of enzyme replacement therapy as a potential therapeutic strategy; however, the current production process remains insufficient for industrial translation. To address this limitation, we developed and implemented dedicated analytical methods to assess both the quantity and functional quality of recombinant hCDKL5. Production yields were evaluated using a customised quantitative sandwich Enzyme-Linked Immunosorbent Assay (ELISA) specifically detecting full-length hCDKL5, while protein functionality was assessed by mass spectrometry-based quantification of autophosphorylation levels, a key indicator of kinase activity. These tools were applied to characterise hCDKL5 expression under different growth conditions. Overall, this work establishes an integrated analytical framework aligned with a Quality by Design approach, providing a robust basis for future process optimisation toward scalable hCDKL5 production.

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ORGANISM(S): Pseudoalteromonas Haloplanktis

SUBMITTER: ilaria iacobucci

LAB HEAD: flora cozzolino

PROVIDER: PXD073800 | Pride | 2026-05-29

REPOSITORIES: Pride

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Publications

From Sea to Therapy: Development and Analytical Control of Recombinant Human CDKL5 Production in the Marine Bacterium Pseudoalteromonas haloplanktis TAC125.

Coletti Andrea A Calvanese Marzia M Cozzolino Flora F Iacobucci Ilaria I Lauro Concetta C Severino Angelica A Monti Maria M Parrilli Ermenegilda E Tutino Maria Luisa ML

Marine drugs 20260424 5

Marine bacteria are increasingly explored as alternative microbial platforms for the production of high-value biopharmaceuticals. In this study, we investigate the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125), an unconventional host capable of yielding soluble and biologically active human cyclin-dependent kinase-like 5 (hCDKL5). This serine/threonine kinase plays a crucial role in neuronal development, and its deficiency causes CDKL5 Deficiency Disor ...[more]

PMID: 42188286

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Project description:In actinomycetes, antibiotic production is often associated with a morpho-physiological differentiation program that is regulated by complex molecular and metabolic networks. Many aspects of these regulatory circuits have been already elucidated and many others still deserve further investigations. In this regard, the possible role of many small open-reading frames(smORFs) in actinomycete morpho-physiological differentiation is still elusive. In Streptomyces coelicolor, inactivation of the smORF trpM (SCO2038) - whose product modulates L-tryptophan metabolism - impairs production of antibiotics and morphological differentiation. Indeed, it was demonstrated that TrpM is able to interact with PepA (SCO2179), a putative cytosol aminopeptidase playing a key role in antibiotic production and sporulation. In this work, a S. coelicolor trpM knock-in (Sco-trpMKI) mutant strain was generated by cloning trpM into overexpressing vector to further investigate the role of trpM in actinomycete growth and morpho-physiological differentiation. Results highlighted that trpM: i) stimulates growth and actinorhodin production; ii) decreases calcium-dependent antibiotic production; iii) has no effect on undecylprodigiosin production. Metabolic pathways influenced by trpM knock-in were investigated by combining two-difference in gel electrophoresis/nanoliquid chromatography coupled to electrospray linear ion trap tandem mass spectrometry (2D-DIGE/nanoLC-ESI-LIT- MS/MS) and by LC-ESI-MS/MS procedures, respectively. These analyses demonstrated that over-expression of trpM causes an over-representation of factors involved in protein synthesis and nucleotide metabolism as well as a down-representation of proteins involved in central carbon and amino acid metabolism. At the metabolic level, this corresponded to a differential accumulation pattern of different amino acids - including aromatic ones but tryptophan – and central carbon intermediates. PepA was also down-represented in Sco-trpMKI. The latter was produced as recombinant His- tagged protein and was originally proven having the predicted aminopeptidase activity. Altogether, these results highlight the stimulatory effect of trpM in S. coelicolor growth and actinorhodin biosynthesis, which are elicited through the modulation of various metabolic pathways and PepA representation, further confirming the complexity of regulatory networks that control antibiotic production in actinomycetes.

Project description:For over 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease up-regulation. In this study, the effects of a serine hydroxamate addition on high cell density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas, recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas, recombinant protein production regulated numerous classical stringent response genes; however, not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses; however, are far from identical. It was hypothesized that recombinant protein production leads to a stringent response due to the high amino acid synthesis demands related to recombinant protein synthesis. A comparison of the transcriptomes during recombinant protein production and a chemical imposed stringent response would assist with determining what portion of the “metabolic burden” associated with recombinant protein production is due to amino acid shortages. In this study, the transcriptome profiles of recombinant E. coli were examined and compared for the three culture conditions: 1) Normal growth, no external stress; 2) L-serine hydroxamate addition (to mediate a stringent response); and 3) IPTG-induction to produce the recombinant protein chloramphenicol acetyltransferase (CAT). The transcriptome profiles from these three conditions were analyzed using Affymetrix Anti-sense E. coli GeneChip® microarrays.

			Action	DRS
	CDKL5_KD.raw	Raw
	F044652.dat	Other
	F045176.dat	Other
	F045177.dat	Other
	F045178.dat	Other

Dataset Information

From Sea to Therapy: Development and analytical control of recombinant human CDKL5 production in the marine bacterium Pseudoalteromonas haloplanktis TAC125

Dataset's files

Publications

From Sea to Therapy: Development and Analytical Control of Recombinant Human CDKL5 Production in the Marine Bacterium <i>Pseudoalteromonas haloplanktis</i> TAC125.

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Dataset Information

From Sea to Therapy: Development and analytical control of recombinant human CDKL5 production in the marine bacterium Pseudoalteromonas haloplanktis TAC125

Dataset's files

Publications

From Sea to Therapy: Development and Analytical Control of Recombinant Human CDKL5 Production in the Marine Bacterium &lt;i&gt;Pseudoalteromonas haloplanktis&lt;/i&gt; TAC125.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

From Sea to Therapy: Development and Analytical Control of Recombinant Human CDKL5 Production in the Marine Bacterium <i>Pseudoalteromonas haloplanktis</i> TAC125.