Hot isopropanol quenching procedure for automated microtiter plate scale 13C-labeling experiments

Abstract

Background: Currently, the generation of genetic diversity for microbial cell factories outpaces the screening of strain variants with omics-based phenotyping methods. Especially isotopic labeling experiments, which constitute techniques aimed at elucidating cellular phenotypes and supporting rational strain design by growing microorganisms on substrates enriched with heavy isotopes, suffer from comparably low throughput and the high cost of labeled substrates.

Results: We present a miniaturized, parallelized, and automated approach to ¹³C-isotopic labeling experiments by establishing and validating a hot isopropanol quenching method on a robotic platform coupled with a microbioreactor cultivation system. This allows for the first time to conduct automated labeling experiments at a microtiter plate scale in up to 48 parallel batches. A further innovation enabled by the automated quenching method is the analysis of free amino acids instead of proteinogenic ones on said microliter scale. Capitalizing on the latter point and as a proof of concept, we present an isotopically instationary labeling experiment in Corynebacterium glutamicum ATCC 13032, generating dynamic labeling data of free amino acids in the process.

Conclusions: Our results show that a robotic liquid handler is sufficiently fast to generate informative isotopically transient labeling data. Furthermore, the amount of biomass obtained from a sub-milliliter cultivation in a microbioreactor is adequate for the detection of labeling patterns of free amino acids. Combining the innovations presented in this study, isotopically stationary and instationary automated labeling experiments can be conducted, thus fulfilling the prerequisites for ¹³C-metabolic flux analyses in high-throughput.

Keywords: 13C-labeling; Boiling solvent quenching; Corynebacterium glutamicum; Isotopic labeling; Isotopically transient experiment; Lab automation; Metabolic quenching; Microbioreactor cultivation.

Fig. 1

Complete experimental workflow of the automated cultivation, sacrifice sampling, quenching, and sample processing. 1) A pre-heating step for the hot isopropanol quenching is initiated upon exceeding the cultivation time threshold (t_threshold), whereas the start of the sampling procedure is dependent on crossing a backscatter threshold (BS_threshold). 2) The time for heating the isopropanol solution and the residence time of cellular material inside the heated vials depend on the pipetting scheme of the particular variant of the workflow so the given values are to be understood as lower practical limits. Depending on the subsequent analyses, the extracts may be stored in a sealed deep well plate (a) or Eppendorf tubes (b).

Fig. 2

Combined results of the spiking experiment for validation (bar diagrams) and the isotopically instationary ILE as a proof of concept (line diagrams) accompanied by a simplified portrayal of the metabolic network of C. glutamicum ATCC 13032. The numbers in brackets pertain to each amino acid’s number of carbon atoms. In both experiments, the cultivation began on unlabeled d-glucose but in the former case, U¹³C d-glucose was added to the quenching reagent, and in the latter case, U¹³C d-glucose was pulsed before the transient sampling