7 research outputs found
Einfluss des Hintergrundgenoms und der Signalpeptide auf die Cutinasesekretion durch Corynebacterium glutamicum in Mikrobioreaktorsystemen
Microaerobic growthâdecoupled production of αâketoglutarate and succinate from xylose in a oneâpot process using Corynebacterium glutamicum
BackgroundLignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacterium glutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2evo. The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process.Methods and ResultsC. glutamicum WMB2evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C. glutamicum WMB2evo. Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4 mmol Lâ1 (11.45 g Lâ1) α-ketoglutarate and 96.2 mmol Lâ1 (11.36 g Lâ1) succinate.ConclusionThe developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose.AbstractThe Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of xylose to α-ketoglutarate and a shortcut route to succinate as established platform chemical. In this study, we employed the recently engineered and evolved strain C. glutamicum WMB2evo to establish a one-pot cultivation process for co-production of α-ketoglutarate and succinate from xylose
Less Sacrifice, More Insight: Repeated Low-Volume Sampling of Microbioreactor Cultivations Enables Accelerated Deep Phenotyping of Microbial Strain Libraries
With modern genetic engineering tools, high number of potentially improved production strains can be created in a short time. This results in a bottleneck in the succeeding step of bioprocess development, which can be handled by accelerating quantitative microbial phenotyping. Miniaturization and automation are key technologies to achieve this goal. In this study, a novel workflow for repeated lowâvolume sampling of BioLectorâbased cultivation setups is presented. Six samples of 20âÎŒL each can be taken automatically from shaken 48âwell microtiter plates without disturbing cell population growth. The volume is sufficient for quantification of substrate and product concentrations by spectrophotometricâbased enzyme assays. From transient concentration data and replicate cultures, valid performance indicators (titers, rates, yields) are determined through process modeling and random error propagation analysis. Practical relevance of the workflow is demonstrated with a set of five genomeâreduced Corynebacterium glutamicum strains that are engineered for Secâmediated heterologous cutinase secretion. Quantitative phenotyping of this strain library led to the identification of a strain with a 1.6âfold increase in cutinase yield. The prophageâfree strain carries combinatorial deletions of three gene clusters (Î3102â3111, Î3263â3301, and Î3324â3345) of which the last two likely contain novel target genes to foster rational engineering of heterologous cutinase secretion in C. glutamicum
Evaluation of a library of genome-reduced C. glutamicum strains for optimization of heterologous cutinase secretion
Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion
Hemmerich J, Labib M, Steffens C, et al. Screening of a genome-reduced Corynebacterium glutamicum strain library for improved heterologous cutinase secretion. Microbial Biotechnology. 2020;13(6):2020-2031.The construction of microbial platform organisms by means of genome reduction is an ongoing topic in biotechnology. In this study, we investigated whether the deletion of single or multiple gene clusters has a positive effect on the secretion of cutinase from Fusarium solani pisi in the industrial workhorse Corynebacterium glutamicum. A total of 22 genomeâreduced strain variants were compared applying two Sec signal peptides from Bacillus subtilis. Highâthroughput phenotyping using roboticsâintegrated microbioreactor technology with automated harvesting revealed distinct cutinase secretion performance for a specific combination of signal peptide and genomic deletions. The biomassâspecific cutinase yield for strain GRS41_51_NprE was increased by ~ 200%, although the growth rate was reduced by ~ 60%. Importantly, the causative deletions of genomic clusters cg2801âcg2828 and rrnCâcg3298 could not have been inferred a priori. Strikingly, bioreactor fedâbatch cultivations at controlled growth rates resulted in a complete reversal of the screening results, with the cutinase yield for strain GRS41_51_NprE dropping by ~ 25% compared to the reference strain. Thus, the choice of bioprocess conditions may turn a âhighâperformanceâ strain from batch screening into a âlowâperformanceâ strain in fedâbatch cultivation. In conclusion, future studies are needed in order to understand metabolic adaptations of C. glutamicum to both genomic deletions and different bioprocess conditions