Biology subjects

Kuipers, O. P.

Publications and source records attributed to Kuipers, O. P..

2 recordsLinked to original sources

bioRxiv2018

Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis

Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been solved to achieve production strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a {beta}-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CcpA and CodY that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic gel mobility shift analyses showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain.\n\nHighlightsO_LIThe global transcription machinery engineering (gTME) technique was applied to build mutational libraries of the pleiotropic regulators CodY and CcpA in Bacillus subtilis\nC_LIO_LISpecific point mutations within the DNA-binding domains of CodY and CcpA elicited alterations of the binding specificity and regulatory activity\nC_LIO_LIChanges in the transcriptome evoked the reprogramming of networks that gear the carbon and nitrogen metabolism\nC_LIO_LIThe rewired metabolic networks provided a higher building block capacity for heterologous protein production by adjusting the nutrient uptake and channeling its utilization for protein overexpression\nC_LI

bioengineering↗

bioRxiv2017

Unleashing natural competence in Lactococcus lactis by induction of the competence regulator ComX

In biotechnological work horses like Streptococcus thermophilus and Bacillus subtilis natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis natural competence has not been established to date. However, in silico analysis of complete genome sequences of 43 L. lactis strains revealed complete late-competence gene-sets in 2 L. lactis subspecies cremoris strains (KW2 and KW10) and 8 L. lactis subspecies lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon, and elicited a state of natural competence in this strain. By contrast, comX expression in L. lactis NZ9000, predicted to encode an incomplete competence gene-set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147, abolished the comX driven natural competence, underpinning the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence also in these strains, expanding this phenotype to other L. lactis strains of both subspecies.\n\nSignificance statementSpecific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end-product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria, and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

microbiology↗