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Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes.

ABSTRACT: In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in Escherichia coli host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained its catalytic activity at the neutral pH and showed a 60% activity reduction at pH 10.5. This reduction in the alkaline domain is desirable for enhancing the stability of the enzyme in the liquid laundary detergent, whereas the wild-type molecule showed a 20% increase in response to the same pH shift. The engineered pH dependency of the activity of the variant was ascribed partly to a lowered substrate affinity under the alkaline conditions, in which the incorporated 3-nitrotyrosine was probably charged negatively due to the phenolic pK a lower than that of tyrosine.

SUBMITTER: Osamura T

PROVIDER: S-EPMC6295607 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes.

Osamura Tatsuya T Okuda Mitsuyoshi M Yamaguchi Atsushi A Ohtake Kazumasa K Sakamoto Kensaku K Takimura Yasushi Y

Biochemistry and biophysics reports 20181212

In the present study, we attempted to control the pH profile of the catalytic activity of the industrially relevant alkaline protease KP-43, by incorporating 3-nitro-l-tyrosine and 3-chloro-l-tyrosine at and near the catalytic site. Thirty KP-43 variants containing these non-natural amino acids at the specific positions were synthesized in <i>Escherichia coli</i> host cells with expanded genetic codes. The variant with 3-nitrotyrosine at position 205, near the substrate binding site, retained it ...[more]

PMID: 30582013

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Project description:Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence.We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism's ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions.We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.

Project description:BackgroundEconomically viable biobased production of bulk chemicals and biofuels typically requires high product titers. During microbial bioconversion this often leads to product toxicity, and tolerance is therefore a critical element in the engineering of production strains.ResultsHere, a systems biology approach was employed to understand the chemical stress response of Escherichia coli, including a genome-wide screen for mutants with increased fitness during chemical stress. Twelve chemicals with significant production potential were selected, consisting of organic solvent-like chemicals (butanol, hydroxy-γ-butyrolactone, 1,4-butanediol, furfural), organic acids (acetate, itaconic acid, levulinic acid, succinic acid), amino acids (serine, threonine) and membrane-intercalating chemicals (decanoic acid, geraniol). The transcriptional response towards these chemicals revealed large overlaps of transcription changes within and between chemical groups, with functions such as energy metabolism, stress response, membrane modification, transporters and iron metabolism being affected. Regulon enrichment analysis identified key regulators likely mediating the transcriptional response, including CRP, RpoS, OmpR, ArcA, Fur and GadX. These regulators, the genes within their regulons and the above mentioned cellular functions therefore constitute potential targets for increasing E. coli chemical tolerance. Fitness determination of genome-wide transposon mutants (Tn-seq) subjected to the same chemical stress identified 294 enriched and 336 depleted mutants and experimental validation revealed up to 60 % increase in mutant growth rates. Mutants enriched in several conditions contained, among others, insertions in genes of the Mar-Sox-Rob regulon as well as transcription and translation related gene functions.ConclusionsThe combination of the transcriptional response and mutant screening provides general targets that can increase tolerance towards not only single, but multiple chemicals.

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Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes.

Publications

Variants of the industrially relevant protease KP-43 with suppressed activity under alkaline conditions developed using expanded genetic codes.

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