Recombinant Ashbya gossypii Pyruvate kinase (PYK1), partial

Introduction

Ashbya gossypii is a filamentous fungus known for its ability to produce riboflavin (vitamin B2) and has been explored for various biotechnological applications . Pyruvate kinase (PYK) is a crucial enzyme in glycolysis, catalyzing the final, irreversible step of converting phosphoenolpyruvate (PEP) to pyruvate . In A. gossypii, as in other organisms, PYK plays a central metabolic regulatory role . Recombinant PYK1, partial, refers to a version of the A. gossypii PYK1 enzyme that has been produced using recombinant DNA technology, but only includes a portion of the full-length protein sequence. This may be done for research purposes, such as studying the structure and function of specific domains within the enzyme.

Role of Pyruvate Kinase in Ashbya gossypii Metabolism

PYK is a key enzyme in the glycolytic pathway, which is responsible for the breakdown of glucose to produce energy and metabolic intermediates. The reaction catalyzed by PYK is essential for energy production and provides precursors for various biosynthetic pathways.

• Glycolysis: PYK catalyzes the transfer of a phosphoryl group from PEP to ADP, yielding pyruvate and ATP. This is a critical step in glycolysis, generating ATP and providing pyruvate for further metabolism .

• Metabolic Regulation: PYK activity is regulated by various factors, including allosteric effectors and genetic control. This regulation allows the cell to adjust its metabolic flux in response to changing environmental conditions .

• Connection to other Metabolic Pathways: Pyruvate, the product of the PYK reaction, is a precursor for several important metabolic pathways, including the citric acid cycle, gluconeogenesis, and amino acid biosynthesis .

Recombinant Production and Uses

Recombinant production of A. gossypii PYK1, partial, involves cloning the gene encoding the enzyme into a suitable expression vector, introducing the vector into a host organism (e.g., Escherichia coli or Saccharomyces cerevisiae), and culturing the host under conditions that allow for high-level expression of the recombinant protein. The recombinant protein can then be purified for downstream applications.

• Enzyme Characterization: Recombinant PYK1, partial, can be used to study the enzyme's biochemical properties, including its substrate specificity, kinetics, and regulation .

• Structural Studies: Recombinant production allows for the preparation of large quantities of the enzyme, which is essential for structural studies using techniques such as X-ray crystallography or cryo-electron microscopy.

• Metabolic Engineering: Modified versions of PYK1 can be introduced into A. gossypii or other organisms to alter metabolic fluxes and improve the production of desired metabolites .

• Drug Discovery: PYK is a potential drug target in various diseases, including cancer and infectious diseases. Recombinant PYK1, partial, can be used in drug screening assays to identify compounds that inhibit or modulate enzyme activity .

4.1. Metabolic Re-annotation of Ashbya gossypii

Genome-wide re-annotation studies of A. gossypii have provided insights into its metabolic capabilities, including the identification and characterization of enzymes involved in various pathways. Such studies enhance understanding of the organism's unique metabolic features and its potential for biotechnological applications .

4.2. Overexpression for Metabolic Engineering

Overexpression of specific genes, including PYK1, has been used to enhance the production of desired metabolites in A. gossypii. For example, overexpression of genes in the kynurenine pathway for NAD biosynthesis has been shown to improve nicotinic acid production .

4.3. Sabinene Production

A. gossypii has been engineered to produce sabinene, a valuable terpene, by introducing a sabinene synthase gene from S. pomifera. Overexpression of ERG12, encoding mevalonate kinase, further enhances sabinene production .

4.4. Role in Hyphal Maturation

Research has identified genes in A. gossypii that are important for hyphal maturation, contributing to our understanding of fungal development and morphogenesis .

Tables

The tables below represent data from research findings and applications related to Recombinant Ashbya gossypii Pyruvate kinase (PYK1), partial.

Table 1: In vitro inhibitors of recombinant Babesia microti PYK1 .

Table 2: Optical density measurements of modified Saccharomyces cerevisiae strains .