Recombinant Saccharomyces cerevisiae Phosphoglycerate kinase (PGK1)

Basic Research Questions

• What is the function of phosphoglycerate kinase (PGK1) in S. cerevisiae metabolism?

Phosphoglycerate kinase (PGK) is a well-conserved glycolytic enzyme that catalyzes one of the two ATP-producing reactions in the glycolytic pathway. As a monomeric enzyme of approximately 45 kDa, PGK1 converts 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA) while producing ATP . It also participates in gluconeogenesis by catalyzing the reverse reaction .

Beyond its primary metabolic functions, PGK1 exhibits various moonlighting functions including pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death regulation, and viral replication . This multifunctionality makes PGK1 an important enzyme for understanding both basic metabolism and complex cellular processes in yeast.

Methodological approach: To study PGK1's metabolic functions, researchers typically employ gene deletion/mutation studies and metabolic flux analysis. Creating pgk1 mutant strains reveals its impact on metabolism under different carbon source conditions. For instance, Pgk- cells cannot effectively metabolize glycerol, limiting their growth during respiratory metabolism .

• How does codon usage affect PGK1 expression in S. cerevisiae?

S. cerevisiae exhibits strong codon bias with preference for 25 of the 61 possible coding triplets. This bias is particularly pronounced in highly expressed genes like PGK1, which uses these 25 major codons almost exclusively .

Systematic experimental codon replacement studies have demonstrated that replacing major codons with synonymous minor ones at the 5' end of the PGK1 coding sequence dramatically reduces expression levels. Specific findings include:

• PGK protein levels dropped 10-fold

• Steady-state mRNA levels declined 3-fold

• The reduction in mRNA levels was attributed to destabilization caused by impaired translation elongation at minor codons

Methodological approach: To study codon effects, researchers construct PGK1 variants with systematic codon replacements and analyze protein and mRNA levels using Western blotting and Northern blotting, respectively. Expression can be studied using both high-copy-number plasmids and single-copy genes integrated into the chromosome .

• What are the common methods for using the PGK1 promoter in heterologous protein expression?

The PGK1 promoter is frequently utilized for recombinant protein production in S. cerevisiae due to its strong constitutive expression characteristics. Common methodological approaches include:

Methodological consideration: While strong constitutive promoters like PGK1 can achieve high expression levels, they may also lead to lower secretion efficiency due to aggregation of misfolded proteins, as reported in the expression of insulin precursor and α-amylase . For proteins prone to misfolding, inducible promoters like GAL1 and GAL10 may be preferable to control expression levels.

• What factors affect plasmid stability in PGK1-expressing strains?

Plasmid stability in S. cerevisiae strains expressing recombinant PGK1 is influenced by several factors:

• Growth phase: Plasmid stability paradoxically increases during the respiratory phase of growth in batch cultures

• Carbon source: Selective advantages conferred by PGK1 under specific metabolic conditions affect plasmid maintenance

• Selection pressure: Presence of selective markers and their expression levels

• Metabolic burden: The energetic cost of plasmid maintenance and recombinant protein production

Methodological approach: To study plasmid stability, researchers typically employ cultivation methods that track plasmid retention over time:

• Prolonged chemostat cultivation at glucose-limited conditions

• Batch cultures monitoring plasmid distribution through growth phases

• Selective growth conditions that create differential advantages for plasmid-containing cells

Research has shown that Pgk+ plasmid-containing cells have a selective advantage during the respiratory phase since they can utilize both glycerol and ethanol, while Pgk- cells cannot effectively metabolize glycerol .

Advanced Research Questions

• How can recombinant PGK1 be engineered for improved expression and stability?

Engineering recombinant PGK1 for improved expression requires multiple approaches targeting gene sequence, protein structure, and host cell physiology:

Codon optimization methodology:

• Identify and replace rare codons with preferred codons in S. cerevisiae, particularly at the 5' end of the gene

• Maintain mRNA secondary structures important for stability

• Avoid creating new regulatory elements or binding sites

Protein engineering methodology:

• Introduction of stabilizing mutations based on structural analysis

• Optimization of N-terminal sequence to improve translation initiation

• Engineering of post-translational modification sites

Experimental data: Replacing up to 39% of major codons with synonymous minor ones at the 5' end of the coding sequence caused a 10-fold drop in protein levels and a 3-fold decline in mRNA levels . This indicates that codon optimization efforts should prioritize the 5' region of the gene.

• What are the molecular mechanisms linking PGK1 translation to mRNA stability?

The stability of PGK1 mRNA in S. cerevisiae is tightly coupled to translation efficiency through several mechanisms:

• Preventing translation of PGK mRNAs by introducing a stop codon adjacent to the start codon dramatically decreases steady-state mRNA levels

• Active translation appears to protect mRNA from degradation pathways

• Slow translation at rare codons may expose mRNA to degradation machinery

• The 5' end of the transcript is particularly important for this coupling

Methodological approach: To study these mechanisms, researchers employ techniques such as:

• Polysome profiling to analyze ribosome loading on mRNAs

• Construction of translation-inhibited variants (early stop codons, frameshifts)

• mRNA decay assays following transcriptional shut-off

• RNA-seq and ribosome profiling to map translation efficiency genome-wide

This research has significant implications for heterologous gene expression in yeast, suggesting that efficient translation initiation and elongation are crucial for maintaining mRNA stability and achieving high protein yields.

• How do double mutations in sod1/pgk1 affect cellular physiology and recombinant protein production?

Double mutant sod1/pgk1 strains of S. cerevisiae provide insights into the complex interplay between metabolism, oxidative stress, and recombinant protein production:

Experimental findings:

• These strains have been specifically constructed to investigate the effects of different environmental conditions on yeast physiology, plasmid stability, and superoxide dismutase (SOD) production

• When transformed with yeast episomal plasmids (YEp) containing both PGK1 and SOD1 genes and grown on fermentable carbon sources under vigorous aeration, both genes are efficiently expressed

• The presence of the PGK1 gene becomes essential for growth under these conditions

• Interestingly, plasmid-borne PGK1 was found not to increase the stability of YEp vectors in batch cultures of Pgk- cells

Methodological approach: Researchers analyze these strains through:

• Growth studies under varying environmental conditions

• Measurement of SOD activity and expression levels

• Analysis of respiratory vs. fermentative metabolism

• Tracking of plasmid stability through growth phases

• Quantification of reactive oxygen species and oxidative stress markers

These studies illuminate the complex relationships between central carbon metabolism, oxidative stress responses, and heterologous protein production in yeast.

• How does evolutionary history of the PGK1 gene influence its function in modern S. cerevisiae?

The evolutionary history of PGK1 in S. cerevisiae provides important context for understanding its function and regulation:

Evolutionary context:

• PGK1 evolved through a wholesale genome duplication that occurred during the evolutionary path leading to modern S. cerevisiae

• This duplication event created paralogs for many genes

• Selection pressure has optimized codon usage in PGK1 to maximize expression efficiency

• The enzyme structure is highly conserved across diverse species, indicating its fundamental importance in metabolism

Methodological approach: To study the evolutionary aspects of PGK1, researchers employ:

• Comparative genomics across yeast species

• Phylogenetic analysis of PGK sequences

• Functional complementation studies between orthologs

• Experimental evolution to study adaptive changes in PGK1 sequence or regulation

Understanding PGK1's evolutionary history provides insights into its optimal expression parameters, regulatory mechanisms, and integration with other metabolic pathways.

• What methodologies are most effective for studying the moonlighting functions of PGK1?

PGK1 exhibits several non-glycolytic "moonlighting" functions including roles in pathogenesis, nucleic acid interactions, tumorigenesis, cell death regulation, and viral replication . Studying these functions requires specialized approaches:

Effective methodologies:

• Proteomics approaches to identify non-glycolytic interaction partners

• Subcellular localization studies using fluorescent protein fusions

• Creation of separation-of-function mutants that maintain glycolytic function but disrupt specific moonlighting roles

• Comparative studies between different organisms to identify conserved non-glycolytic functions

• Conditional expression systems to manipulate PGK1 levels in specific cellular compartments

Research strategy: Effective investigation requires differentiating between direct effects of PGK1 and indirect consequences of disrupting glycolysis. This typically involves creating mutants that maintain glycolytic flux while disrupting specific protein-protein interactions or cellular localizations.

• How can the PGK1 promoter be optimized for heterologous protein production in different S. cerevisiae strains?

Optimizing the PGK1 promoter for heterologous protein production requires consideration of strain background, growth conditions, and target protein characteristics:

Optimization methodology:

• Comparative analysis with other strong promoters (TEF1, TPI1, HXT7, PYK1, ADH1, TDH3)

• Creation of hybrid promoters combining elements of PGK1 with other regulatory sequences

• Promoter engineering to modify transcription factor binding sites

• Integration of inducible elements to enable controlled expression

Strain-specific considerations:

• Industrial strains often show different promoter performance compared to laboratory strains

• Diploid industrial strains may exhibit different expression dynamics than haploid laboratory strains

• Promoter function may vary between respiratory and fermentative growth conditions