Humanized Glycolysis Pathway in Yeast: Index #56

Glycolysis is highly conserved across organisms, but transplanting the full pathway from humans to yeast still required an engineering tour-de-force.

Happy July 4th to American readers.

It’s easy to take a gene from human cells and insert it into yeast. We’ve done the same for entire pathways, though it’s more difficult. But full humanization of the yeast glycolysis pathway, as in this new study in Cell Reports, is truly astounding.

Human and yeast proteins share, on average, just 32 percent identity. For proteins involved in glycolysis — the process by which nearly all living organisms convert glucose to pyruvate — yeast and humans share between 43 and 66 percent identity.

Yeast and humans are separated by about a billion years of evolution. By swapping out each of yeast’s glycolytic enzymes, one by one, and swapping in a human version, this new study reveals life's remarkable modularity and adaptability.

To start, researchers from TU Delft, in the Netherlands, codon-optimized 25 different human orthologs and individually expressed each of them in yeast. Straight away, 22 out of the 25 genes worked as expected. Some of the human genes caused a sharp drop in growth rate.

One of the unsuccessful human genes, hexokinase 1, could not convert glucose into G6P in yeast. But three separate yeast strains with human hexokinase 1 all mutated this gene in the same way, by swapping out a single letter encoding the glucose-6-phosphate (G6P) binding site of the protein. Though evolution made the human version compatible with yeast, the enzyme was still self-inhibited by its own product, G6P, because its intracellular concentration is about 50-200x higher in yeast than in human cells.

A fully humanized glycolysis pathway was next built in the yeast by expressing ten genes from human skeletal muscle: ALDOA, GPI1, PGK1, PFKM, PKM1, GAPDH, ENO3, HK4 (or HK2), PGAM2 and TPI1.

The humanized yeast strains metabolized both galactose and glucose, as expected. Apart from GPI1, the human enzymes were 2–50x slower than their yeast orthologs. In the humanized strain, metabolic bottlenecks swiftly emerged; hexokinase, aldolase, and PGAM2 were particularly slow. Overexpressing these genes caused the humanized yeast strains to grow 55 percent faster in glucose, but still 30 to 40 percent slower than control yeast.

Laboratory evolution resolved the growth defects a bit. After 630 generations of growth in glucose, the humanized yeast grew two-fold faster than their slow-growing predecessors.

Glycolysis is a particularly difficult pathway to ‘humanize’ because most of its enzymes moonlight in other roles. Yeast aldolase and enolase, for instance, also play roles in ATPase assembly, vacuolar fusion, and the import of mitochondrial tRNA. Humanizing the glycolysis pathway in yeast would surely eliminate many of these moonlighting functions, right? Right?

Not so. The secondary function of yeast aldolase and enolase was fully complemented by the human versions of these enzymes. Even the authors seemed surprised:

This extraordinary conservation of glycolytic moonlighting functions challenges our understanding of the underlying molecular mechanisms and reveals evolutionarily conserved functions.

So why does all this matter? Why am I so excited about a fully-humanized, metabolic pathway? Because it will help with basic science, mainly.

As if the aldolase and enolase ‘moonlighting’ finding wasn’t cool enough, researchers can now study human glycolysis in a simplified organism. Many complex interactions normally found in human cells are absent in yeast. This means that yeast with humanized glycolysis can be used to study this pathway in a ‘clean’ background strain, according to the authors. Humanized pathways can also be used to screen for chemical inhibition of protein drug targets. And, for diabetes, “transplantation to the yeast context enables dissection of metabolic from signaling-related mechanisms in control and regulation of glucose metabolism.” In other words, there’s lots of exciting science to be done.

Read more at Cell Reports.

Other Papers

(↑ = recommended article, * = open access, † = review article )

Assembly, Synthesis & Sequencing

*Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination. Zhao Y…Zhu Q. Plant Biotechnology Journal. Link

Basic Research

↑Prebiotic synthesis and triphosphorylation of 3′-amino-TNA nucleosides. Whitaker D & Powner MW. Nature Chemistry. Link

↑Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation. Pinglay S…Boeke JD. Science. Link

Metabolic reprogramming of skeletal muscle by resident macrophages points to CSF1R inhibitors as muscular dystrophy therapeutics. Babaeijandaghi F…Rossi FMV. Science Translational Medicine. Link

*Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites. Tao J…Chiarle R. Nature Communications. Link

*In vivo multidimensional CRISPR screens identify Lgals2 as an immunotherapy target in triple-negative breast cancer. Ji P…Shao Z. Science Advances. Link

*Cell surface glycan engineering reveals that matriglycan alone can recapitulate dystroglycan binding and function. Sheikh MO…Boons G-J. Nature Communications. Link

Biomanufacturing & Metabolic Engineering

*CRISPR/Cas9-mediated point mutations improve α-amylase secretion in Saccharomyces cerevisiae. Wang Y…Siewers V. FEMS Yeast Research. Link

↑*†Challenges and opportunities in bringing nonbiological atoms to life with synthetic metabolism. Haas R & Nikel PI. Trends in Biotechnology. Link

*Integration of synthetic microbial consortia based bioprocessing with pyrolysis for efficient conversion of cellulose to valuables. Joshi C…Masakapalli SK. bioRxiv (preprint). Link

*A bacteria-based carbon sequestration and waste recycling system. La YH…Song JY. Scientific Reports. Link

Biosensors

↑*A GPCR-based yeast biosensor for biomedical, biotechnological, and point-of-use cannabinoid determination. Miettinen K…Kampranis SC. Nature Communications. Link

*Efficient delivery of a DNA aptamer-based biosensor into plant cells for glucose sensing through thiol-mediated uptake. Mou Q…Lu Y. Science Advances. Link

Engineering a Synthetic Dopamine-Responsive Riboswitch for In Vitro Biosensing. Harbaugh SV…Chávez JL. ACS Synthetic Biology. Link

Circuits

*A microfluidic optimal experimental design platform for forward design of cell-free genetic networks. van Sluijs B…Huck WTS. Nature Communications. Link

Model-Based Design of a Synthetic Oscillator Based on an Epigenetic Methylation Memory System. Klingel V…Radde NE. ACS Synthetic Biology. Link

Computational Tools & Models

↑pySBOL3: SBOL3 for Python Programmers. Mitchell T, Beal J & Bartley B. ACS Synthetic Biology. Link

*Co-optimization of therapeutic antibody affinity and specificity using machine learning models that generalize to novel mutational space. Makowski EK…Tessier PM. Nature Communications. Link

*Recurrent neural networks enable design of multifunctional synthetic human gut microbiome dynamics. Baranwal M…Venturelli OS. eLife. Link

*Protein–protein interaction and non-interaction predictions using gene sequence natural vector. Zhao N…Gong X. Communications Biology. Link

EcoliGD: An Online Tool for Designing Escherichia coli Genome. Guo H…Guo F. ACS Synthetic Biology. Link

CRISPR & Central Dogma

*Repurposing CRISPR RNA-guided integrases system for one-step, efficient genomic integration of ultra-long DNA sequences. Chen Z-H…Yu H-Q. Nucleic Acids Research. Link

Directed Evolution

↑*Laboratory evolution of synthetic electron transport system variants reveals a larger metabolic respiratory system and its plasticity. Anand A…Palsson BO. Nature Communications. Link

Medicine & Diagnostics

*Endowing universal CAR T-cell with immune-evasive properties using TALEN-gene editing. Jo S…Valton J. Nature Communications. Link

Enhanced Myogenesis by Silencing Myostatin with Nonviral Delivery of dCas9 Ribonucleoprotein Complex. Chen Y…Lue TF. The CRISPR Journal. Link

*Clinical enrollment assay to detect preexisting neutralizing antibodies to AAV6 with demonstrated transgene expression in gene therapy trials. Cao L…Meyer K. Gene Therapy. Link

*Characterization of a thermostable Cas13 enzyme for one-pot detection of SARS-CoV-2. Mahas A…Mahfouz M. PNAS. Link

*Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells. Tasca F…Gonçalves MAFV. Nucleic Acids Research. Link

†Engineering the human gut commensal Bacteroides thetaiotaomicron with synthetic biology. Lai Y, Hayashi N & Lu TK. Current Opinion in Chemical Biology. Link

Plants

*Increasing the resilience of plant immunity to a warming climate. Kim JH…He SY. Nature. Link

*An Intein-Mediated Split–nCas9 System for Base Editing in Plants. Yuan G…Yang X. ACS Synthetic Biology. Link

Protein & Molecular Engineering

*SpySwitch enables pH- or heat-responsive capture and release for plug-and-display nanoassembly. Vester SK…Howarth M. Nature Communications. Link

*Switchable Control of Scaffold Protein Activity via Engineered Phosphoregulated Autoinhibition. Nikroo AH…Brunsveld L. ACS Synthetic Biology. Link

Synthetic Cells

Phase-specific RNA accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles. Choi S…Keating CD. Nature Chemistry. Link

Tools & Technology

*High-throughput total RNA sequencing in single cells using VASA-seq. Salmen F…van Oudenaarden A. Nature Biotechnology. Link

*Robotic search for optimal cell culture in regenerative medicine. Kanda GN…Natsume T. eLife. Link

*Coupling cell communication and optogenetics: Implementation of a light-inducible intercellular system in yeast. Rojas V & Larrondo LF. bioRxiv (preprint). Link

*A multiplexed, three-dimensional pooling and next-generation sequencing strategy for creating barcoded mutant arrays: construction of a Schizosaccharomyces pombe transposon insertion library. Li Y…Runge KW. Nucleic Acids Research. Link

Optimal LentiCRISPR-Based System for Sequential CRISPR/Cas9 Screens. Hutcheson RL, Hayes M & Sugden B. ACS Synthetic Biology. Link

Miscellaneous

*Insights from one thousand cloned dogs. Olsson PO…Hwang WS. Scientific Reports. Link

A genome and gene catalog of glacier microbiomes. Liu Y…Yao T. Nature Biotechnology. Link

*Enteric viruses replicate in salivary glands and infect through saliva. Ghosh S…Altan-Bonnet N. Nature. Link

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More soon,

— Niko

Twitter: @NikoMcCarty // Email: niko@newscience.org