Yeast Central Carbon Metabolism and Metabolic Engineering

Yeast central carbon metabolism (CCM) plays a pivotal role in cellular energy production and the synthesis of key metabolites. As an industrial workhorse, yeast's efficient conversion of carbon sources like glucose into energy and biosynthetic precursors makes it ideal for biotechnological applications, particularly in biofuel, pharmaceutical, and chemical production.

Yeast Central Carbon Metabolism: Pathways and Key Mechanisms

Yeast central carbon metabolism involves several interconnected pathways. These pathways convert glucose, the primary carbon source, into energy, essential cellular precursors, and metabolites. The three core processes that define CCM in yeast are glycolysis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, all of which contribute to maintaining cellular homeostasis and providing critical metabolites for biosynthesis.

Glycolysis: The Entry Point for Carbohydrate Catabolism

Glycolysis, also known as the Embden–Meyerhof–Parnas pathway, is the first step in yeast's breakdown of glucose to extract energy. In this anaerobic process, glucose is converted into two molecules of pyruvate, yielding a net gain of 2 ATP and 2 NADH molecules.

Key Enzymes:

• Hexokinase: Converts glucose to glucose-6-phosphate, initiating glycolysis.
• Phosphofructokinase: The rate-limiting enzyme of glycolysis, controlling the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate.
• Pyruvate kinase: Catalyzes the final step of glycolysis, converting phosphoenolpyruvate into pyruvate, generating ATP in the process.

Pentose Phosphate Pathway (PPP): NADPH and Ribose Biosynthesis

The PPP serves two primary functions: the generation of NADPH for reductive biosynthetic reactions, and the production of ribose-5-phosphate, an essential precursor for nucleotide biosynthesis. This pathway is particularly important for supporting cellular processes like fatty acid and isoprenoid biosynthesis.

Key Enzymes:

• Glucose-6-phosphate dehydrogenase: The rate-limiting enzyme in the oxidative branch of PPP, producing NADPH from glucose-6-phosphate.
• Transketolase: Facilitates the transfer of carbon units, contributing to the generation of ribose-5-phosphate and erythrose-4-phosphate.

The Tricarboxylic Acid Cycle: Energy Production and Biosynthesis

The TCA cycle, or Krebs cycle, occurs in the mitochondria and plays a central role in aerobic metabolism. It oxidizes pyruvate to CO₂ and water. This process generates high-energy intermediates like NADH and FADH₂, which are used in the electron transport chain to produce ATP.

Key Enzymes:

• Pyruvate dehydrogenase: Converts pyruvate into acetyl-CoA, the gateway to the TCA cycle.
• Citrate synthase: Initiates the TCA cycle by condensing acetyl-CoA with oxaloacetate to form citrate.
• Succinate dehydrogenase: Links the TCA cycle with oxidative phosphorylation through the production of FADH₂.

Fermentation: Anaerobic Metabolism and By-Product Formation

In the absence of oxygen, yeast shifts from oxidative phosphorylation to fermentation. Pyruvate is converted into ethanol and CO₂, a process that regenerates NAD⁺, allowing glycolysis to continue. This metabolic shift is critical for yeast survival under anaerobic conditions and is exploited in industrial fermentation processes, including alcohol production.

Key Enzymes:

• Pyruvate decarboxylase: Converts pyruvate to acetaldehyde, the first step in alcoholic fermentation.
• Alcohol dehydrogenase: Reduces acetaldehyde to ethanol.

Overall strategy of HIF-complex-mediated CCM redirection for the biosynthesis of triterpenoids (Lin et al., 2023).

Metabolic Engineering for Industrial Isoprenoid Production

The demand for sustainable alternatives to petrochemical-based products has driven the need for industrial-scale production of bio-based chemicals, particularly isoprenoids. These compounds, which include terpenes, carotenoids, and steroids, are valuable for their use in pharmaceuticals, biofuels, fragrances, and other specialty chemicals. Yeast, due to its efficient central carbon metabolism, is an ideal host for engineered isoprenoid production.

The Mevalonate and Methylerythritol Phosphate Pathways

Isoprenoids in yeast are primarily synthesized via two distinct pathways: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Both pathways produce isoprenoid precursors, such as isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are then condensed to form various isoprenoids.

• The Mevalonate Pathway (MVA):
  This pathway, primarily found in yeast, begins with the conversion of acetyl-CoA to mevalonate. Key enzymes in this pathway, such as HMG-CoA reductase and mevalonate kinase, are often overexpressed in engineered strains to enhance isoprenoid production.
• The Methylerythritol Phosphate Pathway (MEP):
  The MEP pathway, present in plants and some bacteria, operates in the plastids and uses glucose-derived metabolites (such as pyruvate and glyceraldehyde-3-phosphate) to generate IPP and DMAPP. This pathway is typically less active in yeast but can be engineered into yeast strains for isoprenoid production.

Metabolic Engineering Approaches

The efficiency of isoprenoid production in yeast is heavily influenced by metabolic engineering. Strategies to optimize yeast central carbon metabolism for high-yield production include:

• Overexpression of Key Enzymes:
  Enhancing the expression of enzymes involved in the MVA or MEP pathways, such as HMG-CoA reductase (for MVA) or 1-deoxy-D-xylulose-5-phosphate synthase (for MEP), can increase the availability of precursors for isoprenoid biosynthesis.
• Carbon Flux Optimization:
  Redirecting carbon flux from glycolysis and the TCA cycle towards isoprenoid biosynthesis is essential for maximizing yield. This often involves manipulating key regulatory enzymes to optimize precursor supply while minimizing the production of by-products like ethanol.
• Engineering Co-Factor Balance:
  Many isoprenoid biosynthetic reactions require specific co-factors, such as NADPH. Engineering pathways that enhance NADPH production, either by upregulating the PPP or through engineered enzymes, is crucial for improving isoprenoid yield.
• Overcoming By-Product Toxicity:
  Accumulation of by-products such as ethanol and acetate can inhibit yeast growth and productivity. Strategies to mitigate this include the engineering of stress-tolerant strains, optimization of fermentation conditions, and the development of novel fermentation strategies.

Industrial Applications of Isoprenoids

Isoprenoids are valuable in a wide range of industries:

• Pharmaceuticals: Compounds like taxol (a chemotherapy agent) and artemisinin (an anti-malarial drug) are derived from isoprenoids.
• Biofuels: Isoprenoids such as farnesene hold promise as renewable biofuels.
• Fragrance and Flavor: Terpenes like limonene and pinene are widely used in fragrances and flavorings.

Yeast's central carbon metabolism, when engineered to efficiently produce these compounds, provides a sustainable and scalable alternative to traditional chemical synthesis.

Biosynthesis and Metabolic Engineering: Interplay and Optimization

Engineering Yeast for High-Yield Biosynthesis

The industrial application of engineered yeast strains heavily depends on the ability to redirect metabolic flux toward desired biosynthetic pathways while maintaining cellular growth and energy production. This requires an integrated approach, combining classical metabolic engineering with modern synthetic biology tools, to optimize the flow of carbon and energy through key metabolic networks.

Redirection of Carbon Flux

A key goal in metabolic engineering of yeast is to redirect carbon flux toward biosynthetic pathways, such as isoprenoid production. This is typically achieved by overexpressing specific enzymes or entire pathways, including shifting glycolytic intermediates toward acetyl-CoA. Balancing fermentation and oxidative phosphorylation also plays a crucial role in directing energy toward anabolic processes.

A key strategy is to downregulate or knock out competing pathways. For example, knocking out the gene encoding pyruvate decarboxylase can reduce the formation of ethanol, thereby increasing the pool of pyruvate available for other biosynthetic processes. Similarly, enzymes involved in fermentation can be repressed under aerobic conditions to prioritize energy production through the TCA cycle, which is more efficient for cellular growth and precursor generation.

Overexpression of Biosynthetic Pathways

Metabolic engineering of yeast for isoprenoid production involves enhancing rate-limiting enzymes within the MVA and MEP pathways, such as overexpressing HMG-CoA reductase to boost precursor availability. Additionally, synthetic biology enables the creation of non-native biosynthetic pathways by introducing heterologous genes, such as plant-derived MEP pathways or terpene synthases from other microorganisms, to expand the range of isoprenoids and other bio-based chemicals that yeast can produce.

Metabolic engineering for the production of isoprenoid-based biofuels in a prokaryotic host (Phularaet al., 2016)

Metabolic Flux Analysis and Modeling

To efficiently engineer yeast for industrial-scale production, researchers need to understand how metabolic pathways are interconnected and how carbon flows through these pathways.

Metabolic flux analysis (MFA) is essential for understanding how carbon moves through yeast metabolism. It helps identify bottlenecks that limit production. When combined with flux balance analysis (FBA), these tools model metabolic networks, predict how genetic changes impact metabolism, and optimize pathways to increase the production of target molecules like isoprenoids, without harming cell growth.

For example, FBA can be used to identify which enzymes are limiting in the pathway leading to IPP production, allowing for targeted engineering efforts that increase enzyme expression or alter pathway regulation. These computational models guide the engineering process, ensuring that changes in one part of the metabolic network do not have unintended, negative consequences on overall cell metabolism.

Balancing Co-Factors and By-Products

Balancing target molecule production with cellular homeostasis is a key challenge in biosynthesis. Many biosynthetic reactions require co-factors like NADPH for reductive reactions and ATP for energy-intensive processes. Ensuring sufficient availability of these co-factors is crucial for high-yield production.

Engineering Co-Factor Regeneration

In engineered yeast, enhancing the regeneration of NADPH and ATP is essential for maintaining biosynthetic activity. The PPP, which generates NADPH, can be upregulated to support high demands for this co-factor in the MVA or MEP pathways. Similarly, optimizing the TCA cycle and mitochondrial function can increase the production of ATP, especially when large amounts of energy are required for biosynthesis.

Mitigating By-Product Formation

As metabolic flux is redirected toward the production of target compounds, by-product formation often becomes a significant issue. Common by-products like ethanol, acetic acid, and glycerol can accumulate and inhibit the growth of yeast cells, reducing overall productivity. Engineering strategies that reduce or eliminate the formation of unwanted by-products can significantly improve the yield of target metabolites.

For instance, knockout or downregulation of genes involved in by-product formation, such as alcohol dehydrogenase (which is involved in ethanol production), can free up carbon flux for isoprenoid biosynthesis. Additionally, adaptive laboratory evolution (ALE) can be used to select strains that tolerate higher concentrations of by-products, enhancing strain robustness.

Systems Biology Approaches

Systems biology plays an increasingly important role in optimizing metabolic engineering strategies. This approach combines high-throughput omics technologies (such as genomics, transcriptomics, proteomics, and metabolomics) to analyze the entire metabolic network. By integrating these data with computational models, systems biology allows for a more holistic understanding of how genetic modifications influence global metabolism.

Omics Technologies

• Genomics provides detailed insights into the entire genome of engineered yeast strains, identifying potential targets for modification.
• Transcriptomics reveals gene expression changes that occur in response to modifications in the metabolic network, highlighting bottlenecks in specific pathways.
• Proteomics and metabolomics allow for the identification of proteins and metabolites that play a key role in the production of isoprenoids, facilitating the detection of enzymes or intermediates that limit productivity.

Dynamic Control Systems

Systems biology also allows for the development of dynamic control systems that can continuously optimize the metabolic network in response to real-time changes in environmental conditions or metabolic flux. This can be particularly useful in continuous fermentation processes, where yeast must adjust its metabolism to shifting nutrient concentrations or process parameters.

By integrating omics data with machine learning algorithms, researchers can predict the impact of genetic and environmental changes on product yield, enabling a more efficient and iterative process of strain optimization.

High-Throughput Screening and Synthetic Biology Tools

Finally, high-throughput screening (HTS) and synthetic biology tools are crucial for optimizing biosynthesis pathways and identifying optimal strains. HTS allows researchers to rapidly test thousands of genetic variants, enabling the identification of yeast strains with the highest production rates or tolerance to by-products.

Synthetic biology tools, such as modular pathway design and gene synthesis, provide a streamlined approach to engineer complex biosynthetic pathways. These tools enable the creation of novel biosynthetic routes that can be fine-tuned for maximum efficiency, providing an avenue for producing a wider variety of bio-based chemicals.

Future Directions in Yeast Central Carbon Metabolism and Industrial Applications

As yeast continues to be a critical platform for the production of bio-based chemicals, including isoprenoids, ongoing research and development are essential to overcome existing limitations and meet the ever-growing industrial demands. The future of yeast central carbon metabolism and its industrial applications will be shaped by advanced technologies in metabolic engineering, systems biology, and synthetic biology.

Multitrophic Metabolism: Expanding Yeast's Capabilities

One of the most promising directions in metabolic engineering is the optimization of yeast for multitrophic metabolism. This approach allows engineered strains to simultaneously produce multiple bio-based chemicals from a single carbon feedstock. Rather than being constrained to a single biosynthetic pathway, these strains could be designed to dynamically distribute carbon flux across several pathways, maximizing the overall yield of valuable metabolites.

For example, the ability to produce both biofuels and high-value industrial chemicals like isoprenoids or amino acids simultaneously would allow for more efficient utilization of available carbon. This strategy may require integrated metabolic networks that are highly flexible, enabling yeast to shift carbon allocation in response to changing growth conditions, nutrient availability, or production needs.

Advanced Metabolic Engineering Tools and Strategies

The field of metabolic engineering is rapidly advancing with new tools that allow for more precise and efficient manipulation of metabolic pathways. Techniques such as CRISPR/Cas9, base editing, and prime editing enable researchers to perform high-fidelity gene editing, leading to more stable and reproducible genetic modifications in yeast. This opens new possibilities for designing yeast strains with enhanced isoprenoid production capacity or improved tolerance to metabolic by-products.

In addition to genome editing, adaptive laboratory evolution (ALE) is a promising approach to optimizing yeast strains for industrial applications. By subjecting yeast to selective pressures (such as high concentrations of ethanol or other toxic metabolites), researchers can evolve strains with improved resilience, productivity, and resistance to metabolic bottlenecks.

Furthermore, synthetic biology is playing a significant role in creating novel yeast strains capable of producing non-native compounds, including engineered isoprenoids and other bio-based chemicals. By assembling synthetic pathways and incorporating new genetic circuits, researchers can introduce heterologous genes from other organisms or even design entirely new pathways to meet specific industrial requirements.

Metabolic Flux Analysis and Optimization

MFA is a crucial tool in understanding the dynamics of central carbon metabolism in engineered yeast strains. By quantitatively analyzing the flow of metabolites through different pathways, MFA provides insights into bottlenecks, inefficiencies, and areas where metabolic flux can be redirected for optimized production. This enables researchers to design more efficient strains by targeting key enzymes or regulatory mechanisms that control carbon allocation.

Incorporating computational models, such as flux balance analysis (FBA) or constraint-based optimization, can guide the rational design of engineered strains, allowing for the prediction and fine-tuning of metabolic networks. These models can simulate the impact of genetic modifications, fermentation conditions, and nutrient supply on the overall metabolic performance, ultimately helping to scale up production processes for industrial applications.

Sustainability and Green Chemistry in Industrial Biotechnology

As industries seek to reduce fossil fuel dependence, yeast-based biotechnology offers a sustainable solution. Engineered yeast can produce biofuels, chemicals, and materials from renewable resources, like plant-derived sugars, without toxic solvents or harsh conditions. By optimizing central carbon metabolism, yeast can efficiently produce bio-based isoprenoids and other chemicals, aligning with the demand for cleaner production methods. Additionally, the scalability of yeast fermentation processes facilitates the rapid transition from lab-scale to large-scale industrial production, making bio-based isoprenoid production both cost-effective and environmentally sustainable.

Yeast as a Platform for Circular Economy

Yeast-based biotechnology also supports the circular economy by utilizing waste carbon sources, such as agricultural residues or CO₂ from industrial emissions. Engineered yeast strains can convert these waste streams into valuable biofuels and isoprenoids, helping reduce greenhouse gas emissions and reliance on fossil raw materials. This approach not only mitigates environmental impact but also contributes to sustainable bio-based chemical production.

Integration of Microbial Consortia and Co-Cultures

Optimizing individual yeast strains is one approach, but microbial consortia or co-cultures also show great promise for improving industrial isoprenoid production. By combining yeast with other microorganisms like bacteria or fungi, we can create systems where each organism complements the metabolic abilities of the others. For example, certain bacteria may efficiently utilize lignocellulosic biomass, while yeast can convert the intermediate metabolites into isoprenoids.

These microbial consortia can enable more efficient and versatile bioprocesses, as each organism can be engineered to perform specific tasks, reducing metabolic load and enhancing overall productivity. This approach also allows for the use of a broader range of feedstocks, including complex or heterogeneous materials, which may not be suitable for a single microbial species.

References

1. Meadows, Adam L., et al. "Rewriting yeast central carbon metabolism for industrial isoprenoid production." Nature 537.7622 (2016): 694-697.
2. Lin, Xiaona, et al. "Optimization of central carbon metabolism by Warburg effect of human cancer cell improves triterpenes biosynthesis in yeast." Advanced Biotechnology 1.4 (2023): 4.
3. Phulara, Suresh & Chaturvedi, Preeti & Gupta, Pratima. Isoprenoid-based Biofuels: Homologous and Heterologous Expressions in Prokaryotes. Applied and Environmental Microbiology. (2016).82. AEM.01192-16.