Microbial production of the low-caloric sweetener D-allulose from D-glucose by evolutionary engineering

Through evolutionary engineering of *Corynebacterium glutamicum*, researchers developed a high-performance strain with enhanced XylA and IolT1 activities that enables efficient whole-cell conversion of D-glucose to the low-calorie sweetener D-allulose at mesophilic temperatures, rivaling traditional high-temperature immobilized enzyme processes.

The Gist

The Big Picture: Making a "Magic" Sweetener

Imagine you want to make a low-calorie sweetener called D-allulose. It tastes like sugar but has almost no calories, making it great for people watching their weight or blood sugar.

Currently, factories make this sweetener using giant machines with purified enzymes (biological catalysts) that need to be heated to very high temperatures (like a hot oven, around 60°C/140°F). This is expensive and energy-intensive.

The scientists in this paper asked: "Can we get tiny bacteria to do this job for us inside a simple tank at room temperature?"

The answer is yes, but they had to play "evolutionary tag" with the bacteria to make them fast enough.

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The Problem: The Bacteria Were Too Slow

The bacteria they chose, Corynebacterium glutamicum, is a workhorse used in industry for decades. However, it had two major problems when trying to turn Glucose (sugar) into Allulose:

1. The Doorkeeper was picky: The bacteria's "doors" (transporters) that let sugar inside were designed for a specific type of sugar. They didn't let enough Glucose or Fructose (the intermediate step) in fast enough.
2. The Worker was lazy: The enzyme inside the bacteria responsible for changing Glucose into Fructose (called XylA) was very slow and clumsy at room temperature. It usually works best in a hot oven, which the bacteria can't survive.

If the bacteria couldn't get sugar in fast enough, or if the worker inside couldn't process it quickly, the whole factory would shut down.

The Solution: Evolutionary Training Camp (ALE)

Instead of trying to design a perfect enzyme from scratch (which is like trying to build a Ferrari engine by hand), the scientists used Adaptive Laboratory Evolution (ALE).

Think of this as a survival training camp for the bacteria:

1. The Trap: They created a special strain of bacteria that could not eat sugar normally. The only way they could survive and grow was if they could turn Fructose back into Glucose using the slow "lazy" enzyme (XylA).
2. The Pressure: They put these bacteria in a tank with Fructose. If they were too slow, they starved. If they were fast enough, they multiplied.
3. The Evolution: Over a few weeks, the scientists kept taking the fastest-growing bacteria and moving them to a fresh tank. It was like a game of "survival of the fittest." The bacteria that happened to have random mutations making them faster survived and reproduced.

The Winners: Two Key Upgrades

After the training camp, the scientists looked at the DNA of the super-fast bacteria and found they had upgraded two specific parts of their machinery:

1. The "Super-Doors" (IolT1 Mutations)

The bacteria mutated their sugar transporters (the doors).

• The Analogy: Imagine the original door was a narrow, single-lane gate that clogged easily. The mutation turned it into a wide, high-speed turnstile that let sugar rush in 10 times faster.
• The Result: The inside of the bacteria became flooded with sugar, giving the workers plenty of raw material to work with.

2. The "Super-Worker" (XylA Mutations)

The enzyme that converts the sugar also got an upgrade.

• The Analogy: The original worker was like a slow, sleepy librarian sorting books. The mutation made them 9 times faster and more efficient.
• The Result: They could process the incoming sugar almost instantly.

The "Aha!" Moment: A Sneaky Side Effect

While testing the final factory, the scientists noticed something weird. The bacteria started growing too well on Glucose, which they weren't supposed to be able to do.

It turned out the bacteria had found a "backdoor." A different transporter, usually used for sucrose (table sugar), had mutated to accidentally let Glucose and Fructose in together.

• The Fix: The scientists simply "welded shut" this backdoor (deleted the gene) to force the bacteria to stick to the main production line. This ensured all the sugar went toward making the sweetener, not just growing the bacteria.

The Final Result: A Room-Temperature Factory

By combining these upgrades, the scientists created a bacterial strain that:

• Takes Glucose.
• Turns it into Fructose (using the Super-Worker).
• Turns Fructose into Allulose (using a second enzyme).
• All at 30°C (room temperature).

The Yield: They achieved a 15% yield. This is the same efficiency as the current industrial methods that use expensive, hot, purified enzymes.

Why This Matters

• Cheaper: No need to purify enzymes or heat the tanks to 60°C.
• Greener: Uses less energy.
• Simpler: It's a "one-pot" process where the bacteria do all the work.

In short: The scientists didn't build a better machine; they bred a better worker. They trained a microscopic factory to run a complex sugar conversion at room temperature, offering a cheaper and greener way to make low-calorie sweeteners for our food and drinks.

Technical Summary

1. Problem Statement

The industrial production of D-allulose (a low-calorie sweetener) from D-glucose typically requires a two-step enzymatic process:

1. Isomerization: D-glucose $\rightarrow$ D-fructose (catalyzed by D-glucose isomerase, XylA).
2. Epimerization: D-fructose $\rightarrow$ D-allulose (catalyzed by D-allulose 3-epimerase).

Key Bottlenecks:

• Low Mesophilic Activity: Native D-glucose isomerases have very low affinity ($K_m > 100$ mM) and poor activity toward D-glucose at mesophilic temperatures (30°C). Industrial processes currently operate at high temperatures (60–70°C) to compensate, which requires energy-intensive conditions and enzyme immobilization to prevent thermal degradation of the epimerase.
• Metabolic Constraints: In microbial hosts like Corynebacterium glutamicum, D-glucose is rapidly phosphorylated by the Phosphotransferase System (PTS) into D-glucose-6-phosphate, which is not a substrate for XylA. Furthermore, D-fructose uptake is often inefficient or results in export/re-import cycles.
• Goal: Develop a whole-cell biocatalyst capable of converting D-glucose to D-allulose efficiently at 30°C without enzyme purification or immobilization.

2. Methodology

The researchers employed Adaptive Laboratory Evolution (ALE) combined with Reverse Engineering and Molecular Dynamics (MD) Simulations.

• Host Strain Engineering:
  • Created a selection strain (Fruneg) of C. glutamicum where growth on D-fructose was strictly dependent on the isomerization of D-fructose to D-glucose by an exogenous XylA.
  • Deleted PTS transporters (ptsG, ptsF) to prevent phosphorylation of imported sugars.
  • Constitutively expressed the promiscuous H+-coupled transporter IolT1 to allow uptake of unphosphorylated D-glucose and D-fructose.
• Evolutionary Engineering:
  • Introduced seven different XylA genes into Fruneg.
  • Selected the best performers (Xanthomonas campestris XylA and Arthrobacter sp. XylA) and subjected them to ALE in D-fructose minimal media to select for faster growth.
• Reverse Engineering & Validation:
  • Sequenced evolved clones to identify mutations.
  • Reconstructed strains with specific mutations (single and combined) to confirm their individual and synergistic effects on growth and sugar uptake.
• Biophysical Analysis:
  • Kinetic Assays: Measured $k_{cat}$ and $K_m$ of purified XylA variants.
  • MD Simulations: Performed all-atom unbiased MD simulations (2 µs per replica) to understand how mutations in IolT1 and XylA affect structural stability, allosteric regulation, and ligand binding.
• Final Production Strain Construction:
  • Engineered a "Glucose-Negative" strain (Gluneg) by deleting glucokinases (glk, ppgK, nanK) and inositol dehydrogenase clusters to prevent D-glucose metabolism.
  • Integrated evolved XylA and IolT1 variants along with a D-allulose 3-epimerase (daeAT).
  • Identified and deleted a suppressor mutation in the sucrose transporter (ptsS) that inadvertently allowed D-glucose utilization.

3. Key Contributions & Results

A. Discovery of Mutations via ALE

Evolution of the selection strain yielded clones with significantly improved growth on D-fructose. Genomic analysis revealed mutations in only two targets:

1. IolT1 (Sugar Transporter): Two distinct mutations were found: G87S and T351P.
   • Effect: These mutations increased the transport activity for both D-glucose and D-fructose by approximately 10-fold.
   • Kinetics: Reduced the $K_s$ (half-saturation constant) for D-fructose from ~107 mM to ~70–90 mM and increased $\mu_{max}$ significantly.
   • Mechanism (MD Simulations): The mutations are distal to the binding site but act allosterically. They stabilize the "inward-open" conformation of the transporter and destabilize the "outward-open" state, facilitating the transport cycle. They also improved carbohydrate binding energy.
2. XylA (D-Glucose Isomerase):
   • Arthrobacter variant: Mutations (S2F or 5'-UTR change) led to a 9-fold increase in protein expression levels, thereby increasing total activity.
   • Xanthomonas variant: A single amino acid substitution W147S increased the catalytic efficiency ($k_{cat}/K_m$) for D-glucose by 9-fold (from 0.7 to ~6.3 $s^{-1}M^{-1}$) while slightly reducing xylose activity. A subsequent mutation (G15A) provided a marginal further improvement.
   • Mechanism (MD Simulations): W147S stabilizes the binding of D-glucose within the active site.

B. Synergistic Effect

Reverse engineering confirmed that neither the transporter nor the enzyme mutations alone were sufficient to match the performance of the evolved strains. The combination of improved uptake (IolT1) and improved isomerization (XylA) was required to overcome the intracellular bottlenecks.

C. D-Allulose Production

• Strain Construction: A Gluneg strain (unable to metabolize D-glucose) was equipped with the evolved IolT1-G87S, XylA-W147S (or G15A-W147S), and DaeAT (from Agrobacterium tumefaciens).
• Unexpected Phenomenon: The production strain initially regained the ability to grow on D-glucose due to a suppressor mutation in the sucrose transporter PtsS (A129G), which allowed co-transport of D-glucose and D-fructose.
• Optimization: Deleting ptsS prevented this parasitic growth, ensuring all D-glucose was directed toward D-allulose production.
• Performance:
  • Yield: Achieved a D-allulose yield of 15.1% from D-glucose at 30°C.
  • Comparison: This yield rivals industrial immobilized enzyme processes that operate at ~60°C.
  • High-Cell-Density: In high-cell-density biotransformations, a yield of ~11.4% was maintained.

4. Significance

• Process Innovation: This study demonstrates the first successful whole-cell microbial conversion of D-glucose to D-allulose at mesophilic temperatures (30°C) with yields comparable to high-temperature immobilized enzyme processes.
• Cost Reduction: By avoiding enzyme purification, immobilization, and high-temperature operation, the process significantly reduces energy consumption and capital/operational costs.
• Methodological Advance: It showcases the power of Adaptive Laboratory Evolution to simultaneously optimize enzyme kinetics and transporter promiscuity, a strategy that can be applied to other metabolic engineering challenges.
• Scientific Insight: The work provides deep mechanistic insights into how distal mutations in membrane transporters (IolT1) and enzymes (XylA) can allosterically enhance function, validated by extensive MD simulations.
• Future Applications: The evolved XylA variants and IolT1 transporters offer new tools for producing other D-fructose-derived products (e.g., disaccharides, furan-based chemicals) from D-glucose.

In conclusion, the authors successfully engineered a robust C. glutamicum chassis that overcomes the kinetic and thermodynamic limitations of D-glucose isomerization, enabling a sustainable, low-cost, and energy-efficient route to produce the low-calorie sweetener D-allulose.