Engineering Microbes to Create Powerful Natural Preservatives
Imagine a natural food preservative so effective it can inhibit the growth of mold, bacteria, and yeast while being produced through sustainable manufacturing methods.
This isn't science fiction—it's the reality of D-phenyllactic acid (D-PLA), a compound with extraordinary antimicrobial properties that's found naturally in honey and some fermented foods 4 . For decades, scientists have recognized its potential to revolutionize food safety, but there was one significant challenge: producing it efficiently in its most active form.
Like left and right hands, molecules can have mirror-image forms with different properties
Traditional methods produced mixtures of both D and L forms, reducing effectiveness
The problem lies in molecular handedness, a property known as chirality. Like your left and right hands, some molecules exist in two mirror-image forms that are structurally identical but functionally different. This is the case with phenyllactic acid, where the D-form demonstrates significantly higher antimicrobial activity than its L-form counterpart 4 . While traditional methods produced mixtures of both forms, recent breakthroughs in protein engineering have enabled scientists to create highly efficient biological factories that produce exclusively the powerful D-PLA.
This article explores how researchers used cutting-edge structure-guided enzyme design to transform a common bacterial enzyme into a highly specialized factory for producing enantiomerically pure D-phenyllactate, paving the way for broader application of this natural preservative in our food supply and beyond.
D-phenyllactic acid (D-PLA) represents a new generation of antimicrobial compounds that align with consumer demands for natural, safe food ingredients. Research has demonstrated its broad-spectrum activity against diverse microorganisms, including both bacteria and fungi 1 .
Natural Antimicrobial Compound
Effective against diverse microorganisms including bacteria and fungi 1 .
Found naturally in honey and fermented foods, ideal for clean-label products 4 .
Used in pharmaceuticals, animal feed, and biodegradable plastics 2 .
Unlike many chemical preservatives, D-PLA occurs naturally in foods like honey and fermented products, making it particularly attractive for "clean-label" products 4 .
The compound's antimicrobial mechanism involves damaging microbial cell membranes and preventing biofilm formation, effectively controlling unwanted growth without the potential side effects associated with synthetic preservatives 4 . This broad activity profile means it could protect against a wide range of food spoilage organisms and pathogens with a single compound.
Beyond food preservation, D-PLA shows promising applications in pharmaceuticals, animal feed, and even as a building block for biodegradable plastics 2 . Its unique structure makes it suitable for creating poly(phenyllactic acid), a biopolymer with excellent thermal stability and ultraviolet absorption properties 5 . These diverse applications have fueled significant interest in developing efficient production methods for this versatile compound.
In nature, lactic acid bacteria serve as miniature factories for phenyllactic acid production. These bacteria, commonly found in fermented foods and dairy products, possess a two-step enzymatic pathway for converting phenylalanine to PLA 2 :
The amino acid phenylalanine is converted to phenylpyruvic acid (PPA) through the transfer of an amino group, catalyzed by aminotransferase enzymes.
PPA is transformed into PLA through the action of lactate dehydrogenase (LDH) enzymes, which specifically produce either the D- or L-form depending on the enzyme type.
While this natural pathway works efficiently for the bacteria's own metabolic needs, it doesn't produce the volumes required for commercial applications. Traditional microbial fermentation without precursor supplementation yields relatively low amounts of PLA—the highest reported titer reaching only about 400 mg/L 2 . This limitation prompted scientists to look for ways to enhance and optimize the production process.
To overcome nature's production limitations, researchers turned to protein engineering, specifically focusing on D-lactate dehydrogenase (D-LDH), the enzyme responsible for the final step of D-PLA production 1 . This enzyme naturally converts pyruvate to D-lactate but can also process the bulkier phenylpyruvate to produce D-PLA, albeit with lower efficiency.
The engineering strategy employed structure-guided design, a sophisticated approach that combines computational modeling with experimental validation:
Researchers first docked phenylpyruvate molecules into the active center of D-LDH to visualize how the substrate interacts with the enzyme 1 .
They identified amino acid residues surrounding the benzene ring of phenylpyruvate that might create tight packing constraints.
Based on this analysis, they selected specific residues for mutation to create more space for the bulkier phenylpyruvate substrate.
Through this process, the research team discovered that a single mutation—M307L—created an enzyme with significantly increased activity toward phenylpyruvate 1 . By replacing methionine with leucine at position 307, they reduced steric hindrance in the substrate-binding pocket, allowing the enzyme to more efficiently accommodate and process the phenylpyruvate substrate.
In the groundbreaking 2016 study that advanced the field, researchers undertook a systematic approach to engineer and validate an improved D-lactate dehydrogenase 1 . This experiment demonstrated how a single precise mutation could dramatically enhance the enzyme's capability to produce D-PLA.
The researchers began with DLDH744, a D-lactate dehydrogenase from Sporolactobacillus inulinus CASD. Using protein structure visualization software, they created a three-dimensional model of the enzyme's active site with phenylpyruvate docked in position.
Analysis revealed that methionine at position 307 (M307) created potential steric clashes with the benzene ring of phenylpyruvate. They hypothesized that replacing this bulky methionine with smaller leucine (M307L) would create more favorable packing.
The researchers introduced the M307L mutation into the DLDH744 gene and expressed both the wild-type and mutant enzymes in Escherichia coli cells, then purified them for characterization.
The team compared the ability of wild-type and mutant enzymes to convert phenylpyruvate to D-PLA in bioconversion experiments, measuring both the rate of conversion and the final concentration achieved.
The engineered M307L variant demonstrated remarkable superiority over the wild-type enzyme. When employed in fed-batch biotransformation processes, the mutant enzyme achieved a D-PLA titer of 21.43 g/L with a productivity of 1.58 g/L/h—ranking as the highest production by a D-lactate dehydrogenase reported at that time 1 .
Perhaps even more impressive was the exceptional optical purity of the product, with an enantiomeric excess value greater than 99.7% 1 . This demonstrated that the mutation not only improved efficiency but maintained perfect stereospecificity—a crucial requirement for obtaining the more potent D-enantiomer.
| Parameter | Wild-type Enzyme | M307L Mutant |
|---|---|---|
| D-PLA Production | Lower efficiency | 21.43 g/L |
| Productivity | Not reported | 1.58 g/L/h |
| Optical Purity | Not specified | >99.7% ee |
| Conversion Yield | Not optimal | High efficiency |
The success of engineering D-LDH for efficient D-PLA production represents more than just a technical achievement—it demonstrates the power of rational protein design to overcome limitations in natural biocatalysts. This approach has implications far beyond this specific application, providing a template for optimizing enzymes for various industrial processes 1 .
Metal-organic frameworks enhance enzyme stability and reusability 7 .
Direct production from glucose rather than expensive precursors .
Template for optimizing enzymes for various industrial processes.
Recent advances have built upon this foundation, exploring innovative production systems such as metal-organic framework (MOF)-encapsulated enzymes that enhance stability and reusability 7 , and metabolic engineering strategies that enable microbes to produce D-PLA directly from glucose rather than expensive precursors . These developments promise to make D-PLA more accessible and cost-effective for widespread application.
The growing demand for natural preservatives and sustainable manufacturing processes ensures that research in this field will continue to advance. As one team of researchers noted, structure-guided design of enzymes like D-LDH "will provide referential information for further engineering other 2-hydroxyacid dehydrogenases, which are useful for a wide range of fine chemical synthesis" 1 .
The journey to efficient D-phenyllactate production illustrates how interdisciplinary approaches combining computational modeling, biochemistry, and microbial engineering can solve complex challenges in sustainable manufacturing. By understanding and optimizing the tiny molecular machines that nature provides, scientists have developed an efficient process to produce a powerful natural preservative with applications spanning food safety, medicine, and materials science.
This breakthrough in enzyme engineering represents more than just a technical achievement—it's a step toward a future where we can harness nature's sophistication to create effective, safe, and sustainable alternatives to synthetic chemicals. As research progresses, we can expect to see more examples of this powerful approach applied to other challenges at the intersection of biotechnology and sustainable manufacturing.