A Green Recipe for Malic Acid
How metabolic engineering transforms Mannheimia succiniciproducens into an efficient malic acid factory using DMSO as an electron acceptor
Explore the ScienceImagine a world where the chemicals in our everyday products—from the tangy taste in our candy to the effective ingredients in our skincare—are no longer derived from petrochemicals, but are instead brewed sustainably by microscopic bacteria.
This is not a scene from a science fiction novel, but the reality being crafted today in biotechnological laboratories around the world.
At the forefront of this revolution is a remarkable bacterium known as Mannheimia succiniciproducens, a microbe originally isolated from the rumen of a Korean cow. Recent scientific breakthroughs have supercharged this natural factory, enabling it to produce large quantities of malic acid, a key industrial chemical, with unprecedented efficiency. The secret to this enhanced production? An unexpected substance called dimethylsulfoxide (DMSO), which helps the bacterium breathe new life into green chemistry 1 4 .
This article delves into the fascinating science of metabolic engineering, exploring how researchers are reshaping the very core of microbial metabolism to turn them into efficient cell factories for a more sustainable future.
Mannheimia succiniciproducens is no ordinary microbe. It is a capnophilic bacterium, meaning it "loves" carbon dioxide (CO₂). This trait is a golden ticket for sustainable bioproduction, as it allows the bacterium to capture and utilize this greenhouse gas as a key raw material 5 6 .
Naturally, M. succiniciproducens possesses a exceptionally strong reductive branch of the TCA cycle (the tricarboxylic acid cycle, a central hub of metabolism in most living cells). This natural metabolic pathway is like a biological superhighway for producing C4 dicarboxylic acids, including succinic and malic acid 1 8 . For the bacterium, this pathway is crucial for survival, as it is directly linked to generating energy (ATP) under anaerobic (oxygen-free) conditions 1 . Scientists saw the potential to re-route this native superhighway to make it an industrial-scale production line for malic acid.
Capnophilic nature allows utilization of greenhouse gas as raw material
Natural superhighway for producing C4 dicarboxylic acids
To transform M. succiniciproducens into a malic acid specialist, researchers employed a sophisticated strategy known as systems metabolic engineering. This approach goes beyond traditional genetic tinkering, integrating tools from systems biology and synthetic biology to overhaul the cell completely 2 . The process involved three key interventions, which are summarized in the table below.
| Engineering Strategy | Genetic Modification | Purpose and Function |
|---|---|---|
| Pathway Reconstruction | Elimination of the fumarase enzyme | To block the conversion of malic acid into fumarate, effectively trapping the desired product in the cell 1 4 . |
| Respiration Reconstruction | Introduction of DMSO reductase from A. succinogenes | To provide a new pathway for electron transport, using DMSO as an electron acceptor to boost cell growth and energy levels 1 4 . |
| Membrane Engineering | Employment of cis-trans isomerase from P. aeruginosa | To increase the fluidity and stability of the cell membrane, enhancing the bacterium's tolerance to high levels of malic acid 1 4 . |
The use of Dimethylsulfoxide (DMSO) is a particularly clever part of this strategy. Under anaerobic conditions, the bacterium's growth is limited because it struggles to dispose of the electrons generated during metabolism. Its natural solution is to use fumarate reduction, a step that unfortunately converts our target product, malic acid, into something else 1 .
By introducing a DMSO reductase gene from another efficient bacterium, Actinobacillus succinogenes, engineers gave M. succiniciproducens a new and highly efficient "electron sink" 1 4 . The engineered bacterium could now use DMSO as an electron acceptor, a process that supported better growth and, crucially, uncoupled energy production from the consumption of malic acid. This meant more malic acid could accumulate without hindering the cell's vitality.
DMSO serves as an alternative electron acceptor, enabling the bacterium to maintain energy production without consuming the valuable malic acid product.
The proof of any engineering feat is in its performance. A pivotal study published in Biotechnology and Bioengineering detailed the fed-batch fermentation of the final engineered strain, putting it to the ultimate test of productivity and efficiency 1 4 .
The researchers cultivated the engineered M. succiniciproducens in a controlled bioreactor, using a high-inoculum fed-batch fermentation process. This method involves starting with a large number of cells and carefully feeding them nutrients throughout the fermentation to maintain optimal production conditions 1 .
The results were record-breaking. The final engineered strain achieved a remarkable malic acid titer of 61 g/L and, most importantly, an overall productivity of 2.27 g/L/h 1 4 . This productivity was the highest ever reported for malic acid at the time of the study, demonstrating a highly efficient and potentially industrially viable process.
| Final Malic Acid Titer | 61 g/L |
| Overall Productivity | 2.27 g/L/h |
| Electron Acceptor | Dimethylsulfoxide (DMSO) |
The success of this experiment hinged on the synergistic effect of all three engineering strategies. The elimination of fumarase ensured the produced malic acid was not consumed. The introduction of the DMSO reductase enabled robust cell growth by providing a new electron acceptor, addressing the fundamental metabolic limitation. Finally, the membrane engineering allowed the cells to withstand the high concentrations of malic acid they were producing, preventing self-toxicity and allowing for accumulation to high levels 1 4 .
This holistic approach is a hallmark of modern systems metabolic engineering, where the cell is optimized not just for a single reaction, but as an entire production system.
Building an efficient microbial factory requires a suite of specialized tools and reagents. The following table outlines some of the key components used in this field, drawing from the featured experiment and broader metabolic engineering practices.
| Tool/Reagent | Function and Application |
|---|---|
| Dimethylsulfoxide (DMSO) | Acts as an alternative electron acceptor in anaerobic respiration, enhancing microbial growth and metabolic efficiency 1 4 . |
| Genome-Scale Metabolic Models (GEMs) | Computational models that simulate entire cellular metabolism, used to predict gene knockout targets and optimize metabolic fluxes 2 . |
| Serine Recombinase Toolkit | Enables efficient, marker-free integration of multiple DNA constructs into bacterial genomes, even in non-model organisms 2 . |
| Alkaline Neutralizers (e.g., Mg(OH)₂) | Used in fermentation to maintain optimal pH. Mg²⁺ ions also act as essential cofactors for enzymes and can significantly boost production 6 8 . |
| Ion Transporters (e.g., CorA) | Membrane proteins that facilitate the uptake of crucial ions like Mg²⁺. Engineering more efficient transporters can enhance cofactor supply and overall process robustness 6 . |
Identification of Mannheimia succiniciproducens as a promising host due to its natural metabolic capabilities 5 6 .
Using genome-scale models to identify key metabolic bottlenecks and engineering targets 2 .
Implementation of three key engineering strategies: pathway reconstruction, respiration reconstruction, and membrane engineering 1 4 .
The successful engineering of Mannheimia succiniciproducens to produce malic acid using DMSO is more than just a laboratory achievement; it is a beacon for the future of industrial biotechnology. It demonstrates a powerful framework for manipulating the complex machinery of life to serve human needs in an environmentally conscious way.
The strategies pioneered here—pathway reconstruction, respiration engineering, and membrane fortification—are now being applied to produce a wide array of industrially important chemicals 1 2 . As tools like machine learning and advanced genome editing continue to evolve, the design and optimization of these microbial cell factories will only accelerate 2 . The humble rumen bacterium, enhanced by human ingenuity, is helping to pave the way for a world where our chemicals are not extracted from the ground, but are grown sustainably in a vat.
Reduces reliance on petrochemicals
Record-breaking productivity of 2.27 g/L/h
Uses CO₂ as a raw material