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Microorganisms rely on proteins as an essential carbon and energy source, particularly in environments with limited polysaccharides or lipids. However, proteins are too large to cross the plasma membrane unaided, necessitating enzymatic degradation. Microbes secrete extracellular proteases and peptidases that hydrolyze proteins into peptides, which can then be transported across the membrane. Once inside the cell, intracellular proteases degrade these peptides into free amino acids, which...
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Amino acid biosynthesis is essential for cell growth, protein synthesis, and metabolic regulation. Cells generate essential and non-essential amino acids from metabolic intermediates to sustain vital biological functions. These intermediates originate from key metabolic pathways: glycolysis, the tricarboxylic acid (TCA) cycle, and the pentose phosphate pathway. Important precursors include α-ketoglutarate, pyruvate, oxaloacetate, phosphoenolpyruvate, and erythrose-4-phosphate, which...
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Carbon Negative Synthesis of Amino Acids Using a Cell-Free-Based Biocatalyst.

Shaafique Chowdhury1, Ray Westenberg1,2, Kimberly Wennerholm3

  • 1School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States.

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Summary

Researchers developed a cell-free biocatalyst for carbon-negative amino acid synthesis from CO2 equivalents. This system efficiently produces glycine and serine, overcoming limitations of traditional biological methods for industrial applications.

Keywords:
carbon negative synthesiscell-free expression systemsmetabolic engineeringvalue-added chemicals

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Area of Science:

  • Synthetic biology and metabolic engineering.
  • Biocatalysis and enzyme engineering.
  • Carbon capture and utilization (CCU).

Background:

  • Biological systems can convert carbon dioxide (CO2) into chemicals, but natural CO2 fixation rates are too slow for industrial use.
  • Engineered metabolic pathways in organisms have limitations, including slow carbon fixation and carbon allocation between growth and synthesis.
  • Previous attempts using engineered organisms for CO2 fixation resulted in cell growth but no chemical synthesis.

Purpose of the Study:

  • To engineer a cell-free expression (CFE)-based multienzyme biocatalyst for carbon-negative synthesis of glycine and serine from CO2 equivalents.
  • To overcome the limitations of slow CO2 fixation rates and inefficient product synthesis seen in whole-cell systems.
  • To establish a novel biocatalytic platform for producing valuable chemicals from CO2.

Main Methods:

  • Developed a lysate-based cell-free expression (CFE) system utilizing a multienzyme biocatalyst.
  • Incorporated tetrahydrofolate (THF)-dependent formate fixation, reductive glycine synthesis, and serine synthesis pathways.
  • Implemented phosphite dehydrogenase-dependent NAD(P)H regeneration to drive reactions and optimized gene ratios and cofactor recycling.

Main Results:

  • Achieved a 30% conversion of formate to serine and glycine using the CFE-based biocatalyst, exceeding previous purified enzyme system efficiency (22%).
  • Demonstrated biocatalyst activity even after 200-fold dilution, allowing higher substrate loading without increased cell lysate cost.
  • Identified NAD(P)H regeneration as crucial for driving reactions near thermodynamic equilibrium and efficient THF recycling reduced cofactor costs.

Conclusions:

  • This study presents the first synthesis of amino acids using a CFE-based biocatalyst that captures CO2 equivalents for carbon-negative production.
  • The CFE system offers advantages in efficiency, substrate loading, and cost-effectiveness compared to whole-cell or purified enzyme systems.
  • The CFE platform holds potential for extension to other valuable chemicals like pyruvate, enabling broader applications in chemical synthesis from CO2.