# Cell-Free-Based Thermophilic Biocatalyst for the Synthesis of Amino Acids from One-Carbon Feedstocks

## TL;DR

This paper presents a new cell-free biocatalyst that efficiently converts one-carbon feedstocks into amino acids like serine and glycine.

## Contribution

The first use of a thermophilic pathway in E. coli lysate-based cell-free systems to create a high-yield biocatalyst for amino acid synthesis.

## Key findings

- The thermophilic biocatalyst achieved 97% stoichiometric yield of serine and glycine from formate and bicarbonate.
- The system outperformed previous mesophilic and purified enzyme systems by tripling and quadrupling the amino acid yield, respectively.
- Cofactor usage for THF and NADPH could be reduced fivefold without significantly affecting product yields.

## Abstract

Bioproduction from
one-carbon compounds, such as formate, is an
attractive prospect due to reduced energy requirements and the possibility
for using CO2 as a sustainable feedstock. Formate-fixing
pathways engineered using Escherichia coli lysate-based cell-free expression (CFE) biocatalysts have the potential
to route 100% of feedstock carbon toward chemical synthesis but are
undermined by siphoning of in-pathway metabolites and cofactors by
the CFE background metabolism. To address this limitation, we engineer
a CFE-based thermophilic multienzyme biocatalyst for the synthesis
of serine and glycine from formate, bicarbonate, and ammonia. After
expression of the thermophilic formate-to-serine pathway in a one-pot
reaction, the mesophilic E. coli CFE
background machinery is removed by simple heat denaturation, eliminating
the siphoning of cofactors, in-pathway metabolites, and products.
After bioprocess optimization, including pathway gene expression duration
and chemical synthesis temperature, we achieve near stoichiometric
conversion of formate and bicarbonate to serine and glycine, reaching
97% of stoichiometric yield. The use of a moderately thermophilic
biocatalyst allowed chemical synthesis to take place at mesophilic
temperatures, enabling the balance of optimal enzyme activity with
minimal metabolite/cofactor thermal degradation. In a fed-batch experiment,
the biocatalyst shows sustained chemical synthesis rates for 8 h,
paving the way toward a continuous bioprocess. Finally, a sensitivity
analysis of cofactor usage revealed that the most expensive cofactors,
THF and NADPH, can be reduced by 5-fold without significantly lowering
product yields. To the best of our knowledge, this is the first instance
of expressing a thermophilic pathway in an E. coli lysate-based CFE system to generate a thermophilic biocatalyst for
use at mesophilic temperatures. The CFE-based thermophilic formate-to-serine
biocatalyst triples the combined serine and glycine yield previously
obtained by a CFE-based mesophilic formate-to-serine biocatalyst (30%),
and quadruple the yield obtained by a purified enzyme system (22%).
Ultimately, this work opens the door to using E. coli lysate-based CFE for thermophilic biocatalyst generation to achieve
high chemical synthesis yields.