# Deletion of succinic semialdehyde dehydrogenase sad and chromosomal expression of phosphoenolpyruvate carboxylase as metabolic requirements for improved production of 2,4-dihydroxybutyric acid via malyl-P pathway using E. coli

## TL;DR

This paper describes how deleting a specific enzyme and modifying another in E. coli improves the production of a chemical precursor using a synthetic metabolic pathway.

## Contribution

The study identifies key metabolic modifications for efficient DHB production via the MalP pathway in E. coli.

## Key findings

- Deleting the Sad gene increased DHB production by 3-fold compared to the wildtype host.
- Chromosomal expression of the mutant ppc K620S gene further improved DHB yield to 0.22 mol mol-1.
- The optimized pathway achieved 17% of the theoretical maximum yield under aerobic conditions.

## Abstract

The fermentative production of the functional precursor 2,4-dihydroxybutyrate (DHB) enables sustainable synthesis of the methionine analogue hydroxy-4-(methylthio) butyrate, which is currently still produced from fossil fuels. In this work, we aimed to optimize the aerobic production of DHB from glucose through the synthetic malyl phosphate (MalP) pathway, which comprises the conversion of the natural TCA cycle intermediate malate into MalP and the subsequent reactions to yield malate semialdehyde (MalSA) and finally DHB. We first implemented the synthetic pathway in an engineered Escherichia coli strain previously reported to over-produce malate through the oxidative TCA cycle. However, DHB was only detected in trace amounts, while acetate and malate were secreted in high quantities. Subsequent construction of strains producing malate, but negligible amounts of acetate, revealed that an increased supply of malate alone is not sufficient for improved production of DHB. Instead, we discovered metabolic inefficiencies in the DHB pathway as we found that deleting the endogenous succinate semialdehyde dehydrogenase Sad, whose natural substrate is structurally similar to MalSA, strongly improved performance of the DHB pathway. Specifically, with the single knock-out of sad we could achieve a 3-fold increase in DHB production with a yield of 0.15 mol mol-1 compared to the wildtype host in shake flask experiments. With additional chromosomal expression of the mutant ppc

K620S
gene encoding the malate-insensitive phosphoenolpyruvate carboxylase under control of a weak constitutive promoter, we achieved a DHB yield of 0.22 mol mol-1, which corresponds to 17% of the maximal yield under aerobic conditions.