Metabolic engineering of Halomonas campaniensis strain XH26 to remove competing pathways to enhance ectoine production

Abstract

Ectoine has gained considerable attention as a high-value chemical with significant application potential and market demand. This study aimed to increase ectoine yields by blocking the metabolic shunt pathway of L-aspartate-4-semialdehyde, the precursor substrate in ectoine synthesis. The homoserine dehydrogenase encoded by hom in H. campaniensis strain XH26 is responsible for the metabolic shunt of L-aspartate-4-semialdehyde to glycine. CRISPR/Cas9 technology was used to seamlessly knockout hom, blocking the metabolic shunt pathway to increase ectoine yields. The ectoine yield of XH26/Δhom was 351.13 mg (g CDW)^-1 after 48 h of incubation in 500 mL shake flasks using optimal medium with 1.5 mol L^-1 NaCl, which was significantly higher than the 239.18 mg (g CDW)^-1 of the wild-type strain. Additionally, the absence of the ectoine metabolic shunt pathway affects betaine synthesis, and thus the betaine yields of XH26/Δhom was 19.98 mg (g CDW)^-1, considerably lower than the 69.58 mg (g CDW)^-1 of the wild-type strain. Batch fermentation parameters were optimized, and the wild-type strain and XH26/Δhom were fermented in 3 L fermenters, resulting in a high ectoine yield of 587.09 mg (g CDW)^-1 for the defective strain, which was significantly greater than the ectoine yield of 385.03 mg (g CDW)^-1 of the wild-type strain. This study showed that blocking the metabolic shunt of synthetic substrates effectively increases ectoine production, and a reduction in the competitively compatible solute betaine appears to promote increased ectoine synthesis.

Figure 1

Hypothesized effects of hom knock out on the ectoine biosynthesis pathway of Halomonas strain XH26. lysC: aspartate kinase; asd: aspartate-semialdehyde dehydrogenase; ectB: diaminobutyrate-2-oxoglutarate transaminase; ectA: L-2,4-diaminobutyric acid acetyltransferase; ectC: L-ectoine synthase; ectD: ectoine hydroxylase; hom: homoserine dehydrogenase; thrB: homoserine kinase; thrC: threonine synthase; ltaE: threonine aldolase. The dashed lines represent substances exported or imported by other metabolic pathways. The colored boxes correspond to metabolic pathways.

Figure 2

Diagram of the ectoine metabolism-related pathway of H. campaniensis XH26 in this study. The ectoine metabolism-related pathway of H. campaniensis XH26 was reconstructed based on salt-induced transcriptome analysis.

Figure 3

Mapping of differential expression levels of genes related to ectoine synthesis. RNA-seq and qRT-PCR results showing the expression changes of ectoine synthesis under salt stress. For qRT-PCR, data are presented as the mean ± standard error (n > 3).

Figure 4

Growth (a), ectoine accumulation (b) and betaine accumulation (c) of the wild-type strain XH26 and the defective strain XH26/Δhom under various salinity conditions. Cultures were grown in 500 mL baffled Erlenmeyer flasks, and each experiment was performed in triplicate.

Figure 5

Growth (a) and ectoine accumulation (b) of the wild-type strain XH26 and the defective strain XH26/Δhom under batch fermentation. Each experiment was performed in duplicate.