Systematic Metabolic Engineering of Escherichia coli for High-Level Production of trans-4-Hydroxy-L-proline.

Abstract

As a high-value-added amino acid derivative, trans-4-hydroxy-L-proline (T-4-Hyp) faces key bottlenecks in its microbial production from glucose, including insufficient precursor supply and an imbalance between cell growth and product biosynthesis. In this study, we successfully constructed an engineered Escherichia coli strain QF-27 for efficient T-4-Hyp production. First, the L-proline (L-Pro) biosynthetic pathway was enhanced by overexpressing the feedback-resistant γ-glutamyl kinase, glutamate-γ-semialdehyde dehydrogenase, and pyrroline-5-carboxylate reductase, and by knocking out the L-proline dehydrogenase. The resulting strain QF-9 produced 15.75 ± 0.56 g/L of L-Pro. Subsequently, the expression level of proline-4-hydroxylase from Dactylosporangium sp. RH1 was optimized in strain QF-9, and the resulting strain QF-14 produced 5.32 ± 0.26 g/L of T-4-Hyp. To address the insufficient supply of α-ketoglutarate (α-KG), a multi-modular synergistic strategy (i.e., blocking byproduct pathways, enhancing α-KG flux, and relieving global transcriptional repression) increased T-4-Hyp production to 11.02 ± 0.27 g/L. Moreover, a dynamic switch combining PrpsT promoter and DAS+4 degradation tag was designed to repress expression and promote degradation of the α-ketoglutarate dehydrogenase complex during the stationary phase, thereby balancing cell growth and T-4-Hyp production. Consequently, T-4-Hyp production reached 14.37 ± 0.46 g/L, and residual L-Pro fell to 0.38 ± 0.17 g/L. In fed-batch fermentation, the final strain QF-27 produced 105.72 ± 0.84 g/L of T-4-Hyp with productivity of 2.20 g/L/h and carbon yield of 0.364 g/g glucose. To our knowledge, this is the best performance reported for T-4-Hyp production by microbial fermentation, and the first study to enhance it via systematic modification of central carbon metabolism.