Multidimensional engineering of Escherichia coli for efficient biosynthesis of cis-3-hydroxypipecolic acid

Cis-3-hydroxypipecolic acid (cis-3-HyPip) is the crucial part of many alkaloids and drugs. However, its bio-based industrial production remains challenging. Here, lysine cyclodeaminase from Streptomyces malaysiensis (SmLCD) and pipecolic acid hydroxylase from Streptomyces sp. L-49973 (StGetF) were screened to achieve the conversion of L-lysine to cis-3-HyPip. Considering the high-cost of cofactors, NAD(P)H oxidase from Lactobacillus sanfranciscensis (LsNox) was further overexpressed in chassis strain Escherichia coli W3110 ΔsucCD (α-ketoglutarate-producing strain) to construct the NAD⁺ regeneration system, thus realizing the bioconversion of cis-3-HyPip from low-cost substrate L-lysine without NAD⁺ and α-ketoglutarate addition. To further accelerate the transmission efficiency of cis-3-HyPip biosynthetic pathway, multiple-enzyme expression optimization and transporter dynamic regulation via promoter engineering were conducted. Through fermentation optimization, the final engineered strain HP-13 generated 78.4 g/L cis-3-HyPip with 78.9% conversion in a 5-L fermenter, representing the highest production level achieved so far. These strategies described herein show promising potentials for large-scale production of cis-3-HyPip.