Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient <i>Halomonas elongata</i>

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. <i>Halomonas elongata</i> OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, <i>H. elongata</i> OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of <i>H. elongata</i> OUT30018's Ect biosynthetic operon with the artificial self-cloned <i>proB_m1AC</i> gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GK^D118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the <i>putA</i> gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate <i>H. elongata</i> HN6. While the Ect-deficient <i>H. elongata</i> KA1 could not grow in minimal media containing more than 4% NaCl, <i>H. elongata</i> HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in <i>H. elongata</i> OUT30018 in response to salt stress. With its genetic background, <i>H. elongata</i> HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for <i>de novo</i> biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient <i>Halomonas elongata</i>. Remarkably, the concentration of Pro accumulated in <i>H. elongata</i> HN6 (<i>∆ectABC::mCherry-proB_m1AC ∆putA</i>) is comparable to that of ectoine accumulated in <i>H. elongata</i> OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GK^D118N/D119N and γ-GK^D154A/E155A) designed to resemble the two known <i>Escherichia coli</i> feedback-inhibition insensitive γ-GK^D107N and γ-GK^E143A, the γ-GK^D118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in <i>H. elongata</i>. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered <i>H. elongata</i> HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.