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5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), copolymer P(3HB- co -5HV) of 3 -hydroxybutyrate (3HB) and 5HV were produced from L-lysine as a substrate by recombinant Halomonas bluephagenesis constructed based on codon optimization, deletions of competitive pathway and L-lysine export protein, and three copies of davBA genes encoding L-lysine monooxygenase (DavB) and 5-aminovaleramide amidohydrolase (DavA) inserted into its genome to form H. bluephagenesis YF117Δ gabT 1+2 , which produced 16.4 g L−1 and 67.4 g L−1 5-AVA in flask cultures and in 7 L bioreactor, respectively. It was able to de novo synthesize 5-AVA from glucose by L-lysine-overproducing H. bluephagenesis TD226. Corn steep liquor was used instead of yeast extract for cost reduction during the 5-AVA production. Using promoter engineering based on P porin mutant library for downstream genes, H. bluephagenesis YF117 harboring pSEVA341-P porin 42 - yqhD EC produced 6 g L−1 5HV in shake flask growth , while H. bluephagenesis YF117 harboring pSEVA341-P porin 42 - yqhD EC -P porin 278 - phaC RE -abfT synthesized 42 wt% P(3HB- co -4.8 mol% 5HV) under the same condition. Thus, H. bluephagenesis was successfully engineered to produce 5-AVA and 5HV in supernatant and intracellular P(3HB- co -5HV) utilizing L-lysine as the substrate. [Display omitted] • 5-Aminovaleric acid (5-AVA), 5-hydroxyvalerate (5HV), and P(3HB- co -5HV) were produced by Halomonas bluephagenesis. • The engineered strain YF117Δ gabT 1+2 , produced 16.4and 67.4 g L−1 5-AVA in flask cultures and in 7 L bioreactor, respectively. • It was able to de novo synthesize around 600 mg L−1 5-AVA from glucose by L-lysine- overproducing H. bluephagenesis TD226. • H. bluephagenesis YF117 harboring pSEVA341-P porin 42 - yqhD EC produced 6 g L−1 5HV in shake flasks. • YF117 harboring pSEVA341-P porin 42 - yqhD EC -P porin 278 - phaC RE - abfT synthesized 42 wt% P(3HB- co -4.8 mol% 5HV). [ABSTRACT FROM AUTHOR]

Ligand-induced system plays an important role for microbial engineering due to its tunable gene expression control over timings and levels. An oleic acid (OA)-induced system was recently constructed based on protein FadR, a transcriptional regulator involved in fatty acids metabolism, for metabolic control in Escherichia coli. In this study, we constructed a synthetic FadR-based OA-induced systems in Halomonas bluephagenesis by hybridizing the porin promoter core region and FadR-binding operator (fadO). The dynamic control range was optimized over 150-fold, and expression leakage was significantly reduced by tuning FadR expression and positioning fadO , forming a series of OA-induced systems with various expression strengths, respectively. Additionally, ligand orthogonality and cross-species portability were also studied and showed highly linear correlation among Halomonas spp. , Escherichia coli and Pseudomonas spp. Finally, OA-induced systems with medium- and small-dynamic control ranges were employed to dynamically control the expression levels of morphology associated gene minCD , and monomer precursor 4-hydroxybutyrate-CoA (4HB-CoA) synthesis pathway for polyhydroxyalkanoates (PHA), respectively, in the presence of oleic acid as an inducer. As a result, over 10 g/L of poly-3-hydroxybutyrate (PHB) accumulated by elongated cell sizes, and 6 g/L of P(3HB- co -9.57 mol% 4HB) were obtained by controlling the dose and induction time of oleic acid only. This study provides a systematic approach for ligand-induced system engineering, and demonstrates an alternative genetic tool for dynamic control of industrial biotechnology. • Transcriptional factor FadR-based oleic acid-induced system was firstly constructed in Halomonas bluephagenesis. • Series of oleic acid-induced systems with various dynamic control ranges and species portability were achieved. • The oleic acid-induced systems were employed in cell morphology engineering and P34HB synthesis dynamic control. [ABSTRACT FROM AUTHOR]