Engineering Halomonas bluephagenesis for L-Threonine production.

Halophilic Halomonas bluephagenesis (H. bluephagenesis), a chassis for cost-effective Next Generation Industrial Biotechnology (NGIB), was for the first time engineered to successfully produce L-threonine, one of the aspartic family amino acids (AFAAs). Five exogenous genes including thrA*BC, lysC* and rhtC encoding homoserine dehydrogenase mutant at G433R, homoserine kinase, L-threonine synthase, aspartokinase mutant at T344M, S345L and T352I, and export transporter of threonine, respectively, were grouped into two expression modules for transcriptional tuning on plasmid- and chromosome-based systems in H. bluephagenesis, respectively, after pathway tuning debugging. Combined with deletion of import transporter or/and L-threonine dehydrogenase encoded by sstT or/and thd, respectively, the resulting recombinant H. bluephagenesis TDHR3-42-p226 produced 7.5 g/L and 33 g/L L-threonine when grown under open unsterile conditions in shake flasks and in a 7 L bioreactor, respectively. Engineering H. bluephagenesis demonstrates strong potential for production of diverse metabolic chemicals.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates.

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its ß-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of ß-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV).

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a promising biopolyester with good mechanical properties and biodegradability. Large-scale production of PHBV is still hindered by the high production cost. CRISPR/Cas9 method was used to engineer the TCA cycle in Halomonas bluephagenesis on its chromosome for production of PHBV from glucose as a sole carbon source. Two TCA cycle related genes sdhE and icl encoding succinate dehydrogenase assembly factor 2 and isocitrate lysase were deleted, respectively, in H. bluephagenesis TD08AB containing PHBV synthesis genes on the chromosome, to channel more flux to increase the 3-hydroxyvalerate (3HV) ratio of PHBV. Due to a poor growth behavior of the mutant strains, H. bluephagenesis TY194 equipped with a medium strength Pporin-194 promoter was selected for further studies. The sdhE and/or icl mutant strains of H. bluephagenesis TY194 were constructed to show enhanced cell growth, PHBV synthesis and 3HV molar ratio. Gluconate was used to activate ED pathway and thus TCA cycle to increase 3HV content. H. bluephagenesis TY194 (ΔsdhEΔicl) was found to synthesize 17mol% 3HV in PHBV. Supported by the synergetic function of phosphoenolpyruvate carboxylase and Vitreoscilla hemoglobin encoded by genes ppc and vgb inserted into the chromosome of H. bluephagenesis TY194 (ΔsdhE) serving to enhance TCA cycle activity, a series of strains were generated that could produce PHBV containing 3-18mol% 3HV using glucose as a sole carbon source. Shake flask studies showed that H. bluephagenesis TY194 (ΔsdhE, G7::Pporin-ppc) produced 6.3 g/L cell dry weight (CDW), 65% PHBV in CDW and 25mol% 3HV in PHBV when grown in glucose and gluconate. 25mol% 3HV was the highest reported via chromosomal expression system. PHBV copolymers with different 3HV molar ratios were extracted and characterized. Next-generation industrial biotechnology (NGIB) based on recombinant H. bluephagenesis grown under unsterile and continuous conditions, allows production of P(3HB-0∼25mol% 3HV) in a convenient way with reduced production complexity and cost.