Engineered microbial biosensors based on bacterial two-component systems as synthetic biotechnology platforms in bioremediation and biorefinery.

Two-component regulatory systems (TCRSs) mediate cellular response by coupling sensing and regulatory mechanisms. TCRSs are comprised of a histidine kinase (HK), which serves as a sensor, and a response regulator, which regulates expression of the effector gene after being phosphorylated by HK. Using these attributes, bacterial TCRSs can be engineered to design microbial systems for different applications. This review focuses on the current advances in TCRS-based biosensors and on the design of microbial systems for bioremediation and their potential application in biorefinery.

Metabolic engineering of Ralstonia eutropha for the production of polyhydroxyalkanoates from sucrose.

A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes ß-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that ß-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5 mol% LA) to a polymer content of 19.5 wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

Development of rice bran treatment process and its use for the synthesis of polyhydroxyalkanoates from rice bran hydrolysate solution.

Rice bran treatment process for the production of 43.7 kg of hydrolysate solution containing 24.41 g/L of glucose and small amount of fructose from 5 kg of rice bran was developed and employed to produce polyhydroxyalkanoates in recombinant Escherichia coli and Ralstonia eutropha strains. Recombinant E. coli XL1-Blue expressing R. eutropha phaCAB genes and R. eutropha NCIMB11599 could produce poly(3-hydroxybutyrate) with the polymer contents of 90.1 wt% and 97.2 wt%, respectively, when they were cultured in chemically defined MR medium and chemically defined nitrogen free MR medium containing 10 mL/L of rice bran hydrolysate solution, respectively. Also, recombinant E. coli XL1-Blue and recombinant R. eutropha 437-540, both of which express the Pseudomonas sp. phaC1437 gene and the Clostridium propionicum pct540 gene could produce poly(3-hydroxybutyrate-co-lactate) from rice bran hydrolysate solution. These results suggest that rice bran may be a good renewable resource for the production of biomass-based polymers by recombinant microorganisms.

High-level conversion of L-lysine into 5-aminovalerate that can be used for nylon 6,5 synthesis.

L-Lysine is a potential feedstock for the production of bio-based precursors for engineering plastics. In this study, we developed a microbial process for high-level conversion of L-lysine into 5-aminovalerate (5AVA) that can be used as a monomer in nylon 6,5 synthesis. Recombinant Escherichia coli WL3110 strain expressing Pseudomonas putida delta-aminovaleramidase (DavA) and lysine 2-monooxygenase (DavB) was grown to high density in fed-batch culture and used as a whole cell catalyst. High-density E. coli WL3110 expressing DavAB, grown to an optical density at 600 nm (OD600 ) of 30, yielded 36.51 g/L 5AVA from 60 g/L L-lysine in 24 h. Doubling the cell density of E. coli WL3110 improved the conversion yield to 47.96 g/L 5AVA from 60 g/L of L-lysine in 24 h. 5AVA production was further improved by doubling the L-lysine concentration from 60 to 120 g/L. The highest 5AVA titer (90.59 g/L; molar yield 0.942) was obtained from 120 g/L L-lysine by E. coli WL3110 cells grown to OD600 of 60. Finally, nylon 6,5 was synthesized by bulk polymerization of ϵ-caprolactam and δ-valerolactam prepared from microbially synthesized 5AVA. The hybrid system demonstrated here has promising possibilities for application in the development of industrial bio-nylon production processes.