Jia Jian

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli.

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 gl(-1) cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5-2 gl(-1) alpha-ketoglutarate or 1.0 gl(-1) citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6l fermentor study, a 23.5 gl(-1) cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.

Enhanced production of medium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockout mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 is a medium-chain-length polyhydroxyalkanoates (PHA) producer. One of the main shortages in the production of PHA has been the intracellular PHA degradation caused by its endogenous PHA depolymerase. The aim of this study was to improve PHA production via removing the PHA degradation mechanism. PHA depolymerase phaZ knockout mutant P. putida KTMQ01 was successfully constructed, which accumulated 86 wt% medium-chain-length PHA (mcl PHA) when cultured in mineral medium containing sodium octanoate as the carbon source compared with P. putida KT2442 which produced only 66 wt% of its cell dry weight (CDW). P. putida KTMQ01 cultured over a five-day period on sodium octanoate produced 4.5 g L(-1)-4.0 g L(-1) CDW containing approximately 80 wt% PHA without degradation. In contrast, P. putida KT2442 was observed with decreasing CDW and PHA from over 4 to less than 2 g L(-1) over the same period of time, indicating the function of PHA depolymerases which reduced the amount of PHA from around 50 wt% to none over the incubation period. RT-PCR analysis showed that phaC2 transcriptional level of P. putida KTMQ01 was higher than that of P. putida KT2442, indicating the possibility of relief on negative control of phaC2 transcription by the deletion of phaZ, which combined with the lack of in vivo PHA degradation, led to more PHA accumulation. P. putida KTMQ01 contained PHA granules with larger sizes and smaller numbers than those of P. putida KT2442.

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight (M(w)) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 degrees C and a glass transition temperature of -15.8 degrees C. The degradation temperature at 5% weight loss of the PHV was around 258 degrees C.

Metabolic engineering for microbial production of polyhydroxyalkanoates consisting of high 3-hydroxyhexanoate content by recombinant Aeromonas hydrophila.

Polyhydroxyalkanoate synthase gene phaC(ah) in Aeromonas hydrophila strain 4AK4 was deleted and its function was replaced by phaC1(ps) cloned from Pseudomonas stutzeri strain 1317 which favors 3-hydroxyhexanoate (3HHx) and longer chain length monomers. Genes fadD and fadL encoding Escherichia coli acyl-CoA synthase and Pseudomonas putida KT2442 fatty acid transport protein, respectively, were introduced into the recombinant with new phaC1(ps). Accumulation of a series of novel medium-chain-length polyhydroxyalkanoates (mcl-PHA) consisting of 80-94 mol% 3HHx were observed. The recombinant accumulated 54% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) in cell dry weight consisting of 94.5 mol% 3HHx or 51% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate) consisting of 82 mol% 3HHx and 16 mol% of 3HO during a two-step cultivation process under nitrogen limitation when grown on sodium hexanoate or sodium octanoate. The two polyesters containing high percentage of 3HHx are physically characterized. They could be used as biodegradable pressure sensitive adhesives, coatings, polymer binding agents in organic-solvent-free paints or a source for chiral R-3-hydroxyhexanoate.

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Recombinant Pseudomonas stutzeri 1317 from Unrelated Carbon Sources

Abstract Synthetic biology promises to simplify the construction of metabolic pathways by assembling the detached modules of the whole pathway. This gives new approaches for the microbial production of industrial products such as polyhydroxyalkanoates (PHA). In this study, to produce poly(3-hydroxybutyrate- co -3-hydroxyhexanoate) (PHBHHx) by Pseudomonas stutzeri 1317 from unrelated carbon sources such as glucose, the phaC 1- phaZ - phaC 2 operon of P. stutzeri 1317 was knocked out to generate the PHA deficient mutant P. stutzeri 1317LF. Then three modules containing phaC Ah A Re B Re , phaC Ah B Re G Pp and phaC Ah P Ah were introduced into P. stutzeri 1317LF separately. The shake flask results indicated that the precursor supply and PHA synthase activity were the vital factors for the PHBHHx accumulation of P. stutzeri 1317LF. Furthermore, the PHBHHx accumulation of the recombinants from different carbon resources were performed. The highest PHBHHx content was 23.7% (by mass) with 58.6% (by mole) 3HB fraction. These results provide basis for further improving the PHBHHx accumulation of P. stutzeri from unrelated carbon sources.