Co-reporter:Tian Li, Dina Elhadi, Guo-Qiang Chen
Metabolic Engineering 2017 Volume 43, Part A(Volume 43, Part A) pp:
Publication Date(Web):1 September 2017
DOI:10.1016/j.ymben.2017.07.007
•PHA synthesis affects production of many metabolic intermediates.•PHA coproduction with other chemicals is desirable.•Coproduction includes small molecules such as amino acids, hydrogen, alcohols and ALA.•Coproduction includes macromolecules such as proteins, surfactants, polysaccharides.•Many coproductions benefit each other.Engineering microorganisms capable of simultaneously accumulating multiple products are economically attractive for biotechnology. Polyhydroxyalkanoates (PHA) or microbial bioplastics are promising as biodegradable plastics to address environmental concerns resulted from plastic wastes accumulation. Unfortunately, PHA production is still limited and cannot compete with the chemically synthesized plastics due to their high production cost. Efforts have been devoted to reduce PHA production cost by employing PHA co-production with other valuable chemicals. Successful co-productions of PHA have been demonstrated with amino acids, proteins, alcohols, hydrogen, biosurfactants, exopolysaccharides and several fine chemicals. The strategy allows recovering PHA from the cells and other value-added products from the no-cells broths. Numerous successful strategies have been developed for minimizing the substrate cost and improving the product yields. This paper reviews the recent strategies developed in PHA co-production with other compounds, discusses the challenges and prospective during the scale up of the co-production strategies.
Co-reporter:Han Zhao, Haoqian M. Zhang, Xiangbin Chen, Teng Li, ... Guo-Qiang Chen
Metabolic Engineering 2017 Volume 39(Volume 39) pp:
Publication Date(Web):1 January 2017
DOI:10.1016/j.ymben.2016.11.007
•Novel T7-like expression systems were developed for non-model bacteria including Halomonas and Pseudomonas entomophila.•The expression systems displayed low crosstalk, tight regulation and high efficiency.•Cross-species transferring of pathway control was enabled by the T7-like systems.•Ultrahigh PHB accumulation (92% of CDW) was achieved in Halomonas TD01 using T7-like MmP1.To engineer non-model organisms, suitable genetic parts must be available. However, biological parts are often host strain sensitive. It is therefore necessary to develop genetic parts that are functional regardless of host strains. Here we report several novel phage-derived expression systems used for transcriptional control in non-model bacteria. Novel T7-like RNA polymerase-promoter pairs were obtained by mining phage genomes, followed by in vivo characterization in non-model strains Halomonas spp TD01 and Pseudomonas entomophila. Three expression systems, namely, MmP1, VP4, and K1F, were developed displaying orthogonality (crosstalk<0.7%), tight regulation (3085-fold induction), and high efficiency (2.5-fold of Ptac) in Halomonas sp. TD01, a chassis strain with a high industrial value. The expression under the corresponding T7-like promoter libraries persisted with striking correlations (R2 >0.94) between Escherichia coli and Halomonas sp. TD01, implying suitability of broad-host range. Three Halomonas TD strains were then constructed based upon these expression systems that enabled interchangeable and controllable gene expression. One of the strains termed Halomonas TD-MmP1 was used to express the cell-elongation cassette (minCD genes) and polyhydroxybutyrate (PHB) biosynthetic pathway, resulting in a 100-fold increase in cell lengths and high levels of PHB production (up to 92% of cell dry weight), respectively. We envision these T7-like expression systems to benefit metabolic engineering in other non-model organisms.
Co-reporter:Dan Li;Li Lv;Jin-Chun Chen
Applied Microbiology and Biotechnology 2017 Volume 101( Issue 14) pp:5861-5867
Publication Date(Web):15 June 2017
DOI:10.1007/s00253-017-8374-6
Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20–23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47–75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.
Co-reporter:Xiao-Ran Jiang, Zhi-Hao Yao, Guo-Qiang Chen
Metabolic Engineering 2017 Volume 44(Volume 44) pp:
Publication Date(Web):1 November 2017
DOI:10.1016/j.ymben.2017.09.004
•80% PHB yield increase was achieved via controllable morphology manipulated Halomonas.•PyrF- based gene deletion method was constructed in Halomonas.•Larger spherical cells was achieved via inactivation actin-like protein MreB.•Longer filamentous cells was achieved via disruption Z-ring formation protein FtsZ.•Cell sizes can be controlled by regulating mreB or ftsZ gene expression.Bacterial morphology is decided by cytoskeleton protein MreB and cell division protein FtsZ encoded by essential genes mreB and ftsZ, respectively. Inactivating mreB and ftsZ lead to increasing cell sizes and cell lengths, respectively, yet seriously reduce cell growth ability. Here we develop a temperature-responsible plasmid expression system for compensated expression of relevant gene(s) in mreB or ftsZ disrupted recombinants H. campaniensis LS21, allowing mreB or ftsZ disrupted recombinants to grow normally at 30 °C in a bioreactor for 12 h so that a certain cell density can be reached, followed by 36 h cell size expansions or cell shape elongations at elevated 37 °C at which the mreB and ftsZ encoded plasmid pTKmf failed to replicate in the recombinants and thus lost themselves. Finally, 80% PHB yield increase was achieved via controllable morphology manipulated H. campaniensis LS21. It is concluded that controllable expanding cell volumes (widths or lengths) provides more spaces for accumulating more inclusion body polyhydroxybutyrate (PHB) and the resulting cell gravity precipitation benefits the final separation of cells and product during downstream.
Co-reporter:Guo-Qiang Chen, Xiao-Ran Jiang
Synthetic and Systems Biotechnology 2017 Volume 2, Issue 3(Issue 3) pp:
Publication Date(Web):1 September 2017
DOI:10.1016/j.synbio.2017.09.001
Polyhydroxyalkanoates (PHA) have been produced by some bacteria as bioplastics for many years. Yet their commercialization is still on the way. A few issues are related to the difficulty of PHA commercialization: namely, high cost and instabilities on molecular weights (Mw) and structures, thus instability on thermo-mechanical properties. The high cost is the result of complicated bioprocessing associated with sterilization, low conversion of carbon substrates to PHA products, and slow growth of microorganisms as well as difficulty of downstream separation. Future engineering on PHA producing microorganisms should be focused on contamination resistant bacteria especially extremophiles, developments of engineering approaches for the extremophiles, increase on carbon substrates to PHA conversion and controlling Mw of PHA. The concept proof studies could still be conducted on E. coli or Pseudomonas spp. that are easily used for molecular manipulations. In this review, we will use E. coli and halophiles as examples to show how to engineer bacteria for enhanced PHA biosynthesis and for increasing PHA competitiveness.
Co-reporter:Tian Li, Ying-Ying Guo, Guan-Qing Qiao, and Guo-Qiang Chen
ACS Synthetic Biology 2016 Volume 5(Issue 11) pp:1264
Publication Date(Web):May 29, 2016
DOI:10.1021/acssynbio.6b00105
5-Aminolevulinic acid (ALA), an important cell metabolic intermediate useful for cancer treatments or plant growth regulator, was produced by recombinant Escherichia coli expressing the codon optimized mitochondrial 5-aminolevulinic acid synthase (EC: 2.3.1.37, hem1) from Saccharomyces cerevisiae controlled via the plasmid encoding T7 expression system with a T7 RNA polymerase. When a more efficient autoinduced expression approach free of IPTG was applied, the recombinant containing antibiotic-free stabilized plasmid was able to produce 3.6 g/L extracellular ALA in shake flask studies under optimized temperature. A recombinant E. coli expressing synthesis pathways of poly-3-hydroxybutyrate (PHB) and ALA resulted in coproduction of 43% PHB in the cell dry weights and 1.6 g/L extracellular ALA, leading to further reduction on ALA cost as two products were harvested both intracellularly and extracellularly. This was the first study on coproduction of extracellular ALA and intracellular PHB for improving bioprocessing efficiency. The cost of ALA production could be further reduced by employing a Halomonas spp. TD01 able to grow and produce ALA and PHB under continuous and unsterile conditions even though ALA had the highest titer of only 0.7 g/L at the present time.Keywords: 5-aminolevulinic acid; Escherichia coli; Halomonas; PHB; Saccharomyces cerevisiae; synthetic biology; T7 RNA polymerase
Co-reporter:Teng Li, Jianwen Ye, Rui Shen, Yeqing Zong, Xuejin Zhao, Chunbo Lou, and Guo-Qiang Chen
ACS Synthetic Biology 2016 Volume 5(Issue 11) pp:1308
Publication Date(Web):May 2, 2016
DOI:10.1021/acssynbio.6b00083
As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%–92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (Mw) of 2.7–6.8 × 106 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.Keywords: high-throughput screening; PHB; polyhydroxyalkanoates; ribosomal binding site calculator
Co-reporter:Shaoqin Zhang;Lei Fang;Zhengjun Li;Yingying Guo
Science China Chemistry 2016 Volume 59( Issue 11) pp:1390-1396
Publication Date(Web):2016 November
DOI:10.1007/s11426-016-0194-x
With the help of Tn5 transposon technique, gene yfjB encoding NAD kinase in Escherichia coli (E. coli) was inserted into chromosome of recombinant E. coli polyhydroxybutyrate (PHB) containing PHB synthesis operon integrated in the host genome. After successful transposition of an extra yfjB gene copy into genome, the selected recombinant named E. coli PHBTY4 showed stronger NAD kinase activity than that of E. coli PHB. Shake flask studies suggested that both cell dry weight and PHB accumulation were significantly increased in E. coli PHBTY4 compared with that of the control. E. coli PHBTY4 produced approximately 23 g/L PHB compared with its control which synthesized only 10 g/L PHB when grown under the same conditions in a 6 L fermentor after 32 h of cultivation. In addition, E. coli PHBTY4 maintained high genetic stability during the cultivation processes. These results revealed a practical method to construct genetically stable strains harboring extra NAD kinase gene to enhance NADP(H)-dependent bio-reactions.
Co-reporter:Ivan Hajnal;Xiangbin Chen
Applied Microbiology and Biotechnology 2016 Volume 100( Issue 21) pp:9103-9110
Publication Date(Web):2016 November
DOI:10.1007/s00253-016-7669-3
The industrial production of low value-added biological products poses significant challenges due to cost pressures. In recent years, it has been argued that synthetic biology approaches will lead to breakthroughs that eliminate price bottlenecks for the production of a wide range of biological products including bioplastics and biofuels. One significant bottleneck lies in the necessity to break the tough cell walls of microbes in order to release intracellular products. We here report the implementation of the first synthetic biology standard part based on the lambda phage SRRz genes and a synthetic ribosome binding site (RBS) that works in Escherichia coli and Halomonas campaniensis, which enables the producer strains to induce lysis after the addition of small amounts (1–5 %) of solvents or to spontaneously lyse during the stresses of downstream processing, and thus has the potential to eliminate the mechanical cell disruption step as both an efficiency bottleneck and a significant capex barrier when implementing downstream bioprocesses.
Co-reporter:Hong Wu;Jinchun Chen
Applied Microbiology and Biotechnology 2016 Volume 100( Issue 23) pp:9907-9916
Publication Date(Web):2016 December
DOI:10.1007/s00253-016-7715-1
E. coli JM109∆envC∆nlpD deleted with genes envC and nlpD responsible for degrading peptidoglycan (PG) led to long filamentous cell shapes. When cell fission ring location genes minC and minD of Escherichia coli were deleted, E. coli JM109∆minCD changed the cell growth pattern from binary division to multiple fissions. Bacterial morphology can be further engineered by overexpressing sulA gene resulting in inhibition on FtsZ, thus generating very long cellular filaments. By overexpressing sulA in E. coli JM109∆envC∆nlpD and E. coli JM109∆minCD harboring poly(3-hydroxybutyrate) (PHB) synthesis operon phbCAB encoded in plasmid pBHR68, respectively, both engineered cells became long filaments and accumulated more PHB compared with the wild-type. Under same shake flask growth conditions, E. coli JM109∆minCD (pBHR68) overexpressing sulA grown in multiple fission pattern accumulated approximately 70 % PHB in 9 g/L cell dry mass (CDM), which was significantly higher than E. coli JM109∆envC∆nlpD and the wild type, that produced 7.6 g/L and 8 g/L CDM containing 64 % and 51 % PHB, respectively. Results demonstrated that a combination of the new division pattern with elongated shape of E. coli improved PHB production. This provided a new vision on the enhanced production of inclusion bodies.
Co-reporter:Cui-Ling Dong;William R. Webb;Qiang Peng;James Z. Tang;Nicholas R. Forsyth;Alicia J. El Haj
Journal of Biomedical Materials Research Part A 2015 Volume 103( Issue 1) pp:282-288
Publication Date(Web):
DOI:10.1002/jbm.a.35149
Abstract
This study aimed to design a growth factor loaded copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) nanoparticles containing 3D collagen matrix to achieve growth factor sustained release for long-term stimulation of human mesenchymal stem cells (hMSCs) proliferation/differentiation for tissue engineer application. Platelet-derived growth factor-BB (PDGF-BB), which is known to enhance hMSCs proliferation in human serum, was selected as a model growth factor, and biodegradable copolyester of PHBHHx was chosen to be the sustained release vehicle. PDGF-BB phospholipid complex encapsulated PHBHHx nanoparticles were fabricated, and their effect on hMSCs proliferation was investigated via assays of CCK-8 and live-dead staining to cells inoculated in 2D tissue culture plates and 3D collagen gel scaffolds, respectively. The resulting spherical PHBHHx nanoparticles were stable in terms of their mean particle size, polydispersity index and zeta potential before and after lyophilization. In vitro study revealed a sustained release of PDGF-BB with a low burst release. Furthermore, sustained released PDGF-BB was revealed to significantly promote hMSCs proliferation in both cell monolayer and cell seeded 3D collagen scaffolds inoculated in serum-free media. Therefore, the 3D collagen matrices with locally sustained release growth factor nanoparticles hold promise to be used for stem cell tissue engineering. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 282–288, 2015.
Co-reporter:Jin Yin;Huan Wang;Xiao-Zhi Fu;Xue Gao
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 13) pp:5523-5534
Publication Date(Web):2015 July
DOI:10.1007/s00253-015-6510-8
Chromosomal integration and expression of heterologous gene(s) are favored in industrial biotechnology due to the inheriting expression stability. Yet, chromosomal expression is commonly weaker than plasmid one. The effect on gene expression level at 13 chromosomal locations in Escherichia coli was investigated using the polyhydroxybutyrate (PHB) synthesis pathway encoded by a phaCAB operon as a reporter. When 11 copies of phaCAB were randomly integrated into 11 of the 13 chromosomal locations, respectively, 5.2 wt% of PHB was produced. PHB (34.1 wt%) was accumulated by a recombinant E. coli inserted chromosomally with 50 copies of phaCAB in the active asnB site using a Cre-loxP recombination method. This PHB accumulation level was equivalent to a medium-copy-number plasmid expression system, suggesting the importance of chromosomal gene copy number for PHB production by E. coli. This result was used to manipulate a Halomonas strain. One copy of genes scpAB encoding methylmalonyl-CoA mutase and methylmalonyl-CoA decarboxylase was inserted into the strongest expression site porin in the chromosome of the 2-methylcitrate synthase (prpC) deleted mutant Halomonas TD08, leading to the synthesis of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) from glucose as the sole carbon source. The chromosome-engineered strain produced PHBV consisting of 5–12 mol% 3-hydroxyvalerate (3HV) stably compared with unstable fluctuation of 7–25 mol% 3HV by a medium-copy-number plasmid system. These results demonstrated that chromosome engineering based on active transcriptional site and gene copy number is more feasible for polyhydroxyalkanoate (PHA) synthesis in Halomonas TD08 compared with in E. coli.
Co-reporter:Hong Wu;Huan Wang;Jinchun Chen
Applied Microbiology and Biotechnology 2014 Volume 98( Issue 24) pp:10013-10021
Publication Date(Web):2014 December
DOI:10.1007/s00253-014-6059-y
Micro-aeration is a situation that will be encountered in bacterial cell growth especially when the saturated dissolved oxygen level cannot match the demand from cells grown to a high density. Therefore, it is desirable to separate aerobic growth and micro-aerobic product formation into two stages using methods including anaerobic or micro-aerobic promoters that are inducible under low aeration intensity. Eleven potential low aeration-inducible promoters were cloned and studied for their induction strengths under micro-aerobic conditions. Of them, Vitreoscilla hemoglobin promoter (Pvgb) was found to be the strongest among all 11 promoters. At the same time, six E. coli hosts harboring poly(R-3-hydroxybutyrate) (PHB) synthesis operon phaCAB were compared for their ability to accumulate poly(hydroxyalkanoates) (PHA). E. coli S17-1 was demonstrated to be the best host achieving a 70 % (mass fraction) PHB in the cell dry weigh (CDW) after 48 h under micro-aerobic growth. Cascaded Pvgb repeats (Pnvgb) were investigated for enhanced expression level under micro-aerobic growth. The highest PHA production was obtained when a promoter containing eight cascaded Pvgb repeats (P8vgb) was used, 5.37 g/l CDW containing 90 % PHB was obtained from recombinant in E. coli S17-1. Cells grew further to 6.30 g/l CDW containing 91 % PHB when oxygen-responsive transcription factor ArcA (arcA) was deleted in the same recombinant E. coli S17-1. This study revealed that vgb promoter containing cascaded Pvgb repeats (P8vgb) is useful for product formation under low aeration intensity.
Co-reporter:Rui Shen;LongWei Cai;DeChuan Meng;LinPing Wu;Kai Guo
Science China Life Sciences 2014 Volume 57( Issue 1) pp:4-10
Publication Date(Web):2014 January
DOI:10.1007/s11427-013-4596-8
Microbial synthesis of functional polymers has become increasingly important for industrial biotechnology. For the first time, it became possible to synthesize controllable composition of poly(3-hydroxyalkanoate) (P3HA) consisting of 3-hydroxydodecanoate (3HDD) and phenyl group on the side-chain when chromosome of Pseudomonas entomophila was edited to weaken its β-oxidation. Cultured in the presence of 5-phenylvaleric acid (PVA), the edited P. entomophila produced only homopolymer poly(3-hydroxy-5-phenylvalerate) or P(3HPhV). While copolyesters P(3HPhV-co-3HDD) of 3-hydroxy-5-phenylvalerate (3HPhV) and 3-hydroxydodecanoate (3HDD) were synthesized when the strain was grown on mixtures of PVA and dodecanoic acid (DDA). Compositions of 3HPhV in P(3HPhV-co-3HDD) were controllable ranging from 3% to 32% depending on DDDA/PVA ratios. Nuclear magnetic resonance (NMR) spectra clearly indicated that the polymers were homopolymer of P(3HPhV) and random copolymers of 3HPhV and 3HDD. Their mechanical and thermal properties varied dramatically depending on the monomer ratios. Our results demonstrated the possibility to produce tailor-made, novel functional PHA using the chromosome edited P. entomophila.
Co-reporter:Shijun Li, Longwei Cai, Linping Wu, Guodong Zeng, Jinchun Chen, Qiong Wu, and Guo-Qiang Chen
Biomacromolecules 2014 Volume 15(Issue 6) pp:
Publication Date(Web):May 16, 2014
DOI:10.1021/bm500669s
Functional polyhydroxyalkanoates (PHAs) allow chemical modifications to widen PHA diversity, promising to increase values of these biodegradable and biocompatible polyesters. Among functional PHAs, unsaturated PHA site chains can be easily grafted to add chemical groups, and to cross-link with other PHA polymer chains. However, it has been very difficult to obtain structurally controllable functional homo-, random, or block PHA. For the first time, a β-oxidation deleted Pseudomonas entomophila was used to successfully synthesize random copolymers of 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decenoate (3H9D). Compositions of the random copolymers P(3HDD-co-3H9D) can be adjusted by ratios of dodecanoic acid (DDA) to 9-decenol (9DEO) fed to the culture of P. entomophila. Homopolymer P3H9D was formed when only 9DEO was added to the culture. Diblock copolymers of P3HDD-b-P3H9D were produced by feeding DDA as the first precursor to form a P3HDD block followed by adding 9DEO as the second precursor to form a second P3H9D block. It was demonstrated that random copolymers P(3HDD-co-3H9D) could be crossed-linked under UV-radiation due to the presence of the unsaturated bonds. Thermal and mechanical characterizations of the above homo-, random, and diblock PHA polymers were conducted. It was found that the diblock polymer P3HDD-b-P3H9D increased at least 2-fold on Young’s modulus compared with its random copolymers consisting of similar 3HDD/3H9D ratios. This study demonstrates that PHA functionality could be controlled to meet various requirements.
Co-reporter:Qian Cao, Junyu Zhang, Haitao Liu, Qiong Wu, Jinchun Chen, Guo-Qiang Chen
Biomaterials 2014 35(28) pp: 8273-8283
Publication Date(Web):
DOI:10.1016/j.biomaterials.2014.06.020
Co-reporter:Jin Yin;Xiao-Zhi Fu;Qiong Wu;Jin-Chun Chen
Applied Microbiology and Biotechnology 2014 Volume 98( Issue 21) pp:8987-8997
Publication Date(Web):2014 November
DOI:10.1007/s00253-014-5959-1
Since halophile Halomonas spp. can grow contamination free in seawater under unsterile and continuous conditions, it holds great promise for industrial biotechnology to produce low-cost chemicals in an economic way. Yet, metabolic engineering methods are urgently needed for Halomonas spp. It is commonly known that chromosomal expression is more stable yet weaker than plasmid one is. To overcome this challenge, a novel chromosomal expression method was developed for halophile Halomonas TD01 and its derivatives based on a strongly expressed porin gene as a site for external gene integration. The gene of interest was inserted downstream the porin gene, forming an artificial operon porin-inserted gene. This chromosome expression system was proven functional by some examples: First, chromosomal expression of heterologous polyhydroxybutyrate (PHB) synthase gene phaCRe from Ralstonia eutropha completely restored the PHB accumulation level in endogenous phaC knockout mutant of Halomonas TD01. The integrated phaCRe was expressed at the highest level when inserted at the locus of porin compared with insertions in other chromosome locations. Second, an inducible expression system was constructed in phaC-deleted Halomonas TD01 by integrating the lac repressor gene (lacI) into the porin site in the host chromosome. The native porin promoter was inserted with the key 21 bp DNA of lac operator (lacO) sequence to become an inducible promoter encoded in a plasmid. This inducible system allowed on-off switch of gene expression in Halomonas TD strains. Thus, the stable and strong chromosomal expression method in Halomonas TD spp. was established.
Co-reporter:Lin-Ping Wu, Mingliang You, Danyang Wang, Gongfeng Peng, Zhihui Wang and Guo-Qiang Chen
Polymer Chemistry 2013 vol. 4(Issue 16) pp:4490-4498
Publication Date(Web):30 May 2013
DOI:10.1039/C3PY00668A
Carbon nanotube (CNT)/poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) nanocomposite films were fabricated using a solution mixing and evaporation method. The surface morphology, mechanical and electrical properties of these novel hybrid films were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle determination, tensile tests and electrical measurements. Compared to neat PHBHHx, both the surface roughness and the electrical conductivity of the nanocomposites increased, and mechanical properties showed a significant improvement due to the presence of CNTs. The cell compatibility of the nanocomposites was evaluated by human mesenchymal stem cells (hMSCs). The activity and proliferation of hMSCs were demonstrated to be outstanding when nanocomposite films contained 1% CNTs compared with that on the neat PHBHHx. Levels of osteogenesis differentiation on nanocomposite films were assessed through alkaline phosphatase (ALP) activities, calcium contents and specific osteogenesis genes mRNA expressions, which showed that the 1% CNT/PHBHHx composite film was also suitable for osteogenesis of hMSCs. The results indicated that semi-conductive CNT/PHBHHx biomaterials could be a potential candidate in bone tissue engineering.
Co-reporter:Honglin Xie, Jian Li, Liang Li, Ying Dong, Guo-Qiang Chen, Kevin C. Chen
Acta Biomaterialia 2013 Volume 9(Issue 8) pp:7845-7854
Publication Date(Web):August 2013
DOI:10.1016/j.actbio.2013.04.038
Abstract
Polyhydroxyalkanoates (PHAs) belong to a family of copolyesters with demonstrated biocompatibility. We hypothesize that genetically fusing evolutionarily preserved cell binding motifs, such as RGD or IKVAV, to the PHA-binding protein phasin (PhaP) for surface functionalization of PHA materials could better support the growth and differentiation of neural stem cells (NSCs). This hypothesis is tested on three polyester materials of the same aliphatic family: poly(L-lactic acid) (PLA) and two PHB copolymers, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx). Experimental results indicate that surface coating of the two fusion proteins, PhaP–RGD and PhaP–IKVAV, provides short-term advantages in promoting the adhesion, proliferation and neural differentiation of rat NSCs compared to the PhaP-coated or uncoated material. Among the tested samples, the combination of coating PhaP–IKVAV on an PHBVHHx surface yields the highest levels in cell adhesion and proliferation, while the PLA film coated with PhaP–IKVAV promotes better neural differentiation and neurite outgrowth in the early stage. Because both PhaP–RGD and PhaP–IKVAV could be produced in an inexpensive manner, our data suggest that PhaP–IKVAV is an ideal nonspecific coating agent to functionalize hydrophobic biomaterials in the application of neural tissue engineering.
Co-reporter:Junyu Zhang, Qian Cao, Shaowu Li, Xiaoyun Lu, Yongxi Zhao, Ji-Song Guan, Jin-Chun Chen, Qiong Wu, Guo-Qiang Chen
Biomaterials 2013 34(30) pp: 7552-7562
Publication Date(Web):
DOI:10.1016/j.biomaterials.2013.06.043
Co-reporter:Yang Wang, Xian-Li Jiang, Si-Wu Peng, Xiao-Yong Guo, Guan-Guan Shang, Jin-Chun Chen, Qiong Wu, Guo-Qiang Chen
Biomaterials 2013 34(15) pp: 3737-3746
Publication Date(Web):
DOI:10.1016/j.biomaterials.2013.01.088
Co-reporter:Lakshmi Tripathi, Lin-Ping Wu, Dechuan Meng, Jinchun Chen, and Guo-Qiang Chen
Biomacromolecules 2013 Volume 14(Issue 3) pp:
Publication Date(Web):January 25, 2013
DOI:10.1021/bm3019517
Poly(4-hydroxybutyrate) (P4HB) is a highly elastic polymer, whereas poly(3-hydroxypropionate) (P3HP) is a polymer with enormous tensile strength. This study aimed to biosynthesize a block copolymer consisting of soft P4HB block with a strong P3HP block to gain unique and excellent material properties. A recombinant Escherichia coli strain that produces homopolymers of P3HP and P4HB was employed for the block copolymer synthesis. When the strain was grown in the presence of 1,4-butanediol (BDO) as a 4HB precursor, P4HB block was formed. Sequential supplementation of 1,3-propanediol (PDO) as a 3HP precursor allowed the strain to produce P3HP block. Thermal, NMR, fractionation, and mechanical characterizations confirmed the resulting polymer as a block copolymer of P3HP-b-P4HB. Two block copolymers were formed from this study, including the P3HP-b-29% P4HB and P3HP-b-37% P4HB, they showed superior properties over random copolymers P(3HP-co-4HB). The block copolymers had two glass transition temperatures (Tg) and two melting temperatures (Tm). In comparison to the homopolymers P3HP and P4HB, incorporation of block microstructure resulted in the lowering of Tm, block copolymers were revealed with higher Young’s modulus, yield strengths, and tension strengths much better than the previously reported random copolymers of similar compositions. Block copolymerization of P3HP and P4HB adds a new vision on PHA polymerization by generation of new polymers with superior properties.
Co-reporter:Guo-qiang Chen 陈国强;Yang Wang 汪洋
Chinese Journal of Polymer Science 2013 Volume 31( Issue 5) pp:719-736
Publication Date(Web):2013 May
DOI:10.1007/s10118-013-1280-1
Microbial polyhydroxyalkanoates (PHAs) are a family of biopolyesters produced by many wild type and engineered bacteria. PHAs have diverse structures accompanied by flexible thermal and mechanical properties. Combined with their in vitro biodegradation, cell and tissue compatibility, PHAs have been studied for medical applications, especially medical implants applications, including heart valve tissue engineering, vascular tissue engineering, bone tissue engineering, cartilage tissue engineering, nerve conduit tissue engineering as well as esophagus tissue engineering. Most studies have been conducted in the authors’ lab in the past 20+ years. Recently, mechanism on PHA promoted tissue regeneration was revealed to relate to cell responses to PHA biodegradation products and cell-material interactions mediated by microRNA. Very importantly, PHA implants were found not to cause carcinogenesis during long-term implantation. Thus, PHAs should have a bright future in biomedical areas.
Co-reporter:William R. Webb, Tina P. Dale, Alex J. Lomas, Guodong Zeng, Ian Wimpenny, Alicia J. El Haj, Nicholas R. Forsyth, Guo-Qiang Chen
Biomaterials 2013 34(28) pp: 6683-6694
Publication Date(Web):
DOI:10.1016/j.biomaterials.2013.05.041
Co-reporter:Guo-Qiang Chen and Martin K. Patel
Chemical Reviews 2012 Volume 112(Issue 4) pp:2082
Publication Date(Web):December 21, 2011
DOI:10.1021/cr200162d
Co-reporter:Rui-Yan Wang, Zhen-Yu Shi, Jin-Chun Chen, and Guo-Qiang Chen
ACS Synthetic Biology 2012 Volume 1(Issue 7) pp:291
Publication Date(Web):June 11, 2012
DOI:10.1021/sb300025d
Despite recent advances in genomic sequencing and DNA chemical synthesis, construction of large gene clusters containing DNA fragments is still a difficult and expensive task. To tackle this problem, we developed a gene cluster extraction method based on in vitro single-strand overlapping annealing (SSOA). It starts with digesting the target gene cluster in an existing genome, followed by recovering digested chromosome fragments. Subsequently, the single-strand DNA overhangs formed from the digestion process would be specifically annealed and covalently joined together with a circular and a linear vector, respectively. The SSOA method was successfully applied to clone a 18 kb DNA fragment encoding NADH:ubiquinone oxidoreductase. Genomic DNA fragments of different sizes including 11.86, 18.33, 28.67, 34.56, and 55.99 kb were used to test the cloning efficiency. Combined with genetic information from KEGG and the KEIO strain collection, this method will be useful to clone any specific region of an E. coli genome at sizes less than ∼28 kb. The method provides a cost-effective way for genome assembly, alternative to chemically synthesized gene clusters.Keywords: Escherichia coli; gene cluster; homologous recombination; NADH:ubiquinone oxidoreductase; single-strand overlapping annealing; synthetic biology;
Co-reporter:Cui-Ling Dong, Shi-Yan Li, Yang Wang, Ying Dong, James Zhenggui Tang, Jin-Chun Chen, Guo-Qiang Chen
Biomaterials 2012 33(9) pp: 2593-2599
Publication Date(Web):
DOI:10.1016/j.biomaterials.2011.12.020
Co-reporter:Mingliang You, Gongfeng Peng, Jian Li, Ping Ma, Zhihui Wang, Weiliang Shu, Siwu Peng, Guo-Qiang Chen
Biomaterials 2011 Volume 32(Issue 9) pp:2305-2313
Publication Date(Web):March 2011
DOI:10.1016/j.biomaterials.2010.12.009
Hydrophobic polyhydroxyalkanoate (PHA) scaffolds made of a copolyester of 3-hydroxybutyrate-co-hydroxyhexanoate (PHBHHx) were coated with a fusion protein PHA granule binding protein PhaP fused with RGD peptide (PhaP-RGD). Human bone marrow mesenchymal stem cells (hBMSCs) were inoculated on/in the scaffolds for formation of articular cartilages derived from chondrogenic differentiation of hBMSCs for cartilage tissue engineering. PhaP-RGD coating led to more homogeneous spread of cells, better cell adhesion, proliferation and chondrogenic differentiation in the scaffolds compared with those of PhaP coated or uncoated scaffolds immerging in serum minus chondrogenic induction medium. In addition, more extracellular matrices were produced by the differentiated cells over a period of 14 days on/in the PhaP-RGD coated scaffolds evidenced by scanning electron microscopy imaging, enhanced expression of chondrocyte specific genes including SOX-9, aggrecan and type II collagen, suggesting the positive effect of RGD on extracellular matrix production. Furthermore, cartilage-specific extracellular substances sulphated glycosaminoglycans (sGAG) and total collagen content found on/in the PhaP-RGD coated scaffolds were significantly more compared with that produced by the control and PhaP only coated scaffolds. Homogeneously distributed chondrocytes-like cells forming cartilage-like matrices were observed on/in the PhaP-RGD coated scaffolds after 3 weeks. The results suggested that PhaP-RGD coated PHBHHx scaffold promoted chondrogenic differentiation of hBMSCs and could support cartilage tissue engineering.
Co-reporter:Jian Li, Guanguan Shang, Mingliang You, Siwu Peng, Zhihui Wang, Hongning Wu, and Guo-Qiang Chen
Biomacromolecules 2011 Volume 12(Issue 3) pp:
Publication Date(Web):January 24, 2011
DOI:10.1021/bm101230n
Polyhydroxyalkanoates (PHAs) granule associated protein PhaP has a strong affinity to PHA and other hydrophobic polymers. Human lipopolysaccharide binding protein (hLBP) is a natural endotoxin receptor in plasma. In this study, genes encoding hLBP fused with PhaP were expressed in Pichia pastoris GS115 for production of the fusion protein. The purified rhLBP-PhaP fusion protein was immobilized on particles of polyhydroxybutyrate (PHB), which is a member of microbial polyhydroxyalkanoates (PHA). The rhLBP-PhaP-coated PHB particles were added to endotoxin containing water and protein solutions to study their endotoxin removal and protein recovery efficiencies. The influences of ionic strengths and pH on endotoxin removal and protein recovery in different protein solutions were also studied using acidic proteins including bovine serum albumin (BSA), ovalbumin, and basic protein α-chymotrypsinogen as model proteins. The results showed that rhLBP-PhaP particles could remove endotoxin with an efficiency of over 90%. All endotoxin removal and protein recovery efficiencies were only slightly affected by ionic strengths but were drastically affected by pH changes. Our results demonstrated that rhLBP-PhaP particles with their high efficiency, ease of preparation, and nontoxicity will be a suitable system for endotoxin removal in the protein purification industry.
Co-reporter:Die Hu, Ah-Leum Chung, Lin-Ping Wu, Xin Zhang, Qiong Wu, Jin-Chun Chen, and Guo-Qiang Chen
Biomacromolecules 2011 Volume 12(Issue 9) pp:
Publication Date(Web):August 24, 2011
DOI:10.1021/bm200660k
Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into beta-oxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (Tm) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.
Co-reporter:Ah-Leum Chung, Hong-Liang Jin, Long-Jian Huang, Hai-Mu Ye, Jin-Chun Chen, Qiong Wu, and Guo-Qiang Chen
Biomacromolecules 2011 Volume 12(Issue 10) pp:
Publication Date(Web):August 12, 2011
DOI:10.1021/bm200770m
A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in β-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between β-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L–1 PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between Tg and Tm, which were −49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.
Co-reporter:Xiao-Wen Shen;Zhen-Yu Shi;Ge Song;Zheng-Jun Li
Applied Microbiology and Biotechnology 2011 Volume 91( Issue 3) pp:655-665
Publication Date(Web):2011 August
DOI:10.1007/s00253-011-3274-7
The site-specific mutagenesis for PHA synthase PhaC2Ps1317 from Pseudomonas stutzeri 1317 was conducted for optimizing production of short-chain-length and medium-chain-length polyhydroxyalkanoates (scl-mcl PHA). Recombinant Ralstonia eutropha PHB-4 harboring double mutated phaC2Ps1317 gene (phaC2PsQKST) produced 42 wt.% PHA content in the cell dry weight (CDW) with 93 mol% 3-hydroxybutyrate (HB) as monomer in the PHA copolymer. Compared to that of wild-type phaC2Ps1317, the higher PHA content indicated the effectiveness of the specific point mutations for improvement on PhaC2Ps1317 activity and PHA production. The physical characterization revealed that the PHA produced by the recombinant strain was scl-mcl PHA copolymers with molecular weights and polydispersity reasonable for practical applications. Recombinant R. eutropha PHB-4 containing mutated phaC2Ps1317 termed phaC2PsQKST was demonstrated to be able to produce scl-mcl PHA copolymers consisting of even-numbered, odd-numbered, or a combination of even- and odd-numbered monomers covering the carbon chain lengths from C4 to C12 when related substrates were provided. Recombinant R. eutropha PHB-4 containing phaC2PsQKST could be used as a strain for production of copolymers consisting of dominated HB and medium-chain-length 3-hydroxyalkanoates (HA) with better application properties.
Co-reporter:Si-Wu Peng, Xiao-Yong Guo, Guan-Guan Shang, Jian Li, Xian-Yi Xu, Ming-Liang You, Ping Li, Guo-Qiang Chen
Biomaterials 2011 32(10) pp: 2546-2555
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.12.051
Co-reporter:Ying Dong, Ping Li, Chong-bo Chen, Zhi-hui Wang, Ping Ma, Guo-Qiang Chen
Biomaterials 2010 31(34) pp: 8921-8930
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.08.001
Co-reporter:Jing Zhou, Si-Wu Peng, Yuan-Yuan Wang, Song-Bin Zheng, Yang Wang, Guo-Qiang Chen
Biomaterials 2010 31(29) pp: 7512-7518
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.06.044
Co-reporter:Zheng-Jun Li;Jia Jian;Xiao-Xing Wei
Applied Microbiology and Biotechnology 2010 Volume 87( Issue 6) pp:2001-2009
Publication Date(Web):2010 August
DOI:10.1007/s00253-010-2676-2
A metabolically engineered Escherichia coli has been constructed for the production of meso-2,3-butanediol (2,3-BD) under low oxygen condition. Genes responsible for 2,3-BD formation from pyruvate were assembled together to generate a high-copy plasmid pEnBD, in which each gene was transcribed with a constitutive promoter. To eliminate by-product formation under low oxygen condition, genes including ldhA, pta, adhE, and poxB which functioned for the mixed acid fermentation pathways were deleted in E. coli JM109. Compared with the wild type, the quadruple gene deletion mutant produced smaller amounts of acetate, succinate, and ethanol from glucose when cultivated in LB medium in shake flasks under low-aeration. When 2,3-BD producing pathway was introduced via pEnBD into the mutant, higher glucose consumption and faster 2,3-BD production rate compared with that of the wild-type control were observed under aerobic condition in shake flasks. In a 6-L fermentor supplied with only 3% dissolved oxygen (DO), the mutant harboring pEnBD converted glucose to 2,3-BD much faster than the control did. When DO supply was further lowered to 1% DO, the recombinant mutant grew much slower but produced 2,3-BD as a major fermentation metabolic product. In addition, the 2,3-BD yield showed an increase from 0.20 g BD/g glucose for the control to 0.43 g BD/g glucose for the mixed acid pathway deleted mutant grown in fermentors under 1% DO. These results reveals the potential of production of enantiomerically pure 2,3-BD isomer by recombinant E. coli under low oxygen condition.
Co-reporter:Hai-Mu Ye, Zhen Wang, Hong-Hui Wang, Guo-Qiang Chen, Jun Xu
Polymer 2010 Volume 51(Issue 25) pp:6037-6046
Publication Date(Web):26 November 2010
DOI:10.1016/j.polymer.2010.10.030
Various methods were employed to study the thermal behaviors of a novel microbial polyhydroxyalkanoate (PHA) terpolyester, namely, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx) compared with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). PHBVHHx showed higher crystallization rate and degree of crystallinity. PHBVHHx exhibited also different multiple melting behaviors from PHBHHx. The WAXD results demonstrated that the crystal lattice of PHBVHHx was more compact than that of PHBHHx, suggesting stronger interaction between chain stems. DSC and in-situ heating WAXD studies revealed that PHBVHHx showed a partial melting-lamellar thickening-remelting process during heating, while PHBHHx demonstrated a melting-rapid formation of new crystals-remelting process. It is proposed that the simultaneous introduction of 3-hydroxyvalerate and 3-hydroxyhexanoate monomers into poly(3-hydroxybutyrate) improves the mobility of chain stems along the chain direction, leading to easier intralamellar slip during heating or drawing, further resulting in improvement of mechanical properties, which was supported by the DMA tests. Consequently, we establish a relationship between the thermal behavior and the mechanical properties of biodegradable plastics, which we believe is applicable to other polymers as well.
Co-reporter:Lei Wang, Zhi-Hui Wang, Chong-Yang Shen, Ming-Liang You, Jian-Feng Xiao, Guo-Qiang Chen
Biomaterials 2010 31(7) pp: 1691-1698
Publication Date(Web):
DOI:10.1016/j.biomaterials.2009.11.053
Co-reporter:Xian-Yi Xu, Xiao-Tao Li, Si-Wu Peng, Jian-Feng Xiao, Chao Liu, Guo Fang, Kevin C. Chen, Guo-Qiang Chen
Biomaterials 2010 31(14) pp: 3967-3975
Publication Date(Web):
DOI:10.1016/j.biomaterials.2010.01.132
Co-reporter:Guo-Qiang Chen
Chemical Society Reviews 2009 vol. 38(Issue 8) pp:2434-2446
Publication Date(Web):08 May 2009
DOI:10.1039/B812677C
Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).
Co-reporter:Xiao-Xing Wei;Zhen-Yu Shi;Mei-Qing Yuan
Applied Microbiology and Biotechnology 2009 Volume 82( Issue 4) pp:
Publication Date(Web):2009 March
DOI:10.1007/s00253-008-1816-4
Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (PadhE) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring PadhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of PadhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.
Co-reporter:Ahleum Chung;Qian Liu;Shao-Ping Ouyang
Applied Microbiology and Biotechnology 2009 Volume 83( Issue 3) pp:513-519
Publication Date(Web):2009 June
DOI:10.1007/s00253-009-1919-6
To produce extracellular chiral 3-hydroxyacyl acids (3HA) by fermentation, a novel pathway was constructed by expressing tesB gene encoding thioesterase II into Pseudomonas putida KTOY01, which was a polyhydroxyalkanoate (PHA) synthesis operon knockout mutant. 3HA mixtures of 0.35 g/l consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (3HDD) were produced in shake-flask study using dodecanoate as a sole carbon source. Additional knockout of fadB and fadA genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KTOY01 led to the weakening of the β-oxidation pathway. The fadBA and PHA synthesis operon knockout mutant P. putida KTOY07 expressing tesB gene produced 2.44 g/l 3HA, significantly more than that of the β-oxidation intact mutant. The 3HA mixture contained 90 mol% 3HDD as a dominant component. A fed-batch fermentation process carried out in a 6-l automatic fermentor produced 7.27 g/l extracellular 3HA containing 96 mol% fraction of 3HDD after 28 h of growth. For the first time, it became possible to produce 3HDD-dominant 3HA monomers.
Co-reporter:Zheng-Jun Li;Lei Cai;Qiong Wu
Applied Microbiology and Biotechnology 2009 Volume 83( Issue 5) pp:939-947
Publication Date(Web):2009 July
DOI:10.1007/s00253-009-1943-6
NAD kinase was overexpressed to enhance the accumulation of poly(3-hydroxybutyrate) (PHB) in recombinant Escherichia coli harboring PHB synthesis pathway via an accelerated supply of NADPH, which is one of the most crucial factors influencing PHB production. A high copy number expression plasmid pE76 led to a stronger NAD kinase activity than that brought about by the low copy number plasmid pELRY. Overexpressing NAD kinase in recombinant E. coli was found not to have a negative effect on cell growth in the absence of PHB synthesis. Shake flask experiments demonstrated that excess NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35 wt.% compared with the controls; meanwhile, NADP concentration was enhanced threefold to sixfold. Although the two NAD kinase overexpression recombinants exhibited large disparity on NAD kinase activity, their influence on cell growth and PHB accumulation was not proportional. Under the same growth conditions without process optimization, the NAD kinase-overexpressing recombinant produced 14 g/L PHB compared with 7 g/L produced by the control in a 28-h fermentor study. In addition, substrate to PHB yield YPHB/glucose showed an increase from 0.08 g PHB/g glucose for the control to 0.15 g PHB/g glucose for the NAD kinase-overexpressing strain, a 76% increase for the YPHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.
Co-reporter:Lei Zhang;Zhen-Yu Shi;Qiong Wu
Applied Microbiology and Biotechnology 2009 Volume 84( Issue 5) pp:909-916
Publication Date(Web):2009 October
DOI:10.1007/s00253-009-2023-7
4-Hydroxybutyrate (4HB) was produced by Aeromonas hydrophila 4AK4, Escherichia coli S17-1, or Pseudomonas putida KT2442 harboring 1,3-propanediol dehydrogenase gene dhaT and aldehyde dehydrogenase gene aldD from P. putida KT2442 which are capable of transforming 1,4-butanediol (1,4-BD) to 4HB. 4HB containing fermentation broth was used for production of homopolymer poly-4-hydroxybutyrate [P(4HB)] and copolymers poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-4HB)]. Recombinant A. hydrophila 4AK4 harboring plasmid pZL-dhaT-aldD containing dhaT and aldD was the most effective 4HB producer, achieving approximately 4 g/l 4HB from 10 g/l 1,4-BD after 48 h of incubation. The strain produced over 10 g/l 4HB from 20 g/l 1,4-BD after 52 h of cultivation in a 6-L fermenter. Recombinant E. coli S17-1 grown on 4HB containing fermentation broth was found to accumulate 83 wt.% of intracellular P(4HB) in shake flask study. Recombinant Ralstonia eutropha H16 grew to over 6 g/l cell dry weight containing 49 wt.% P(3HB-13%4HB) after 72 h.
Co-reporter:Zhenguo Li, Hao Lin, Nariaki Ishii, Guo-Qiang Chen, Yoshio Inoue
Polymer Degradation and Stability 2007 Volume 92(Issue 9) pp:1708-1714
Publication Date(Web):September 2007
DOI:10.1016/j.polymdegradstab.2007.06.001
Enzymatic degradation of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) (PHBA) biopolyester consisting of 3-hydroxybutyrate (HB) and 15 mol% medium-chain-length 3-hydroxyalkanoates (HA) was studied using a polyhydroxyalkanoates (PHA) depolymerase produced by Ralstonia pickettii T1. It was found that PHBA films did not lose their weight after 25 h of depolymerase treatment. In contrast, three commercially available PHAs including poly-3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate–19 mol% 3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate–19 mol% 3-hydroxyhexanoate) (PHBHHx) lost 75%, 94% and 39% of their original weights. Slow degradation of PHBA was also confirmed by the absence of HA monomers, dimers or trimers as degradation products in their depolymerase solution compared with abundance of degradation products released by the other three PHAs under the same condition. Surface erosion of PHBA was only observed after 48 h of enzymatic treatment compared with those of PHB, PHBV and PHBHHx which already had obvious surface changes after 7.5 h of same treatment. Although the crystallinities of PHB, PHBV, PHBHHx and PHBA were in the order PHB > PHBV > PHBHHx > PHBA valued at 55.8%, 47.8%, 45.9% and 40.9%, respectively, the order of degradability was PHBV > PHB > PHBHHx > PHBA. It can be proposed that PHA enzymatic degradation using this depolymerase was structure related: longer side-chain PHA including PHBHHx and PHBA was less favorable for the depolymerase degradation, longer the side chain, less the biodegradation.
Co-reporter:Xiang-Hua Qu, Qiong Wu, Kun-Yang Zhang, G.Q. Chen
Biomaterials 2006 Volume 27(Issue 19) pp:3540-3548
Publication Date(Web):July 2006
DOI:10.1016/j.biomaterials.2006.02.015
The in vivo tissue reactions and biodegradations of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide) (PLA), poly(3-hydroxybutyrate) (PHB), blends of PHBHHx (X) and poly(ethylene glycol) (PEG) (E) with ratios of 1:1 (E1X1) and 1:5 (E1X5), respectively, were evaluated by subcutaneous implantation in rabbits. Results revealed that the degradation rate increased in the order of PHB
Co-reporter:Ying-Ying Shangguan, Ya-Wu Wang, Qiong Wu, Guo-Qiang Chen
Biomaterials 2006 Volume 27(Issue 11) pp:2349-2357
Publication Date(Web):April 2006
DOI:10.1016/j.biomaterials.2005.11.024
Strong mechanical properties and controllable biodegradability, together with biocompatibility, are the important requirement for the development of medical implant materials. In this study, an ultraviolet (UV) radiation method was developed to achieve controlled degradation for bacterial biopolyester poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) which has a low biodegradation rate that limits its application for many implant applications required quick degradation. When UV radiation was applied directly to PHBHHx powder, significant molecular weight (Mw) losses were observed with the powder, Mw reduction depended on the UV radiation time. At the same time, a broad PHBHHx Mw distribution was the result of inhomogeneous radiation. Interestingly, this inhomogeneous radiation helped maintain the mechanical properties of films made of the UV-radiated powder. In comparison, the PHBHHx films subjected to direct UV radiation became very brittle although their degradation was faster than that of the PHBHHx powders subjected to direct UV radiation. After 15 weeks of degradation in simulated body fluid (SBF), films prepared from 8 and 16 h UV-treated PHBHHx powders maintained 92% and 87% of their original weights, respectively, while the untreated PHBHHx films lost only 1% of its weight. Significant increases in growth of fibroblast L929 were observed on films prepared from UV-radiated powders. This improved biocompatibility could be attributed to increasing hydrophilic functional groups generated by increasing polar groups C–O and CO. In general, UV-treated PHBHHx powder had a broad Mw distribution, which contributed to fast degradation due to dissolution of low Mw polymer fragments, and strong mechanical property due to high Mw polymer chains. Combined with its improved biocompatibility, PHBHHx is one more step close to become a biomedical implant material.
Co-reporter:Guo-Qiang Chen, Qiong Wu
Biomaterials 2005 Volume 26(Issue 33) pp:6565-6578
Publication Date(Web):November 2005
DOI:10.1016/j.biomaterials.2005.04.036
Polyhydoxyalkanoates (PHA) are polyesters produced by microorganisms under unbalanced growth conditions. They are generally biodegradable and thermoprocessable, making them attractive as biomaterials for applications in both conventional medical devices and tissue engineering. Over the past years, PHA, particularly poly 3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly 4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) and poly 3-hydroxyoctanoate (PHO) and its composites have been used to develop devices including sutures, repair devices, repair patches, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings. The changing PHA compositions also allow favorable mechanical properties, biocompatibility, and degradation times within desirable time frames under specific physiological conditions. This paper reviews what have been achieved in the PHA tissue engineering area and concluded that the PHA prospective will look very bright in the near future.
Co-reporter:Shaoping Ouyang;Yuanzheng Qiu;Lingfang Qin;Song Chen;Qiong Wu;Michael L. Leski;Guoqiang Chen;Jing Han
Macromolecular Symposia 2005 Volume 224(Issue 1) pp:21-34
Publication Date(Web):20 MAY 2005
DOI:10.1002/masy.200550603
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), a copolyester consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx), can be synthesized by Aeromonas hydrophila strain 4AK4 using long chain fatty acids as the carbon source. The wild type A. hydrophila 4AK4 accumulated PHBHHx consisting of 12-15 mol% 3HHx regardless of growth conditions. When phbA, phbB and vgb genes encoding β-ketothiolase, acetoacetyl-CoA reductase and vitreoscilla hemoglobin, respectively, were introduced together into A. hydrophila 4AK4, the recombinant strain grew to over 20 g/L cell dry weight (CDW) after 48 h of shake flask cultivation in co-substrates of dodecanoate and gluconate (weight ratio 1:1), and the CDW contained 50% PHBHHx consisting of 9 mol% 3HHx. Under similar conditions, the wild type strain produced only 12 g/L CDW containing 32% PHBHHx with 15 mol% 3HHx. In comparison, recombinant harboring phbA and phbB produced 35% PHBHHx with 9 mol% 3HHx in 15 g/L CDW under the same conditions. The obvious differences in terms of the cell growth and PHBHHx production were attributed to the expression of vgb in A. hydrophila 4AK4, which was clearly observed in carbon monoxide difference spectra. The expression of vgb in the recombinant not only improved cell growth and PHBHHx accumulation, but also increased the plasmid stability during cell growth, especially under low dissolved oxygen tension in fermentors. PHBHHx production could be further increased to over 60% of the CDW by the over expression of phaC and phaJ from Aeromonas caviae encoding PHBHHx synthase and (R)-specific enoyl-CoA hydratase, respectively. Over expression of phaC, phaJ and phaP, alone or in various combinations, also increased the 3HHx content of PHBHHx from 14-34%. The above results showed that A. hydrophila was amenable to genetic manipulation, and that these modifications could be exploited to produce compounds with different properties for commercial and research applications.
Co-reporter:Zhong Zheng, Feng-Feng Bei, Hong-Lei Tian, Guo-Qiang Chen
Biomaterials 2005 Volume 26(Issue 17) pp:3537-3548
Publication Date(Web):June 2005
DOI:10.1016/j.biomaterials.2004.09.041
As a new member of polyhydroxyalkanoate (PHA) family, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has better mechanical properties compared with poly(3-hydroxybutyrate) (PHB). Still, many properties of PHBHHx and its blend with PHB may be exploited for a wide range of applications, such as tissue engineering and drug delivery. In this study, PHBHHx was found to be completely miscible with PHB in their blends. Crystallization behavior of these polyester blends played an important role in determination of the surface physicochemical properties including surface free energy, chemical states and polarity, and could subsequently govern the cellular responses. Interaction between PHB and PHBHHx influenced the non-dispersion surface free energy component, which governed the total surface free energy. It was shown that the blend with a 1:1 ratio of PHB/PHBHHx possessed the highest surface free energy, which was the most optimal material for chondrocytes adhesion. After 24 h, the amount of chondrocytes adhered on films of PHB/PHBHHx (1:1) was 2.1×104 cells/cm2, 5-times more compared with that on the PHB films (0.4×104 cells/cm2). The polarity of the blends increased with decreasing crystallinity. After 8 days of cultivation, the chondrocytes attached on PHB films were surrounded by both collagen II and collagen X, the amount of extracellular collagen X decreased with increasing polarity contributed by increasing PHBHHx content in the blend, while chondrocytes changed shapes from spherical to flat with increasing polarity. It indicated that endochondral ossification of chondrocytes was remarkably influenced by the crystallinity of the polyesters.
Co-reporter:Yuan-Zheng Qiu;Zhongyao Shen;Shao-Ping Ouyang;Qiong Wu
Macromolecular Bioscience 2004 Volume 4(Issue 3) pp:255-261
Publication Date(Web):15 MAR 2004
DOI:10.1002/mabi.200300090
Summary:Aeromonas hydrophila 4AK4 was able to synthesize copolyesters consisting of 3-hydroxybutyrate (3HB) and about 15 mol-% 3-hydroxyhexanoate (3HHx) (PHBHHx) when grown in long chain fatty acids such as dodecanoate regardless of growth conditions. To regulate the unit fraction in PHBHHx, phbA and phbB genes encoding β-ketothiolase and acetoacetyl-CoA reductase in Ralstonia eutropha, were introduced into A. hydrophila 4AK4. When gluconate was used as cosubstrate of dodecanoate, the recombinant produced PHBHHx containing 3–12 mol-% 3HHx, depending on the gluconate concentration in media. Vitreoscilla hemoglobin gene, vgb, was also introduced into the above recombinant, resulting in improved PHBHHx content from 38 to 48 wt.-% in shake flask study. Fermentor studies also showed that increased gluconate concentration in medium containing dodecanoate promoted the recombinant strain harboring phbA and phbB genes to incorporate more 3HB unit into PHBHHx, resulting in reduced 3HHx fraction. Recombinant A. hydrophila harboring phbA, phbB and vgb genes demonstrated better PHBHHx productivity and higher conversion efficiency from dodecanoate to PHBHHx than those of the recombinant without vgb in fermentation study. Combined with the robust growth property and simple growth requirement, A. hydrophila 4AK4 appeared to be a useful organism for metabolic engineering.
Co-reporter:Ya-Wu Wang, Weike Mo, Huilan Yao, Qiong Wu, Jinchun Chen, Guo-Qiang Chen
Polymer Degradation and Stability 2004 Volume 85(Issue 2) pp:815-821
Publication Date(Web):August 2004
DOI:10.1016/j.polymdegradstab.2004.02.010
To investigate the biodegradability of novel thermoplastics poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), films made of PHBHHx were subjected to degradation in activated sludge and compared with poly(3-hydroxybutyrate) (PHB) and Ecoflex, a biodegradable product of BASF. After 18 days degradation, 40% of P(HB-co-12%-HHx) and 20% of PHB were degraded, while Ecoflex only lost 5% of its weight. Scanning electron microscopy (SEM) revealed that the surface of Ecoflex was much smoother than that of P(HB-co-12%-HHx) and PHB. At the same time, PHBHHx degradation in a simplified system containing 0.1 g/L lipase in phosphate buffer saline was found to be affected by its HHx content. It was found that P(HB-co-12%-HHx) was degraded faster compared with PHB, P(HB-co-5%-HHx) and P(HB-co-20%-HHx). SEM results revealed that P(HB-co-12%-HHx) films had the most porous surface after degradation. All these indicate that surface morphology played an important role in degradation of PHBHHx. P(HB-co-12%-HHx) combining the advantage of low crystallinity and rough surface was degraded the fastest.
Co-reporter:Ying Deng, Xing-Sun Lin, Zhong Zheng, Jin-Guang Deng, Jin-Chun Chen, Hui Ma, Guo-Qiang Chen
Biomaterials 2003 Volume 24(Issue 23) pp:4273-4281
Publication Date(Web):October 2003
DOI:10.1016/S0142-9612(03)00367-3
The present investigation describes the production of extracellular matrix of rabbit articular cartilage chondrocytes grown on scaffolds of polyhydroxybutyrate (PHB) blended with poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for up to 7 days. The mRNA level of type II collagen of chondrocytes seeded on all scaffolds consisting of PHBHHx were obviously higher than that of PHB-only scaffold throughout the culture period, suggesting the positive effect of PHBHHx on extracellular matrix production. Second-harmonic generation (SHG) imaging technique, combined with confocal fluorescence microscopy (CFM) revealed that PHBHHx in PHB scaffold provided better surface properties for anchoring type II collagen filaments and their penetration into internal layers of the scaffolds. Glycosaminoglycan (GAG), a major composition of extracellular matrix, showed a sharp increase in construct of 1:2 PHB/PHBHHx scaffold after 7 day cultivation, while only a small increase was observed in all other tested scaffolds. At the same time, total collagen contents in all scaffolds containing PHBHHx increased with time, with the maximum collagen production of 742.1±99.2 mg/g dry weight observed in construct of 1:2 PHB/PHBHHx scaffold inoculated for 7 days, this was almost 4-fold higher than that in scaffold of PHB only. It appears that the presence of right proportion of PHBHHx in the composite system of PHB/PHBHHx highly favored the production of extracellular matrix of articular cartilage chondrocytes.
Co-reporter:Quan Zhou, Lin Zhang, Xue-Qi Fu, Guo-Qiang Chen
Journal of Chromatography B 2002 Volume 780(Issue 1) pp:161-169
Publication Date(Web):15 November 2002
DOI:10.1016/S1570-0232(02)00466-X
A high-performance liquid chromatograph equipped with an evaporative light scattering detector (ELSD) (HPLC–ELSD) was used to assay the ceramides in yeast cells. The HPLC–ELSD method employed a cyanopropyl bonded column (CN column) that effectively separated the main interfering substance ergosterol without any derivatization process; most other interfering substances were also removed. The method can be applied for routine assay of ceramide content in yeast.
Co-reporter:Qiong Wu;Ge Tian;Suqin Sun;Isao Noda
Journal of Applied Polymer Science 2001 Volume 82(Issue 4) pp:934-940
Publication Date(Web):15 AUG 2001
DOI:10.1002/app.1925
The premelting behavior of bacterially synthesized polyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as P(HB-co-HHx), was investigated by two-dimensional Fourier-transform infrared (2D FTIR) correlation spectroscopy. The temperature-dependent dynamic spectra were measured over a temperature range of 25–300°C. We focused our study on the thermally induced intensity fluctuations of bands for CO (1700–1760 cm−1), CH (2910–3010 cm−1) and COC groups (1220–1310 cm−1) stretching vibrations. Changes of crystalline conformation due to the thermal perturbation could be detected by the intensity and location variations of those characteristic bands responding to the variations of dipole moments. 2D correlation analysis indicated that the appearance of fully amorphous component did not happen simultaneously with the disappearance of crystalline component, suggesting that there was an intermediate state between ordered crystalline and amorphous states in P(HB-co-HHx). © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 934–940, 2001
Co-reporter:Chen Guo-Qiang, Xu Jun, Wu Qiong, Zhang Zengming, Ho Kwok-Ping
Reactive and Functional Polymers 2001 Volume 48(1–3) pp:107-112
Publication Date(Web):2 May 2001
DOI:10.1016/S1381-5148(01)00042-6
Strain Pseudomonas stutzeri 1317 was able to synthesize a variety of polyhydroxyalkanoates (PHAs) when grown in glucose and/or fatty acids. The monomer structures of the PHAs were found to be dependent on the structures of related fatty acids. Up to ten PHA monomers have been found in PHAs, including 3-hydroxybutyrate (HB), 3-hydroxyvalerate (HV), 3-hydroxyhexanoate (HHx), 3-hydroxyheptanoate (HHp), 3-hydroxyoctanoate (HO), 3-hydroxynonanoate (HN), 3-hydroxydecanoate (HD), 3-hydroxyundecanoate (HU), 3-hydroxydodecanoate (HDD) and 3-hydroxytetradecanoate (HTD). In the presence of butyric acid, valeric acid to nonanoic acid, HB, HV to HN were all synthesized. Mixed substrates of glucose and fatty acids led to the formation of fatty acid structure related PHA monomers. Monomer percentage contents in PHA could be adjusted by regulating ratios of fatty acids in growth substrates. During cell growth in glucose and octanoic acid, the addition of heptanoic acid stimulated the rapid synthesis of HHp in PHA. It can be concluded that PHA structures can be tailor made by using structure-related fatty acids and varying ratios of fatty acids.
Co-reporter:Xiaoxing WEI, Feng LIU, Jia JIAN, Ruiyan WANG, Guoqiang CHEN
Chinese Journal of Chemical Engineering (September 2013) Volume 21(Issue 9) pp:1057-1061
Publication Date(Web):1 September 2013
DOI:10.1016/S1004-9541(13)60549-2
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 phaC1-phaZ-phaC2 operon of P. stutzeri 1317 was knocked out to generate the PHA deficient mutant P. stutzeri 1317LF. Then three modules containing phaCAhAReBRe, phaCAhBReGPp and phaCAhPAh 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.
Co-reporter:Guo-Qiang Chen, Ivan Hajnal
Trends in Biotechnology (October 2015) Volume 33(Issue 10) pp:559-564
Publication Date(Web):1 October 2015
DOI:10.1016/j.tibtech.2015.07.006
Polyhydroxyalkanoates (PHAs) are a family of polyesters synthesized by bacteria. Similarly to the genome, transcriptome, and proteome (the entire array of nucleic acids and proteins present in a cell or population of cells at a given time), the PHA spectrum exhibits diverse and dynamic modifications – the ‘PHAome’ – reflecting not only by the diversity of monomers, homopolymers, random and block copolymers, functional and graft polymers, molecular weights, and combinations of the above, but also the ranges of PHAs with various molecular weights and monomer ratios that are present at a particular timepoint in a bacterial cell. Echoing the Materials Genome Initiative (MGI) launched in 2011 to develop an infrastructure to accelerate advanced materials discovery and deployment, understanding the PHAome and ensuring an ample supply of PHAs based on it will promote the discovery of new properties and applications of this family of advanced materials.
Co-reporter:Guo-Qiang Chen, Ivan Hajnal, Hong Wu, Li Lv, Jianwen Ye
Trends in Biotechnology (October 2015) Volume 33(Issue 10) pp:565-574
Publication Date(Web):1 October 2015
DOI:10.1016/j.tibtech.2015.07.007
Polyhydroxyalkanoates (PHA) are a family of diverse biopolyesters synthesized by bacteria. PHA diversity, as reflected by its monomers, homopolymers, random and block copolymers, as well as functional polymers, can now be generated by engineering the three basic synthesis pathways including the acetoacetyl-CoA pathway, in situ fatty acid synthesis, and/or β-oxidation cycles, as well as PHA synthase specificity. It is now possible to tailor the PHA structures via genome editing or process engineering. The increasing PHA diversity and maturing PHA production technology should lead to more focused research into their low-cost and/or high-value applications.
Co-reporter:De-Chuan Meng, Rui Shen, Hui Yao, Jin-Chun Chen, ... Guo-Qiang Chen
Current Opinion in Biotechnology (October 2014) Volume 29() pp:24-33
Publication Date(Web):1 October 2014
DOI:10.1016/j.copbio.2014.02.013
•Increasing PHA diversity reflects more on polymer microstructures.•Engineering the β-oxidation pathway allows controlled synthesis of PHA compositions.•Grafting of PHA site chains result in endless variation of PHA.Many bacteria have been found to produce various polyhydroxyalkanoates (PHA) biopolyesters. In many cases, it is not easy to control the structures of PHA including homopolymers, random copolymers and block copolymers as well as ratios of monomers in the copolymers. It has become possible to engineer bacteria for controllable synthesis of PHA with the desirable structures by creating new PHA synthesis pathways. Remarkably, the weakening of β-oxidation cycle in Pseudomonas putida and Pseudomonas entomophila led to controllable synthesis of all kinds of PHA structures including monomer ratios in random and/or block copolymers when fatty acids are used as PHA precursors. Introduction of functional groups into PHA polymer chains in predefined proportions has become a reality provided fatty acids containing the functional groups are taken up by the bacteria for PHA synthesis. This allows the formation of functional PHA for further grafting. The PHA diversity is further widened by the endless possibility of controllable homopolymerization, random copolymerization, block copolymerization and grafting on functional PHA site chains.Download high-res image (92KB)Download full-size image
Co-reporter:Xiao-Ran Jiang, Guo-Qiang Chen
Biotechnology Advances (July–August 2016) Volume 34(Issue 4) pp:435-440
Publication Date(Web):July–August 2016
DOI:10.1016/j.biotechadv.2015.12.007
Co-reporter:Guo-Qiang Chen, Xiao-Ran Jiang, Yingying Guo
Synthetic and Systems Biotechnology (December 2016) Volume 1(Issue 4) pp:236-242
Publication Date(Web):1 December 2016
DOI:10.1016/j.synbio.2016.09.006
Microbial polyhydroxyalkanoates (PHA) have been produced as bioplastics for various purposes. Under the support of China National Basic Research 973 Project, we developed synthetic biology methods to diversify the PHA structures into homo-, random, block polymers with improved properties to better meet various application requirements. At the same time, various pathways were assembled to produce various PHA from glucose as a simple carbon source. At the end, Halomonas bacteria were reconstructed to produce PHA in changing morphology for low cost production under unsterile and continuous conditions. The synthetic biology will advance the PHA into a bio- and material industry.
Co-reporter:Ying Wang, Jin Yin, Guo-Qiang Chen
Current Opinion in Biotechnology (December 2014) Volume 30() pp:59-65
Publication Date(Web):1 December 2014
DOI:10.1016/j.copbio.2014.06.001
•The development of super PHA production strain using systems and synthetic biology approaches is urgent.•Open and continuous bioprocesses that allow PHA production from lower cost mixed substrates lead to lower cost PHA.•Functional PHA that provide high values should be an important strategy for PHA industry.Microbial polyhydroxyalkanoates (PHA) have been developed as biodegradable plastics for the past many years. However, PHA still have only a very limited market. Because of the availability of large amount of shale gas, petroleum will not raise dramatically in price, this situation makes PHA less competitive compared with low cost petroleum based plastics. Therefore, two strategies have been adopted to meet this challenge: first, the development of a super PHA production strain combined with advanced fermentation processes to produce PHA at a low cost; second, the construction of functional PHA production strains with technology to control the precise structures of PHA molecules, this will allow the resulting PHA with high value added applications. The recent systems and synthetic biology approaches allow the above two strategies to be implemented. In the not so distant future, the new technology will allow PHA to be produced with a competitive price compared with petroleum-based plastics.Open and continuous production of PHA by the constructed super PHA producer.Download high-res image (122KB)Download full-size image
Co-reporter:Xue Gao, Jin-Chun Chen, Qiong Wu, Guo-Qiang Chen
Current Opinion in Biotechnology (December 2011) Volume 22(Issue 6) pp:768-774
Publication Date(Web):1 December 2011
DOI:10.1016/j.copbio.2011.06.005
Microbial polyhydroxyalkanoates (PHA) are a family of structurally diverse polyesters produced by many bacteria. Deleting key steps from the beta-oxidation cycle in Pseudomonas putida makes it possible to achieve precise substrate based design of PHA homopolymers, copolymers, and block polymers, allowing the study of structure–property relationship in a clear way. The PHA homopolymer synthesis also allows the microbial or chemical production of pure monomers of PHA in a convenient way without separating the mixed monomers. After used as bioplastics, PHA can be methyl esterified to become biofuels, which further extends the PHA application value. The microbial production of PHA with diverse structures is entering a new developing phase.Highlights► Microbial polyhydroxyalkanoates (PHA) are a source of polymers, chemicals and biofuels. ► A Pseudomonas putida based platform for producing PHA homopolymers and block copolymers has opened a new area to diversify PHA monomers and structures. ► Further PHA development appears to be limited only by imagination and practices.
Co-reporter:Guo-Qiang Chen, Romas Kazlauskas
Current Opinion in Biotechnology (December 2011) Volume 22(Issue 6) pp:747-748
Publication Date(Web):1 December 2011
DOI:10.1016/j.copbio.2011.08.003
Co-reporter:Shen Yu Wang, Zhen Wang, Ming Ming Liu, Yu Xu, Xiao Jun Zhang, Guo-Qiang Chen
Biomass and Bioenergy (August 2010) Volume 34(Issue 8) pp:1216-1222
Publication Date(Web):August 2010
DOI:10.1016/j.biombioe.2010.03.020
Co-reporter:Hong-Kun Ma, Ming-Ming Liu, Shi-Yan Li, Qiong Wu, Jin-Chun Chen, Guo-Qiang Chen
Journal of Biotechnology (20 June 2013) Volume 166(Issues 1–2) pp:34-41
Publication Date(Web):20 June 2013
DOI:10.1016/j.jbiotec.2013.04.017
•We showed that PHA synthesis regulatory protein PhaR is a bio-surfactant with excellent emulsification ability and strong bactericidal capacity.•PhaR was found highly stable even at 95 °C.•PhaR could be used in areas including food, beverage, pharmaceutical and cosmetics industries.Polyhydroxyalkanoates (PHA), a family of diverse bio-polyesters, are produced by many bacteria as an energy and carbon storage material. PHA synthesis regulatory protein PhaR was reported to attach on the surface of intracellular PHA granules for convenience of synthesis regulation. PhaR was found to have an amphiphilic property. However, no study was conducted to exploit this property for applications as bio-surfactant and bactericide agent. Purified PhaR showed a higher emulsification ability than that of the widely used chemical surfactants including SDS, Tween 20, sodium oleate, and liquefied detergent (LD). PhaR also showed a higher emulsification ability than bio-surfactants rhamnose and PHA granules associated protein termed phasin or PhaP. Non-purified PhaR, namely, the native inclusion bodies and cell lysates, also demonstrated to be an excellent surfactant. PhaR was found highly stable even at 95 °C. In addition, PhaR was revealed to be a promising bactericidal agent against Gram positive and negative bacteria. PhaR can be conveniently produced by recombinant Escherichia coli. It has shown to be a bio-surfactant with excellent emulsification ability and strong bactericidal capacity at elevated temperature as high as 95 °C. Therefore, PhaR could be used in areas including food, beverage, pharmaceutical and cosmetics industries.
Co-reporter:Lei Cai, Mei-Qing Yuan, Zheng-Jun Li, Jin-Chun Chen, Guo-Qiang Chen
Journal of Biotechnology (20 January 2012) Volume 157(Issue 2) pp:320-325
Publication Date(Web):20 January 2012
DOI:10.1016/j.jbiotec.2011.12.004
Folate derivatives are crucial growth factors for Ketogulonigenium vulgare which is used in mixed culture with Bacillus megaterium for the industrial production of 2-keto-l-gulonic acid (2-KGA), the precursor of l-ascorbic acid (l-AA) or vitamin C (Vc). To improve the growth and 2-KGA production, five genes involved in folate biosynthesis identified in a folate gene cluster from Lactococcus lactis MG1363, including folB, folKE, folP, folQ and folC, were over-expressed in K. vulgare. Intracellular folate concentration in the recombinant strain harboring folate biosynthesis genes cluster under the control of Psdh (sorbose dehydrogenase gene sdh promoter from K. vulgare) was 8 times higher than that of the wildtype K. vulgare DSM 4025 (P < 0.001). In shake flask studies, the cell density and 2-KGA production of the recombinant K. vulgare Rif (pMCS2PsdhfolBC) were increased by 18% (P < 0.001) and 14% (P < 0.001), respectively, under a relatively stable pH 7 condition. In fermentor studies, enhancements around 25% cell density (P < 0.001) and approximately 35% 2-KGA productivity (P < 0.001) were observed in comparison with the controls without over-expressing the folate biosynthesis genes. This was the first successful study of metabolic engineering on K. vulgare for enhanced 2-KGA production.Highlights► Over-expression of folate gene cluster increased folate concentration 8 times. ► Cell density and 2-KGA production increased by 18% and 14% in shake flasks. ► A 25% cell density and 35% 2-KGA productivity enhancement was observed in fermentor. ► This was the first successful study of metabolic engineering on K. vulgare.
Co-reporter:Jin Yin, Jin-Chun Chen, Qiong Wu, Guo-Qiang Chen
Biotechnology Advances (15 November 2015) Volume 33(Issue 7) pp:
Publication Date(Web):15 November 2015
DOI:10.1016/j.biotechadv.2014.10.008
•Industrial biotechnology is less competitive compared with chemical processes.•Halophiles allow fermentation processes to run contamination free under unsterile conditions and continuous way.•Halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants.•Genetic manipulation methods have been developed for halophiles.Industrial biotechnology aims to produce chemicals, materials and biofuels to ease the challenges of shortage on petroleum. However, due to the disadvantages of bioprocesses including energy consuming sterilization, high fresh water consumption, discontinuous fermentation to avoid microbial contamination, highly expensive stainless steel fermentation facilities and competing substrates for human consumption, industrial biotechnology is less competitive compared with chemical processes. Recently, halophiles have shown promises to overcome these shortcomings. Due to their unique halophilic properties, some halophiles are able to grow in high pH and high NaCl containing medium under higher temperature, allowing fermentation processes to run contamination free under unsterile conditions and continuous way. At the same time, genetic manipulation methods have been developed for halophiles. So far, halophiles have been used to produce bioplastics polyhydroxyalkanoates (PHA), ectoines, enzymes, and bio-surfactants. Increasing effects have been made to develop halophiles into a low cost platform for bioprocessing with advantages of low energy, less fresh water consumption, low fixed capital investment, and continuous production.
Co-reporter:Guo-Qiang Chen
Chemical Society Reviews 2009 - vol. 38(Issue 8) pp:NaN2446-2446
Publication Date(Web):2009/05/08
DOI:10.1039/B812677C
Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).
