Metabolic engineering of Aeromonas hydrophila for the enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Wild-type Aeromonas hydrophila 4AK4 produced 35–45 wt.% poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) consisting of 10–15 mol% 3-hydroxyhexanoate (3HHx). To enhance PHBHHx production, vgb gene encoding Vitreoscilla haemoglobin or fadD gene encoding Escherichia coli acyl-CoA synthase was co-expressed with polyhydroxyalkanoates (PHA) synthesis-related genes including phbAB from Wautersia eutropha and phaPCJ from A. hydrophila. Expression of vgb increased PHBHHx content from 46 to 53 wt.% without affecting the polymer monomers composition, whereas fadD increased both PHBHHx content from 46 to 64 wt.% and its 3HHx fraction from 15 to 24 mol%. Co-expression of vgb or fadD gene with PHA-synthesis-related genes generally increased PHBHHx content over 60 wt.%. Co-expression of phbAB with vgb increased PHBHHx content and concentration up to about 70 wt.% and 4.0 g l⁻¹, respectively. Fermentor study also showed that in the recombinants harboring vgb, CDW, PHBHHx concentration and productivity were significantly elevated up to 54 g l⁻¹, 28.5 g l⁻¹ and 0.791 g l⁻¹ h⁻¹, respectively, suggesting that vgb could promote PHA synthesis. In this strain, lac promoter could be used to constitutively express foreign genes such as phbA and phbB encoding β-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase of W. eutropha, respectively, without use of IPTG. The results showed that combined expression of different genes was a successful strategy to enhance PHA production, which could be useful for strain development to construct other recombinant PHA-producing strains.     

Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition

Aeromonas hydrophila 4AK4 produces poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) containing 3-hydroxybutyrate (3HB) and about 15 mol% 3-hydroxyhexanoate (3HHx) from dodecanoate. To study the factors affecting the monomer composition and PHBHHx content, genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase (phaJ) from Aeromonas punctata (formerly named Aeromonas caviae) were introduced individually or jointly into A. hydrophila 4AK4. The phaC gene increased 3HHx fraction more significantly than phaP, while phaJ had little effect. Expression of phaC alone increased the 3HHx fraction from 14 to 22 mol%. When phaC was co-expressed with phaP and phaJ, the 3HHx fraction increased from 14 to 34 mol%. Expression of phaP or phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight (CDW) as much as 4.4 gL(-1) and PHBHHx concentration to 2.7 gL(-1) after 48 h in shake flask culture. The results suggest that a higher PHA synthase activity could lead to a higher 3HHx fraction and PHBHHx content. Co-expression of phaJ with phaC or phaP would favor PHA accumulation, although over-expression of phaJ did not affect PHA synthesis much. In addition, inhibition of beta-oxidation by acrylate in A. hydrophila 4AK4 enhanced PHBHHx content. However, no monomers longer than 3HHx were detected. The results show that genetic modification of A. hydrophila 4AK4 enhanced PHBHHx production and altered monomer composition of the polymer.     

Production and characterization of terpolyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by recombinant Aeromonas hydrophila 4AK4 harboring genes phaAB

Recombinant Aeromonas hydrophila 4AK4 harboring phbA and phbB (phaAB) genes encoding β-ketothiolase and acetoacetyl-CoA reductase of Ralstonia eutropha produced a terpolyester of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from mixtures of dodecanoic acid and propionic acid. Depending on the concentration of propionic acid in bacterial cultures, cell growth represented by cellular dry weight (CDW), P(3HB-co-3HV-co-3HHx) contents in dry cells and 3HV molar percentage in the terpolyester ranged from 0.43 g l⁻¹ to 3.29 g l⁻¹, 20.7% to 35.6%, 2.3 mol% to 7.1 mol%, respectively. Number average molecular (M_n) weights of the terpolyesters were 303,000–800,000, independent from monomer fraction content. This terpolyester was characterized by nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and stress–strain measurement studies. Results showed that the terpolyester had higher thermal stability and elongation at break compared with that of homopolymer poly(3-hydroxybutyrate) (PHB) and its copolymers P(3HB-co-5 mol%3HV) or P(3HB-co-12 mol%3HHx). In addition, the terpolyester had lower melting (T_m) temperatures and enthalpy of fusions (ΔH_m) than PHB did.     

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.     

重组嗜水气单胞菌Aeromonas hydrophila 4AK4中3-羟基丁酸3-羟基己酸共聚酯PHBHHx的发酵生产

3-羟基丁酸和3-羟基己酸共聚酯(PHBHHx)是一种性能优良的新型生物可降解材料,其机械和加工性能与3-羟基己酸(3HHx)在共聚物中的含量密切相关。在嗜水气单孢菌Aeromonas hydrophila 4AK4中引入了编码β-酮基硫解酶(β-ketothiolase)的phbA基因和编码乙酰乙酰辅酶A还原酶(Acetoacetyl-CoA reductase)的phbB基因,使重组菌增加了一条利用乙酰辅酶A合成3-羟基丁酸-CoA的代谢途径,这使得利用非相关性碳源调控PHBHHx的单体组成比例成为可能。利用葡萄糖酸钠和月桂酸作为碳源,对重组Aeromonas hydrophila 4AK4进行了摇瓶培养及5L发酵罐培养的研究。在摇瓶实验中,通过改变碳源中两种组分的比例,可以使A,hydrophila 4AK4合成的PHBHHx中的3HHx摩尔含量由原来的15％左右降低到3％～12％,成功地实现了对PHBHHx单体组成的调控;当以月桂酸为唯一碳源时,在5L发酵罐中,经过56h的培养,获得了51．5g／L的细胞干重(CDW),其中62％为PHBHHx,3HHx在PHBHHx中的摩尔含量为9．7％;当以1：1的葡萄糖酸钠和月桂酸为碳源时,48h的5L发酵罐培养获得了32．8g／L的CDW和52％的PHBHHx含量,其中3HHx在PHBHHx中的摩尔含量为6．7％。结果证明了该重组菌在大规模生产单体组成可控PHBHHx方面具有很大的应用潜力。     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains

The polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(HB-co-HHx)] has been shown to have potential to serve as a commercial bioplastic. Synthesis of P(HB-co-HHx) from plant oil has been demonstrated with recombinant Ralstonia eutropha strains expressing heterologous PHA synthases capable of incorporating HB and HHx into the polymer. With these strains, however, short-chain-length fatty acids had to be included in the medium to generate PHA with high HHx content. Our group has engineered two R. eutropha strains that accumulate high levels of P(HB-co-HHx) with significant HHx content directly from palm oil, one of the world's most abundant plant oils. The strains express a newly characterized PHA synthase gene from the bacterium Rhodococcus aetherivorans I24. Expression of an enoyl coenzyme A (enoyl-CoA) hydratase gene (phaJ) from Pseudomonas aeruginosa was shown to increase PHA accumulation. Furthermore, varying the activity of acetoacetyl-CoA reductase (encoded by phaB) altered the level of HHx in the polymer. The strains with the highest PHA titers utilized plasmids for recombinant gene expression, so an R. eutropha plasmid stability system was developed. In this system, the essential pyrroline-5-carboxylate reductase gene proC was deleted from strain genomes and expressed from a plasmid, making the plasmid necessary for growth in minimal media. This study resulted in two engineered strains for production of P(HB-co-HHx) from palm oil. In palm oil fermentations, one strain accumulated 71% of its cell dry weight as PHA with 17 mol% HHx, while the other strain accumulated 66% of its cell dry weight as PHA with 30 mol% HHx.     

Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Large scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by Aeromonas hydrophila 4AK4 was examined in a 20,000 l fermentor. Cells were first grown using glucose as a carbon source, and polyhydroxyalkanoate (PHA) biosynthesis was triggered by the addition of lauric acid under conditions of limited nitrogen or phosphorus. When cells first grown in a medium containing 50 g glucose l(-1) were further cultivated after the addition of 50 g lauric acid l(-1) under phosphorus limitation, a final cell concentration, PHA concentration and PHA content of 50 g l(-1), 25 g l(-1), and 50 wt%, respectively, were obtained in 46 h, equivalent to PHA productivity of 0.54 g l(-1)t h(-1). The copolymer produced was found to be a random copolymer, and the 3HHx fraction was 11 mol%.     

Cloning and analysis of the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae.

A 5.0-kbp EcoRV-EcoRI restriction fragment was cloned and analyzed from genomic DNA of Aeromonas caviae, a bacterium producing a copolyester of (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyhexanoate (3HHx) [P(3HB-co-3HHx)] from alkanoic acids or oils. The nucleotide sequence of this region showed a 1,782-bp poly (3-hydroxyalkanoate) (PHA) synthase gene (phaC(Ac) [i.e., the phaC gene from A. caviae]) together with four open reading frames (ORF1, -3, -4, and -5) and one putative promoter region. The cloned fragments could not only complement PHA-negative mutants of Alcaligenes eutrophus and Pseudomonas putida, but also confer the ability to synthesize P(3HB-co-3HHx) from octanoate or hexanoate on the mutants' hosts. Furthermore, coexpression of ORF1 and ORF3 genes with phaC(Ac) in the A. eutrophus mutant resulted in a decrease in the polyester content of the cells. Escherichia coli expressing ORF3 showed (R)-enoyl-coenzyme A (CoA) hydratase activity, suggesting that (R)-3-hydroxyacyl-CoA monomer units are supplied via the (R)-specific hydration of enoyl-CoA in A. caviae. The transconjugant of the A. eutrophus mutant expressing only phaC(Ac) effectively accumulated P(3HB-co-3HHx) up to 96 wt% of the cellular dry weight from octanoate in one-step cultivation.     

Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) polymers

In support of programs to identify polyhydroxyalkanoates with improved materials properties, we report on our efforts to characterize the mechanical and thermal properties of copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). The copolyesters, having molar fraction of 3HHx ranging from 2.5 to 35 mol % and average molecular weights ranging from 1.15 x 10(5) to 6.65 x 10(5), were produced by fermentation using Aeromonas hydrophila and a recombinant strain of Pseudomonas putida GPp104. The polymers were chloroform extracted and characterized by solution-state and solid-state nuclear magnetic resonance (NMR) spectroscopy and a variety of mechanical and thermal tests. Solution-state (1)H NMR data were used to determine polymer composition-of-matter, while solution-state (13)C NMR data provided polymer-sequence information. Solvent fractionation and NMR spectroscopic characterization of these polymers showed that polymers containing up to 9.5 mol % 3HHx had a Bernoullian compositional distribution. By contrast, polymers containing more than 9.5 mol % 3HHx had a bimodal polymer composition. Solvent fractionation of these 3HHx-rich polyesters produced two polymer fractions, each of which was again consistent with Bernoullian polymerization statistics. Solid-state NMR relaxation experiments provided insight into aging in poly(3HB-co-3HHx) copolymers, demonstrating increased polymer-chain motion with increasing 3HHx content. The elongation-to-break ratio in the polyesters increased with increasing molar fraction of 3HHx monomers. Aging properties of the poly(3HB-co-3HHx) copolymers were very similar to copolymers of 3HB and 3-hydroxyvalerate (3HV). However, poly(3HB-co-3HHx) exhibited increased activation energy to thermal degradation with increasing 3HHx content.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations

Improved production costs will accelerate commercialization of polyhydroxyalkanoate (PHA) polymer and PHA-based products. Plant oils are considered favorable feedstocks, due to their high carbon content and relatively low price compared to sugars and other refined carbon feedstocks. Different PHA production strategies were compared using a recombinant strain of Ralstonia eutropha that produces high amounts of P(HB-co-HHx) when grown on plant oils. This R. eutropha strain was grown to high cell densities using batch, extended batch, and fed batch fermentation strategies, in which PHA accumulation was triggered by nitrogen limitation. While extended batch culture produced more biomass and PHA than batch culture, fed batch cultivation was shown to produce the highest levels of biomass and PHA. The highest titer achieved was over 139 g/L cell dry weight (CDW) of biomass with 74% of CDW as PHA containing 19 mol% HHx. Our data suggest that the fermentation process is scalable with a space time yield (STY) better than 1 g PHA/L/h. The achieved biomass concentration and PHA yield are among the highest reported for the fermentation of recombinant R. eutropha strains producing P(HB-co-HHx).     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) by metabolically engineered Escherichia coli strains

The recombinant Escherichia coli strain, equipped with the newly cloned Aeromonas PHA biosynthesis genes, could produce a terpolymer of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) [P(3HB-co-3HV-co-3HHx)] from dodecanoic acid plus odd carbon number fatty acid. In addition, the orf1 gene of Aeromonas hydrophila was found to play a critical role in assimilating the 3HV monomer and in regulating the monomer fraction in the terpolymer.     

Gelatin blending improves the performance of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films for biomedical application

To improve the performance of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), gelatin was blended with PHBHHx at different ratios. With increasing gelatin content, the weight loss of gelatin/PHBHHx blend in simulated body fluid at 37 degrees C was accelerated. After 2 months, there was about 15% weight loss in PHBHHx blending with 30% gelatin. Scanning electron microscopy and X-ray diffraction results showed that gelatin blending increased the surface porosity and decreased the crystallinity, which may be responsible for the acceleration of the weight loss. Second harmonic generation results indicated that 10% gelatin blending had less disruption to PHBHHx spatial structure, resulting in better tensile mechanical properties. At the same time, increased surface porosity and decreased crystallinity caused by gelatin incorporation may be beneficial for cell growth compared with pure PHBHHx. All these indicated that gelatin incorporation may improve the performances of PHBHHx to meet the need of different situations during medical implantation.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Alcaligenes latus

Summary Copolymers of (R)-3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) were efficiently produced by Alcaligenes latus in the culture solution containing sucrose and -butyrolactone. The conversion yield of -butyrolactone into 4HB unit of copolymer by A. latus was as high as 60%.     

Production of (3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer from coffee waste oil using engineered Ralstonia eutropha

Bioprocess and Biosystems Engineering - Polyhydroxyalkonate (PHA) is a type of polymer that has the potential to replace petro-based plastics. To make PHA production more economically feasible,...     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) byPseudomonas acidovorans

Summary Terpolyesters of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvarelate (3HV) were produced byPseudomonas acidovorans in nitrogen-free culture solutions of 1,4-butanediol and pentanol. When 1,4-butanediol was used as the sole carbon source, a polyester with an unusually high 4HB fraction of 99 mol% was produced.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

嗜水气单胞菌WQ中PHBHHx的合成及其分子基础研究

聚羟基脂肪酸酯(Polyhydroxyalkanoate,PHA)是一系列生物合成的高分子材料,其单体可由多种3-羟基脂肪酸(3-hydroxyalkanoate,3HA)构成^[1]。PHA物理和机械性能的变化很大,从高脆性到弹性体,这跟它们的单体成分有很大关系^[2]。短链和中长链单体共聚的PHA比短链单体或中长链单体聚合得到的PHA有着更好的性能^[3]。在1994年,豚鼠气单胞菌(Aeromonas caviae)FA440被发现能以偶数碳原子数脂肪酸或植物油作为碳源在体内积累PHBHHx^[4]其PHA生物合成基因被成功克隆^[5]。根据亚基数目和底物特异性,PHA合成的关键酶,即PHA合酶或PhaC,被分成了3种类型。A．caviae的PHA合酶属于第1类PHA合酶^[6]。PHA合酶的一些类型含有一些保守的基因序列,该特征可被用于克隆,特别是第Ⅱ类PHA合酶^[2,8]。嗜水气单胞菌(Aeromonas hydrophila)WQ和A．hydrophila 4AK4是能够合成PHBHHx的另外两种菌株,其中A．hydrophila 4AK4已被用作大规模生产PHBHHx。就目前来说,不管生长条件怎么改变,其合成的PHBHHx中3羟基己酸单体(3-hydroxyhexanoate,3HHx)的含量始终在12％～17％之间变化^[9]。而A．hydrophila WQ合成的PHBHHx中则含有6％～14％ 3HHx。本论文研究了A．hydrophila WQ的PHA生物合成及其分子基础。     

Fed-batch culture ofAeromonas hydrophila for the production of poly(3-hydroxubutyrate-co-3-hydroxyhexanoate) using two carbon sources

To produce polyhydroxyalkanoate (PHA) copolymer which consists of 3-hydroxy-butyrate (3HB) and 3-hydroxyhexanoate (3HHx) by cultivation ofAeromonas hydrophila, fed-batch cultures were done under several nutrient limiting conditions. With the results from flask cultures, fed-batch cultures were carried out to produce large amounts of PHA. In the fed-batch culture, firstly glucose was fed to grow cell, and then, oleic acid fed to stimulate PHA in the cell. The final cell concentration, PHA content, PHA concentration, and 3-hydroxy-hexanoate fraction in 38 hr were 48.9 g/L, 15.05 wt%, 7.36 g/L and 12.2 wt%, respectively, resulting in the productivity of 0.19 g/L-h under phosphate-limiting condition.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).