Hyperproduction of PHA copolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors

Bacterial outer membrane (OM) is a self-protective and permeable barrier, while having many non-negligible negative effects in industrial biotechnology. Our previous studies revealed enhanced properties of Halomonas bluephagenesis based on positive cellular properties by OM defects. This study further expands the OM defect on membrane compactness by completely deleting two secondary acyltransferases for lipid A modification in H. bluephagenesis, LpxL and LpxM, and found more significant advantages than that of the previous lpxL mutant. Deletions on LpxL and LpxM accelerated poly(3-hydroxybutyrate) (PHB) production by H. bluephagenesis WZY229, leading to a 37% increase in PHB accumulation and 84-folds reduced endotoxin production. Enhanced membrane permeability accelerates the diffusion of γ-butyrolactone, allowing H. bluephagenesis WZY254 derived from H. bluephagenesis WZY229 to produce 82wt% poly(3-hydroxybutyrate-co-23mol%4-hydroxybutyrate) (P(3HB-co-23mol%4HB)) in shake flasks, showing increases of 102% and 307% in P(3HB-co-4HB) production and 4HB accumulation, respectively. The 4HB molar fraction in copolymer can be elevated to 32 mol% in the presence of more γ-butyrolactone. In a 7-l bioreactor fed-batch fermentation, H. bluephagenesis WZY254 supported a 84 g l⁻¹ dry cell mass with 81wt% P(3HB-co-26mol%4HB), increasing 136% in 4HB molar fraction. This study further demonstrated that OM defects generate a hyperproduction strain for high 4HB containing copolymers.     

Biosynthesis of functional polyhydroxyalkanoates by engineered Halomonas bluephagenesis

Polyhydroxyalkanoates (PHA) have found widespread medical applications due to their biocompatibility and biodegradability, while further chemical modification requires functional groups on PHA. Halomonas bluephagenesis, a non-model halophilic bacterium serving as a chassis for the Next Generation Industrial Biotechnology (NGIB), was successfully engineered to express heterologous PHA synthase (PhaC) and enoyl coenzyme-A hydratase (PhaJ) from Aeromonas hydrophila 4AK4, along with a deletion of its native phaC gene to synthesize the short chain-co-medium chain-length PHA copolymers, namely poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), poly(3-hydroxybutyrate-co-3-hydroxyhex-5-enoate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyhex-5-enoate). After optimizations of the expression cassette and ribosomal binding site combined with introduction of endogenous acyl-CoA synthetase (fadD), the resulting recombinant strain H. bluephagenesis TDR4 achieved a remarkably high 3-hydroxyhexenoate (3HHxE) molar ratio of 35% when grown on glucose and 5-hexenoic acid as co-substrates. The total ratio of side chain consisting of 3HHx and 3HHxE monomers in the terpolymer can approach 44 mol%. H. bluephagenesis TDR4 was grown to a cell dry mass (CDM) of 30 g/L containing approximately 20% poly(3-hydroxybutyrate-co-22.75 mol% 3-hydroxy-5-hexenoate) in a 48-h of open and unsterile fermentation with a 5-hexenoic acid conversion efficiency of 91%. The resulted functional PHA containing 12.5 mol% 3-hydroxy-5-hexenoate exhibits more than 1000% elongation at break. The engineered H. bluephagenesis TDR4 can be used as an experimental platform to produce functional PHA.     

Microbial production of poly-β-hydroxybutyrate by marine microbes isolated from various marine environments

Considering the industrial interest of Poly-β-hydroxybutyrate (PHB), bacteria isolated from the various marine arenas were screened for their ability to accumulate PHB and were compared with Wausteria eutropha (MTCC-1285). Among the 42 isolates, four strains showed the accumulation of PHB. The maximum PHB producer Vibrio sp. (MK4) was further studied in detail. To increase the productivity, steps were taken to evaluate the effect of carbon sources, nitrogen sources, pH and sodium chloride concentration on PHB productivity by MK4. The optimized conditions were further used for the batch fermentation over a period of 72 h. Significantly higher maximum biomass of 9.1 g/L with a PHB content of 4.223 g/L was obtained in a laboratory-scale bioreactor at 64 h, thus giving a productivity of 0.065 g/L/h. The extracted polymer was compared with the authentic PHB and was confirmed to be PHB using FTIR analysis and ¹H NMR analysis. Thus, the study highlights the potential of the use of Vibrio sp (MK4) in the commercial production of PHB.     

Microbial biodegradable plastic production from a wheat-based biorefining strategy

Restructuring the current fermentation and recovery practices employed for the production of polyhydroxyalkanoates is essential for the commercialisation of environmentally benign and cost competitive biodegradable plastics. This study presents the potential of a wheat-based biorefinery for the production of poly(3-hydroxybutyrate) (PHB). Fed-batch bioconversions using Wautersia eutropha growing on wheat-derived media led to the production of 162.8 g/l PHB. A high PHB to total dry weight (TDW) yield of 93% (w/w) was achieved due to microbial autolysis at the end of fermentation. Images of bacterial cells taken with a Transmission Electron Microscope (TEM) indicated the potential of bacterial autolysis as a mean to shorten downstream processing for PHB purification. The consumption of amino acids and peptides derived from wheat gluten hydrolysis resulted in a high glucose to PHB conversion yield of 0.47 g/g. The respective yield regarding the amount of wheat used for the production of enzymes and PHB was around 0.3 g PHB/g wheat, which corresponds to 82.8% of the maximum theoretical conversion yield. The productivity achieved was around 0.9 g/l h. Fermentations carried out on wheat-derived media and media formulated with various commercial sources of nutrients (glucose, yeast extract, soy-protein acid hydrolysate, casein hydrolysates, corn steep liquor and various inorganic chemicals) showed that the proposed wheat-based biorefinery strategy enhanced PHB production.     

Response coefficient analysis of a fed-batch bioreactor to dissolved oxygen perturbation in complementary cultures during PHB production

Background  

Although the production of poly-β-hydroxybutyrate (PHB) has many biological, energetic and environmental advantages over chemically synthesized polymers, synthetic polymers continue to be produced industrially since the productivities of fermentation processes fr PHB are not yet economically competitive. Improvement of a PHB fermentation requires good understanding and optimization under the realistic conditions of large bioreactors.     

Overexpression of NAD kinase in recombinant <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> harboring the <Emphasis Type="BoldItalic">phbCAB</Emphasis> operon improves poly(3-hydroxybutyrate) production

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 Y _PHB/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 Y _PHB/glucose. These results clearly showed that the overexpression of NAD kinase could be used to enhance the PHB synthesis.     

Engineering the permeability of Halomonas bluephagenesis enhanced its chassis properties

Bacterial outer membrane (OM), an asymmetric lipid bilayer functioning as a self-protective barrier with reduced permeability for Gram-negative bacteria, yet wasting nutrients and energy to synthesize, has not been studied for its effect on bioproduction. Here we construct several OM-defected halophile Halomonas bluephagenesis strains to investigate the effects of OM on bioproduction. We achieve enhanced chassis properties of H. bluephagenesis based on positive cellular properties among several OM-defected strains. The OM-defected H. bluephagenesis WZY09 demonstrates better adaptation to lower salinity, increasing 28%, 30% and 12% on dry cell mass (DCM), poly(3-hydroxybutyrate) (PHB) accumulation and glucose to PHB conversion rate, respectively, including enlarged cell sizes and 21-folds reduced endotoxin. Interestingly, a poly(3-hydroxybutyrate-co-21mol%4-hydroxybutyrate) (P(3HB-co-21mol%4HB)) is produced by H. bluephagenesis WZY09 derivate WZY249, increasing 60% and 260% on polyhydroxyalkanoate (PHA) production and 4HB content, respectively. Furthermore, increased electroporation efficiency, more sensitive isopropyl β-D-1-thio-galactopyranoside (IPTG) induction, better oxygen uptake, enhanced antibiotics sensitivity and ectoine secretion due to better membrane permeability are observed if OM defected, demonstrating significant OM defection impacts for further metabolic engineering, synthetic biology studies and industrial applications.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.     

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains

In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i _Bl and i _Ec , respectively) were expressed in lycopene producing E. coli strains by pTlyci_Bl and pTlyci_Ec plasmids, under the control of tac promoter. The results showed that the overexpression of i _Ec improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci_Ec. In contrast, the expression of i _Bl increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci_Bl. The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i _Ec in E. coli Tlyci_Ec-mev and 181 ± 9 mg/gDCW with i _Bl in E. coli Tlyci_Bl-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci_Bl-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens

Summary Poly--hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly--hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 10⁵ Da was obtained for three PHB samples using two different measurement methods.Publication number NRCC No. 33672 Offprint requests to: D. Groleau     

Production of PHB from Crude Glycerol

Crude glycerol – a by‐product of the large scale production of diesel oil from rape – is examined for its possible use as a cheap feedstock for the biotechnological synthesis of poly(3‐hydroxybutyrate) (PHB). The glycerol samples of various manufacturers differ in their contamination with salts (NaCl or K₂SO₄), methanol or fatty acids. At high cell density fermentation these pollutants could possibly accumulate to inhibiting concentrations. The bacteria used were Paracoccus denitrificans and Cupriavidus necator JMP 134, which accumulate PHB from pure glycerol to a content of 70 % of cell dry mass. When using crude glycerol containing 5.5 % NaCl, a reduced PHB content of 48 % was observed at a bacterial dry mass of 50 g/L. Furthermore the PHB yield coefficient was reduced, obviously due to osmoregulation. The effect of glycerol contaminated with K₂SO₄ was less pronounced. The molecular weight of PHB produced with P. denitrificans or C. necator from crude glycerol varies between 620000 and 750000 g/mol which allows the processing by common techniques of the polymer industry.     

Effect of total nutrient feed on production of poly-3-hydroxybutyrate by Methylobacterium sp. ZP24 grown on sugars

Methylobacterium sp. ZP24 is able to produce poly-3-hydroxybutyrate (PHB) from sucrose and lactose. As the production of PHB is growth-associated, a strategy of intermittent feeding of sugars and other nutrients was assessed for obtaining high yields of the polymer. Higher PHB synthesis was obtained at increased sugar feed rates. Cellular PHB contents of 63% and 71%, with productivities of up to 0.354 and 0.645 g PHB/l h were obtained from sucrose and lactose, respectively. A short-duration semicontinuous production level of up to 2.4 g PHB/l h was achieved in the lactose fermentation. Journal of Industrial Microbiology & Biotechnology (2000) 25, 276–279. Received 06 June 2000/ Accepted in revised form 31 August 2000     

Production of poly-β-hydroxybutyrate (PHB) by <Emphasis Type="Italic">Alcaligenes latus</Emphasis> from maple sap

Maple sap, an abundant natural product especially in Canada, is rich in sucrose and thus may represent an ideal renewable feedstock for the production of a wide variety of value-added products. In the present study, maple sap or sucrose was employed as a carbon source to Alcaligenes latus for the production of poly-β-hydroxybutyrate (PHB). In shake flasks, the biomass obtained from both the sap and sucrose were 4.4 ± 0.5 and 2.9 ± 0.3 g/L, and the PHB contents were 77.6 ± 1.5 and 74.1 ± 2.0%, respectively. Subsequent batch fermentation (10 L sap) resulted in the formation of 4.2 ± 0.3 g/L biomass and a PHB content of 77.0 ± 2.6%. The number average molecular weights of the PHB produced by A. latus from maple sap and pure sucrose media were 300 ± 66 × 10³ and 313 ± 104 × 10³ g/mol, respectively. Near-infrared, ¹H magnetic resonance imaging (MRI), and ¹³C-MRI spectra of the microbially produced PHB completely matched those obtained with a reference material of poly[(R)-3-hydroxybutyric acid]. The polymer was found to be optically active with [α]²⁵ _D equaled to −7.87 in chloroform. The melting point (177.0°C) and enthalpy of fusion (77.2 J/g) of the polymer were also in line with those reported, i.e., 177°C and 81 J/g, respectively.     

A process for the production of ectoine and poly(3-hydroxybutyrate) by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L⁻¹) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L⁻¹) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L⁻¹, respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH₄⁺, and PO₄³⁻. In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L⁻¹ h⁻¹ and ectoine concentration, content, and volumetric productivity of 4.3 g L⁻¹, 7.2 wt.%, and 2.8 g L⁻¹ day⁻¹, respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L⁻¹ day⁻¹.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Engineering Halomonas bluephagenesis via small regulatory RNAs

Halomonas bluephagenesis, a robust and contamination-resistant microorganism has been developed as a chassis for “Next Generation Industrial Biotechnology”. The non-model H. bluephagenesis requires efficient tools to fine-tune its metabolic fluxes for enhanced production phenotypes. Here we report a highly efficient gene expression regulation system (PrrF1-2-HfqPa) in H. bluephagenesis, small regulatory RNA (sRNA) PrrF1 scaffold from Pseudomonas aeruginosa and a target-binding sequence that downregulate gene expression, and its cognate P. aeruginosa Hfq (HfqPa), recruited by the scaffold to facilitate the hybridization of sRNA and the target mRNA. The PrrF1-2-HfqPa system targeting prpC in H. bluephagenesis helps increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to 21 mol% compared to 3.1 mol% of the control. This sRNA system repressed phaP1 and minD simultaneously, resulting in large polyhydroxybutyrate granules. Further, an sRNA library targeting 30 genes was employed for large-scale target identification to increase mevalonate production. This work expands the study on using an sRNA system not based on Escherichia coli MicC/SgrS-Hfq to repress gene expression, providing a framework to exploit new powerful genome engineering tools based on other sRNAs.     

Biosynthesis of poly-3-hydroxybutyrate by Sphaerotilus natans

A sheathed bacterium Sphaerotilus natans could not survive at 4°C for 2 months, and mutants that exhibited different colony phenotypes were obtained only by repeating the short period of storage at 4 °C. The ability of these mutants and the parent strain to produce poly-3-hydroxybutyrate (PHB) was compared in batch cultures. The parent strain accumulated 30% (w/w) PHB, while one of the mutants defective in sheath formation, designated as T2, accumulated over 50% PHB. Because T2 did not require strict air or nitrogen limitation for polymer accumulation, its production was growth-associated, allowing one-stage fermentation. In a pH-controlled fermentation using a jar fermentor, 10 g/l glucose was converted into 2.0 g/l PHB in 24 h.