重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

Mechanistic investigation of ultrasonic enhancement of glycerol bioconversion by immobilized Clostridium pasteurianum on silica support

Glycerol, the principal byproduct of biodiesel production, can be a valuable carbon source for bioconversion into diverse class of compounds. This article attempts to investigate the mechanistic aspects of ultrasound mediated bioconversion of glycerol to ethanol and 1,3‐propanediol (1,3‐PDO) by immobilized Clostridium pasteurianum cells on silica support. Our approach is of coupling experimental results with simulations of cavitation bubble dynamics and enzyme kinetics. In addition, the statistical analysis (ANOVA) of experimental results was also done. The glycerol uptake by cells was not affected by either immobilization or with ultrasonication. Nonetheless, both immobilization and ultrasonication were found to enhance glycerol consumption. The enhancement effect of ultrasound on glycerol consumption was most marked (175%) at the highest glycerol concentration of 25 g/L (271.7 mM). The highest glycerol consumption (32.4 mM) was seen for 10 g/L (108.7 mM) initial glycerol concentration. The immobilization of cells shifted the metabolic pathway almost completely towards 1,3‐PDO. No formation of ethanol was seen with mechanical shaking, while traces of ethanol were detected with ultrasonication. On the basis of analysis of enzyme kinetics parameters, we attribute these results to increased substrate‐enzyme affinity and decreased substrate inhibition for 1,3‐PDO dehydrogenase in presence of ultrasound that resulted in preferential conversion of glycerol into 1,3‐PDO. Biotechnol. Bioeng. 2013; 110: 1637–1645. © 2013 Wiley Periodicals, Inc.     

Isolation and characterization of the new <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B strain showing improved growth characteristics with reduced lipopolysaccharide formation

Klebsiella pneumoniae is a suitable biocatalyst for the production of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) from glycerol. However, its commercial applications have been impeded due to its poor growth characteristics and the excessive production of lipopolysaccharide (LPS). To overcome these limitations, a new K. pneumoniae J2B (KpJ2B) strain was isolated from municipal waste anaerobic digester samples. The shake flask cultivation of this new strain under aerobic conditions showed a specific growth rate of 0.92/h, which is 1.13 times higher than that achieved using the well studied K. pneumoniae DSMZ2026 (KpDSMZ). When the new strain was grown in a bioreactor under aerobic conditions using a fed-batch mode for 36 h, the biomass concentration (4.03 g/L CDW) and productivity (0.15 g/L/h) were almost 2.2 times higher than the corresponding values with KpDSMZ. Growth was accompanied by the production of 1,3-PDO (186 mM), lactic acid (235 mM), ethanol (170 mM), and acetic acid (92.2 mM) at significant levels, indicating the resistance of the strain to the inhibitory effects of these metabolites. A comparison of the SEM images and 2-keto-3-deoxyoctonate content (KpJ2B, 1.4 μg/g CDW; KpDSMZ, 1.9 μg/g CDW) confirmed the lower LPS content in the KpJ2B strain. Furthermore, this new isolate exhibited higher sensitivity towards a range of antibiotics and better sedimentation properties than the KpDSMZ strain. This suggests that KpJ2B is an attractive strain for industrial applications.     

Production of high amounts of 3-hydroxypropionaldehyde from glycerol by Lactobacillus reuteri with strongly increased biocatalyst lifetime and productivity

3-hydroxypropionaldehyde (3HPA) is a promising versatile substance derived from the renewable feedstock glycerol. It is a product of glycerol metabolism in Lactobacillus reuteri. Because of toxic effects, the biotechnological production is poor. In this work the biocatalyst lifetime and product formation could be drastically increased. In the established two-step process already applied, cells are grown in the first step under anaerobic conditions, and in the second step the immobilised or suspended biocatalyst is used for 3HPA-production under strict anaerobic conditions. In the first step it was possible to reach a biomass concentration of 5.5g CDW/L (OD(600)≈23.4). In the second step, normally, 3HPA accumulates to a toxic concentration and the reaction stops in less than 60min because of the interaction of 3HPA with cell components. To prevent this, the toxic product is bound to the newly found scavenger carbohydrazide to form the hydrazone. For the first time it was possible to recycle the immobilised biocatalyst for at least ten cycles (overall life time>33hours) in a repeated batch biotransformation with an overall production of 67g 3HPA. The optimal pH-value was between 6.8 and 7.2 at an optimal temperature of 40-45°C. In a single batch biotransformation with suspended resting cells it was possible to produce 150g/L 3HPA as carbohydrazone at an overall productivity of 10.7gL(-1)hours(-1). In a single fed-batch biotransformation at 45°C 138g/L glycerol was converted into 108g/L 3HPA with an overall productivity of 21.6gL(-1)hours(-1). This is the highest 3HPA concentration and productivities reported so far for the microbial production of 3HPA from glycerol.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli arcA</Emphasis> mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the 'one-factor-at-a-time' technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett-Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box-Wilson design. Under such optimized conditions (22.02 g l(-1) glycerol, 1.78 g l(-1) CAS, and 1.83 g l(-1) inoculum) microaerobic batch cultures gave rise to 8.37 g l(-1) CDW and 3.52 g l(-1) PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l(-1). After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l(-1), respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures.     

静息细胞转化生产3-羟基丙酸的条件优化

本文研究了静息细胞生物转化生产3-羟基丙酸的反应体系。考察了以甘油为底物,利用静息细胞转化生产3一羟基丙酸的相关因素,确定了最佳的转化条件：细胞浓度20g／L,甘油浓度20g／L,辅酶VB12浓度10mg／L,NAD＋浓度0．15mmol／L,温度35℃,反应体系为0．05mol／LpH7．0Tris—HCl缓冲液。在上述条件下反应6h后,3-羟基丙酸的产量达到为3．17g／L,底物转化率为28．33％。由上述结果可知,采用静息细胞转化法为3-HP的生物合成提供了一种可能的方法。     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> growing on biodiesel-derived crude glycerol through a non-sterilized fermentation process

The aim of the present study was to investigate the production of 1,3-propanediol (PDO) under non-sterile fermentation conditions by employing the strain Clostridium butyricum VPI 1718. A series of batch cultures were performed by utilizing biodiesel-derived crude glycerol feedstocks of different origins as the sole carbon source, in various initial concentrations. The strain presented similarities in terms of PDO production when cultivated on crude glycerol of various origins, with final concentrations ranging between 11.1 and 11.5 g/L. Moreover, PDO fermentation was successfully concluded regardless of the initial crude glycerol concentration imposed (from 20 to 80 g/L), accompanied by sufficient PDO production yields (0.52–0.55 g per gram of glycerol consumed). During fed-batch operation under non-sterile culture conditions, 67.9 g/L of PDO were finally produced, with a yield of 0.55 g/g. Additionally, the sustainability of the bioprocess during a continuous operation was tested; indeed, the system was able to run at steady state for 16 days, during which PDO effluent level was 13.9 g/L. Furthermore, possible existence of a microbial community inside the chemostat was evaluated by operating a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, and DGGE results revealed the presence of only one band corresponding to that of C. butyricum VPI 1718. Finally, non-sterile continuous cultures were carried out at different dilution rates (D), with inlet glycerol concentration at 80 g/L. Maximum PDO production was achieved at low D values (0.02 h⁻¹) corresponding to 30.1 g/L, while the elaboration of kinetic data from continuous cultures revealed the stability of the bioprocess proposed, with global PDO production yield corresponding to 0.52 g/g.     

Production of 3‐hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae ΔdhaTΔyqhD which can produce vitamin B12 naturally

3‐Hydroxypropionic acid (3‐HP) is an important platform chemical that can be used to synthesize a range of chemical compounds. A previous study demonstrated that recombinant Escherichia coli stains can produce 3‐HP from glycerol in the presence of vitamin B₁₂ (coenzyme B₁₂), when overexpressed with a coenzyme B₁₂‐dependent glycerol dehydratase (DhaB) and an aldehyde dehydrogenase. The present study examined the production of 3‐HP in recombinant Klebsiella pneumoniae strains, which naturally synthesizes vitamin B₁₂ and does not require supplementation of the expensive vitamin. The NAD⁺‐dependent gamma‐glutamyl‐gamma‐aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae alone or with its DhaB was overexpressed homologously, and two major oxidoreductases, DhaT and YqhD, were disrupted. Without vitamin B₁₂ addition, the recombinant K. pneumoniae ΔdhaTΔyqhD overexpressing PuuC could produce ～3.8 g/L 3‐HP in 12 h of flask culture. However, this was possible only under the appropriate aeration conditions; 1,3‐propanediol (1,3‐PDO) (instead of 3‐HP) was mainly produced when aeration was insufficient, whereas a very small amount of both 3‐HP and 1,3‐PDO were produced when aeration was too high. The production of a small amount of 3‐HP under improper aeration conditions was attributed to either slow NAD⁺ regeneration (under low aeration) or reduced vitamin B₁₂ synthesis (under high aeration). In a glycerol fed‐batch bioreactor experiment under a constant DO of 5%, the strain, K. pneumoniae ΔdhaTΔyqhD, overexpressing both PuuC and DhaB could produce >28 g/L 3‐HP in 48 h with a yield of >40% on glycerol. Only small amount of 3‐HP was produced when cultivation was carried out at a constant aeration of 1 vvm or constant 10% DO. These results show that K. pneumoniae is potentially useful for the production of 3‐HP in an economical culture medium that does not require vitamin B₁₂. The results also suggest that the aeration conditions should be optimized carefully for the efficient production of 3‐HP while using this strain. Biotechnol. Bioeng. 2013; 110: 511–524. © 2012 Wiley Periodicals, Inc.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the ‘one-factor-at-a-time’ technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett–Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box–Wilson design. Under such optimized conditions (22.02 g l⁻¹ glycerol, 1.78 g l⁻¹ CAS, and 1.83 g l⁻¹ inoculum) microaerobic batch cultures gave rise to 8.37 g l⁻¹ CDW and 3.52 g l⁻¹ PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l⁻¹. After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l⁻¹, respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

Effect of crude glycerol-derived inhibitors on ethanol production by Enterobacter aerogenes

In this study, ethanol production from pure and crude glycerol using Enterobacter aerogenes ATCC 29007 was evaluated under anaerobic culture conditions. Inhibitory effects of substrate concentrations, pH, and salt concentrations were investigated based on crude glycerol components. Ethanol production was performed with pure glycerol concentrations ranging from 5 to 30 g/L to evaluate the effects of substrate concentration and osmotic pressure. The consumed glycerol was 5-14.33 g/L, and the yield of ethanol was higher than 0.75 mol ethanol/mol glycerol after 24 h of cultivation. To evaluate the inhibitory effects of salts (NaCl and KCl), experiments were performed with 0-20 g/L of each salt. Inhibitory effects of salts were strongest at high salt concentrations. The inhibitory effect of pH was performed in the pH range 4-10, and cell growth and ethanol production were highest at pH 5-6. Also, ethanol production was slightly inhibited at low concentration of crude glycerol comparison with pure glycerol. However, significant inhibitory effects were not observed at 1.5 and 2% crude glycerol which showed higher ethanol production compared to pure glycerol.     

Regulation of 3-hydroxypropionaldehyde accumulation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes

3-Hydroxypropionaldehyde (3-HPA), an important intermediary metabolite of 1,3-propanediol (PDO) production, would be toxic to the cell growth and led to the abnormal cessation of the fermentation process. In this study, the dhaD gene encoding glycerol dehydrogenase (GDH) and dhaT gene encoding 1,3-propanediol oxidoreductase (PDOR) were overexpressed in Klebsiella pneumoniae ACCC 10082 to decrease the 3-HPA accumulation and increase the coenzyme NADH supply. By the construction of pTD plasmid, GDH and PDOR were both overexpressed and their enzyme activities were increased by 2.6- and 3.2-fold, respectively. The enzyme activity ratio of PDOR/GDHt (glycerol dehydratase) also was increased. On the other hand, NADH production was enhanced and the ratio of NADH/NAD⁺ exceeded 1 after the inducement of IPTG for the constructed strain. The two factors enhanced the transformation of 3-HPA to PDO. In the batch and fed-batch fermentation by the constructed strain, the peak of 3-HPA accumulation reduced by 52.2% and 33.3%, respectively, compared with the control. The PDO concentration and yield reached 59.2 g/L and 0.48 mol/mol, respectively. Furthermore, the fed-batch fermentation process appeared easier to be regulated. This work is considered helpful for the further understanding on the PDO metabolic mechanism of K. pneumoniae and also useful for the PDO fermentation in a large-scale bioreactor.     

Efficient bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate to γ-aminobutyric acid by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> resting cells

This work investigated the efficient bioconversion process of l-glutamate to GABA by Lactobacillus brevis TCCC 13007 resting cells. The optimal bioconversion system was composed of 50 g/L 48 h cultivated wet resting cells, 0.1 mM pyridoxal phosphate in glutamate-containing 0.6 M citrate buffer (pH 4.5) and performed at 45 °C and 180 rpm. By 10 h bioconversion at the ratio of 80 g/L l-glutamic acid to 240 g/L monosodium glutamate, the final titer of GABA reached 201.18 g/L at the molar bioconversion ratio of 99.4 %. This process presents a potential for industrial and commercial applications and also offers a promising feasibility of continuous GABA production coupled with fermentation. Besides, the built kinetics model revealed that the optimum operating conditions were 45 °C and pH 4.5, and the bioconversion kinetics at low ranges of substrate concentration (0 < S < 80 g/L) was assumed to follow the classical Michaelis–Menten equation.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

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 (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE 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 P _adhE 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.     

Isolation of microorganisms able to produce 1,3-propanediol and optimization of medium constituents for Klebsiella pneumoniae AJ4

Microbial fermentation under anaerobic and microaerobic conditions has been used for the production of 1,3-propanediol (1,3-PD), a monomer used to produce polymers such as polytrimethylene terephthalate. In this study, we screened microorganisms using the high throughput screening method and isolated the Klebsiella pneumoniae AJ4 strain, which is able to produce 1,3-PD under aerobic conditions. To obtain the maximum 1,3-PD concentration from glycerol, the response surface methodology based on a central composite design was chosen to show the statistical significance of the effects of glycerol, peptone, and (NH₄)₂SO₄ on 1,3-PD production by K. pneumoniae AJ4. The optimal culture medium factors for achieving maximum concentrations of 1,3-PD included glycerol, 108.5 g/L; peptone, 2.72 g/L; and (NH₄)₂SO_4, 4.38 g/L. Under this optimum condition, the maximum concentration of 1,3-PD, 54.76 g/L, was predicted. A concentration of about 52.59 g/L 1,3-PD was obtained using the optimized medium during 26-h batch fermentation, a finding that agreed well with the predicted value.     

Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions

The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH₂) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle (δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism (γ). The maximum ratio of 0.32 mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.     

Dehydrogenation of ribitol with Gluconobacter oxydans: production and stability of L-ribulose

l-Ribulose is an important chiral lead molecule used for the synthesis of, among others, l-ribose, a high-value rare sugar used in the preparation of antiviral drugs. These drugs--nucleoside-analogues--gain importance in the treatment of severe viral diseases, like those caused by the HIV or hepatitis virus. In this study, factors that may have an impact on l-ribulose production with Gluconobacter oxydans and on the stability of l-ribulose were investigated. A bioconversion-type process, using washed resting cells, was chosen to produce l-ribulose from ribitol. In this process, the cell production and bioconversion phase were separated. The former was first optimized and a maximum cell mass of 1.5 g CDWL(-1) could be produced. For the bioconversion phase, the aeration level of the system proved to be one of the most critical factors; a maximal production rate of 15.7 g L(-1)h(-1) or 5.9 g(g CDW)(-1)h(-1) of l-ribulose could be reached. Furthermore, resting cells were found capable of completely converting ribitol solutions of up to 300 g L(-1) within 30 h, although the kinetics indicated a rather low affinity of the dehydrogenase enzymes for the substrate.     

Respiratory glycerol metabolism of Actinobacillus succinogenes 130Z for succinate production

Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. A few studies have demonstrated that A. succinogenes can anaerobically metabolize glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock, to produce high yields of succinate. However, all these studies were performed in the presence of yeast extract, which largely removes the redox constraints associated with fermenting glycerol, a highly reduced molecule. We demonstrated that A. succinogenes cannot ferment glycerol in minimal medium, but that it can metabolize glycerol by aerobic or anaerobic respiration. These results were expected based on the A. succinogenes genome, which encodes respiratory enzymes, but no pathway for 1,3-propanediol production. We investigated A. succinogenes’s glycerol metabolism in minimal medium in a variety of respiratory conditions by comparing growth, metabolite production, and in vitro activity of terminal oxidoreductases. Nitrate inhibited succinate production by inhibiting fumarate reductase expression. In contrast, growth in the presence of dimethylsulfoxide and in microaerobic conditions allowed high succinate yields. The highest succinate yield was 0.75 mol/mol glycerol (75 % of the maximum theoretical yield) in continuous microaerobic cultures. A. succinogenes could also grow and produce succinate on partially refined glycerols obtained directly from biodiesel manufacture. Finally, by expressing a heterologous 1,3-propanediol synthesis pathway in A. succinogenes, we provide the first proof of concept that A. succinogenes can be engineered to grow fermentatively on glycerol.     

Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3‐hydroxypropionic acid from glycerol

3‐Hydroxypropionic acid (3‐HP) is a commercially valuable chemical with the potential to be a key building block for deriving many industrially important chemicals. However, its biological production has not been well documented. Our previous study demonstrated the feasibility of producing 3‐HP from glycerol using the recombinant Escherichia coli SH254 expressing glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH), and reported that an “imbalance between the two enzymes” and the “instability of the first enzyme DhaB” were the major factors limiting 3‐HP production. In this study, the efficiency of the recombinant strain(s) was improved by expressing DhaB and AldH in two compatible isopropyl‐thio‐β‐galactoside (IPTG) inducible plasmids along with glycerol dehydratase reactivase (GDR). The expression levels of the two proteins were measured. It was found that the changes in protein expression were associated with their enzymatic activity and balance. While cloning an alternate aldehyde dehydrogenase (ALDH), α‐ketoglutaric semialdehyde dehydrogenase (KGSADH), instead of AldH, the recombinant E. coli SH‐BGK1 showed the highest level of 3‐HP production (2.8 g/L) under shake‐flask conditions. When an aerobic fed‐batch process was carried out under bioreactor conditions at pH 7.0, the recombinant SH‐BGK1 produced 38.7 g 3‐HP/L with an average yield of 35%. This article reports the highest level of 3‐HP production from glycerol thus far. Biotechnol. Bioeng. 2009; 104: 729–739 © 2009 Wiley Periodicals, Inc.     

Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae

Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH(4)Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g.L(-1) glycerol, 5.7 g.L(-1) KCl, 13.8 g.L(-1) NH(4)Cl, 1.5 g.L(-1) CaCl(2) and 3.0 g.L(-1) yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L.h). Furthermore, 1,3-propanediol (6.7 g.L(-1)) was also obtained from the liquid medium as a by-product.