Enhanced production of ethanol from glycerol by engineered Hansenula polymorpha expressing pyruvate decarboxylase and aldehyde dehydrogenase genes from Zymomonas mobilis

To improve production of ethanol from glycerol, the methylotrophic yeast Hansenula polymorpha was engineered to express the pdc and adhB genes encoding pyruvate decarboxylase and aldehyde dehydrogenase II from Zymomonas mobilis, respectively, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The ethanol yield was 3.3-fold higher (2.74 g l^?1) in the engineered yeast compared with the parent strain (0.83 g l^?1). Further engineering to stimulate glycerol utilization in the recombinant strain via expression of dhaD and dhaKLM genes from Klebsiella pneumoniae encoding glycerol dehydrogenase and dehydroxyacetone kinase, respectively, resulted in a 3.7-fold increase (3.1 g l^?1) in ethanol yield.     

Purification and primary structure of pyruvate decarboxylase from Zymomonas mobilis.

Pyruvate decarboxylase (E.C. 4.1.1.1), the key enzyme in the glycolytic pathway to ethanol, was isolated in gram amounts from Zymomonas mobilis for structural studies. The primary structure was determined by automated Edman degradation and compared with that deduced from the DNA sequence of the structural gene, previously published by two groups (A. D. Neale, R. K. Scopes, R. E. H. Wettenhall, and N. J. Hoogenraad, 1987, Nucleic Acids Res. 15, 1753-1761; M. Reynen, and H. Sahm, 1988, J. Bacteriol. 170, 3310-3313). The peptide data differ from the published DNA sequences, which also deviate from each other. Crystals diffracting to about 0.3 nm resolution have been obtained by the hanging drop vapor diffusion method. The space group was identified as P4(1)22 or its enantiomorphs containing presumably one tetramer per asymmetric unit.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3-propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.     

Microbial production of 1,3-propanediol by Klebsiella pneumoniae using crude glycerol from biodiesel preparations

1,3-Propanediol (1,3-PD) was produced by Klebsiella pneumoniae using crude glycerol obtained from biodiesel production. The 1,3-PD concentration of 51.3 g/l⁻¹ on crude glycerol from alkali-catalyzed methanolysis of soybean oil was comparable to that of 53 g/l⁻¹ on crude glycerol derived from a lipase-catalyzed process. The productivities of 1.7 g l⁻¹ h⁻¹ on crude glycerol were comparable to that of 2 g l⁻¹ h⁻¹ on pure glycerol. It could be concluded that the crude glycerol could be directly converted to 1,3-PD without any prior purification.     

Formation of acetyl-CoA in Zymomonas mobilis by a pyruvate dehydrogenase complex

In the gram negative, obligately ethanologenic bacterium Zymomonas mobilis a pyruvate dehydrogenase complex was identified and the complex was enriched from cell extracts. This multienzyme complex is responsible for acetyl-CoA biosynthesis from pyruvate. No activities of related multienzyme complexes, 2-ketoglutarate dehydrogenase and branched chain keto acid dehydrogenase, could be detected.     

Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l^–1 h^–1, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l^–1 h^–1, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l^–1 h^–1, respectively.     

Promoter and nucleotide sequences of the Zymomonas mobilis pyruvate decarboxylase.

DNA sequence analysis showed that pyruvate decarboxylase (one of the most abundant proteins in Zymomonas mobilis) contains 559 amino acids. The promoter for the gene encoding pyruvate decarboxylase was not recognized by Escherichia coli, although the cloned gene was expressed at relatively high levels under the control of alternative promoters. The promoter region did not contain sequences which could be identified as being homologous to the generalized promoter structure for E. coli. Hydropathy plots for the amino acid sequence indicated that pyruvate decarboxylase contains a large number of hydrophobic domains which may contribute to the thermal stability of this enzyme.     

Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis.

The optimization of a continuous enzymatic reaction yielding (R)-phenylacetylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presented. We compare the suitability of three pyruvate decarboxylases (PDC), PDC from Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent mutant of the latter, PDCW392M, with respect to their application in the biotransformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction conditions of the carboligation reaction were investigated by determining initial rate velocities with varying substrate concentrations of both aldehydes. From the resulting data a kinetic model was inferred which fits the experimental data with sufficient reliability to deduce the optimal concentrations of both substrates for the enzymatic process. The results demonstrate that the carboligation is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L(-1). d(-1) using a substrate concentration of 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fermentative process.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

The effects of cell recycling on the production of 1,3-propanediol by Klebsiella pneumoniae

The effects of both biomass age and cell recycling on the 1,3-propanediol (1,3-PDO) production by Klebsiella pneumoniae were investigated in a membrane-supported bioreactor using hollow-fiber ultrafiltration membrane module in two separate experiments. It was determined that older cells have a negative effect on 1,3-PDO production. The concentrations of by-products, such as acetic acid and ethanol, increased in cultures with older cells, whereas the concentrations of succinic acid, lactic acid and 2,3-butanediol decreased. The effect of cell recycling was comparatively studied at a cell recycling ratio of 100 %. The results showed that cell recycling had also negative effects on 1,3-PDO fermentation. It was hypothesized that both cell recycling and biomass age caused metabolic shifts to undesired by-products which then inhibited the 1,3-PDO production. On the other hand, the use of hollow-fiber ultrafiltration membrane module was found to be very effective in terms of removal of cells from the fermentation broth.     

不同pH调节剂对补料批式发酵产1,3-丙二醇的影响

对肺炎克雷伯氏菌（Klebsiellapneumoniae）发酵生产1,3-丙二醇（1,3-Propanediol,1,3．PD）的补碱策略进行了研究。分别利用NaOH、氨水、KOH三种溶液作为pH调节剂,优化三种pH调节剂并得到按一定比例混合的混合碱。当采用混合碱调控发酵pH值为7．0时,1,3-丙二醇的产量达到了55dL,比无pH调控（对照）发酵过程发酵水平提高了10．6倍。     

Ethanol production from xylose with a pyruvate-formate-lyase mutant of Klebsiella planticola carrying a pyruvate-decarboxylase gene from Zymomonas mobilis

Summary Grown anaerobically on d-xylose, Klebsiella planticola ATCC 33531 produced acetate, formate, lactate, CO₂ and ethanol as major end-products. A Mu-insertion mutant which lacked pyruvate-formate-lyase showed among its fermentation products more than 70% d-lactate with residual acetate, 2,3-butanediol, and traces of ethanol, formate, and CO₂. After the introduction of a plasmid carrying the gene for the enzyme pyruvate decarboxylase from Zymomonas mobilis, this Klebsiella mutant became an efficient ethanol producer. The recombinant strain produced 387 mM ethanol from 275 mM xylose in 80 h, about 83% of the theoretical maximal yield. Furthermore, this mutant consumed more than double the amount of xylose (41 g/l) compared to the wild-type, due to reduced production of inhibiting acids during growth.Dedicated to Professor Dr. Zähner on the occasion of his 60th birthday     

Effects of different pH regulators on 1,3-propanediol production by Klebsiella pneumoniae in fed-batch culture

对肺炎克雷伯氏菌(Klebsiella pneumoniae)发酵生产1,3-丙二醇(1,3-Propanediol,1,3-PD)的补碱策略进行了研究.分别利用NaOH、氨水、KOH三种溶液作为pH调节剂,优化三种pH调节剂并得到按一定比例混合的混合碱.当采用混合碱调控发酵pH值为7.0时,1,3-丙二醇的产量达到了55 g/L,比无pH调控(对照)发酵过程发酵水平提高了10.6倍.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Genetic improvement of Escherichia coli for ethanol production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II

Zymomonas mobilis genes for pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) were integrated into the Escherichia coli chromosome within or near the pyruvate formate-lyase gene (pfl). Integration improved the stability of the Z. mobilis genes in E. coli, but further selection was required to increase expression. Spontaneous mutants were selected for resistance to high level of chloramphenicol that also expressed high levels of the Z. mobilis genes. Analogous mutants were selected for increased expression of alcohol dehydrogenase on aldehyde indicator plates. These mutants were functionally equivalent to the previous plasmid-based strains for the fermentation of xylose and glucose to ethanol. Ethanol concentrations of 54.4 and 41.6 g/liter were obtained from 10% glucose and 8% xylose, respectively. The efficiency of conversion exceeded theoretical limits (0.51 g of ethanol/g of sugar) on the basis of added sugars because of the additional production of ethanol from the catabolism of complex nutrients. Further mutations were introduced to inactivate succinate production (frd) and to block homologous recombination (recA).