Microbial production of diols as platform chemicals: recent progresses

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.     

Microbial production of 1,3-propanediol: Recent developments and emerging opportunities

1,3-Propanediol, a valuable bifunctional molecule, can be produced from renewable resources using microorganisms. It has several promising properties for many synthetic reactions, particularly for polymer and cosmetic industries. By virtue of being a natural product, relevant biochemical pathways can be harnessed into fermentation processes to produce 1,3-propanediol. Various strategies for the microbial production of 1,3-propanediol are reviewed and compared in this article with their promises and constraints. Furthermore, genetic and metabolic engineering could significantly improve product yields and overcome the limitations of fermentation technology. Present review gives an overview on 1,3-propanediol production by wild and recombinant strains. It also attempts to encompass the various issues concerned in utilization of crude glycerol for 1,3-propanediol production, with particular emphasis laid on biodiesel industries. This review also summarizes the present state of strategies studied for the downstream processing and purification of biologically produced 1,3-propanediol. The future prospect of 1,3-propanediol and its potential as a major bulk chemical are discussed under the light of the current research.     

Microbial formation, biotechnological production and applications of 1,2-propanediol

This short review covers metabolic pathways, genetics and metabolic engineering of 1,2-propanediol formation in microbes. 1,2-Propanediol production by bacteria and yeasts has been known for many years and two general pathways are recognized. One involves the metabolism of deoxyhexoses, where lactaldehyde is formed during the glycolytic reactions and is then reduced to 1,2-propanediol. The second pathway derives from the formation of methylglyoxal from dihydroxyacetonephosphate and its subsequent reduction to 1,2-propanediol. The enzymes involved in the reduction of methylglyoxal can generate isomers of lactaldehyde or acetol, which can be further reduced by specific reductases, giving chiral 1,2-propanediol as the product. The stereospecificity of the enzymes catalyzing the two reduction steps is important in deriving a complete pathway. Through genetic engineering, appropriate combinations of enzymes have been brought together in Escherichia coli and yeast to generate 1,2-propanediol from glucose. The optimization of these strains may yield microbial processes for the production of this widely used chemical. Received: 25 May 2000 / Received revision: 24 July 2000 / Accepted: 25 July 2000     

Microbial metabolism of 1,2-propanediol studied by the Rumen Simulation Technique (Rusitec)

C zerkawski J.W., P iatkova M. & B reckenridge , G. 1984. Microbial metabolism of 1,2-propanediol studied by the Rumen. Simulation Technique (Rusitec). Journal of Applied Bacteriology , 56 , 81–94.
A series of experiments with the Rumen Simulation Technique (Rusitec) showed that 1,2-propanediol was metabolized efficiently by rumen micro-organisms and that the main end-products of fermentation were propionic and 2-methylbutyric acids. 'Propionaldehyde and n-propanol were also formed as intermediate compounds.' The effect of the diol on digestion of the basal diet appeared to be small with concentrate, or when the roughage was supplemented with additional nitrogen (urea). The decrease in the output of acetic and butyric acids was consistent with utilization of C₂ units for synthesis of 2-methylbutyric acid. The fermentation of 1,2-propanediol resulted in little or no increase in the output of additional microbial matter. The distribution of radioactivity from [1-¹⁴C] 1,2-propanediol confirmed that propionaldehyde and n -propanol were the primary products of metabolism of the diol and that the end-products were propionic and 2-methylbutyric acids, with very little labelling of microbial matter. Between 2% and 3% of radioactivity was found in gases and surprisingly the specific radioactivity of methane was higher than that of carbon dioxide, particularly during the initial stages of incubation. Possible pathways in the degradation of 1,2-propanediol by rumen micro-organisms are suggested and discussed in relation to similar reactions established in other systems.     

Anaerobic metabolism of the L-rhamnose fermentation product 1,2-propanediol in Salmonella typhimurium.

When grown anaerobically on L-rhamnose, Salmonella typhimurium excreted 1,2-propanediol as a fermentation product. Upon exhaustion of the methyl pentose, 1,2-propanediol was recaptured and further metabolized, provided the culture was kept under anaerobic conditions. n-Propanol and propionate were found in the medium as end products of this process at concentrations one-half that of 1,2-propanediol. As in Klebsiella pneumoniae (T. Toraya, S. Honda, and S. Fukui, J. Bacteriol. 139:39-47, 1979), a diol dehydratase which transforms 1,2-propanediol to propionaldehyde and the enzymes involved in a dismutation that converts propionaldehyde to n-propanol and propionate were induced in S. typhimurium cultures able to transform 1,2-propanediol anaerobically.     

Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase.

Klebsiella pneumoniae ATCC 25955 (formerly named Aerobacter aerogenes PZH 572, Warsaw), which is known to produce coenzyme-B12-dependent glycerol dehydratase when grown anaerobically in a glycerol medium, formed coenzyme-B12-dependent diol dehydratase in a 1,2-propanediol-containing medium. Both the diol dehydratase and the glycerol dehydratase produced by the organism catalyzed the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes and underwent concomitant inactivation during the catalysis of glycerol dehydration, as does the diol dehydratase of K. pneumoniae (A. aerogenes) ATCC 8724. However, the two enzymes were distinguishable from each other by the monovalent-cation-selectivity pattern and by substrate specificity; that is, glycerol dehydratase preferred glycerol to 1,2-propanediol as a substrate, whereas diol dehydratase preferred 1,2-propanediol to glycerol, as judged from initial velocity studies. Ouchterlony double-diffusion analysis and immunochemical titration with rabbit antiserum against diol dehydratase of K. pneumoniae ATCC 8724 established clearly that the diol dehydratase of K. pneumoniae ATCC 25955 is immunologically similar to that of K. pneumoniae ATCC 8724, while the glycerol dehydratase of the former is different from the diol dehydratase of both strains. Both the enzymes were found to be distributed in several bacteria of the family Enterobacteriaceae.     

Fermentation of 1,2-Propanediol and 1,2-Ethanediol by Some Genera of Enterobacteriaceae, Involving Coenzyme B12-Dependent Diol Dehydratase

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.     

Conversion of sugars to 1,2-propanediol by Thermoanaerobacterium thermosaccharolyticum HG-8

The purpose of this study was to explore a fermentation route for the production of 1,2-propanediol (1,2-PD) from renewable sugars: lactose found in cheese whey, and D-glucose, D-galactose, L-arabinose, and D-xylose found in corn and wood byproducts. Thermoanaerobacterium thermosaccharolyticum, a naturally occurring organism, was found to ferment a wider range of sugars to 1,2-PD than previously reported. The specific sugar had a significant effect on the selectivity for 1,2-PD vs other fermentation products such as ethanol, D- and L-lactate, and acetate. T. thermosaccharolyticum potentially provides an environmentally friendly route to a major commodity chemical now made from petrochemicals.     

Fermentation mechanism of fucose and rhamnose in Salmonella typhimurium and Klebsiella pneumoniae.

An equimolar amount of 1,2-propanediol was detected in the medium when Salmonella typhimurium or Klebsiella pneumoniae fermented L-fucose or L-rhamnose. These metabolic conditions induced a propanediol oxidoreductase that converted the lactaldehyde formed in the dissimilation of either sugar into the diol. The enzyme was further identified by cross-reaction with antibodies against Escherichia coli propanediol oxidoreductase. This indicates that L-fucose and L-rhamnose fermentation takes place in these species by 1,2-propanediol production and excretion.     

A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli

Anaerobic fermentation of glycerol in the Enterobacteriaceae family has long been considered a unique property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that Escherichia coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol (1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as its role in the metabolism of glycerol. We also showed that the trunk pathway responsible for the conversion of glycerol into glycolytic intermediates is composed of two enzymes: a type II glycerol dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown physiological role. Based on our findings, we propose a new model for glycerol fermentation in enteric bacteria in which: (i) the production of 1,2-PDO provides a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via substrate-level phosphorylation. The activity of the formate hydrogen-lyase and F(0)F(1)-ATPase systems were also found to facilitate the fermentative metabolism of glycerol, and along with the ethanol and 1,2-PDO pathways, were considered auxiliary or enabling. We demonstrated that glycerol fermentation in E. coli was not previously observed due to the use of medium formulations and culture conditions that impair the aforementioned pathways. These include high concentrations of potassium and phosphate, low concentrations of glycerol, alkaline pH, and closed cultivation systems that promote the accumulation of hydrogen gas.     

Glycerol fermentation by (open) mixed cultures: a chemostat study

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.     

PduA is a shell protein of polyhedral organelles involved in coenzyme B(12)-dependent degradation of 1,2-propanediol in Salmonella enterica serovar typhimurium LT2

Salmonella enterica forms polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. These organelles are thought to consist of a proteinaceous shell that encases coenzyme B(12)-dependent diol dehydratase and perhaps other enzymes involved in 1,2-propanediol degradation. The function of these organelles is unknown, and no detailed studies of their structure have been reported. Genes needed for organelle formation and for 1,2-propanediol degradation are located at the 1,2-propanediol utilization (pdu) locus, but the specific genes involved in organelle formation have not been identified. Here, we show that the pduA gene encodes a shell protein required for the formation of polyhedral organelles involved in coenzyme B(12)-dependent 1,2-propanediol degradation. A His(6)-PduA fusion protein was purified from a recombinant Escherichia coli strain and used for the preparation of polyclonal antibodies. The anti-PduA antibodies obtained were partially purified by a subtraction procedure and used to demonstrate that the PduA protein localized to the shell of the polyhedral organelles. In addition, electron microscopy studies established that strains with nonpolar pduA mutations were unable to form organelles. These results show that the pduA gene is essential for organelle formation and indicate that the PduA protein is a structural component of the shell of these organelles. Physiological studies of nonpolar pduA mutants were also conducted. Such mutants grew similarly to the wild-type strain at low concentrations of 1,2-propanediol but exhibited a period of interrupted growth in the presence of higher concentrations of this growth substrate. Growth tests also showed that a nonpolar pduA deletion mutant grew faster than the wild-type strain at low vitamin B(12) concentrations. These results suggest that the polyhedral organelles formed by S. enterica during growth on 1,2-propanediol are not involved in the concentration of 1,2-propanediol or coenzyme B(12), but are consistent with the hypothesis that these organelles moderate aldehyde production to minimize toxicity.     

The metabolism of 1,2-propanediol by the propylene oxide utilizing bacterium Nocardia A60

A bacterium designated Nocardia A60 was isolated for its capacity to utilize propylene oxide (1,2-epoxypropame) aerobically as a carbon and energy source for growth. Extracts of cells grown on the epoxide catalyzed the conversion of propylene oxide to 1,2-propanediol This epoxidase activity was absent in cells grown on 1,2-propanediol or succinate. During growth of the organism on propylene oxide and 1,2-propanediol it contained high levels of diol dehydratase (EC 4.2.1.28). Enhanced levels of propionyl-CoA carboxylase during growth on propylene oxide and 1,2-propanediol suggest that these compounds are metabolized via propionate and succinate.     

The Propanediol Utilization (pdu) Operon of Salmonella enterica Serovar Typhimurium LT2 Includes Genes Necessary for Formation of Polyhedral Organelles Involved in Coenzyme B12-Dependent 1,2-Propanediol Degradation

The propanediol utilization (pdu) operon of Salmonella enterica serovar Typhimurium LT2 contains genes needed for the coenzyme B(12)-dependent catabolism of 1,2-propanediol. Here the completed DNA sequence of the pdu operon is presented. Analyses of previously unpublished pdu DNA sequence substantiated previous studies indicating that the pdu operon was acquired by horizontal gene transfer and allowed the identification of 16 hypothetical genes. This brings the total number of genes in the pdu operon to 21 and the total number of genes at the pdu locus to 23. Of these, six encode proteins of unknown function and are not closely related to sequences of known function found in GenBank. Two encode proteins involved in transport and regulation. Six probably encode enzymes needed for the pathway of 1,2-propanediol degradation. Two encode proteins related to those used for the reactivation of adenosylcobalamin (AdoCbl)-dependent diol dehydratase. Five encode proteins related to those involved in the formation of polyhedral organelles known as carboxysomes, and two encode proteins that appear distantly related to those involved in carboxysome formation. In addition, it is shown that S. enterica forms polyhedral bodies that are involved in the degradation of 1,2-propanediol. Polyhedra are formed during either aerobic or anaerobic growth on propanediol, but not during growth on other carbon sources. Genetic tests demonstrate that genes of the pdu operon are required for polyhedral body formation, and immunoelectron microscopy shows that AdoCbl-dependent diol dehydratase is associated with these polyhedra. This is the first evidence for a B(12)-dependent enzyme associated with a polyhedral body. It is proposed that the polyhedra consist of AdoCbl-dependent diol dehydratase (and perhaps other proteins) encased within a protein shell that is related to the shell of carboxysomes. The specific function of these unusual polyhedral bodies was not determined, but some possibilities are discussed.     

Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation

1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied. The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation. μ _max was inversely proportional to the initial concentration of 1,3-propanediol (0–65 g l⁻¹). For glycerol at 20 g l⁻¹, the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l⁻¹. However, for an initial 1,3-propanediol concentration of 50 g l⁻¹ and glycerol at 70 g l⁻¹, the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l⁻¹(1.1 M), with complete consumption of the glycerol. Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l⁻¹). The addition of 1,2-propanediol or 2,3-butanediol (50 g l⁻¹) in the presence of glycerol (50–100 g l⁻¹), showed that 2-diols reduced the μ _max in a similar way to 1,3-propanediol. The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds. The hypothesis of diol inhibition was discussed. Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered. Received: 15 November 1999 / Revision received: 1 February 2000 / Accepted: 4 February 2000     

基于甘油的1,3-丙二醇生物合成的代谢局限及其改造策略

粗甘油是生物柴油生产中的主要副产物,一些微生物可将甘油转化为重要化工原料1,3-丙二醇(1,3-PD),而利用这些微生物野生菌株生物合成1,3-PD会存在一些局限性,如底物抑制、产物抑制等。文中从1,3-丙二醇的甘油生物转化途径与这些局限性出发,总结了生物合成中存在的问题,并针对这些问题提出了一些基于基因敲除或基因过表达等基因工程技术的改造方法,综述了利用基因工程菌生物转化甘油生成1,3-丙二醇的最新研究进展。     

Biotechnological routes based on lactic acid production from biomass

Lactic acid, the most important hydroxycarboxylic acid, is now commercially produced by the fermentation of sugars present in biomass. In addition to its use in the synthesis of biodegradable polymers, lactic acid can be regarded as a feedstock for the green chemistry of the future. Different potentially useful chemicals such as pyruvic acid, acrylic acid, 1,2-propanediol, and lactate ester can be produced from lactic acid via chemical and biotechnological routes. Here, we reviewed the current status of the production of potentially valuable chemicals from lactic acid via biotechnological routes. Although some of the reactions described in this review article are still not applicable at current stage, due to their “greener” properties, biotechnological processes for the production of lactic acid derivatives might replace the chemical routes in the future.     

Microbial Production of Hydrogen by Mixed Culture Technologies: A Review

With its high energy content and clean combustion, hydrogen is recognized as a renewable clean energy source with enormous potential. Biological hydrogen production is a promising alternative with significant advantages over conventional petroleum‐derived chemical processes. Sustainable hydrogen production from renewable resources such as cassava, wastewater, and other agricultural waste is economically feasible for industrial applications. So far, the major bottlenecks in large‐scale biological hydrogen production are the low production rate and yield. This review discusses the various factors that affect the metabolic pathways of dark hydrogen production, and highlights the state‐of‐the‐art development of mixed culture technology. The aim of this review is to provide suggestions for the future directions of mixed culture technology, as well as by‐product valorization in dark fermentation.     

甘油歧化为1,3-丙二醇的代谢及关键酶研究进展

微生物发酵生产1,3-丙二醇因对环境友好而成为研究热点。通过对发酵菌种、代谢途径、调节子和关键酶的分析,阐述了微生物转化甘油为1,3-丙二醇的分子机理。尤其对还原途径的限速酶-甘油脱水酶的分子结构及再激活因子进行了详细分析,为菌种的遗传改造提供了理论依据。     

Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures

To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions. The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain. In this way, two positive E. coli clones out of 560,000 tested clones were obtained. In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes. The screening of 158,000 E. coli clones by this method yielded five positive clones. Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further. The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria. Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases. We were able to perform high-level production and purification of three of these dehydratases. The glycerol dehydratases purified from E. coli Bl21/pAK2.1 and E. coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E. coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C. freundii. The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C. freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol. Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E. coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.