Oxidative phosphorylation in Zymomonas mobilis

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H⁺-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - CCCP carbonyl cyanide m-chlorophenylhydrazone     

Production of Acetaldehyde by Zymomonas mobilis

Mutants of Zymomonas mobilis were selected for decreased alcohol dehydrogenase activity by using consecutively higher concentrations of allyl alcohol. A mutant selected by using 100 mM allyl alcohol produced acetaldehyde at a level of 4.08 g/liter when the organism was grown in aerated batch cultures on a medium containing 4.0% (wt/wt) glucose. On the basis of the amount of glucose utilized, this level of acetaldehyde production represents nearly 40% of the maximum theoretical yield. Acetaldehyde produced during growth was continuously air stripped from the reactor. Acetaldehyde present in the exhaust stream was then trapped as the acetaldehyde-bisulfite addition product in an aqueous solution of sodium bisulfite and released by treatment with base. Acetaldehyde was found to inhibit growth of Z. mobilis at concentrations as low as 0.05% (wt/wt) acetaldehyde. An acetaldehyde-tolerant mutant of Z. mobilis was isolated after both mutagenesis with nitrosoguanidine and selection in the presence of vapor-phase acetaldehyde. The production of acetaldehyde has potential advantages over that of ethanol: lower energy requirements for product separation, efficient separation of product from dilute feed streams, continuous separation of product from the reactor, and a higher marketplace value.     

R-Plasmid Transfer in Zymomonas mobilis

Conjugal transfer of three IncP1 plasmids and one IncFII plasmid into strains of the ethanol-producing bacterium Zymomonas mobilis was obtained. These plasmids were transferred at high frequencies from Escherichia coli and Pseudomonas aeruginosa into Z. mobilis and also between different Z. mobilis strains, using the membrane filter mating technique. Most of the plasmids were stably maintained in Z. mobilis, although there was some evidence of delayed marker expression. A low level of chromosomal gene transfer, mediated by plasmid R68.45, was detected between Z. mobilis strains. Genetic evidence suggesting that Z. mobilis may be more closely related to E. coli than to Pseudomonas or Rhizobium is discussed.     

Complete Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. mobilis Centrotype ATCC 29191

Zymomonas mobilis is an ethanologenic bacterium that has been studied for use in biofuel production. Of the sequenced Zymomonas strains, ATCC 29191 has been described as the phenotypic centrotype of Zymomonas mobilis subsp. mobilis, the taxon that harbors the highest ethanol-producing Z. mobilis strains. ATCC 29191 was isolated in Kinshasa, Congo, from palm wine fermentations. This strain is reported to be a robust levan producer, while in recent years it has been employed in studies addressing Z. mobilis respiration. Here we announce the finishing and annotation of the ATCC 29191 genome, which comprises one chromosome and three plasmids.     

Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

Continuous ethanol fermentation in molasses medium using Zymomonas mobilis immobilized in photo-crosslinkable resin gels

Zymomonas mobilis B-69 147, an ethanol-producing bacterium, was immobilized in photo-crosslinkable resin gels to form a biocatalyst system. Continuous ethanol fermentation with this immobilized Zymomonas was carried out in molasses and compared to that with immobilized yeast. As a result of operating this process for two weeks, a productivity of 60 g/l·h based on immobilized gel was obtained with improvement in the poor tolerance to salts of Zymomonas. The productivity of immobilized Z. mobilis was superior to that of immobilized yeast.     

Comparison of pyruvate decarboxylases from Saccharomyces cerevisiae and Komagataella pastoris (Pichia pastoris)

Pyruvate decarboxylases (PDCs) are a class of enzymes which carry out the non-oxidative decarboxylation of pyruvate to acetaldehyde. These enzymes are also capable of carboligation reactions and can generate chiral intermediates of substantial pharmaceutical interest. Typically, the decarboxylation and carboligation processes are carried out using whole cell systems. However, fermentative organisms such as Saccharomyces cerevisiae are known to contain several PDC isozymes; the precise suitability and role of each of these isozymes in these processes is not well understood. S. cerevisiae has three catalytic isozymes of pyruvate decarboxylase (ScPDCs). Of these, ScPDC1 has been investigated in detail by various groups with the other two catalytic isozymes, ScPDC5 and ScPDC6 being less well characterized. Pyruvate decarboxylase activity can also be detected in the cell lysates of Komagataella pastoris, a Crabtree-negative yeast, and consequently it is of interest to investigate whether this enzyme has different kinetic properties. This is also the first report of the expression and functional characterization of pyruvate decarboxylase from K. pastoris (PpPDC). This investigation helps in understanding the roles of the three isozymes at different phases of S. cerevisiae fermentation as well as their relevance for ethanol and carboligation reactions. The kinetic and physical properties of the four isozymes were determined using similar conditions of expression and characterization. ScPDC5 has comparable decarboxylation efficiency to that of ScPDC1; however, the former has the highest rate of reaction, and thus can be used for industrial production of ethanol. ScPDC6 has the least decarboxylation efficiency of all three isozymes of S. cerevisiae. PpPDC in comparison to all isozymes of S. cerevisiae is less efficient at decarboxylation. All the enzymes exhibit allostery, indicating that they are substrate activated.     

Roles of alcohol dehydrogenases of Zymomonas mobilis (ZADH): characterization of a ZADH-2-negative mutant

Zymomonas mobilis is a potential candidate for fuel ethanol production because of its high ethanol productivity. A key enzyme in ethanol production is alcohol dehydrogenase (ADH). Z. mobilis possesses two isozymes, ZADH-1 and ZADH-2. To clarify their physiological roles, mutants with modified ADH were isolated by selection based on resistance to allyl alcohol. From the physiological characteristics of a ZADH-2-negative mutant, it is suggested that ZADH-1 functions as the major ADH, while ZADH-2 could become functional in the latter stages of fermentation.     

The macromolecular composition and essential amino acid profiles of strains of Zymomonas mobilis

Summary From continuous culture studies it has been shown that the protein concentrations of strains of Z. mobilis (62–68%) were appreciably higher than for the yeast S.uvarum (45–50%). The DNA and RNA contents were similar for the two species. Comparison of the essential amino acids indicated that Z.mobilis did not exhibit the deficiency in methionine which was apparent in the yeast. Such a study of the macromolecular composition of cells of Z.mobilis is important in assessing its by-product nutritional value for animal feed supplementation.     

The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.     

A derivative of Zymomonas mobilis ATCC 10988 with impared ethanol production

Summary A derivative of Zymomonas mobilis ATCC 10988 has been isolated from cells treated with acridine orange. This derived strain, designated CU1, was found to have markedly decreased ethanol production and concomitant glucose utilisation capabilities when grown on high concentrations of glucose. Additionally, it was found that CU1 had altered alcohol dehydrogenase activity and also lacks at least one of the natural plasmids of Z.mobilis, the 3kb species.     

Characteristics of Zymomonas mobilis immobilized by photo-crosslinkable resin in ethanol fermentation

Zymomonas mobilis, an ethanol-producing bacterium, was immobilized in hydrophilic photo-crosslinked resin gels to form a biocatalyst. The molecular structure of the photo-crosslinkable resin could be modulated so as to minimize a disadvantage of this bacterium—poor-tolerance to salts in molasses. Characteristics of Z. mobilis immobilized by photo-crosslinkable resin gel, such as fermentability, cell growth in gel, the potential of gel materials, diffusion of materials, and salt distribution are discussed. ENTG-3800 photo-crosslinkable resin was selected as the most suitable entrapping material for Z. mobilis, especially in using molasses.     

Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation

Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.     

Expression of a xylose-specific transporter improves ethanol production by metabolically engineered Zymomonas mobilis

Zymomonas mobilis is a promising organism for biofuel production as it can produce ethanol from glucose at high rates. However, Z. mobilis does not natively ferment C5 sugars such as xylose. While it has been engineered to do so, the engineered strains do not metabolize these sugars at high rates. Previous research has identified some of the bottlenecks associated with xylose metabolism in Z. mobilis. In this work, we investigated transport as a possible bottleneck. In particular, we hypothesized that the slow uptake of xylose through the promiscuous Glf transporter may limit the efficiency of xylose metabolism in Z. mobilis. To test this hypothesis, we expressed XylE, the low-affinity xylose transporter from Escherichia coli, in a xylose-utilizing strain of Z. mobilis. Our results show that the expression of this pentose-specific transporter improves the rate of xylose utilization in Z. mobilis; however, this enhancement is seen only at high xylose concentrations. In addition, we also found that overexpression of the promiscuous Z. mobilis transporter Glf yielded similar results, suggesting that the transport bottleneck is not due to the specificity, but rather the capacity for sugar uptake.     

Computational and functional analysis of β-lactam resistance in Zymomonas mobilis

Zymomonas mobilis, a Gram-negative ethanologenic non-pathogenic bacterium, is reported to exhibit resistance to high concentrations of β-lactam antibiotics. In the present study, Z. mobilis was found to be resistant to I-IV generations of cephalosporins and carbapenems, i.e. narrow, broad and extended spectrum β-lactam antibiotics. We have analysed the genome of Z. mobilis (GenBank accession No.: NC 006526) harbouring multiple genes coding for β-lactamases (BLA), β-lactamase domain containing proteins (BDP) and penicillin binding proteins (PBP). The conserved domain database analysis of BDPs predicted them to be members of metallo β-lactamase superfamily. Further, class C specific multidomain AmpC (β-lactamase C) was found in the three β-lactamases. The β-lactam resistance determinants motifs, HXHXD, KXG, SXXK, SXN, and YXN are present in the BLAs, BDPs and PBPs of Z. mobilis. The predicted theoretical pI and aliphatic index values suggested their stability. One of the PBPs, PBP2, was predicted to share functional association with rod shape determining proteins (GenBank accession Nos. YP_162095 and YP_162091). Homology modelling of three dimensional structures of the β-lactam resistance determinants and further docking studies with penicillin and other β-lactam antibiotics indicated their substrate-specificity. Semi-quantitative PCR analysis indicated that the expression of all BLAs and one BDP are induced by penicillin. Disk diffusion assay, SDS-PAGE and zymogram analysis confirms the substrate specificity of the β-lactam resistance determinants. This study gives a broader picture of the β-lactam resistance determinants of a non-pathogenic ethanologenic Z. mobilis bacterium that could have implications in laboratories since it is routinely used in many research laboratories in the world for ethanol, fructooligosaccharides, levan production and has also been reported to be present in wine and beer as a spoilage organism.     

Heterologous Expression and Extracellular Secretion of Cellulolytic Enzymes by Zymomonas mobilis

Development of the strategy known as consolidated bioprocessing (CBP) involves the use of a single microorganism to convert pretreated lignocellulosic biomass to ethanol through the simultaneous production of saccharolytic enzymes and fermentation of the liberated monomeric sugars. In this report, the initial steps toward achieving this goal in the fermentation host Zymomonas mobilis were investigated by expressing heterologous cellulases and subsequently examining the potential to secrete these cellulases extracellularly. Numerous strains of Z. mobilis were found to possess endogenous extracellular activities against carboxymethyl cellulose, suggesting that this microorganism may harbor a favorable environment for the production of additional cellulolytic enzymes. The heterologous expression of two cellulolytic enzymes, E1 and GH12 from Acidothermus cellulolyticus, was examined. Both proteins were successfully expressed as soluble, active enzymes in Z. mobilis although to different levels. While the E1 enzyme was less abundantly expressed, the GH12 enzyme comprised as much as 4.6% of the total cell protein. Additionally, fusing predicted secretion signals native to Z. mobilis to the N termini of E1 and GH12 was found to direct the extracellular secretion of significant levels of active E1 and GH12 enzymes. The subcellular localization of the intracellular pools of cellulases revealed that a significant portion of both the E1 and GH12 secretion constructs resided in the periplasmic space. Our results strongly suggest that Z. mobilis is capable of supporting the expression and secretion of high levels of cellulases relevant to biofuel production, thereby serving as a foundation for developing Z. mobilis into a CBP platform organism.The biological conversion of lignocellulosic biomass to ethanol represents a potential major source of future domestic transportation fuels, but the current cost of converting biomass to fermentable sugars still needs to be reduced further (12). Most current strategies for ethanol production via biochemical conversion of lignocellulosic feedstocks utilize simultaneous saccharification and fermentation (SSF) or simultaneous saccharification and cofermentation (SSCF) processes (8, 21, 22). The process configuration known as consolidated bioprocessing (CBP) (20) would alleviate the financial strain of producing saccharolytic enzyme cocktails by combining the necessary steps for ethanol production as the action of one microorganism.A particularly attractive microbial candidate for the development of a CBP microorganism is the Gram-negative fermentative bacterium Zymomonas mobilis. Z. mobilis has been studied for its exceptionally high ethanol production rate, yield, and tolerance to the toxicity of the final product (15-17, 20, 31-33, 35, 43). In addition, Z. mobilis has the ability to ferment sugars at low pH and has a naturally high tolerance to many of the inhibitory compounds found in hydrolysates derived from lignocellulosic biomass (45, 46) Furthermore, the use of the Entner-Doudoroff pathway (37) allows Z. mobilis to achieve the near-theoretical maximum ethanol yields during fermentation while achieving relatively low biomass formation. Accordingly, Z. mobilis has been used successfully in SSF and SSCF processes (14, 24, 36). Additionally, Z. mobilis has been successfully engineered to ferment the pentose (C₅) sugars, xylose (45) and arabinose (10).A necessary prerequisite to establishing Z. mobilis as a CBP host is the ability to achieve high levels of cellulolytic enzyme expression. However, there is not yet a strong consensus on how to achieve maximal heterologous protein expression in Z. mobilis. Multiple groups have attempted heterologous expression of numerous genes, including cellulolytic enzymes in Z. mobilis with various degrees of success (6, 7, 9, 19, 27, 42, 44). Unfortunately, there are no obvious correlations between the expression strategies employed compared to the results obtained. Intriguingly, however, when researchers used the tac promoter (P_tac) to drive expression of native Z. mobilis genes, they were able to express several genes to extremely high levels (2). The results from this study (2) suggest that while the potential to achieve high levels of heterologous cellulase expression in Z. mobilis certainly exists, the ability to do so on a consistent basis will need further investigation.While achieving high-level expression of cellulases is an important hurdle to overcome in the development of the CBP technology, it is imperative that these enzymes additionally be translocated to the extracellular medium in order to directly contact the lignocellulosic substrate. The most obvious means by which to achieve this translocation is by harnessing the host cell''s protein secretion apparatus. There is, however, in general, very little fundamental knowledge regarding the capacity of Z. mobilis to secrete proteins. There is only one account to our knowledge of fusing secretion signals native to Z. mobilis onto proteins from exogenous sources, where extracellular secretion of a recombinant β-glucosidase reached only 11% of the total amount of enzyme synthesized (43).We initially report the finding that several Z. mobilis strains natively produce an endogenous activity against carboxymethyl cellulose (CMC) and that this activity can be detected extracellularly. Together, these results suggest that Z. mobilis may be adept at producing and secreting cellulolytic enzymes, and as this attribute is essential for a CBP organism, Z. mobilis serves as an ideal candidate for further investigation.We next describe the expression of two cellulolytic enzymes (E1 and GH12) in both Escherichia coli and Z. mobilis. E1 (locus tag Acel_0614) and GH12 (locus tag Acel_0619) are both from the acidothermophile Acidothermus cellulolyticus and are representative of glycoside hydrolase families 5 and 12, respectively (10a, 38a). E1 is an endo-1,4-β-glucanase, and GH12 is an uncharacterized enzyme that has a very high sequence identity to the GH12 domain of GuxA (Acel_0615) from A. cellulolyticus. GuxA has activities against a wide variety of substrates, including carboxymethyl cellulose, arabinoxylan, xylan, and xyloglucan (W. Adney, unpublished results). While GH12 has yet to be fully characterized, we used homology modeling (3) to predict the enzyme class of GH12 and found that it strongly resembles an endo-1,4-β-glucanase. These enzymes were chosen because of their relatively low molecular weight, high stability, and activity over a broad temperature and pH range using only the catalytic domains (Adney, unpublished). We report the successful expression of both enzymes in Z. mobilis by addressing several variables related to gene expression. Additionally, the use of codon optimization was explored as a way of enhancing heterologous expression in Z. mobilis. After successfully demonstrating the intracellular expression of E1 and GH12 in Z. mobilis, we further show that Z. mobilis is capable of secreting these proteins extracellularly through the use of native secretion signals predicted to utilize two separate protein translocation pathways in Z. mobilis, the SecB-dependent and twin arginine translocation (TAT) pathways. This finding should prove valuable beyond the production of cellulases and could include all classes of recombinant proteins.     

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

Summary Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac⁺ plasmids have been constructed. Although -galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.