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Transposons are mobile genetic elements that can give rise to gene mutation and genome rearrangement. Due to their mobility, transposons have been exploited as genetic tools for modification of plants, animals, and microbes. Although a plethora of reviews have summarized families of transposons, the transposons from fermentation bacteria have not been systematically documented, which thereby constrain the exploitation for metabolic engineering and synthetic biology purposes. In this review, we summarize the transposons from the most used fermentation bacteria including Escherichia coli, Bacillus subtilis, Lactococcus lactis, Corynebacterium glutamicum, Klebsiella pneumoniae, and Zymomonas mobilis by literature retrieval and data mining from GenBank and KEGG. We also outline the state-of-the-art advances in basic research and industrial applications especially when allied with other genetic tools. Overall, this review aims to provide valuable insights for transposon-mediated strain improvement. KEY POINTS: • The transposons from the most-used fermentation bacteria are systematically summarized. • The applications of transposons in strain improvement are comprehensively reviewed.

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated β-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6 g L

Zymomonas mobilis may encounter various types of stress during ethanol fermentation, which reduces ethanol production efficiency. This situation may be mitigated by molecular chaperones, including the chaperonin GroESL, which confers enhanced protection against various stresses. In this study, we successfully developed a Z. mobilis strain R301 that harbors groESL genes and can be used for high-temperature ethanol production from sweet sorghum juice. Sequence analyses of GroES and GroEL from Z. mobilis TISTR548 demonstrated conserved residues at specific positions within GroES and conserved glycine-glycine-methionine (GGM) repeats at the C-terminus of GroEL. The Z. mobilis wild-type and R301 strains were then evaluated for their tolerance to stresses, including high temperatures, high sugar concentrations, and high ethanol concentrations up to 40°C, 300 g/L, and 13% (v/v), respectively. Z. mobilis R301 exhibited better growth performance than the wild-type strain under all stress conditions. This is the first report on ethanol production at 40°C by recombinant Z. mobilis using sweet sorghum juice; this strain produced an ethanol concentration of 41.66 g/L, with a productivity of 0.87 g/L/h and a theoretical ethanol yield of 88.9%. Overexpression of groESL resulted in increased ethanol production, with values approximately 11% higher than those of the wild type at 40°C. Additionally, at 37°C, Z. mobilis R301 gave a higher theoretical ethanol yield (92.6%) than that shown in previous research. This work illustrates the potential for future enhancement of industrial-scale ethanol production at high temperatures utilizing Z. mobilis R301 in the bioconversion of sweet sorghum juice, a promising energy crop. KEY POINTS: • The groESL-overexpressing Z. mobilis strain was successfully constructed. • The recombinant Z. mobilis exhibited higher stress tolerance than the wild-type strain. • Overexpression of groESL genes improved ethanol production efficiency at high temperatures.

Biological ethylene production is a promising sustainable alternative approach for fossil-based ethylene production. The high glucose utilization of Z. mobilis makes it as a promising bioethylene producer. In this study, Zymomonas mobilis has been engineered to produce ethylene through the introduction of the synthetic ethylene-forming enzyme (EFE). We also investigated the effect of systematically knocking out the competitive metabolic pathway of pyruvate in an effort to improve the availability of pyruvate for ethylene production in Z. mobilis expressing EFE. Guided by these results, we tested a number of conjectures that could improve the α-ketoglutarate supply. Optimization of these pathways and different substrate supplies resulted in a greater production of ethylene (from 1.36 to 12.83 nmol/OD600/mL), which may guide future engineering work on ethylene production using other organisms. Meanwhile, we achieved an ethylene production of 5.8 nmol/OD600/mL in the ZM532-efe strain using enzymatic straw hydrolysate of corn straw as the sole carbon source. As a preferred host in biorefinery technologies using lignocellulosic biomass as feedstock, heterologous expression of EFE in Z. mobilis converts the non-ethylene producing strain into an ethylene-producing one using a metabolic engineering approach, which is of great significance for the utilization of cellulosic biomass in the future. KEY POINTS: • Heterologous expression of EFE in Z. mobilis successfully converted the non-ethylene producing strain into an ethylene producer (1.36 nmol/OD600/mL). Targeted modifications of the central carbon metabolism can effectively improve ethylene production (peak production: 8.3 nmol/OD600/mL). • The addition of nutrients to the medium can further increase the production of ethylene (peak production: 12.8 nmol/OD600/mL). • The ZM532-efe strain achieved an ethylene production of 5.8 nmol/OD600/mL when enzymatic hydrolysate of corn straw was used as the sole carbon source.

Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.

Bioethanol has been considered as a potentially renewable energy source, and metabolic engineering plays an important role in the production of biofuels. As an efficient ethanol-producing bacterium, Zymomonas mobilis has garnered special attention due to its high sugar uptake, ethanol yield, and tolerance. Different metabolic engineering strategies have been used to establish new metabolic pathways for Z. mobilis to broaden its substrate range, remove competing pathways, and enhance its tolerance to ethanol and lignocellulosic hydrolysate inhibitors. Recent advances in omics technology, computational modeling and simulation, system biology, and synthetic biology contribute to the efficient re-design and manipulation of microbes via metabolic engineering at the whole-cell level. In this review, we summarize the progress of some new technologies used for metabolic engineering to improve bioethanol production and tolerance in Z. mobilis. Some successful examples of metabolic engineering used to develop strains for ethanol production are described in detail. Lastly, some important strategies for future metabolic engineering efforts are also highlighted.

We describe here the characterization of a novel enzyme called aldose-aldose oxidoreductase (Cc AAOR; EC 1.1.99) from Caulobacter crescentus. The Cc AAOR exists in solution as a dimer, belongs to the Gfo/Idh/MocA family and shows homology with the glucose-fructose oxidoreductase from Zymomonas mobilis. However, unlike other known members of this protein family, Cc AAOR is specific for aldose sugars and can be in the same catalytic cycle both oxidise and reduce a panel of monosaccharides at the C1 position, producing in each case the corresponding aldonolactone and alditol, respectively. Cc AAOR contains a tightly-bound nicotinamide cofactor, which is regenerated in this oxidation-reduction cycle. The highest oxidation activity was detected on D-glucose but significant activity was also observed on D-xylose, L-arabinose and D-galactose, revealing that both hexose and pentose sugars are accepted as substrates by Cc AAOR. The configuration at the C2 and C3 positions of the saccharides was shown to be especially important for the substrate binding. Interestingly, besides monosaccharides, Cc AAOR can also oxidise a range of 1,4-linked oligosaccharides having aldose unit at the reducing end, such as lactose, malto- and cello-oligosaccharides as well as xylotetraose. (1)H NMR used to monitor the oxidation and reduction reaction simultaneously, demonstrated that although D-glucose has the highest affinity and is also oxidised most efficiently by Cc AAOR, the reduction of D-glucose is clearly not as efficient. For the overall reaction catalysed by Cc AAOR, the L-arabinose, D-xylose and D-galactose were the most potent substrates.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

The ethanologenic bacterium Zymomonas mobilis is usually tolerant to high concentrations of glucose. The addition of sorbitol decreases the lag phase and increases ethanol yield and productivity of the bacteria in high glucose concentrations. The molecular mechanisms of adaptation to high glucose concentrations and the effect of sorbitol are still unclear. In this study, microarray analysis was used to study the global transcriptional adaptation responses of Z. mobilis to high glucose concentrations. A total of 235 genes were differentially expressed when 220 g/L glucose was added with or without 10 mM sorbitol. These genes are involved in diverse aspects of cell metabolism and regulation, including membrane transporters, nitrogen metabolism, and plasmid-encoded genes. However, most differentially expressed genes were downregulated when sorbitol was added. Notably, the transcription of almost all genes involved in the Entner-Doudoroff and ethanol production pathways was not significantly affected. In addition, a prophage and a nitrogen-fixation cluster were significantly induced. These results revealed that Z. mobilis cells responded to high glucose concentrations by regulating the transcriptional levels of genes related to membrane channels and transporters, stress response mechanisms, and metabolic pathways. These data provide insight into the intracellular adaptation responses to high glucose concentrations and reveal strategies to engineer efficient ethanol fermentation in Z. mobilis.

Cyanobacteria, which perform oxygenic photosynthesis, have drawn attention as hosts for the direct production of biofuels and commodity chemicals from CO

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.

Two genes from Zymomonas mobilis that are responsible for ethanol production, pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhII), were heterologously expressed in the Gram-positive bacterium Streptomyces lividans TK24. An examination of carbon distribution revealed that a significant portion of carbon metabolism was switched from biomass and organic acid biosynthesis to ethanol production upon the expression of pdc and adhII. The recombinant S. lividans TK24 produced ethanol from glucose with a yield of 23.7 % based on the carbohydrate consumed. The recombinant was able to produce ethanol from xylose, l-arabinose, mannose, l-rhamnose, galactose, ribose, and cellobiose with yields of 16.0, 25.6, 21.5, 33.6, 30.6, 14.6, and 33.3 %, respectively. Polymeric substances such as starch and xylan were directly converted to ethanol by the recombinant with ethanol yields of 18.9 and 8.8 %, respectively. The recombinant S. lividans TK24/Tpet developed in this study is potentially a useful microbial resource for ethanol production from various sources of biomasses, especially microalgae.

The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l−1) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l−1) together with sorbitol (65 g l−1) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l−1, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) of Escherichia coli was usually inactivated to increase PEP supply for succinate production. However, cell growth and glucose utilization rate decreased significantly with PTS inactivation. In this work, two glucose transport proteins and two glucokinases (Glk) from E. coli and Zymomonas mobilis were recruited in PTS(-) strains, and their impacts on glucose utilization and succinate production were compared. All PTS(-) strains recruiting Z. mobilis glucose facilitator Glf had higher glucose utilization rates than PTS(-) strains using E. coli galactose permease (GalP), which was suggested to be caused by higher glucose transport velocity and lower energetic cost of Glf. The highest rate obtained by combinatorial modulation of glf and glk E. coli (2.13 g/L•h) was 81 % higher than the wild-type E. coli and 30 % higher than the highest rate obtained by combinatorial modulation of galP and glk E. coli . On the other hand, although glucokinase activities increased after replacing E. coli Glk with isoenzyme of Z. mobilis, glucose utilization rate decreased to 0.58 g/L•h, which was assumed due to tight regulation of Z. mobilis Glk by energy status of the cells. For succinate production, using GalP led to a 20 % increase in succinate productivity, while recruiting Glf led to a 41 % increase. These efficient alternative glucose utilization pathways obtained in this work can also be used for production of many other PEP-derived chemicals, such as malate, fumarate, and aromatic compounds.

Furfural from lignocellulosic hydrolysates is the prevalent inhibitor to microorganisms during cellulosic ethanol production, but the molecular mechanisms of tolerance to this inhibitor in Zymomonas mobilis are still unclear. In this study, genome-wide transcriptional responses to furfural were investigated in Z. mobilis using microarray analysis. We found that 433 genes were differentially expressed in response to furfural. Furfural up- or down-regulated genes related to cell wall/membrane biogenesis, metabolism, and transcription. However, furfural has a subtle negative effect on Entner-Doudoroff pathway mRNAs. Our results revealed that furfural had effects on multiple aspects of cellular metabolism at the transcriptional level and that membrane might play important roles in response to furfural. This research has provided insights into the molecular response to furfural in Z. mobilis, and it will be helpful to construct more furfural-resistant strains for cellulosic ethanol production.

Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.

To better understand the DNA restriction-modification (R-M) systems for more amenable strain development of the alternative industrial ethanologen, Zymomonas mobilis, three gene knockout mutants were constructed. The gene knockout mutants were tested for their DNA restriction activities by the determination of transformation efficiency using methylated and unmethylated foreign plasmid DNAs. Inactivation of a putative mrr gene encoded by ZMO0028 (zmrr) resulted in a 60-fold increase in the transformation efficiency when unmethylated plasmid DNA was used. This indicated that the putative mrr gene may serve as a type IV restriction-modification system in Z. mobilis ZM4. To assign the function of a putative type I DNA methyltransferase encoded by ZMO1933 (putative S subunit) and ZMO1934 (putative M subunit), the putative S subunit was inactivated. The gene inactivation of ZMO1933 resulted in a 30-fold increase in the transformation efficiency when methylated plasmid DNA was introduced, indicating that the putative S subunit possibly serves as a part of functional type I R-M system(s). Growth studies performed on the mutant strains indicate inactivation of the type I S subunit resulted in a lower maximum specific glucose consumption rate and biomass yield, while inactivation of the type IV Zmrr had the opposite effect, with an increase in the maximum specific growth rate and biomass yield.

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.

A recombinant oxidation/reduction cycle for the conversion of D-fructose to D-mannitol was established in resting cells of Corynebacterium glutamicum. Whole cells were used as biocatalysts, supplied with 250 mM sodium formate and 500 mM D-fructose at pH 6.5. The mannitol dehydrogenase gene (mdh) from Leuconostoc pseudomesenteroides was overexpressed in strain C. glutamicum ATCC 13032. To ensure sufficient cofactor [nicotinamide adenine dinucleotide (reduced form, NADH)] supply, the fdh gene encoding formate dehydrogenase from Mycobacterium vaccae N10 was coexpressed. The recombinant C. glutamicum cells produced D-mannitol at a constant production rate of 0.22 g (g cdw)(-1) h(-1). Expression of the glucose/fructose facilitator gene glf from Zymomonas mobilis in C. glutamicum led to a 5.5-fold increased productivity of 1.25 g (g cdw)(-1) h(-1), yielding 87 g l(-1) D-mannitol from 93.7 g l(-1) D-fructose. Determination of intracellular NAD(H) concentration during biotransformation showed a constant NAD(H) pool size and a NADH/NAD(+) ratio of approximately 1. In repetitive fed-batch biotransformation, 285 g l(-1) D-mannitol over a time period of 96 h with an average productivity of 1.0 g (g cdw)(-1) h(-1) was formed. These results show that C. glutamicum is a favorable biocatalyst for long-term biotransformation with resting cells.

Furfural from lignocellulosic hydrolysates is the key inhibitor for bio-ethanol fermentation. In this study, we report a strategy of improving the furfural tolerance in Zymomonas mobilis on the transcriptional level by engineering its global transcription sigma factor (σ(70), RpoD) protein. Three furfural tolerance RpoD mutants (ZM4-MF1, ZM4-MF2, and ZM4-MF3) were identified from error-prone PCR libraries. The best furfural-tolerance strain ZM4-MF2 reached to the maximal cell density (OD600) about 2.0 after approximately 30 h, while control strain ZM4-rpoD reached its highest cell density of about 1.3 under the same conditions. ZM4-MF2 also consumed glucose faster and yield higher ethanol; expression levels and key Entner-Doudoroff (ED) pathway enzymatic activities were also compared to control strain under furfural stress condition. Our results suggest that global transcription machinery engineering could potentially be used to improve stress tolerance and ethanol production in Z. mobilis.

Immobilised cells of the bacterium Zymomonas mobilis were used to remove glucose, fructose, and sucrose from food-grade oligosaccharide mixtures. Unpurified fructo-, malto-, isomalto-, gentio-, and inulinoligosaccharides, containing total carbohydrate concentrations of 300 g l(-1), were added to immobilised cells, in 100 ml batch reactors. No pH control or nutrient additions were required. Contaminating glucose, fructose, and sucrose within the mixtures was completely fermented within 12 h. The fermentation end products were ethanol and carbon dioxide. A minor amount of sorbitol was also produced as a fermentation by-product in the inulin-oligosaccharide mixture. No degradation of the oligosaccharides in the mixtures was observed.

A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201-3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).

Ethanol is a potential energy source and its production from renewable biomass has gained lot of popularity. There has been worldwide research to produce ethanol from regional inexpensive substrates. The present study deals with the optimization of process parameters (viz. temperature, pH, initial total reducing sugar (TRS) concentration in sugar cane molasses and fermentation time) for ethanol production from sugar cane molasses by Zymomonas mobilis using Box–Behnken experimental design and genetic algorithm (GA). An empirical model was developed through response surface methodology to analyze the effects of the process parameters on ethanol production. The data obtained after performing the experiments based on statistical design was utilized for regression analysis and analysis of variance studies. The regression equation obtained after regression analysis was used as a fitness function for the genetic algorithm. The GA optimization technique predicted a maximum ethanol yield of 59.59 g/L at temperature 31 °C, pH 5.13, initial TRS concentration 216 g/L and fermentation time 44 h. The maximum experimental ethanol yield obtained after applying GA was 58.4 g/L, which was in close agreement with the predicted value.

Ribitol dehydrogenase (RDH) catalyzes the conversion of ribitol to D-ribulose. A novel RDH gene was cloned from Zymomonas mobilis subsp. mobilis ZM4 and overexpressed in Escherichia coli BL21(DE3). DNA sequence analysis revealed an open reading frame of 795 bp, capable of encoding a polypeptide of 266 amino acid residues with a calculated molecular mass of 28,426 Da. The gene was overexpressed in E. coli BL21(DE3) and the protein was purified as an active soluble form using glutathione S-transferase affinity chromatography. The molecular mass of the purified enzyme was estimated to be approximately 28 kDa by sodium dodecyl sulfate-polyacrylamide gel and approximately 58 KDa with gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 9.5 and 65 degrees C, respectively. Unlike previously characterized RDHs, Z. mobilis RDH (ZmRDH) showed an unusual dual coenzyme specificity, with a k(cat) of 4.83 s(-1) for NADH (k(cat)/K(m) = 27.3 s(-1) mM(-1)) and k(cat) of 2.79 s(-1) for NADPH (k(cat)/K(m) = 10.8 s(-1) mM(-1)). Homology modeling and docking studies of NAD+ and NADP+ into the active site of ZmRDH shed light on the dual coenzyme specificity of ZmRDH.

Escherichia coli BL21, expressing a quintuple mutant of P450(BM-3), oxyfunctionalizes alpha-pinene in an NADPH-dependent reaction to alpha-pinene oxide, verbenol, and myrtenol. We optimized the whole-cell biocatalyst by integrating a recombinant intracellular NADPH regeneration system through co-expression of a glucose facilitator from Zymomonas mobilis for uptake of unphosphorylated glucose and a NADP(+)-dependent glucose dehydrogenase from Bacillus megaterium that oxidizes glucose to gluconolactone. The engineered strain showed a nine times higher initial alpha-pinene oxide formation rate corresponding to a sixfold higher yield of 20 mg g(-1) cell dry weight after 1.5 h. The initial total product formation rate was 1,000 micromol h(-1) micromol(-1) P450 leading to a total of 32 mg oxidized products per gram cell of dry weight after 1.5 h. The physiological functioning of the heterologous cofactor regeneration system was illustrated by a sevenfold increased alpha-pinene oxide yield in the presence of glucose compared to glucose-free conditions.

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce D-mannitol from D-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up D-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD(+)-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y(D-mannitol/D-fructose) was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.

Sorbitol, a polyol found in many fruits, is of increasing industrial interest as a sweetener, humectant, texturizer and softener. At present, it is produced chemically. The bacterium Zymomonas mobilis is able to produce sorbitol and gluconic acid from fructose and glucose, respectively. This is possible in a one-step reaction via a glucose-fructose oxidoreductase so far only known from Z. mobilis. The possibilities for the industrial production of sorbitol by Z. mobilis are discussed, and compared with the current chemical production method as well as other microbiological processes.

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures were carried out at different operational conditions (aeration, feed rate and substrate concentration) to test their effects on the system productivity. In these fermentations, the main products were ethanol and sorbitol. Kinetic parameters were calculated using the experimental data. However, parameters in the sorbitol synthesis rate were estimated from data recorded in different experiments in order to avoid the effect of the simultaneous cell growth and ethanol synthesis. In this case, the crude cell extract was used as source of the enzyme responsible for the sorbitol synthesis. The highest degree of conversion of fructose into sorbitol obtained with the extract was equal to 71% in a sugar mixture with an initial concentration of 200 g/l. Results obtained in the fed-batch fermentations showed that aeration of the culture has a positive effect on the final biomass concentration. However, final ethanol concentration is lower under aerated conditions. The best sugar yields to biomass and ethanol were 0.032 and 0.411 g/g, respectively. On the other hand, the highest sorbitol yield in the fed-batch fermentations was 0.148 g/g.

Different concentrations of corn steep liquor (CSL) were tested in the cultivation of Zymomonas mobilis. Cell growth, ethanol production, and the formation of glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL), the enzymes responsible for the bio-production of gluconic acid and sorbitol, were examined. The cell yields using 25 g CSL l(-1) and 40 g CSL l(-1) (Y(X,S) approximately 0.031 g g(-1)) were close to that obtained with 5 g yeast extract (YE) l(-1). With 5 g CSL l(-1) and 15 g CSL l(-1), the nutritional limitation led to smaller Y(X/S). Using 100 g CSL l(-1) produced an inhibitory effect on cell growth. Similar ethanol yields (92-95%) were calculated for each concentration of CSL and also for YE medium. The highest specific GFOR/GL activities (13.2-13.5 U g(-1) dry cell) were reached with 25 g CSL l(-1) and 40 g CSL l(-1), values comparable to that achieved with 5 g YE l(-1). The results confirm that CSL is an effective and cheap supplement for Z. mobilis medium, increasing the economic potential of a large-scale bio-production of sorbitol and gluconic acid by untreated Z. mobilis cells. The economic feasibility of the process is discussed.

Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l-1 h-1. No levan was produced even if a sucrose-based medium was used.