Draft Genome Sequence of the Flocculating Zymomonas mobilis Strain ZM401 (ATCC 31822)

Zymomonas mobilis ZM401 is a flocculating strain which can be self-immobilized within fermentors for a high-cell-density culture to improve ethanol productivity, as well as high-gravity fermentation to increase ethanol titer, due to its improved ethanol tolerance associated with the morphological change. Here, we report its draft genome sequence.     

Kinetic studies on a flocculent strain of Zymomonas mobilis

Summary A flocculent mutant of Zymomonas mobilis has been isolated and kinetic studies carried out in batch and continuous culture. By comparison with the parent strain the specific rates of glucose uptake and ethanol production were decreased by 20%. Cell recycle and semibatch cultures with the flocculent strain resulted in relatively high productivities (viz. 50 g/l/h). However semibatch culture had the additional advantages of an increased ethanol concentration (viz. 82 g/l) and a more stable and controlled environment for cell separation.     

Flocculating Zymomonas mobilis is a promising host to be engineered for fuel ethanol production from lignocellulosic biomass

Whereas Saccharomyces cerevisiae uses the Embden‐Meyerhof‐Parnas pathway to metabolize glucose, Zymomonas mobilis uses the Entner‐Doudoroff (ED) pathway. Employing the ED pathway, 50% less ATP is produced, which could lead to less biomass being accumulated during fermentation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific surface area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. We performed ethanol fermentations using these two species under comparable conditions to validate these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanol productivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing ～100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation bythe flocculating Z. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yield and productivity, the flocculation of the bacterial species enabled biomass recovery by cost‐effective sedimentation, instead of centrifugation with intensive capital investment and energy consumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment, particularly acetic acid and vanillin, was improved. These experimental results indicate that Z. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineered for fuel ethanol production from lignocellulosic biomass.     

Continuous ethanol production by a flocculent strain of Zymomonas mobilis

Summary Cell retention and ethanol production using the flocculent bacterium Zymomonas mobilis NRRL B-12526 were studied in three bioreactor configurations. The flocculent growth characteristic of this strain and a special reactor design were combined to achieve relatively high cell concentrations in a continuous bioreactor for the conversion of glucose to ethanol.Research sponsored by the Office of Energy Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation.     

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD’s SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD’s SP were also higher than the other two strains containing PhoC or ZMO0331’s SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.     

Ethanol production from fructose in continuous culture by free and flocculent cells of Zymomonas mobilis

Summary Zymomonas mobilis strain ZM4 was used for ethanol production from fructose (100 g/l) in continuous culture with a mineral (containing Ca pantothenate) or a rich (containing yeast extract) mediium. With both media high conversion yields were observed but the ethanol productivity was limited by the low biomass content of the fermentor. A new flocculent strain of Z.mobilis (ZM4F) was cultivated in a CSTR with an internal settler and showed a maximal productivity of 93 g/l.h (fructose conversion of 80%). When the fructose conversion was 96% an ethanol productivity of 85.6 g/l.h with an ethanol yield of 0.49 g/g (96% of theoretical) was observed.     

DNA restriction-modification systems in the ethanologen, Zymomonas mobilis ZM4

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.     

High productivity ethanol fermentation on a mineral medium using a flocculent strain ofZymomonas mobilis

Summary A flocculent strain ofZymomonas mobilis (ZM4F JM1) was isolated in continuous culture. The parent strain, ZM4F, had lost its flocculating properties. The isolation was done in a conical fermentor at high dilution rate. Ethanol production by the new strain was then compared on a rich and mineral medium. The mineral medium showed high performance and could be used for industrial production of ethanol since it reduced one hundred fold the vitamin cost of the fermentation.     

Intracellular accumulation of c-di-GMP and its regulation on self-flocculation of the bacterial cells of Zymomonas mobilis

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner–Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.     

生物电化学系统对运动发酵单胞菌代谢的影响北大核心CSCD

生物电化学系统能促进微生物与电极间的相互作用,从而改变微生物的代谢状态。本工作为研究运动发酵单胞菌(Zymomonas mobilis)在电环境中的代谢表现,在外接3V电源的H型电化学发酵装置中测试了其发酵效能。结果表明,相比于无电压的对照,阳极甘油产量上升24%,阴极葡萄糖消耗上升16%,产物乙醇和琥珀酸的产量也分别上升13%和8%。转录组分析表明,代谢物的显著改变归因于电环境导致的有机酸代谢、氧化还原平衡、电子传递等通路的改变。从表达差异显著的基因中挑选了代表胞内氧化还原平衡、生物膜形成和电子传递的3个基因ZMO1060(编码超氧化物歧化酶)、ZMO0401(编码二鸟苷酸磷酸二酯酶)和ZMO1819(编码固氮蛋白)进行验证,结果表明过表达ZMO1060和ZMO1819能够更显著地改变生物电化学系统中Z.mobilis的代谢。本工作为应用生物电化学系统调控微生物代谢物生产提供了参考。     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Isolation and characterization of Zymomonas mobilis mutants resistant to octadecyltrimethylammonium chloride,a detergent acting on hopanoid-producing bacteria

Growth of the hopanoid-producing bacterium Zymomonas mobilis was inhibited at low concentrations of the cationic detergent octadecyltrimethylammoniumchloride (OTAC). A relationship between sensitivity of Zymomonas mobilis to OTAC, presence of hopanoids and ethanol tolerance was postulated. Mutants resistant to OTAC were isolated from strains ZM1 and ZM4. They did not present any alteration of the hopanoid content and their squalene cyclases showed the same sensitity to OTAC as the parent enzymes. Resistance to OTAC paralleled pleiotropic effects including, enhanced accessibility of the membrane-bound alkaline phosphatase, important release of proteins from cells by Tris/HCl treatment, increased resistance to antibiotics and increased sensitivity to ethanol. In addition, OTAC^R mutants were also characterized by the synthesis or the overproduction of an outer membrane protein (F53) not detected on 2D-PAGE maps of parent strains and by a normal heat shock response. The role of hopanoids, heat shock proteins, protein F53 and membrane organization in ethanol tolerance is discussed.Abbreviations OTAC octadecyltrimethylammoniumchloride - SLS sodium lauryl sarcosinate     

Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

The Zymomonas mobilis ZM4 strain with excellent ethanol‐producing capabilities was the first strain of Z. mobilis, which was sequenced. This strain is resistant to transformation, and no previous study has shown a detailed protocol for electrotransfer of ZM4 with foreign DNA. In this work, many electrical and biological parameters were selected and evaluated in order to optimize the electrotransformation of ZM4. First, improved transformation efficiencies of 11 896, 99, 96 and 5989 transformants/μg DNA were separately achieved with shuttle plasmid pZB21‐mini (3082 bp), pZB21 (5930 bp), pZA22 (6994 bp) and broad‐host‐range vector pBBR1MCS‐2 (5144 bp) all prepared from Escherichia coli JM110. The crucial factors affecting the transformation efficiency included the source of the plasmid (the best strain was ZM4), origin and size of the plasmids, growth phase of the cells (the most ideal phase was early log phase with OD₆₀₀ of 0.3–0.4), the electric field strength (generally 11.75 kV/cm–13.25 kV/cm) and the recovery time (3–24 h). Further, based upon the optimal transformation protocol mentioned above for replicative plasmids in ZM4, (i) the electrotransformation by recombinant plasmid pBBR1MCS‐2‐Pgap‐FLP (6880 bp) was an immediate success with the transformation efficiency 10² transformants/μg DNA; (ii) the site‐specific integration efficiencies (expressed in terms of “per μg of DNA”) of 3–6 integrating transformants was obtained using the integrating plasmid pBR328‐ldhR‐cml‐ldhL (7447 bp). This study will assist genetic and biotechnological research of ZM4 and other Z. mobilis strains by providing information about suitable vectors and a more universal and reliable procedure for introducing DNA into this strain.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

Alcoholic fermentation of cellulose hydrolysate by Zymomonas mobilis

Summary Growth and ethanol production by three strains of the bacterium Zymomonas mobilis were tested, at 40°C, in a medium containing cellulose hydrolysate (hexose fraction) as carbon source. The thermotolerant mutant C107 exhibited the best growth compared to wild type ZM4 and to the osmotolerant mutant SBE15. When cultivated in media supplemented with various nutrients, growth was only observed in presence of yeast extract (10 g/l) which acted both as a vitamin supplier and pH stabilizer. Using calcium pantothenate instead of yeast extract and sodium acetate to control pH resulted in growth inhibition by the high medium osmolality. Batch fermentation with pH control (KOH addition) showed good growth and ethanol production with the mineral medium.     

Stability and expression of a cloned α-glucosidase gene inZymomonas mobilis grown in batch and continuous culture

Summary Strains ofZymomonas mobilis containing an -glucosidase gene cloned fromBacillus brevis strain 27-7 (NRRL B-4389) on the plasmid pNSW358 showed varying degrees of stability in batch culture under non-selective conditions. After 45 generations of growth in continuous culture, pNSW358 was stable inZ.mobilis strain ZM6100 and the specific activity of -glucosidase in these cells was 2.7 nmol/min/mg protein. Lysed cell extracts confirmed the activity of the -glucosidase enzyme in ZM6100(pNSW358) with 21 g/1 ethanol in 50 (82% theoretical conversion of maltose to ethanol). ZM6100(pNSW358) whole cells showed a very slow conversion rate on maltose as a sole carbon source with only 5.3 g/1 ethanol after 30 days on 100 g/l maltose medium.     

Engineering endoglucanase-secreting strains of ethanologenic Klebsiella oxytoca P2

Klebsiella oxytoca P2 was developed as a biocatalyst for the simultaneous saccharification and fermentation (SSF) of cellulose by chromosomally integrating Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. This strain contains the native ability to transport and metabolize cellobiose, eliminating the need to supplement with β-glucosidase during SSF. To increase the utility of this biocatalyst, we have now chromosomally integrated the celZ gene encoding the primary endoglucanase from Erwinia chrysanthemi. This gene was expressed at high levels by replacing the native promoter with a surrogate promoter derived from Z. mobilis DNA. With the addition of out genes encoding the type II protein secretion system from E. chrysanthemi, over half of the active endoglucanase (EGZ) was secreted into the extracellular environment. The two most active strains, SZ2(pCPP2006) and SZ6(pCPP2006), produced approximately 24 000 IU L⁻¹ of CMCase activity, equivalent to 5% of total cellular protein. Recombinant EGZ partially depolymerized acid-swollen cellulose and allowed the production of small amounts of ethanol by SZ6(pCPP2006) without the addition of fungal cellulase. However, additional endoglucanase activities will be required to complete the depolymerization of cellulose into small soluble products which can be efficiently metabolized to ethanol. Received 14 December 1998/ Accepted in revised form 04 March 1999     

On the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis

Summary In this paper the effect of temperature and pH on the settling behaviour of a flocculent strain ofZymomonas mobilis is studied by using the old fashioned batch settling technique. Plots are given to show the influence of the above mentioned parameters on the settling curve behaviour.