Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora.

The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.     

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.     

Production of ethanol from sugar cane molasses by Zymomonas mobilis

Summary Zymomonas mobilis strains were compared with each other and with a Saacharomyces cerevisiae strain for the production of ethanol from sugar cane molasses in batch fermentations. The effect of pH and temperature on ethanol production by Zymomonas was studied. The ability of Z. mobilis to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae. However, at higher sugar concentrations the yeast produced considerably more ethanol than Zymomonas.     

Expression and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine biosynthesis genes

Phosphatidylcholine (PC), or lecithin, is the major phospholipid in eukaryotic membranes, whereas only 10% of all bacteria are predicted to synthesize PC. In Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium tumefaciens, PC is essential for the establishment of a successful host-microbe interaction. A. tumefaciens produces PC via two alternative pathways, the methylation pathway and the Pcs pathway. The responsible genes, pmtA (coding for a phospholipid N-methyltransferase) and pcs (coding for a PC synthase), are located on the circular chromosome of A. tumefaciens C58. Recombinant expression of pmtA and pcs in Escherichia coli revealed that the individual proteins carry out the annotated enzyme functions. Both genes and a putative ABC transporter operon downstream of PC are constitutively expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes reaches around 23% of total membrane lipids. We show that PC is distributed in both the inner and outer membranes. Loss of PC results in reduced motility and increased biofilm formation, two processes known to be involved in virulence. Our work documents the critical importance of membrane lipid homeostasis for diverse cellular processes in A. tumefaciens.     

Production of D- and L-xylulose by mutants of Klebsiella pneumoniae and Erwinia uredovora

D-Xylulose and L-xylulose were produced biologically by the oxidation of a corresponding pentitol. A Klebsiella pneumoniae mutant was constructed for the oxidation of D-arabitol to D-xylulose. This mutant constitutively synthesized the D-arabitol permease system and D-arabitol dehydrogenase but was unable to produce the D-xylulokinase of the D-arabitol pathway or the D-xylose isomerase and D-xylulokinase of the D-xylose pathway. An Erwinia uredovora mutant which constitutively synthesized a novel xylitol-4-dehydrogenase but could not synthesize L-xylulokinase was used for the oxidation of xylitol to L-xylulose. Washed cell suspensions of either mutant incubated with 0.5% pentitol would oxidize 60 to 65% of the pentitol to the corresponding ketopentose in 18 h and excrete the ketopentose into the medium. Ketopentoses were rapidly purified from the remaining pentitol by hydroxyl affinity chromatography.     

Production of D- and L-xylulose by mutants of Klebsiella pneumoniae and Erwinia uredovora.

D-Xylulose and L-xylulose were produced biologically by the oxidation of a corresponding pentitol. A Klebsiella pneumoniae mutant was constructed for the oxidation of D-arabitol to D-xylulose. This mutant constitutively synthesized the D-arabitol permease system and D-arabitol dehydrogenase but was unable to produce the D-xylulokinase of the D-arabitol pathway or the D-xylose isomerase and D-xylulokinase of the D-xylose pathway. An Erwinia uredovora mutant which constitutively synthesized a novel xylitol-4-dehydrogenase but could not synthesize L-xylulokinase was used for the oxidation of xylitol to L-xylulose. Washed cell suspensions of either mutant incubated with 0.5% pentitol would oxidize 60 to 65% of the pentitol to the corresponding ketopentose in 18 h and excrete the ketopentose into the medium. Ketopentoses were rapidly purified from the remaining pentitol by hydroxyl affinity chromatography.     

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis.

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase II activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression of adhA, adhB, or pdc at high specific activities (above 8 IU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50% decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.     

Production of sorbitol and gluconic acid by permeabilized cells of Zymomonas mobilis

Summary Zymomonas mobilis is able to convert glucose and fructose to gluconic acid and sorbitol. The enzyme, glucose-fructose oxidoreductase, catalysing the intermolecular oxidation-reduction of glucose and fructose to gluconolactone and sorbitol, was formed in high amounts [1.4 units (U)·mg^-1] when Z. mobilis was grown in chemostats with glucose as the only carbon source under non-carbon-limiting conditions. The activity of a gluconolactone-hydrolysing lactonase was constant at 0.2 U·mg^-1. Using glucose-grown cells for the conversion of equimolar fructose and glucose mixtures up to 60% (w/v), a maximum product concentration of only 240 g·1^-1 of sorbitol was found. The gluconic acid accumulated was further metabolized to ethanol. After permeabilizing the cells using cationic detergents, maximum sorbitol and gluconic acid concentrations of 295 g·1^-1 each were reached; no ethanol production occurred. In a continuous process with -carrageenan-immobilized and polyethylenimin-hardened, permeabilized cells no significant decrease in the conversion yield was observed after 75 days. The specific production rates for a high yield conversion ( > 98%) in a continuous two-stage process were 0.19 g·g^-1·h^-1 for sorbitol and 0.21 g·g^-1·h^-1 for gluconic acid, respectively. For the sugar conversion of cetyltrimethylammonium bromide-treated -carrageenan-immobilized cells a V _max of 1.7 g·g^-1·h^-1 for sorbitol production and a K _m of 77.2 g·1^-1 were determinedOffprint requests to: B. Rehr     

Formation of levan and sorbitol from sucrose by Zymomonas mobilis

Summary Ethanol yields produced by Zymomonas strains from sucrose are significantly lower than from glucose or fructose. The low yield is a consequence of the formation of both levan and sorbitol as by-products. Most of the levan is in a non-precipitable form, indicating low molecular weight. Formation of sorbitol was observed with both the Zymomonas strains studied. The measured amounts of levan and sorbitol were 8% and 11% of the original sucrose content, respectively.     

Conditions for Production of 3-Ketomaltose from Agrobacterium tumefaciens

Up to 39% yields of 3-ketomaltose were achieved in 18 to 22 hr when Agrobacterium tumefaciens NRRL B-36 was cultured at 25 to 28 C in a simple medium containing 4.0 to 8.0% maltose, 0.09% urea, 0.5% CaCO(3), 0.6% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O. For maximum production of 3-ketomaltose the culture had to be maintained approximately at pH 7.0.     

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.Abbreviations CDRP O-(carboxyphenol amino)-1 deoxyribulose 5-phosphate - IGPS indoleglycerol phosphate synthase - TS tryptophan synthase Dedicated in memory of Dr. O. H. Smith     

Effect of azasqualene on hopanoid biosynthesis and ethanol tolerance of Zymomonas mobilis

Pathogenic and non-pathogenic isolates of Streptococcus suis type 2 were screened to determine whether differences in superoxide dismutase (SOD) synthesis could explain the observed differences in their pathogenicity and intracellular fate in macrophages. A single band of SOD activity of similar Rf value was visualised in PAGE gels in all isolates and inhibition studies suggested that the cofactor present was manganese. There was no correlation between specific SOD activity and virulence. It is unlikely, therefore, that SOD produced by S. suis type 2 mediates intracellular survival of pathogenic isolates in macrophages.     

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

Levansucrase (EC 2.4.1.10.) of Zymomonas mobilis 113S can perform the polymerisation of fructose moiety from raffinose to levan concomitantly with a release of non‐catabolised melibiose into the medium. The kinetic parameters of the levansucrase‐catalysed reaction provide even higher reaction velocities on raffinose as compared to sucrose, particularly at low substrate concentrations. A decreased value in the number of the average molecular mass (M_n = 1693 kDa), an increased intrinsic viscosity (η = 49.47 cm³/g), and a diminished Huggin's constant (K^' = 0.67) are intrinsic to the levan synthesis from raffinose, indicating certain structural peculiarities compared to a polysaccharide obtained from sucrose (M_n = 1851 kDa, [η] = 42.47 cm³/g, K' = 1.21).     

Structure and biosynthesis of the ribosomal ribonucleic acids from the oncogenic bacterium Agrobacterium tumefaciens.

The rRNA of the oncogenic bacterium Agrobacterium tumefaciens was extracted by several methods and analysed by polyacrylamide-gel electrophoresis. The large rRNA of this bacterium is degraded in vivo during the maturation of the ribosome. The influence of Mg2+ and denaturation on degradation of 23S RNA was studied. In pulse and chase experiments, we identified two precursors of the rRNA with mol.wts. of 1.04 x 10(6) and 0.70 x 10(6). From studies of the structure of the large rRNA, we propose that it could have arisen from a gene duplication. This structure is discussed in relation to a recent hypothesis involving such gene duplication as a means of origin of 23S rRNA.     

Cloning and expression in Escherichia coli of mercuric ion resistance coding genes from Zymomonas mobilis.

From a genomic library of Zymomonas mobilis prepared in Escherichia coli, two clones (carrying pZH4 and pZH5) resistant to the mercuric ion were isolated. On partial restriction analysis these two clones appeared to have the same 2.9 kb insert. Mercuric reductase activity was assayed from the Escherichia coli clone carrying pZH5 and it was Hg(2+)-inducible, NADH dependent and also required 2-mercaptoethanol for its activity. The plasmid pZH5 encoded three polypeptides, mercuric reductase (merA; 65 kDa), a transport protein (merT 18-17 kDa) and merC (15 kDa) as analysed by SDS-PAGE. Southern blot analysis showed the positive signal for the total DNA prepared from Hgr Z. mobilis but not with the Hgs strain which was cured for a plasmid (30 kb). These results were also confirmed by isolating this plasmid from Hgr Z. mobilis and transforming into E. coli. Moreover the plasmid pZH5 also hybridized with the mer probes derived from Tn21.     

Formation and degradation of gluconate by Zymomonas mobilis

Summary Zymomonas mobilis ATCC 29191 is able to degrade gluconate but cannot use it as a single carbon and energy source. Gluconate is phosphorylated by a gluconate kinase (EC 2.7.1.12) and the resulting 6-phosphogluconate is further catabolized to yield about 0.8 mol ethanol per mol of gluconate, considerable amounts of acetate and acetoin. This product spectrum agrees with the theoretical yield of only one reduction equivalent if gluconate is phosphorylated by a kinase and subsequently metabolized via the Entner-Doudoroff pathway.Furthermore, Z. mobilis contains a membrane-bound enzyme system which is able to oxidize glucose to gluconate. Cell-free extracts were active in an assay system with Wurster's blue as electron acceptor, and various aldoses as well as maltose, mannitol and sorbitol could be oxidized. The affinity for sorbitol was very low (K _m =330 mM) but reasonable for glucose (K _m =2.8 mM). The pH optimum for the glucose-oxidizing reaction was 6.5, while that for sorbitol oxidation was 5.5.Dedicated to Prof. Dr. H. Dörfel on the occasion of his 60th birthday     

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

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