Microbial production of l-leucine from α-ketoisocaproate by Corynebacterium glutamicum

Summary A process for l-leucine production was studied using Corynebacterium glutamicum for the conversion of -ketoisocaproate. When this precursor was added to the culture medium in a concentration of 20 g/l about 16 g/l l-leucine were formed after a fermentation time of 57 h and the molar yield was 91%. Using a fed-batch culture it was possible to produce 24 g/l of l-leucine from 32 g/l of -ketoisocaproate within 23 h. Enzymatic studies indicate that in this glutamate-producing bacterium -ketoisocaproate is converted into l-leucine via the transaminase B reaction and l-glutamate is regenerated by the glutamate dehydrogenase. By the addition of -ketoisocaproate to the culture medium the specific activity of transaminase B was increased threefold.     

Regulation of acetohydroxy acid synthase in Corynebacterium glutamicum during fermentation of α-ketobutyrate to l-isoleucine

Summary Corynebacterium glutamicum ATCC 13 032 produces 13 g/l l-isoleucine from 200 mM -ketobutyrate as a synthetic precursor. In fed batch cultures up to 19 g/l l-isoleucine is formed. For optimal conversion the addition of 0.3 mM l-valine plus 0.3 mM l-leucine to the fermentation medium is required. The affinity constants for the acetohydroxy acid synthase (AHAS) were determined. (This enzyme directs the flow of -ketobutyrate plus pyruvate towards l-isoleucine and that of two moles of pyruvate to l-valine and l-leucine, respectively.) For -ketobutyrate the K _m is 4.8×10^-3 M, and V _max 0.58 U/mg, for pyruvate the K _m is 8.4×10^-3 M, and V _max 0.37 U/mg. Due to these characteristics the presence of high -ketobutyrate concentrations apparently results in a l-valine, l-leucine deficiency. This in turn leads to a derepression of the AHAS synthesis from 0.03 U/mg to 0.29 U/mg and high l-isoleucine production is favoured. The derepression of the AHAS synthesis induced by the l-valine, l-leucine shortage was directly proven with a l-valine, l-leucine, l-isoleucine auxotrophic mutant where the starvation of each amino acid resulted in an increased AHAS level. This is in accordance with the fact that only one AHAS enzyme could be verified by chromatographic and electrophoretic separations as being responsible for the synthesis of all three branched-chain amino-acids.     

Living cell reaction process forl-isoleucine andl-valine production

Summary A new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and -ketobutyric acid (-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l^–1 · day^–1 (molecular yield to -KB: 95%) and 300 mmol · l^–1 · day^–1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.     

Reducing by-product formation in L-lactic acid fermentation by Rhizopus oryzae

During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l^–1 to 0.6 g l^–1 KH₂PO₄ reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l^–1 to 71 g l^–1 and from 1.36 g l^–1 to 0.18 g l^–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l^–1 KH₂PO₄ was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production.     

Fed-batch production of <Emphasis Type="SmallCaps">d</Emphasis>-ribose from sugar mixtures by transketolase-deficient <Emphasis Type="Italic">Bacillus subtilis</Emphasis> SPK1

d-Ribose, a five-carbon sugar, is used as a key intermediate for the production of various biomaterials, such as riboflavin and inosine monophosphate. A high d-ribose-producing Bacillus subtilis SPK1 strain was constructed by the chemical mutation of the transketolase-deficient strain, B. subtilis JY1. Batch fermentation of B. subtilis SPK1 with 20 g l^–1 xylose and 20 g l^–1 glucose resulted in 4.78 g l^–1 dry cell mass, 23.0 g l^–1d-ribose concentration, and 0.72 g l^–1 h^–1 productivity, corresponding to a 1.5- to 1.7-fold increase when compared with values for the parental strain. A late-exponential phase was chosen as the best point for switching to a fed-batch process. Optimized fed-batch fermentation of B. subtilis SPK1, feeding a mixture of 200 g l^–1 xylose and 50 g l^–1 glucose after the late-exponential phase reduced the residual xylose and glucose concentrations to less than 7.0 g l^–1 and gave the best results of 46.6 g l^–1d-ribose concentration and 0.88 g l^–1 h^–1 productivity which were 2.0- and 1.2-fold higher than the corresponding values in a simple batch fermentation.     

Purification and characterization of thermostable leucine dehydrogenase from Bacillus stearothermophilus

Leucine dehydrogenase (l-leucine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.9) has been purified to homogeneity from a moderate thermophilic bacterium, Bacillus stearothermophilus. Am improved method of preparative slab gel electrophoresis was used effectively to purify it. The enzyme has a molecular mass of about 300,000 and consists of six subunits with identical molecular mass (Mr, 49,000). The enzyme does not lose its activity by heat treatment at 70° C for 20 min, and incubation in the pH range of 5.5–10.0 at 55° C for 5 min. It is stable in 10 mM phosphate buffer (pH 7.2) containing 0.01% 2-mercaptoethanol at over 1 month, and is resistant to detergent and ethanol treatment. The enzyme catalyzes the oxidative deamination of branched-chain l-amino acids and the reductive amination of their keto analogs in the presence of NAD⁺ and NADH, respectively, as the coenzymes. The pH optima are 11 for the deamination of l-leucine, and 9.7 and 8.8 for the amination of -ketoisocaproate and -ketoisovalerate, respectively. The Michaelis constants were determined: 4.4 mM for l-leucine, 3.3 mM for l-valine, 1.4 mM for l-isoleucine and 0.49 mM for NAD⁺ in the oxidative deamination. The B. stearothermophilus enzyme shows similar catalytic properties, but higher activities than that from Bacillus sphaericus.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Optimized production of L-β-hydroxybutyric acid by a mutant of Yarrowia lipolytica

A mutant strain of Yarrowia lipolytica was developed which produced 8.0 g l--hydroxybutyric acid l^–1 from butyric acid in a batch culture. The optimum culture conditions in the fermenter for maintenance of a high cell activity, determined by chemostat analyses, were a specific growth rate of 0.06 h^–1, a glucose concentration of 2.0 g l^–1, and a butyric acid concentration of 8.1 g l^–1. A fed-batch fermentation was performed under these conditions resulting in an l--hydroxybutyric acid yield of 31 g l^–1.     

Formulation of a lactose-free, low-cost culture medium for the production of β- D-galactosidase by Kluyveromyces marxianus

A lactose-free, low-cost culture medium for the production of -d-galactosidase by Kluyveromyces marxianus was formulated. At high aeration rates (2.2 vvm) and concentrations of 100 g sugar cane molasses l^–1 as carbon source and 100 g corn steep liquor l^–1 as vitamin and nitrogen source an enzyme production of 708 U l^–1 h was achieved. This was 20% higher than using a medium that contained lactose which is considered the primary inductor of -d-galactosidase synthesis.     

Improved xylanase production by<Emphasis Type="Italic"> Trichoderma reesei</Emphasis> grown on <Emphasis Type="SmallCaps">l</Emphasis>-arabinose and lactose or <Emphasis Type="SmallCaps">d</Emphasis>-glucose mixtures

Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with d-glucose or lactose. The fungal cells consumed all aldopentoses tested, except l-xylose and l-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on l-arabinose as the main carbon source. The total xylanase activity produced by cells grown on l-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of d-glucose (15 g l^–1) and l-arabinose (5 g l^–1), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only d-glucose (20 g l^–1). In a similar experiment, in which cells were grown on a mixture of lactose and l-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of l-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture.     

Plant regeneration of rose (Rosa hybridia) from embryogenic cell-derived protoplasts

Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l^–1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-D) at a density of 5×10⁴ protoplasts ml^–1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l^–1 myo-inositol was replaced with the same osmolarity of 90 g l^–1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l^–1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media

The optimal growth of Cistanche deserticola callus and formation of phenylethanoid glycosides (PeG) was at 25°C with light irradiation intensity of 24 mol m^–2 s^–1 on solidified B5 media supplemented with 0.5 mg 6-benzylaminopurine l^–1, 10 mg gibberellin l^–1, 800 mg casein hydrolysate l^–1 and 20 g sucrose l^–1. After 30 d culture, the biomass reached 15.5 g dry wt callus l^–1 medium and its PEG content was 10.7% (w/w). The PeG content was 42%–127% higher than those in explants.     

Development and germination of Abies alba somatic embryos

Mature zygotic embryos of Abies alba mull were placed on a modified MCM medium (basal medium-BM) with 2.2 M benzyladenine and 2.3 M kinetin to induce embryogenic suspensor masses (ESM). These ESM proliferated on induction medium supplemented with 0.2 M 2,4-dichlorophenoxyacetic acid. From 61 ESM lines induced, 36 are still in culture after 2 years, of which 18 show embryogenic potential indicated by spontaneous formation of globular somatic embryos on the proliferation medium supplemented with 500–1000 mg l^-1 casein hydrolysate and 500 mg l^-1 l-glutamine. ESMs from cell line 2/56 were conditioned 1 week on BM with 58 mM sucrose and 10 g l^-1 activated charcoal for maturation of somatic embryos. Maturation was achieved on BM containing 20 M (±)cis-trans-abscisic acid in combination with 111 mM maltose. Organic nitrogen supplements improved the proliferation rate of cell line 2/56 as well as the maturation and vitality of the somatic embryos. Partial drying was necessary for subsequent root development. Plantlets with a root, primary needles and a terminal bud developed on BM when a combination of 30 mM sucrose and 50 mM maltose was provided as carbon source.Abbreviations BM basal medium - BA benzyladenine - ESM embryogenic suspensor mass - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - l-gln l-glutamine - ABA (±) cis-trans-abscisic acid     

3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation

Interest in producing biofuels from renewable sources has escalated due to energy and environmental concerns. Recently, the production of higher chain alcohols from 2-keto acid pathways has shown significant progress. In this paper, we demonstrate a mutagenesis approach in developing a strain of Escherichia coli for the production of 3-methyl-1-butanol by leveraging selective pressure toward l-leucine biosynthesis and screening for increased alcohol production. Random mutagenesis and selection with 4-aza-d,l-leucine, a structural analogue to l-leucine, resulted in the development of a new strain of E. coli able to produce 4.4 g/L of 3-methyl-1-butanol. Investigation of the host’s sensitivity to 3-methyl-1-butanol directed development of a two-phase fermentation process in which titers reached 9.5 g/L of 3-methyl-1-butanol with a yield of 0.11 g/g glucose after 60 h.     

Therlt11 andraec1 mutants ofArabidopsis thaliana lack the activity of a basic-amino-acid transporter

The concentration dependence of the influx ofl-lysine in excised roots ofArabidopsis thaliana seedlings was analyzed for the wild-type (WT) and two mutants,rlt11 andraec1, which had been selected as resistant to lysine plus threonine, and to S-2-aminoethyl-l-cysteine, respectively. In the WT three components were resolved: (i) a high-affinity, low-capacity component [K _m = 2.2 M;V _max = 23 nmol·(g FW)^–1·h^–1]; (ii) a low-affinity, high-capacity component [K _m = 159 M;V _max = 742 nmol·(g FW)^–1·h^–1]; (iii) a component which is proportional to the external concentration, with a constant of proportionalityk = 104 nmol·(g FW)^–1 h^–1];·mM^–1. The influx ofl-lysine in the mutants was lower than in the WT, notably in the concentration range 0.1–0.4 mM, where it was only 7% of that in the WT. In both mutants the reduced influx could be fully attributed to the absence of the low-affinity (high-K _m ) component. This component most likely represents the activity of a specific basic-amino-acid transporter, since it was inhibited by several other basic amino acids (arginine, ornithine, hydroxylysine, aminoethylcysteine) but not byl-valine. The high-affinity uptake ofl-lysine may be due to the activity of at least two general amino acid transporters, as it was inhibitable byl-valine, and could be further dissected into two components with a high affinity (K _i = 1–5 M; and a low affinity (K _i = 0.5–1mM) forl-valine, respectively. Therlt11 andraecl mutant have the same phenotype and the corresponding loci were mapped on chromosome 1, but it is not yet clear whether they are allelic.Abbreviations AEC S-2-aminoethyl-l-cysteine - K _i equilibrium constant - WT wild-type     

A repeated batch process for cultivation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×10⁹ and 3.4×10⁹ cfu ml^–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l^–1 h^–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l^–1 h^–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid     

Production of a fusion protein of sweet potato sporamin from recombinant E. coli XL1 Blue by fed-batch fermentations

Glucose-stat and pH-stat control strategies were employed in order to culture a recombinant E. coli XL1 Blue to produce a fusion protein of sweet potato sporamin (SPA) and glutathione S-transferase (GST) from the recombinant E. coli XL1 Blue. Cell densities up to 25 g l^–1 and 28.9 mg fusion protein (GST-SPA) g^–1 cell dry weight (CDW) was achieved from a fed-batch fermentation controlled by glucose-stat strategy. A pH-stat control fermentation using glycerol as a carbon source gave E. coli up to 27 g l^–1 and 31.5 mg GST-SPA g^–1 CDW. Additionally, a pH-stat control strategy using glucose as a carbon source gave E. coli up to 15 g l^–1 and about 22.7 mg g^–1 CDW of GST-SPA.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus on different carbon sources

Poly--hydroxybutyrate was produced in shake cultures by Alcaligenes eutrophus H16 on fructose, xylose, and fumaric, itaconic, lactic and propionic acids in a three-stage process. The maximum polymer concentration of 6.9 g l^–1 (69% of cell dry matter) was obtained with 20g l^–1 of fructose with a volumetric productivity of about 0.22 g l^–1 h^–1 at 24h. Up to about 3 g l^–1 (about 50% of cell dry matter) of polymer was also produced on lactic and propionic acids as the sole carbon source during the production phase. In multivatiate optimization employing an orthogonal 2³-factorial central composite experimental design with fructose as the substrate in a single-stage process, the optimal initial fructose concentration decreased from 35 g l^–1 to 24 g l^–1 when the incubation time was increased from about 35 h to 96 h. The optimal shaking speed range was 90–113 rpm. Correspondence to: S. Linko