The production of L-serine with a methylotrophic microorganism using the L-serine pathway and coupling with an L-tryptophan-producing process

The cultural conditions for the production of enhanced formation of L-serine (up to 7 g/L) are described with the methylotrophic bacterium Pseudomonas 3 ab (DSM 672). The batch process is divided into three parts: (1) the biomass production phase, (2) substrate limitation period, and (3) L-serine accumulation phase. The initial specific production rate of q(p) = 0.1 g L-serine/g dry wt/h is based on the inhibition of the L-serine pathway. This is accomplished by high precursor concentrations (glycine) and a pH shift to pH 8.5. The enzymatic background is discussed. Furthermore, a coupling of the L-serine process with a L-tryptophan-producing process is demonstrated.     

cis-benzeneglycol production using a mutant Pseudomonas strain

The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the invironment, Pseudomonas JI104, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient cis-benzenglycol dehydrogenase able to stably produce cis-benzeneglycol when another carbon source was co-metabolized.We exained the possibility of cis-benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the cis-benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite.The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54g·1⁻¹ of a cell suspension produced 5.1 mmol·1⁻¹cis-benzeneglycol in 27 h, using ethanol as the energy source.     

甲基营养型L-丝氨酸产生菌的选育

以甲基营养型假单胞菌J-12为出发菌株,经硫酸二乙酯（DES）和亚硝基胍（NTG）诱变处理,D-丝氨酸结构类似物平板和高浓度甘氨酸平板定向筛选,获得1株三-丝氨酸高产菌N-13,其发酵液中L-丝氨酸产量较出发菌株提高97．9％。在含有20g/L甘氨酸和7mL／L甲醇的培养基中,L-丝氨酸积累可达4．81g／L。     

Enzymatic production of L-serine

Serine hydroxymethyltransferase (SHMT) in the form of crude extract form a recombinant strain of Klebsiella aerogenes was used to study the production of L-serine from glycine and formaldehyde (HCHO). SHMT activity linearly increased with temperature (30-50 degrees C). Addition of exogenous cofactors, tetrahydrofolic acid and pyridoxal-phosphate, significantly increased SHMT activity. The pH optimum of the SHMT catalyzed L-serine synthesis step was between 8.0 and 8.5. The K(m) for glycine was 11.6mM at 37 degrees C and pH 8.0. A 87% molar conversion of glycine to serine was obtained at equilibrium (37 degrees C, pH 8.0). Tetrahydrofolic acid was stabilized by maintaining the redox potential of the reaction solution below -330 mV through the addition of a reducing reagent such as beta-mercaptoethanol. SHMT stability was very sensitive to HCHO concentration. By carefully balancing the HCHO feed rate against the enzymatic bioconversion rate in order to keep HCHO concentration low, a serine titer of 160 g/L was achieved, the residual glycine concentration was reduced to 40 g/L, a 70% molar conversion of glycine with quantitative yield was obtained, and the overall serine productivity was 5.2 g/L/h.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

Strain PM2, a novel methylotrophic fluorescent Pseudomonas sp

A novel bacterial strain, PM2, capable of growing on methanol, was isolated in alkaline conditions from a soil inoculum. This bacterium was characterized at the physiological, biochemical and molecular level. Based on biochemical and molecular data strain PM2 was classified as a novel member of the group of fluorescent pseudomonads. Evidence for the presence of a pyrroloquinoline quinone (PQQ)-linked alcohol dehydrogenase in this organism is presented. Strain PM2 is, to our knowledge, the first example of a methylotrophic Pseudomonas to be characterized in detail. This novel type of metabolism in Pseudomonas broadens even further the metabolic versatility for which this genus is renowned.     

Reduced folate supply as a key to enhanced L-serine production by Corynebacterium glutamicum

The amino acid L-serine is required for pharmaceutical purposes, and the availability of a sugar-based microbial process for its production is desirable. However, a number of intracellular utilization routes prevent overproduction of L-serine, with the essential serine hydroxymethyltransferase (SHMT) (glyA) probably occupying a key position. We found that constructs of Corynebacterium glutamicum strains where chromosomal glyA expression is dependent on Ptac and lacIQ are unstable, acquiring mutations in lacIQ, for instance. To overcome the inconvenient glyA expression control, we instead considered controlling SHMT activity by the availability of 5,6,7,8-tetrahydrofolate (THF). The pabAB and pabC genes of THF synthesis were identified and deleted in C. glutamicum, and the resulting strains were shown to require folate or 4-aminobenzoate for growth. Whereas the C. glutamicum DeltasdaA strain (pserACB) accumulates only traces of L-serine, with the C. glutamicum DeltapabABCDeltasdaA strain (pserACB), L-serine accumulation and growth responded in a dose-dependent manner to an external folate supply. At 0.1 mM folate, 81 mM L-serine accumulated. In a 20-liter controlled fed-batch culture, a 345 mM L-serine accumulation was achieved. Thus, an efficient and highly competitive process for microbial l-serine production is available.     

Isolation of a mutant strain of Pseudomonas sp ATCC 31461 exhibiting elevated polysaccharide production

A mutant strain of the bacterium Pseudomonas sp. ATCC 31461 that exhibited elevated production of the polysaccharide gellan on glucose or corn syrup as a carbon source was isolated. Gellan production by the mutant strain was about twofold higher than its parent strain on glucose or corn syrup after 48 h of growth, and about 1.4-fold higher after 72 h. An increase in biomass production was not correlated with enhanced gellan synthesis by the mutant strain. The increased gellan production by the mutant strain on either carbon source resulted in an increase in its culture medium viscosity and the viscosity of the isolated polysaccharide produced by glucose-grown cells. No differences in the glucuronic acid content of the polysaccharides produced by the mutant and parent strains were observed. Journal of Industrial Microbiology & Biotechnology (2002) 29, 185–188 doi:10.1038/sj.jim.7000278 Received 13 February 2002/ Accepted in revised form 20 May 2002     

13C-NMR studies of acetate and methanol metabolism by methylotrophic Pseudomonas strains

The metabolism of [2-13C]acetate by Pseudomonas M27(Icl-) and Pseudomonas MA(Icl+) was studied in vivo using 13C-NMR spectroscopy. The flux of 13C-label into bicarbonate, glutamate and citrate was observed in both organisms. In addition 13C-labelled alpha, alpha-trehalose was synthesized as a major metabolite by Pseudomonas M27 but not by Pseudomonas MA. The presence of this disaccharide in cell extracts of Pseudomonas AM1(Icl-) grown with [13C]methanol was also observed. The data from analysis of the trehalose multiplet signal observed in the spectra of Pseudomonas M27 cell extracts were consistent with the absence of the glyoxylate cycle in this methylotroph.     

Naphthalene oxidation by a Pseudomonas putida strain carrying a mutant plasmid

Naphthalene oxidation by a parent and a mutant strain of Pseudomonas putida was studied. The parent strain contained a plasmid NPL-1 which controlled oxidation of naphthalene to salicylic acid and was capable of oxidizing salicylate. The mutant strain did not oxidize salicylate because of a mutation in salicylate hydroxylase; it contained also a mutant plasmid NPL-41 which determined constitutive synthesis of naphthalene oxygenase. Salicylic acid which accumulated as a product of naphthalene catabolism in the cultural broth of the wild strain was found to undergo further oxidation by the population of growing cells. The content of salicylic acid in the cultural broth of the mutant strain reached maximum and then remained constant. An anion-exchange resin was tested in order to prevent the inhibition of naphthalene oxygenase by salicylate and to increase the yield of salicylic acid. The transmissible character of the mutant plasmid NPL-41 makes it possible, with the aid of conjugation, to construct Pseudomonas strains which would oxidize naphthalene to salicylic acid without further degradation of this compound.     

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.     

Biochemical characterization of an l-methionine-enriched mutant of a methylotrophic yeast,Candida boidinii

The effects of an ethionine-resistant mutation in a methylotrophic yeast, Candida boidinii, were studied. In mutant strain E500-78 (ethionine-resistant), SAM synthetase activity was low and was only slightly repressed by l-methionine. Formyltetrahydrofolate synthetase and serine hydroxymethyltransferase were involved in synthesis of the methyl group of l-methionine. The activities of the methyl group transferring enzymes and homocysteine transmethylation were repressed by l-methionine in the wild type strain, but not in the mutant. The activities of the methyl group transferring enzymes were markedly stimulated when the mutant was grown in methanol medium.     

Xylitol production by a Pichia stipitis D-xylulokinase mutant

Xylitol production by Pichia stipitis FPL-YS30, a xyl3-1 mutant that metabolizes xylose using an alternative metabolic pathway, was investigated under aerobic and oxygen-limited culture conditions. Under both culture conditions, FPL-YS30 (xyl3-1) produced a negligible amount of ethanol and converted xylose mainly into xylitol with comparable yields (0.30 and 0.27 g xylitol/g xylose). However, xylose consumption increased five-fold under aerobic compared to oxygen-limited conditions. This suggests that the efficiency of the alternative route of xylose assimilation is affected by respiration. As a result, the FPL-YS30 strain produced 26 g/l of xylitol, and exhibited a higher volumetric productivity (0.22 g xylitol l^–1 h^–1) under aerobic conditions.     

Homocitrate production by a lysine-requiring pichia guilliermondii mutant

Mutants of Pichia guilliermondii were isolated that lacked homoaconitate hydratase (lys4), homoisocitrate dehydrogenase (lys10) or α-aminoadipate reductase (lys2) and were able to excrete homocitrate into the culture medium. The effects of incubation time and lysine concentration in the medium on the excretion of homocitrate were examined. In the presence of 600 mg of L-lysine/1 in a minimum salt medium P. guilliermondii G75 (lys2) produced about 280 mg homocitrate/1 during 48 h of growth. A simple procedure to isolate homocitrate from the medium is described.     

Astaxanthin production by a highly photosensitive Haematococcus mutant

Haematococcus pluvialis synthesizes a high yield of astaxanthin using CO₂ in a photoautotrophic culture without contaminant heterotrophs; however, it takes too long to induce astaxanthin production. In this study, a highly photosensitive mutant strain was attained by conventional random mutagenesis and an efficient isolation method to shorten induction time. Sensitivity to photoinhibition in this mutant was raised by a partial lesion in the photosystem II (PSII) of photosynthesis, thereby prompting a change in cellular morphology as well as stimulating carotenogenesis (astaxanthin production). As a result, the concentrations of cell biomass and astaxanthin were dramatically increased by 27% and 62% under strong light and 79% and 153% under moderate light, respectively. This Haematococcus mutant would be useful for the economical astaxanthin production capable of reducing the light energy cost in a photoautotrophic culture system, even in areas with insufficient sunlight.     

Heterologous protein production in methylotrophic yeasts

The facultative methylotrophic yeasts Candida boidinii, Pichia methanolica, Pichia pastoris and Hansenula polymorpha have been developed as systems for heterologous gene expression. They are based on strong and regulatable promoters for expression control derived from methanol metabolism pathway genes. An increasing number of biotechnological applications attest to their status as preferred options among the various gene expression hosts. The well-established P. pastoris and H. polymorpha systems have been utilized in especially competitive and consistent industrial-scale production processes. Pharmaceuticals and technical enzymes produced in these methylotrophs have either already entered the market or are expected to do so in the near future. The article describes the present status of the methylotrophic yeasts as expression systems, focusing on applied examples of the recent past. Received: 9 May 2000 / Received revision: 20 June 2000 / Accepted: 23 June 2000     

Indole-3-acetic acid production by pink-pigmented facultative methylotrophic bacteria

In examining the presence of indole-3-acetic acid (IAA) in supernatants of pink-pigmented facultativemethylotrophic (PPFMs) bacterial cultures, three out of the 16 isolates tested showed a positive reaction ina colorimetric assay. The presence was further unambiguously con?rmed by high-performance liquidchromatography in combination with NMR. The IAA production was signi?cantly stimulated byL-tryptophan. These results prove that PPFM bacteria are able to produce the plant hormone IAA.     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.