Microbial Production of Long-chain Dicarboxylic Acids from n-Alkanes

Strain M-l which was derived from Candida cloacae 310 as a mutant unable to assimilate dicarboxylic acid (DC) produced large amount of DCs from n-alkanes, as expected. It produced DCs with the same number of carbon atoms as those of n-alkanes used (9 to 18 carbon atoms). Among DCs produced, n-tetradecane ω,φ′-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) was most abundantly accumulated and the highest level of DC-16, i.e., 29.3g/liter was obtained by resting cells.

On the other hand, since the growth rate of strain M-l on n-alkane markedly decreased in comparison with that of the parent strain, other carbon source which supported the growth of strain M-l was necessary for the production of DC from n-alkane by growing cells. When acetic acid was used as carbon source for the growth in DC-16 production from n-C₁₆, the highest level of DC-16, i.e., 21.8 g/liter was obtained ofter 3 days' cultivation.     

发酵法生产长链二元酸研究进展

发酵法生产长链二元酸相对于化工法而言有着无可比拟的优势。本文综述了发酵法生产长链二元酸的微生物源、产酸机理、产酸条件和产物分离技术等方面的研究进展 ,并简要介绍了其工业应用前景。     

Microbial Production of l-Phenylalanine from n-Alkanes

A tyrosine auxotroph derived from a hydrocarbon utilizing bacterium, Corynebacterium sp. KY 4309, was found to accumulate a large amount of l-phenylalanine in the broth. The cultural conditions for l-phenylalanine production were studied. The pH value during cultivations exhibited a remakable effect on l-phenylalanine production. The addition of l-tryptophan enhanced the l-phenylalanine accumulation. Shikimic acid and phenylpyruvic acid are possible precursors of phenylalanine biosynthesis in this bacterium. Production of l-phenylalanine attained to a level of 10 mg per ml for 68 hr under optimal conditions.     

Microbial Incorporation of Fatty Acids Derived From n-Alkanes Into Glycerides and Waxes

When n-alkanes with 13 to 20 carbon atoms were fed to a Nocardia closely related to N. salmonicolor, the produced cellular triglycerides and aliphatic waxes invariably contained fatty acids with an even or an odd number of carbon atoms subject to this feature of the n-alkane substrate. Beta-oxidation and C₂ addition are both operative, as evidenced by the spectra of fatty acids incorporated into the cellular lipid components. There is no distinction in the rate of microbial incorporation of the odd-or even-numbered carbon chains. The fatty acids are apparently directly derived from the long chain n-alkanes, rather than synthesized via the classic C₂-condensation route. The alcohol component of waxes produced by the Nocardia is invariably of the same chain length as the n-alkane substrate.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Methylketones from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Cell suspensions of methane-utilizing bacteria grown on methane oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding methylketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). The product methylketones accumulated extracellularly. The rate of production of methylketones varied with the organism used for oxidation; however, the average rate of acetone, 2-butanone, 2-pentanone, and 2-hexanone production was 1.2, 1.0, 0.15, and 0.025 μmol/h per 5.0 mg of protein in cell suspensions. Primary alcohols and aldehydes were also detected in low amounts as products of n-alkane (propane and butane) oxidation, but were rapidly metabolized further by cell suspensions. The optimal conditions for in vivo methylketone formation from n-alkanes were compared in Methylococcus capsulatus (Texas strain), Methylosinus sp. (CRL-15), and Methylobacterium sp. (CRL-26). The rate of acetone and 2-butanone production was linear for the first 60 min of incubation and directly increased with cell concentration up to 10 mg of protein per ml for all three cultures tested. The optimal temperatures for the production of acetone and 2-butanone were 35°C for Methylosinus trichosporium sp. (CRL-15) and Methylobacterium sp. (CRL-26) and 40°C for Methylcoccus capsulatus (Texas). Metal-chelating agents inhibited the production of methylketones, suggesting the involvement of a metal-containing enzymatic system in the oxidation of n-alkanes to the corresponding methylketones. The soluble crude extracts derived from methane-utilizing bacteria contained an oxidized nicotinamide adenine dinucleotide-dependent dehydrogenase which catalyzed the oxidation of secondary alcohols.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 μmol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 μmol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40°C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 × g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.     

Production of Dicarboxylic Acids by Candida cloacae Mutant Unable to Assimilate n-Alkane

Strain MR-12 which was derived from Candida cloacae M-l as a mutant unable to assimilate n-alkane showed marked increase in dicarboxylic acid (DC) productivity from n-alkane.

Resting cells of strain MR-12 produced 42.7g/liter of n-tetradecane 1,14-dicarboxylic acid (DC-16) from n-hexadecane (n-C₁₆) after 72 hr’ incubation. DC degradation activities of strain M-1 and MR-12 were found to be markedly reduced and their activities against DC-16 decreased to 40% and 10% of that of the parent strain, respectively.

Strain M-1 and MR-12 produced DC from the various oxidized derivatives of n-alkane such as alcohol, diol, aldehyde, fatty acid and methyl- or ethylester of fatty acid other than n-alkane.

The carbon balance in n-C₁₆ oxidation was determined by using resting cells of strain MR-12 and about 60% of utilized carbon was recovered as DC-16 and about 40% was recovered as CO₂.     

烷烃对P450酶的诱导及二元酸发酵工艺改进

α、ω 长链二元酸 (Ｌｏｎｇ ｃｈａｉｎα ,ω ｄｉｃａｒｂｏｘｙｌｉｃａｃｉｄ ,ＤＣＡ)是一种重要的化工原料 ,是合成工程塑料、香料、耐寒性增塑剂、涂料、液晶等物质的重要原料。目前主要利用热带假丝酵母 (Ｃａｎｄｉｄａｔｒｏｐｌｉｃａｌｉｓ)转化烷烃生产[1,2 ] 。在以往发酵的过程中 ,通常在初始培养液中加入 5 %～10 %的烷烃。且有文献表明在发酵初期加入烷烃有利于产酸的提高。但我们的研究表明 ,在发酵初期加入烷烃也有其不利的一面 ,如高浓度的烷烃对于菌体的生长有一定的抑制作用。而且有实验表明 :适当提高细胞的培养液…     

Carboxylic and Dicarboxylic Acids Extracted from Crushed Magnesium Oxide Single Crystals

Carboxylic and dicarboxylic acids (glycolic, oxalic, malonic and succinic) have been extracted with tetrahydrofuran (THF) and H₂O from large synthetic MgO crystals, crushed to a medium fine powder. The extracts were characterized by infrared spectroscopy and ¹H-NMR. The THF extracts were derivatized with tert-butyldimethylsilyl (t-BDMS) for GC-MS analysis. A single crystal separated from the extract was used for an x-ray structure analysis, giving the monoclinic unit cell, space group P2₁/c with a_o = 5.543 Å, b_o = 8.845 Å, c_o = 5.086 Å, and = 91.9°, consistent with -succinic acid, HOOC(CH₂)COOH. The amount of extracted acids is estimated to be of the order of 0.1 to 0.5 mg g^-1 MgO. The MgO crystals from which these organic acids were extracted grew from the 2860 °C hot melt, saturated with CO/CO₂ and H₂O, thereby incorporating small amounts of the gaseous components to form a solid solution (ss) with MgO. Upon cooling, the ss becomes supersaturated, causing solute carbon and other solute species to segregate not only to the surface but also internally, to dislocations and subgrain boundaries. The organic acids extracted from the MgO crystals after crushing appear to derive from these segregated solutes that formed C–C, C–H and C–O bonds along dislocations and other defects in the MgO structure, leading to entities that can generically be described as (H_xC_yO_z)^n-. The processes underlying the formation of these precursors are fundamental in nature and expected to be operational in any minerals, preferentially those with dense structures, that crystallized in H₂O–CO₂-laden environments. This opens the possibility that common magmatic and metamorphic rocks when weathering at the surface of a tectonically active planet like Earth may be an important source of abiogenically formed complex organic compounds.     

转基因植物生产超长链多不饱和脂肪酸研究进展

超长链多不饱和脂肪酸(VLCPUFAs)对人类健康非常重要。日常摄入一定量的VLCPUFAs能够补充人体自身合成的不足, 并对某些疾病起到明显的预防和治疗作用。VLCPUFAs主要源自深海鱼油, 但由于市场需求的迅速增长和海洋可捕捞 鱼类资源的日益减少, 该途径已经远远不能满足市场的需要, 寻找更为持续且稳定的VLCPUFAs来源已经成为当务之急。最近, 人们已经克隆了VLCPUFAs生物合成相关的去饱和酶和延伸酶基因, 并希望在植物特别是油料作物中共表达这些基因, 使其成为生产VLCPUFAs的“绿色细胞工厂”。目前已有多个研究小组在进行转基因植物合成VLCPUFAs的探索, 并取得了突破性的研究成果。本文综述了相关的研究进展, 并对存在的问题和解决策略进行了探讨。     

转基因植物生产超长链多不饱和脂肪酸研究进展

超长链多不饱和脂肪酸（VLCPUFAs）对人类健康非常重要。日常摄入一定量的VLCPUFAs能够补充人体自身合成的不足,并对某些疾病起到明显的预防和治疗作用。VLCPUFAs主要源自深海鱼油,但由于市场需求的迅速增长和海洋可捕捞鱼类资源的日益减少,该途径已经远远不能满足市场的需要,寻找更为持续且稳定的VLCPUFAs来源已经成为当务之急。最近,人们已经克隆了VLCPUFAs生物合成相关的去饱和酶和延伸酶基因,并希望在植物特别是油料作物中共表达这些基因,使其成为生产VLCPUFAs的＂绿色细胞工厂＂。目前已有多个研究小组在进行转基因植物合成VLCPUFAs的探索,并取得了突破性的研究成果。本文综述了相关的研究进展,并对存在的问题和解决策略进行了探讨。     

Benzoic Acid Derivatives Conjugated with Dicarboxylic Acids from Alfalfa (Medicago saliva)

Two compounds were isolated from alfalfa. Their structures were determined as benzoyl meso-tartaric acid (I) and benzoyl (S), (–)-malic acid (II) respectively. They were found in nature for the first time.     

Further Studies on Production of Chloramphenicol Analogs (Corynecins) from n-Alkanes

Further studies on culture condition of Corynebacterium hydrocarboclastus were carried out for more efficient production of corynecins (chloramphenicol analogs). The productivity was affected greatly by the concentration of phosphate and stimulated effectively by organic nutrients, particularly by yeast extract. The most effective production was obtained in the presence of 8 g of yeast extract and 0.2 g of K₂HPO₄ per one liter of chemically defined medium. The maximum amount was 1.42 g equivalent of l-base (free base of chloramphenicol) per liter of the broth culture, which corresponded to 4-fold of that reported in the previous paper.

Among fractions of n-alkanes, n-heptadecane was the best carbon source for the production. The proportion among corynecins homologs varied depending on carbon number of n-alkane used; odd number alkanes gave larger amounts of corynecin II (propionyl derivative), suggesting that carbon flow in the bacterial cells functioned significantly in changing the ratios of corynecin homologs.     

热带假丝酵母细胞内pH的测定及其与生长代谢活性的关系

应用荧光探针5(6)-双醋酸羧基荧光素 (Carboxyfluorescein diacetate) 测定了产长链二元酸热带假丝酵母 (Candida tropicalis) 细胞内pH (pHi) 值,确定了该探针载入C. tropicalis细胞的适宜条件。用摇瓶培养C. tropicalis细胞,考察了细胞外pH和生长碳源对pH_I的影响,实验结果表明：细胞外pH对pH_I略有影响,而生长碳源对pH_I的影响略为明显。利用5L发酵罐进一步研究了细胞生长代谢活性与pHi的关系,结果表明：细胞比生长速率、CO₂比生产速率和葡萄糖比消耗速率与pHi变化密切相关,pH_I的增加伴随着细胞生长活力的增加,反之亦然。在pH6.0条件下用葡萄糖和醋酸钠共作碳源培养C. tropicalis细胞时,测得的pH_I值维持在5.72～6.15范围内。     

Metabolic Engineering for Microbial Production of Aromatic Amino Acids and Derived Compounds

Metabolic engineering to design and construct microorganisms suitable for the production of aromatic amino acids and derivatives thereof requires control of a complicated network of metabolic reactions that partly act in parallel and frequently are in rapid equilibrium. Engineering the regulatory circuits, the uptake of carbon, the glycolytic pathway, the pentose phosphate pathway, and the common aromatic amino acid pathway as well as amino acid importers and exporters that have all been targeted to effect higher productivities of these compounds are discussed.     

液蜡发酵制取混合二元酸的研究

A mutant of Candida tropicalis FYD-2 was obtained from its parental strain SFP-1186 by ultraviolet treatments.On shaking flask,the yield of mixed dicarboxylic acid(DCA) by the mutant was 21.4% higher than that by its ancestor.The amount of mixed DCA reached 156g/L for 120h incubation in a 10 L autoconrolled fermentor where the culture medium contained 25% n-paraffin.The process of induced and screening mutant was introduced and the time course of fermentation in 10 L fermentor was discussed.     

Microbial Production of Xylitol from Glucose

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.     

Production of Microbial Cells from Hydrocarbons

Hydrocarbon-assimilating yeasts and bacteria were isolated from soil and sewage. The optimal conditions of cell yield from liquid paraffine by a Torulopsis yeast and a Pseudomonas strain were studied. A Torulopsis yeast gave, in optimal condition, 70 percent cell yield on a weight conversion basis from light oil fraction. In a strain of Pseudomonas the additions of amino acids, Fe^{+ +}, Mg^{+ +} and Ca^{+ +} ions were effective for cell production. This strain showed, in optimal condition, 80 percent cell yield (wt%) from kerosene.