Enzymatic Regeneration of NAD in Enantioselective Oxidation of Secondary Alcohols: Glutamate Dehydrogenase Versus NADH Dehydrogenase

To improve yield and productivity of ketose in NAD-dependent polyol oxidations, two enzymatic methods for regeneration of the oxidized coenzyme form have been compared and partly optimized for the batch conversion of xylitol into D-xylulose and D-sorbitol into D-fructose. Polyol oxidation was catalyzed by xylitol dehydrogenase from the yeast Galactocandida mastotermitis. Reduction of OM₂ (apparently to H₂O) by partially purified NADH dehydrogenase complex from Corynebacterium callunae could drive alcohol oxidations better than reductive amination of EaL-ketoglutarate by glutamate dehydrogenase. A fed-batch procedure was developed that overcame inhibition of glutamate dehydrogenase by α-ketoglutarate (K_is 25 mM), thus increasing the productivity of ketose almost 2-fold. For D-fructose production from D-sorbitol (0.1-0.3M) yields of < 90% and productivities up to 1.30g/(L.h) have been obtained. High conversion of up to 50g/L xylitol into D-xylulose for which xylitol dehydrogenase exhibits an about 80-fold higher specificity constant than for D-fructose required complexation of the ketose product with borate. In comparison with reductive amination by glutamate dehydrogenase, advantages of using NADH-dehydrogenase catalyzed regeneration of NAD for ketose production are (i) avoidance of byproduct formation, (ii) cheaper substrate (02 versus α-ketoglutarate), and (iii) easier process control (batch versus fed-batch).     

D-mannitol production by resting state whole cell biotrans-formation of D-fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium

An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.     

5-Keto-D-fructose production from sugar alcohol by isolated wild strain Gluconobacter frateurii CHM 43

ABSTRACT

Gluconobacter frateurii CHM 43 have D-mannitol dehydrogenase (quinoprotein glycerol dehydrogenase) and flavoprotein D-fructose dehydrogenase in the membranes. When the two enzymes are functional, D-mannitol is converted to 5-keto-D-fructose with 65% yield when cultivated on D-mannitol. 5-Keto-D-fructose production with almost 100% yield was realized with the resting cells. The method proposed here should give a smart strategy for 5-keto-D-fructose production.     

Production of 8,11‐dihydroxy and 8‐hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11‐linoleate diol synthase from Penicillium chrysogenum

Hydroxy unsaturated fatty acids can be used as antimicrobial surfactants. 8,11‐Linoleate diol synthase (8,11‐LDS) catalyzes the conversion of unsaturated fatty acid to 8‐hydroperoxy unsaturated fatty acid, and it is subsequently isomerized to 8,11‐dihydroxy unsaturated fatty acid by the enzyme. The optimal reaction conditions of recombinant Escherichia coli expressing Penicillium chrysogenum 8,11‐LDS for the production of 8,11‐dihydroxy‐9,12(Z,Z)‐octadecadienoic acid (8,11‐DiHODE), 8,11‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid (8,11‐DiHOTrE), 8‐hydroxy‐9(Z)‐hexadecenoic acid (8‐HHME), and 8‐hydroxy‐9(Z)‐octadecenoic acid (8‐HOME) were pH 7.0, 25°C, 10 g/L linoleic acid, and 20 g/L cells; pH 6.0, 25°C, 6 g/L α‐linolenic acid, and 60 g/L cells; pH 7.0, 25°C, 8 g/L palmitoleic acid, and 25 g/L cells; and pH 8.5, 30°C, 6 g/L oleic acid, and 25 g/L cells, respectively. Under these optimized conditions, the recombinant cells produced 6.0 g/L 8,11‐DiHODE for 60 min, with a conversion of 60% (w/w) and a productivity of 6.0 g/L/h; 4.3 g/L 8,11‐DiHOTrE for 60 min, with a conversion of 72% (w/w) and a productivity of 4.3 g/L/h; 4.3 g/L 8‐HHME acid for 60 min, with a conversion of 54% (w/w) and a productivity of 4.3 g/L/h; and 0.9 g/L 8‐HOME for 30 min, with a conversion of 15% (w/w) and a productivity of 1.8 g/L/h. To best of our knowledge, this is the first report on the biotechnological production of 8,11‐DiHODE, 8,11‐DiHOTrE, 8‐HHME, and 8‐HOME. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:390–396, 2017     

Reduction of acetone to isopropanol using producer gas fermenting microbes

Gasification‐fermentation is an emerging technology for the conversion of lignocellulosic materials into biofuels and specialty chemicals. For effective utilization of producer gas by fermenting bacteria, tar compounds produced in the gasification process are often removed by wet scrubbing techniques using acetone. In a preliminary study using biomass generated producer gas scrubbed with acetone, an accumulation of acetone and subsequent isopropanol production was observed. The effect of 2 g/L acetone concentrations in the fermentation media on growth and product distributions was studied with “Clostridium ragsdalei,” also known as Clostridium strain P11 or P11, and Clostridium carboxidivorans P7 or P7. The reduction of acetone to isopropanol was possible with “C. ragsdalei,” but not with P7. In P11 this reaction occurred rapidly when acetone was added in the acidogenic phase, but was 2.5 times slower when added in the solventogenic phase. Acetone at concentrations of 2 g/L did not affect the growth of P7, but ethanol increased by 41% and acetic acid concentrations decreased by 79%. In the fermentations using P11, growth was unaffected and ethanol concentrations increased by 55% when acetone was added in the acidogenic phase. Acetic acid concentrations increased by 19% in both the treatments where acetone was added. Our observations indicate that P11 has a secondary alcohol dehydrogenase that enables it to reduce acetone to isopropanol, while P7 lacks this enzyme. P11 offers an opportunity for biological production of isopropanol from acetone reduction in the presence of gaseous substrates (CO, CO₂, and H₂). Biotechnol. Bioeng. 2011;108: 2330–2338. © 2011 Wiley Periodicals, Inc.     

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO₂) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO₂ for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO₂ and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO₂ into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO₂ reduction. Structural analysis of the enzyme through homology modeling is also presented.     

α-l-Rhamnosidase Activity in Culture Filtrate of Corticium rolfsii Enzymic Activity at Low pH

A phosphorylation system for formation of ATP from AMP by Zymolyase-treated cells of Candida boidinii (Kloeckera sp.) No. 2201 was developed as an ATP production process. This system was shown to be an energy conversion system, from a reduced C₁ -compound to ATP through reduction of NAD⁺ and oxidative phosphorylation but not substrate level phosphorylation, together with phosphorylation of AMP to ADP.

Reaction conditions for the ATP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pressure, and oxygen supply. Under the optimal conditions, 26 mM (13 g/liter) and 8.5 dim (4g/liter) of ATP were produced with methanol and formate as C₁ -substrate, respectively.     

Box-Behnken响应面法优化司替霉素B产生菌发酵条件

【背景】粗糙链霉菌(Streptomyces scabrisporus) HBERC-53204是本中心自主分离的一株链霉菌,经鉴定,其产生一种活性化合物司替霉素B (steffimycin B,SMB),对多种动植物重要病原菌具有良好生物活性。【目的】提高SMB发酵水平,拓宽放线菌活性天然产物在农牧业领域的研究及应用。【方法】以本实验室筛选出的一株产SMB的粗糙链霉菌HBERC-53204为研究对象,运用单因素试验筛选培养基的主效碳源、氮源、无机盐及各营养成分最适浓度,并基于单因素试验结果,通过Plackett-Burman(PB)试验设计筛选出显著影响因素,再结合最陡爬坡试验、Box-Behnken (BB)响应面法拟合显著因子与产量的非线性方程求解,进一步优化菌株产SMB的最佳发酵培养基配方。【结果】优化后最佳培养基配方为：葡萄糖36.22 g/L,蛋白胨8.00 g/L,酵母粉8.51 g/L,酸水解酪蛋白1.50 g/L,MgSO₄ 0.68 g/L,KNO₃ 1.00 g/L。经摇瓶验证,优化后SMB效价达到477.26 mg/L...     

Fructose production from Jerusalem artichoke by inulinase immobilized on chitin

Summary Inulinase fromAspergillus ficuum was immobilized by cross-linking with glutaraldehyde on chitin. Batch and continuous production of fructose from Jerusalem artichoke tuber was studied using this immobililized inulinase. In a batch reactor, the extent of hydrolysis attained 90% (D-fructose/D-glucose :86/14) in 10h and 77.5g/L of D-fructose was produced from the Jerusalem artichoke tuber juice. In a continuous packed bed column reactor, the maximum volumetric productivity of 61 g/L, h was obtained at residence time of 0.9h and conversion yield of 55%. At a fixed residence time of 2.6 h and 40° C, this could be operated for over two weeks with only a slight loss of activity (4.8%).     

Metabolic rewiring of synthetic pyruvate dehydrogenase bypasses for acetone production in cyanobacteria

Designing synthetic pathways for efficient CO₂ fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate‐acetyl‐CoA/malonyl‐CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl‐CoA carboxyltransferase. We demonstrated direct conversion of CO₂ into acetyl‐CoA‐derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl‐CoA pools. The engineered cyanobacterial strain with the AAM‐bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM‐bypass) is a key factor for the high‐level production of an acetyl‐CoA‐derived chemical in photosynthetic organisms.     

Development of a multi-enzymatic desymmetrization and its application for the biosynthesis of l-norvaline from dl-norvaline

Perindopril is an effective antihypertensive drug in strong demand used to treat hypertension. l-norvaline is a vital intermediate of Perindopril production mainly produced by chemical synthesis with low purity. We developed an environmentally friendly method to produce l-norvaline with high purity based on a desymmetrization process. d-Norvaline was oxidized to the corresponding keto acid by d-amino acid oxidase from the substrate dl-norvaline. Asymmetric hydrogenation of the keto acid to l-norvaline was carried out by leucine dehydrogenase with concomitant oxidation of NADH to NAD⁺. A NADH regeneration system was introduced by overexpressing a formate dehydrogenase. The unwanted H₂O₂ by-product generated during d-norvaline oxidation was removed by adding catalase. A total of 54.09 g/L of l-norvaline was achieved, with an enantiomeric excess over 99% under optimal conditions, with a 96.7% conversion rate. Our desymmetrization method provides an environmental friendly strategy for the production of enantiomerically pure l-norvaline in the pharmaceutical industry.     

Reactor kinetics for submerged aerobic biofilms

Sclerotium rolfsii ATCC 15205 was cultivated in continuous culture (48 l reactor volume) for scleroglucan production. The maximum volumetric productivity (Q_Pvmax) amounted to 7.2 g/ld and was more than twice as high as in comparable batchwise cultivations. In addition, the relation between (a) volumetric productivity (Q_P [g/ld]) and product yield (Y_{Glucan/Glucose} [1]), (b) volumetric productivity and product quality (M_W [g/mol]), and (c) product quality and impeller tip speed (Nd [m/s]) was studied in continuous culture. It was found, that an increase in volumetric productivity led to a decline in product yield and product quality. Furthermore, an impeller tip speed of >0.7 m/s decreased the attainable product quality considerably. Based on these results, the impact of the operational setpoint of the process in terms of oxygen supply and reactor scale on the economics of scleroglucan production was discussed. In contrast to batchwise cultivation, scleroglucan production in continuous culture proved to be not feasible under non-aseptic conditions.     

利用代谢工程构建D-甘露醇生产菌株

D-甘露醇广泛应用于食品、制药、化学品工业等领域。从野生型大肠杆菌出发,将来自假肠膜明串珠菌Leuconostoc pseudomesenteroides ATCC 12291菌株的甘露醇脱氢酶与果糖转运蛋白编码基因整合到大肠杆菌ATCC 8739的染色体中,并失活其他的发酵途径 (丙酮酸甲酸裂解酶、乳酸脱氢酶、富马酸还原酶、乙醇脱氢酶、甲基乙二醛合成酶和丙酮酸氧化酶) ,构建了一株遗传稳定的D-甘露醇生产菌株。使用无机盐培养基和葡萄糖果糖作为混合碳源,厌氧发酵6 d,D-甘露醇产量达1.2 mmol/L。基于细胞生长和D-甘露醇合成的偶联,进一步通过代谢进化技术提高细胞合成D-甘露醇的生产能力。经过80代的驯化,D-甘露醇产量提高了2.6倍,甘露醇脱氢酶的活性提高了2.8倍。构建获得的遗传稳定的工程菌能直接发酵糖生产D-甘露醇,不需添加抗生素、诱导剂和甲酸,在工业化生产时有一定优势。     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

<Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> NAD-dependent, <Emphasis Type="SmallCaps">D</Emphasis>-fructose reducing,polyol dehydrogenases activity: screening,medium optimisation and application for enzymatic polyol production

Gluconobacter oxydans LMG 1489 was selected as the best strain for NAD(P)-dependent polyol dehydrogenase production. The highest enzyme activities were obtained when this strain was cultivated on a medium consisting of 30 g glycerol l^–1, 7.2 g peptone l–1 and 1.8 g yeast extract l^–1. Two D-fructose reducing, NAD-dependent intracellular enzymes were present in the G. oxydans cell-free extract: sorbitol dehydrogenase, and mannitol dehydrogenase. Substrate reduction occurred optimally at a low pH (pH 6), while the optimum for substrate oxidation was situated at alkaline pHs (pH 9.5–10.5). The mannitol dehydrogenase was more thermostable than the sorbitol dehydrogenase. The cell-free extract could be used to produce D-mannitol and D-sorbitol enzymatically from D-fructose. Efficient coenzyme regeneration was accomplished by formate dehydrogenase-mediated oxidation of formate into CO₂.     

Enhanced production of heteropolysaccharide-7 by <Emphasis Type="Italic">Beijerinckia indica</Emphasis> HS-2001 in repeated batch culture with optimized substitution of culture medium

In this study, a process of removing a half volume of culture broth and replacing it with an equal volume of substituted solution was developed to enhance the production of heteropolysaccharide-7 (PS-7) by Beijerinckia indica HS-2001. The optimal substitution time and volume of the substituted solution were found to be 48 h after cultivation and 50% of the initial volume of the culture broth. The optimal composition of the substituted solution was determined to be 20.0 g/L glucose, 10.0 g/L soybean pomace, 0.1 g/L MgSO₄·7H₂O, 0.9 g/L NH₄NO₃, and 5.0 g/L potassium phosphate, which was the same composition as the medium developed in a previous study for the production of PS-7 by B. indica HS-2001. The total amount and productivity of PS-7 by B. indica HS-2001 with a substitution under optimized conditions in a 7 L bioreactor for 96 h were 49.28 g and 0.51 g/h, respectively, which were 1.76 and 1.31-foldgreater values than those without a substitution for 72 h.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

The Auxiliary Substrate Concept: From simple considerations to heuristically valuable knowledge

Microorganisms are used in biotechnology. They are either (i) aim and purpose of a process, e.g. with the production of single cell proteins, or (ii) mean to an end insofar as they serve as a catalyst or “factory” for syntheses (e.g. of products of primary and secondary metabolism, of enzymes and antibiotics) or for the degradation and detoxification of harmful organics and inorganics. In all cases, the efficiency and velocity, finally the productivity, are parameters which essentially determine the economy of the processes. Therefore, search for approaches to optimize these processes is a permanent task and challenge for scientists and engineers. It is shown that the auxiliary substrate concept is suitable to increase the yield coefficients. It is based on the energetic evaluation of organics, on the knowledge that organics as sources of carbon and energy for growth are deficient in ATP and/or reducing equivalents, and says that it is possible to improve the carbon conversion efficiency up to the carbon metabolism determined upper limit. The latter is determined by inevitable losses of carbon along the way of assimilation and anabolism and amounts to about 85% for so‐called glycolytic substrates, e.g. glucose, methanol, and to about 75% for gluconeogenetic substrates, e.g. C₂‐substrates (acetic acid, hexadecane). The approach is explained and some experimental examples are presented. By simultaneous utilization of an extra energy source (auxiliary substrate) the yield coefficient can be increased (i) in glucose from about 0.5 to 0.7 g/g (by means of formate), (ii) in acetate from 0.34–0.4 to 0.5–0.65 g/g (by means of formate and thiosulfate, respectively), and (iii) in hexadecane from about 0.94 to 1.26 g/g (by means of formate). The precalculated yield coefficients and mixing ratios agree well with the experimentally attained ones. The approach is easily feasible and economically valuable.     

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]

In mechanistic studies by isotope-exchange tecniques of the conversion of D-fructose and D-glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D-glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D-glucose-2-³H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D-glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect (k_h/k_t=4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D-glucose to D-fructose, as evidenced by the conversion of D-glucose-2-³H into D-fructose-1-³H in acidified water.     

Succinate production from xylose‐glucose mixtures using a consortium of engineered Escherichia coli

The conversion of variable sugar mixtures into biochemicals poses a challenge for a single microorganism. For example, succinate has not been effectively generated from mixtures of glucose and xylose. In this work, a consortium of two Escherichia coli strains converted xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, the optimal pathway from xylose or glucose to succinate was determined by expressing either heterologous pyruvate carboxylase or heterologous adenosine triphosphate‐forming phosphoenol pyruvate (PEP) carboxykinase. Expression of PEP carboxykinase (pck) resulted in higher yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase (pyc) resulted in higher productivity (76 mg/gh) for glucose conversion. Then, processes using consortia of the two optimal xylose‐selective and glucose‐selective strains were designed for two different feed ratios of glucose/xylose. In each case the consortia generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of microbial consortia for the conversion of sugar mixtures: each sugar‐to‐product pathway can be optimized independently, and the volumetric consumption rate for each sugar can be controlled independently, for example, by altering the biomass concentration of each consortium member strain.