L-Fucose production by engineered Escherichia coli

L -Fucose (6-deoxy-L -galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties and may be an economical way to manufacture L -fucose. Research can extract L -fucose directly from brown algae, or by enzymatic hydrolysis of L -fucose-rich microbial exopolysaccharides. However, these L -fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L -fucose. Specifically, we modified the strain genome to eliminate endogenous L -fucose and lactose metabolism, produce 2′-fucosyllactose (2′-FL), and to liberate L -fucose from 2′-FL. This E. coli strain produced 16.7 g/L of L -fucose with productivity of 0.1 g·L⁻¹·h⁻¹ in a fed-batch fermentation. This study presents an efficient one-pot biosynthesis strategy to produce a monomeric form of L -fucose by microbial fermentation, making large-scale industrial production of L -fucose feasible.     

Influence of the concentrations of d-xylose and yeast extract on ethanol production by Pachysolen tannophilus

To determine the most favorable conditions for the production of ethanol by Pachysolen tannophilus, this yeast was grown in batch cultures with various initial concentrations of two of the constituents of the culture medium: d-xylose (s_o), ranging from 1 g·l⁻¹ to 200 g·l⁻¹, and yeast extract (l_o), ranging from 0 g·l⁻¹ to 8 g·l⁻¹. The most favorable conditions proved to be initial concentrations of S_o=25 g·l⁻¹ and l_o=4 g·l⁻¹, which gave a maximum specific growth rate of 0.26 h⁻¹, biomass productivity of 0.023 g·l⁻¹·h⁻¹, overall biomass yield of 0.094 g·g⁻¹, specific xylose-uptake rate (q_s) of 0.3 g·g⁻¹·h⁻¹ (for t=50 h), specific ethanol-production rate (q_E) of 0.065 g·g⁻¹·h⁻¹ and overall ethanol yield of 0.34 g·g⁻¹; q_s values decreased after the exponential growth phase while q_E remained practically constant.     

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L⁻¹·h⁻¹, respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.     

Does midazolam inhibit the development of acute tolerance to the analgesic effect of alfentanil?

Alfentanil-midazolam analgesic interactions were studied in rats with continuous infusions or bolus injections of the drugs. Analgesia was determined by measuring the threshold of motor response to noxious pressure. The continous constant-rate infusion of alfentanil demonstrated that after an initial peak, the analgesia profoundly declined due to the development of acute tolerance. When alfentanil (250 μg·kg⁻¹·h⁻¹) was given together with midazolam (3 mg·kg⁻¹·h⁻¹), the decline in the analgesic effect of alfentanil was attenuated. Following the 4 h period of the constant-rate (250 μg·kg⁻¹·h⁻¹) infusion of alfentanil, when acute tolerance was already developed, midazolam (3 mg·kg⁻¹) given as a bolus injection enhanced the alfentanil-induced anesthesia. At the same time, when alfentanil was given as a bolus injection (30 μg·kg⁻¹) with or without midazolam (3 mg·kg⁻¹) also by bolus injection, no changes were seen to indicate an enhancement of the analgesic effect of alfentanil by midazolam. The results suggest that midazolam attenuates the development of acute tolerance to the analgesic effect of alfentanil.     

Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation

Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high-salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self-flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self-flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self-flocculating H. campaniensis was developed for growth and the production of poly-3-hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self-flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L ⁻¹·hr ⁻¹ for the batch process compared to 0.82 and 0.33 g·L ⁻¹·hr ⁻¹ for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time.     

裸果木再生体系建立与不定芽发生研究

为探究裸果木再生体系建立的影响因素,确定其不定芽发生的起源,该研究以裸果木健壮植株的茎段为外植体,采用6 BA和IBA不同浓度组合,筛选愈伤增殖及不定芽再生的最佳浓度组合,确定生根诱导的关键影响因素,建立再生体系,并对其不定芽分化进程进行解剖结构分析,以确认其起源。结果表明：(1)裸果木茎段的最佳愈伤增殖培养基为MS+1 mg·L^-1 IBA+1 mg·L^-1 6 BA+30 g·L^-1蔗糖+7 g·L^-1琼脂,主体间效应分析表明IBA为关键影响因素;愈伤大小随IBA浓度增加呈现先升高后下降的趋势。(2)最佳不定芽诱导培养基为MS+0.5 mg·L^-1 6 BA+30 g·L^-1蔗糖+7 g·L^-1琼脂,诱导不定芽数量为4.9个/块,生芽率达92.3%。(3)生根诱导中,SH基本培养基和蔗糖浓度为关键因素,最佳生根培养基为SH+0~10 g·L^-1蔗糖+7 g·L^-1琼脂,生根率达91.3%。(4)解剖结构观察发现,不定芽起源于愈伤表层的分生细胞,为外起源。该研究通过器官发生途径建立了裸果木的再生体系,确定了不定芽为外起源,为裸果木这一珍稀濒危的林木种质资源保护及可持续利用奠定了研究基础,并为其未来的发展利用提供了有效途径。     

Recombinant Protein Production with Pichia pastoris in Continuous Fermentation – Kinetic Analysis of Growth and Product Formation

Continuous fermentation was applied to the production of recombinant human chymotrypsinogen B (hCTRB) by the methylotrophic yeast Pichia pastoris as a tool for the kinetic analysis of growth and product formation. Using methanol as the sole source of carbon, energy, and induction, cell growth could be described by a non‐competitive Monod approach. Maximum growth rate μ_max was determined to 0.084 h^‐‐1 and the K_M‐value for methanol to 0.22 g·L^‐‐1, respectively. With respect to product formation, a similar model was established exhibiting a methanol concentration of 0.13 g·L^‐‐1 as the K_M‐value and a maximum biomass‐specific product‐formation rate of π_max = 0.23 mg·g^‐‐1·h^‐‐1. The production of hCTRB was strictly growth‐coupled. The data provided covers the range of methanol concentrations between 0 and 4 g·L^‐‐1. Substrate concentrations exceeding this upper value led to a complete collapse of product formation. This change in phenotype turned out to be irreversible indicating a genetic instability of transformed Pichia pastoris caused by excess methanol.     

Factors affecting l-arginine production in the continuous culture of an l-arginine producer of Corynebacterium acetoacidophilum

The effects of culture conditions on l-arginine production by continuous culture were studied using a stable l-arginine hyperproducing strain of Corynebacterium aceto-acidophilum, SC-190. Strain SC-190 demonstrated a volumetric productivity of 35 g l⁻¹·h⁻¹ at a dilution rate of 0.083h⁻¹ and feeding sugar concentration of 8%, and a product yield of 29.2% at a dilution rate 0.021h⁻¹ and feeding sugar concentration of 15%. The corresponding values for fed-batch culture are 0.85 g·l⁻¹·h⁻¹ and 26%. However, the product yield decreased with an increase in the volumetric productivity. To achieve stable l-arginine production, aeration and agitation conditions sufficient to maintain an optimal level of redox potential (>−100 mV) were necessary. The addition of phosphate to the feeding medium led to a decrease in l-arginine production. It was confirmed in the steady state that growth and l-arginine formation were inhibited by a high concentration of l-arginine.     

Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine

A five carbon linear chain diamine, cadaverine (1,5‐diaminopentane), is an important platform chemical having many applications in chemical industry. Bio‐based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the petroleum‐based chemical synthesis. Here, we report development of a metabolically engineered strain of Escherichia coli that overproduces cadaverine in glucose mineral salts medium. First, cadaverine degradation and utilization pathways were inactivated. Next, L ‐lysine decarboxylase, which converts L ‐lysine directly to cadaverine, was amplified by plasmid‐based overexpression of the cadA gene under the strong tac promoter. Furthermore, the L ‐lysine biosynthetic pool was increased by the overexpression of the dapA gene encoding dihydrodipicolinate synthase through the replacement of the native promoter with the strong trc promoter in the genome. The final engineered strain was able to produce 9.61 g L⁻¹ of cadaverine with a productivity of 0.32 g L⁻¹ h⁻¹ by fed‐batch cultivation. The strategy reported here should be useful for the bio‐based production of cadaverine from renewable resources. Biotechnol. Bioeng. 2011; 108:93–103. © 2010 Wiley Periodicals, Inc.     

Ethanol fermentation by an efficient strain,NRRL B-4286, of Zymomonas mobilis

We studied the effect of the initial substrate concentration over the range of 100–250 g·l⁻¹ on the fermentation kinetics in batch cultures of Zymomonas mobilis NRRL B-4286 on glucose, fructose, and sucrose, using an adapted initial inoculum. With increasing concentrations of substrate, parameters related to growth were more rapidly and strongly affected than those related to ethanol production. This strain produced 94.0 g·l⁻¹, 76.9 g·l⁻¹, and 66.5 g·l⁻¹ of ethanol at glucose, fructose, and sucrose concentrations of 200 g·l⁻¹, respectively, more than the amount produced by the efficient strain ZM4 (NRRL B-14023).     

Identification of the Orotidine-5′-Phosphate Decarboxylase Gene and Development of a Transformation System in the Yeast Saccharomyces exiguus Yp74L-3

To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura⁺ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5′-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L⁻¹) and lipid production (1.04 g·L⁻¹), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C_16:1, 9.4%), oleic acid (C_18:1, 47.2%), linoleic acid (C_{18:2 n−6}, 9.6%), and linolenic acid (C_{18:3 n−3}, 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L⁻¹ of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.     

稀土-落叶松单宁配合物的合成及抗真菌活性研究

以稀土(Re~(3+))和落叶松单宁(LT)为原料,采用液相合成法合成了5种廉价的稀土-落叶松单宁(Re~(3+)-LT)配合物,并通过红外光谱、X射线光电子能谱、紫外光谱以及配位数测定确定了配合物的结构.采用牛津杯法、琼脂稀释法测定配合物对黑曲霉、红曲霉、白腐菌、毛霉4种真菌的抑制作用.在抑菌方面,5种配合物对上述4种真菌均具有较强的抑制作用,其抑菌活性大小顺序为Ce~(3+)-LTGd~(3+)-LTLa~(3+)-LTNd~(3+)-LTYb~(3+)-LT,其中Ce~(3+)-LT对4种真菌的最小抑菌浓度分别为:1.6、1.6、0.8和1.6 g·L~(-1);Yb~(3+)-LT对4种真菌的最小抑菌浓度分别为:3.2、1.6、3.2和3.2 g·L~(-1).在杀菌方面,Yb~(3+)-LT的杀菌活性最强,其对4种真菌的最小杀菌浓度分别为:6.4、3.2、3.2和6.4 g·L~(-1).此外,尽管Nd~(3+)-LT和Gd~(3+)-LT具有较强的抑菌活性,但对黑曲霉和毛霉的杀菌作用较弱.     

光皮桦组织培养离体再生研究

采用正交试验等设计,系统开展了光皮桦组织培养高效再生体系研究。结果表明:光皮桦茎段外植体最佳诱导培养基及激素组合为MS+0.50mg.L-1 6-BA+0.10mg.L-1 TDZ+30mg.L-1蔗糖+5.50g.L-1琼脂,丛生芽最佳生根培养基为1/2MS+1mg.L-1 IBA+20g.L-1蔗糖+5.50g.L-1琼脂;丛生芽在最佳生根培养基上培养15d后获健壮生根苗,移栽成活率达90%。该实验结果为光皮桦的优良品种快繁以及遗传转化体系建立奠定了良好的基础。     

Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome-scale metabolic network model for trans-4-hydroxyproline production

trans-4-Hydroxy- l -proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti-inflammatory drugs in the pharmaceutical industry. Proline 4-hydroxylase (P4H) can catalyze the conversion of l -proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp. strain RH1. In silico model-based simulation showed that α-ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl-CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD-encoding the succinyl-CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine-tuning of plasmid-borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp-7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L ⁻¹·hr ⁻¹ in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose-minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products.     

Molecularly imprinted electrochemiluminescence sensor based on ZIF-8 doped with CdSe quantum dots for the detection of trace estriol

A composite of the metal–organic framework compound ZIF-8 doped with CdSe quantum dots (QDs) with sensitive and stable luminescence was synthesized, and a molecularly imprinted electrochemiluminescence (ECL) sensor was constructed based on this composite. The ZIF-8@CdSe molecularly imprinted ECL sensor combines the high sensitivity of ECL and the high selectivity of molecular imprinting to realize the sensitive and specific detection of estriol. CdSe QDs and gold nanoparticles were encapsulated within ZIF-8 to obtain the ZIF-8@CdSe QDs/GNP (ZIF@CdSe/GNP) composite. Subsequently, the GNPs were further loaded on the surface of this composite to obtain the GNP/ZIF@CdSe/GNP composite. l -Cysteine was used to immobilize the GNP/ZIF@CdSe/GNP composite on the surface of a gold electrode to obtain the GNP/ZIF@CdSe/GNP-modified gold electrode. A molecularly imprinted polymer (MIP) film was prepared on the surface of the modified electrode by electropolymerization with o-phenylenediamine as the functional monomer and estriol as the template molecule. After elution, estriol could be specifically recognized by the cavities. The readsorption of estriol by the MIP can prevent the coreactant from reaching the electrode surface through the cavities, thereby weakening ECL. A good linear relationship existed between the ∆ECL and lg C of estriol concentrations of 1 × 10⁻¹⁴ to 1 × 10⁻⁹ mol·L⁻¹. The detection limit was as low as 8.9 × 10⁻¹⁶ mol·L⁻¹. The sensor was applied in the determination of estriol in serum samples with a recovery of 97.0–102%.     

Enhanced production of peroxidase in continuous culture of Arthromyces ramosus by selective bleeding of mycelium

A new method of continuous culture with selective bleeding of mycelia using 9-mesh screen was developed to improve the production rate of peroxidase (POD) by Arthromyces ramosus. At the dilution rate of 0.05 h⁻¹ with the mycelium leakage rate of 60%, a high production rate (average value was 1.67 U·ml⁻¹·h⁻¹) was maintained for over 100 h: the rate was 3.2 times that in a glucose-fed batch culture. At the same dilution rate, the volumetric and specific production rates of POD in the continuous culture without the screen were lower than those in the first continuous culture and decreased gradually in the later phase of the culture. In the continuous culture with low mycelium leakage rate of 1.6%, the POD production rate was not improved further, although the mycelial concentration (43 g·l⁻¹) increased 2.9 times. It is suggested that the high agitation rate required to meet the oxygen demand is unfavorable for the POD production.     

马来沉香组织培养技术研究

以马来沉香茎段为外植体,分别对外植体的消毒、启动培养、增殖培养、壮苗培养、生根培养、炼苗移栽环节进行研究,着重探索马来沉香组织培养技术各个环节的最佳培养基配方,为马来沉香的工厂化育苗提供技术指导。结果表明:马来沉香最佳消毒方法是用0.1%升汞消毒4～5min;启动率最高的培养基配方是1/2MS+6-BA 0.2mg·L-1+NAA 0.1mg·L-1+蔗糖30g·L-1+琼脂5.8g·L-1,启动率达70.5%;增殖系数最高的培养基是1/2MS+0.1mg·L-16-BA+25g·L-1蔗糖+5.8g·L-1琼脂,增殖系数达2.9;最佳壮苗培养基是1/2MS+30g·L-1蔗糖+5.8g·L-1琼脂;最佳生根培养基为1/2MS+NAA 5.0mg·L-1+20g·L-1糖+6g·L-1琼脂,培养2d后移入1/2MS培养基继续培养,生根率为83%;马来沉香移栽较难成活,在泥炭土∶黄泥土(2∶1)的基质上成活率最高,移栽成活率65%。     

白花泡桐优树组织培养及产业化快繁技术

以自选育的白花泡桐优树茎段为外植体,进行种苗组培快繁技术研究。结果表明:其最佳的外植体灭菌方法是以0.1%升汞处理7 min;合适的初代诱导培养基为MS+6-BA 2.0 mg·L~(-1)+IBA 0.2 mg·L~(-1)+糖30 g·L~(-1)+琼脂3.5 g·L~(-1)(pH 5.8),培养30 d,芽诱导率70%;合适的继代增殖方法为在高浓度植物生长物质培养基MS+6-BA 4.0 mg·L~(-1)+IBA 0.4 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1)(pH 5.8)和低浓度植物生长物质培养基MS+6-BA 0.4 mg·L~(-1)+IBA 0.04 mg·L~(-1)+蔗糖30 g·L~(-1)+琼脂3.5 g·L~(-1)(pH 5.8)中交替培养,获得的丛生芽长势良好,玻璃化率低于5%,增殖系数大于6.0/25 d;最适的生根培养基为1/2MS+NAA0.2 mg·L~(-1)+蔗糖20 g·L~(-1)+卡拉胶3.4 g·L~(-1)(pH 5.8),培养14 d,得到白花泡桐生根苗,每株长根5~10条,根长3~5 cm,生根率98%,根系洁白、根毛少而短,易于清洗。将生根苗按照常规方法炼苗后移栽于温室大棚中,50 d后即可出圃,此时平均苗高1.0 m、地径1.0~2.0 cm,成活率在90%以上。