Optimization of Two-species Whole-cell Immobilization System Constructed with Marine-derived Fungi and Its Biological Degradation Ability

Mycelia pellet formed spontaneously in the process of cultivation was exploited as a biological carrier for whole-cell immobilization due to its unique structural characteristic. An innovative two-species whole-cell im- mobilization system was achieved by inoculating the marine-derived fungus Pestalotiopsis sp. J63 spores into cul- ture medium containing another fungus Penicillium janthinellum P1 pre-grown mycelia pellets for 2 days without any pretreatment. In order to evaluate the biological degradation capacity of this novel constructed immobilization system, the immobilized pellets were applied to treat paper mill effluent and decolorize dye Azure B. The use of the constructed immobilization system in the effluent resulted in successful and rapid biodegradation of numerous in- soluble fine fibers. The optimum conditions of immobilized procedure for maximum biodegradation capacity were determined using orthogonal design with biomass of P1 pellets 10 g （wet mass）, concentration of J63 spore 2x109 mlq, and immobilization time 2 d. The results demonstrate that immobilized pellets have more than 99% biodegradation capacity in a ten-hour treatment process. The kinetics of biodegradation fits the Michaelis-Menten equation well. Besides, the decolorization capability of immobilized pellets is more superior than that of P1 mycelia pellets. Overall, the present study offers a simple and reproducible way to construct a two-species whole-cell immobiliza- tion system for sewage treatment.

Whole-cell recordings of calcium and potassium currents in acutely isolated smooth muscle cells

AIM： To record calcium and potassium currents in acutely isolated smooth muscle cells of mesenteric arterial branches in rats. METHODS： Smooth muscle cells were freshly isolated by collagenase digest and mechanical trituration with polished pipettes. Patch clamp technique in whole-cell mode was employed to record calcium and potassium currents. RESULTS： The procedure dissociated smooth muscle cells without impairing the electrophysiological characteristics of the cells. The voltage-gated Ca^2＋ and potassium currents were successfully recorded using whole-cell patch clamp configuration. CONCLUSION： The method dissociates smooth muscle cells from rat mesenteric arterial branches. Voltage-gated channel currents can be recorded in this preparation.

Whole-cell recording of the robust nucleus of the arcopallium neurons in the adult zebra finch

BACKGROUND： Electrophysiological properties of the song nucleus have been revealed using conventional techniques, such as intracellular and extracellular recording. Research concerning the neuronal activation properties and regulations of the song system at the cellular and ion channel level may help reveal the neural mechanism of song learning. OBJECTIVE： To perform whole-cell recording of robust nucleus of the arcopallium （RA） neurons in brain slices from adult zebra finches （Taeniopygia guttata） and observe the action potential, sodium/potassium current and the spontaneous postsynaptic current of RA neurons. DESIGN, TIME AND SETTING： Self-controlled, neuroelectrophysiological experiment. The study was performed at the Neurophysiology Laboratory of South China Normal University from April to September 2008. MATERIALS： Flaming/Brown puller P-97 was purchased from Sutter Ins, USA; Axopatch 700B amplifier and Digidata 1332A converter were purchased from Axon Instrument, USA; pClamp software was provided by Axon Instrument, USA. METHODS： RA neurons were acutely isolated from 24 healthy male zebra finches. The action potential, voltage-gate sodium/potassium current and spontaneous postsynaptic current were recorded by whole-cell recording technology. Data were analyzed by pClamp software. MAIN OUTCOME MEASURES： The amplitude and frequency of the action potential, and the amplitude of the voltage-dependent and spontaneous postsynaptic currents, were measured. RESULTS： （1） Testing of action potential： Cells exhibited a stable current-voltage relationship following a series of hyperpolarization stepped currents, and an action potential was triggered by the spike threshold. All the recorded cells displayed repetitive firing following depolarizing current injection, with a frequency beyond 100 Hz. （2） Testing of voltage-gate currents： The inward and outward whole-cell currents were observed after a series of depolarizing voltage steps. The inward current disappeared following the application of tetrodotoxin and the outward current was significantly inhibited by application of 4-aminopyfidione and tetraethylammonium chloride. （3） Testing of spontaneous postsynaptic current： The majority of recorded cells exhibited an inward synaptic current when the membrane potential was maintained at -60 mV, with some cells exhibiting a robustly outward current when the membrane potential was maintained at -30 mV. Tetrodotoxin was unable to affect the spontaneous postsynaptic current. Following application of bicuculline [y-aminobutyric acid （A） receptor antagonist] and high concentration kynurenic acid （ionotropic glutamate receptor antagonist）, the inward and outward currents were completely inhibited. CONCLUSION： Under these experimental conditions, the action potential, sodium/potassium current and spontaneous postsynaptic current were recorded successfully in RA neurons. This indicates that the cells preserved relatively intact synaptic connections and normal physiological activity, which is required for investigating ion channels. The inward and outward whole-cell currents were sodium and potassium currents, respectively. The postsynaptic y-aminobutyric acid （A） receptors and ionotropic glutamate receptors contributed to the spontaneous postsynaptic current.

Whole-cell recordings of voltage-gated Calcium,Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons

Objective：To record Calcium, Potassium and Sodium currents in acutely isolated hippocampal pyramidal neurons. Methods：Hippocampal CA3 neurons were freshly isolated by 1 mg protease/3 ml SES and mechanical trituration with polished pipettes of progressively smaller tip diameters. Patch clamp technique in whole-cell mode was employed to record voltage-gated channel currents. Results：The procedure dissociated hippocampal neurons, preserving apical dendrites and several basal dendrites, without impairing the electrical characteristics of the neurons. Whole-cell patch clamp configuration was successfully used to record voltage-gated Ca^2＋ currents, delayed rectifier K^＋ current and voltage-gated Na^＋ currents. Conclusion：Protease combined with mechanical trituration may be used for the dissociation of neurons from rat hippocampus. Voltage-gated channels currents could be recorded using a patch clamp technique.

Efficient biosynthesis of creatine by whole-cell catalysis from guanidinoacetic acid in Corynebacterium glutamicum

Creatine is a naturally occurring derivative of an amino acid commonly utilized in functional foods and pharmaceuticals.Nevertheless,the current industrial synthesis of creatine relies on chemical processes,which may hinder its utilization in certain applications.Therefore,a biological approach was devised that employs whole-cell biocatalysis in the bacterium Corynebacterium glutamicum,which is considered safe for use in food production,to produce safe-for-consumption creatine.The objective of this study was to identify a guanidinoacetate N-methyltransferase(GAMT)with superior catalytic activity for creatine production.Through employing whole-cell biocatalysis,a gamt gene from Mus caroli(Mcgamt)was cloned and expressed in C.glutamicum ATCC 13032,resulting in a creatine titer of 3.37 g/L.Additionally,the study employed a promoter screening strategy that utilized nine native strong promoters in C.glutamicum to enhance the expression level of GAMT.The highest titer was achieved using the P1676 promoter,reaching 4.14 g/L.The conditions of whole-cell biocatalysis were further optimized,resulting in a creatine titer of 5.42 g/L.This is the first report of successful secretory creatine expression in C.glutamicum,which provides a safer and eco-friendly approach for the industrial production of creatine.

Efficient acetoin production from pyruvate by engineered Halomonas bluephagenesis whole-cell biocatalysis

Acetoin is an important platform chemical,which has a wide range of applications in many industries.Halomonas bluephagenesis,a chassis for next generation of industrial biotechnology,has advantages of fast growth and high tolerance to organic acid salts and alkaline environment.Here,α-acetolactate synthase andα-acetolactate decarboxylase from Bacillus subtilis 168 were co-expressed in H.bluephagenesis to produce acetoin from pyruvate.After reaction condition optimization and further increase ofα-acetolactate decarboxylase expression,acetoin production and yield were significantly enhanced to 223.4 mmol·L^(-1) and 0.491 mol·mol^(-1) from 125.4 mmol·L^(-1) and 0.333 mol·mol^(-1),respectively.Finally,the highest titer of 974.3 mmol·L^(-1)(85.84 g·L^(-1))of acetoin was accumulated from 2143.4 mmol·L^(-1)(188.6 g·L^(-1))of pyruvic acid within 8 h in fed-batch bioconversion under optimal reaction conditions.Moreover,the reusability of the cell catalysis was also tested,and the result illustrated that the whole-cell catalysis obtained 433.3,440.2,379.0,442.8 and 339.4 mmol·L^(-1)(38.2,38.8,33.4,39.0 and 29.9 g·L^(-1))acetoin in five repeated cycles under the same conditions.This work therefore provided an efficient H.bluephagenesis whole-cell catalysis with a broad development prospect in biosynthesis of acetoin.

Improvement of a highly sensitive and specific whole-cell biosensor by adding a positive feedback amplifier

In this study,we designed a Cd^(2+)whole-cell biosensor with both positive and negative feedback cascade am-plifiers in Pseudomonas putida KT2440(LTCM)based on our previous design with only a negative feedback amplifier(TCM).The results showed that the newly developed biosensor LTCM was greatly improved compared to TCM.Firstly,the linear response range of LTCM was expanded while the maximum linear response range was raised from 0.05 to 0.1μM.Meanwhile,adding a positive feedback amplifier further increased the fluorescence output signal of LTCM 1.11–2.64 times under the same culture conditions.Moreover,the response time of LTCM for detection of practical samples was reduced from 6 to 4 h.At the same time,LTCM still retained very high sensitivity and specificity,while its lowest detection limit was 0.1 nM Cd^(2+)and the specificity was 23.29(compared to 0.1 nM and 17.55 in TCM,respectively).In summary,the positive and negative feedback cascade amplifiers effectively improved the performance of the biosensor LTCM,resulting in a greater linear response range,higher output signal intensity,and shorter response time than TCM while retaining comparable sensitivity and specificity,indicating better potential for practical applications.

Efficient stereoselective hydroxylation of deoxycholic acid by the robust whole-cell cytochrome P450 CYP107D1 biocatalyst

Deoxycholic acid(DCA)has been authorized by the Federal Drug Agency for cosmetic reduction of redundant submental fat.The hydroxylated product(6β-OH DCA)was developed to improve the solubility and pharmaceutic properties of DCA for further applications.Herein,a combinatorial catalytic strategy was applied to construct a powerful Cytochrome P450 biocatalyst(CYP107D1,OleP)to convert DCA to 6β-OH DCA.Firstly,the weak expression of OleP was significantly improved using pRSFDuet-1 plasmid in the E.coli C41(DE3)strain.Next,the supply of heme was enhanced by the moderate overexpression of crucial genes in the heme biosynthetic pathway.In addition,a new biosensor was developed to select the appropriate redox partner.Furthermore,a cost-effective whole-cell catalytic system was constructed,resulting in the highest reported conversion rate of 6β-OH DCA(from 4.8%to 99.1%).The combinatorial catalytic strategies applied in this study provide an efficient method to synthesize high-value-added hydroxylated compounds by P450s.

NIR Light-Promoted Whole-Cell Catalysis Based on a Light-Harvesting Blackbody Bioreactor

Whole-cell catalysis,which utilizes enzymes expressed in whole organism(e.g.bacteria and fungi)as the catalyst,is a specific mode of biocatalysis.Compared with pure enzyme catalysis,the catalysis with whole-cell catalysts is more cost-effective.However,in the process of whole-cell catalysis,heat treatment is often necessary due to the high optimum temperature of the enzyme.To enable efficient industrial application of whole-cell catalysis,an environmental friendly heating approach is highly desired.Inspired by the light harvest by blackbody materials,in this paper,we introduced a photothermal approach for harnessing the photon energy for enhanced whole-cell catalysis.A blackbody porous sponge(BPS)with excellent photothermal conversion efficiency was prepared as a bioreactor.Escherichia coli expressed with a thermophilic enzyme(β-glucosidase)was utilized as a model whole-cell catalyst.Moreover,the photothermal properties of the BPS and lightassisted whole-cell catalysis were systematically investigated,demonstrating promising application prospects.

Construction of WCB-11:A novel phiYFP arsenic-resistant whole-cell biosensor

The prediction and assessment of environmental pollution by arsenic are important preconditions of advocating environmental protection and human health risk assessment. A yellow fluorescent protein-based whole-cell biosensor for the detection of arsenite and arsenate was constructed and tested. An arsenic-resistant promoter and the regulatory gene arsR were obtained by PCR from the genome ofEscherichia coli DH5ct, andphiYFP was introduced into E. coli DH5ct as a reporter gene to construct an arsenic-resistant whole-cell biosensor （WCB-11） in which phiYFP was expressed well for the first time. Experimental results demonstrated that the biosensor has a good response to arsenic and the expression ofphiYFP. When strain WCB-11 was exposed to As^3＋ and As^5＋, the expression of yellow fluorescence was time-dependent and dose-dependent. This engineered construct is expected to become established as an inexpensive and convenient method for the detection of arsenic in the field.

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst.Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast.Here,the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae,and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor,recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed.Compared with the wild-type ROL-displaying yeast,the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate.To our knowledge,this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction.Consequently,the yeast whole-cell ROL biocatalyst was constructed with high activity.The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C.Furthermore,this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Development of a whole-cell biosensor based on an ArsR-Pars regulatory circuit from Geobacter sulfurreducens

In this study,an Escherichia coli(E.coli)whole-cell biosensor for the specific detection of bioavailable arsenic was developed by placing a green fluorescent protein(GFP)reporter gene under the control of the ArsR1(GSU2952)regulatory circuit from Geobacter sulfurreducens.E.coli cells only emitted green fluorescence in the presence of arsenite and were more sensitive to arsenite when they were grown in M9 supplemented medium compared to LB medium.Under optimal test conditions,the Geobacter arsR1 promoter had a detection limit of 0.01 mM arsenite and the GFP expression was linear within a range of 0.03-0.1 mM(2.25-7.5 mg/l).These values were well below World Health Organization’s drinking water quality standard,which is 10 mg/l.The feasibility of using this whole-cell biosensor to detect arsenic in water samples,such as arsenic polluted tap water and landfill leachate was verified.The biosensor was determined to be just as sensitive as atomic fluorescence spectrometry.This study examines the potential applications of biosensors constructed with Geobacter ArsR-Pars regulatory circuits and provides a rapid and cost-effective tool that can be used for arsenic detection in water samples.

Construction of a dual fluorescence whole-cell biosensor to detect N-acyl homoserine lactones

Detection of N-acyl homoserine lactones （AHLs） is useful for understanding quorum sensing （QS） behaviors, including biofilm formation, virulence and metabolism. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T1-4 to make a dual fluorescent whole- cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components： constitutively expressed enptll：：gfP for indicating host cells, Pahll：：mcherry that produces red fluorescence in response to AHL, and the ahIR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T1-4 （four tandem copies of a transcriptional terminator） were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5 × 10^-8-1 × 10^-5 mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wild- type Pseudomonas PhTA125 （an AHL-producing bacterium）, it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a mi tal niche

WHOLE-CELL CLAMP STUDY OF XENOPUS EMBRYONIC CHOLINERGIC NEURONS

Whole-cell clamped myoballs are placed into direct visible contact with the growth cones of isolated neurons in embryonic Xenopus culture to serve as probe of acetylchollne (AcCHo) release in order to determine whether these neurons are cholinergic or not. Using a GQ-seal, whole-cell recording method, the electrophysiological properties of these identified cholinergic neurons are studied. It is found that these embryonic neurons, like adult frog motor neurons, exhibit repetitive firings in a certain embryonic developing stage. A development of repetitive firings is observed simultaneously. Tracing the development of one neuron, we find that the development of repetitive firing is completed at the 48th h after fertilization. Tetrodotoxin (TTX) which blocks Na^+ channels can abolish all firings; and tetraethyl ammonium chloride (TEA), the blocker of K^+ channels, reverses this development, i. e. it makes the repetitive firings disappear again. These data show that the nature of the development of repetitive firings is the development of K^+ channels.

Cell surface protein engineering for high-performance whole-cell catalysts

Cell surface protein engineering facilitated by accumulation of information on genome and protein structure involves heterologous production and modifica- tion of cell surface proteins using genetic engineering, and is important for the development of high-performance whole-cell catalysts. In this field, cell surface display is a major technology by exposing target proteins, such as enzymes, on the cell surface using a cartier protein. The target proteins are fused to the carrier proteins that transport and tether them to the cell surface, as well as to a secretion signal. This paper reviews cell surface display systems for prokaryotic and eukaryotic cells from the perspective of carrier proteins, which determine the number of displayed molecules, and the localization, size, and direction （N- or C-terminal anchoring） of the passengers. We also discuss advanced methods for displaying multiple enzymes and a new method for the immobilization of whole-cell catalysts using adhesive surface proteins.

Review of construction methods for whole-cell computational models

The complex mechanisms of the internal operation of cellular functions have not been fully resolved and these functions are regulated by multiple effects,such as transcription regulation,signal transduction,and enzyme catalysis,forming complex interactive mechanisms.This makes the construction of a whole-cell computational model,containing various complex cellular functions,very challenging.However,biological models have played a significant role in the field of systems biology,such as guiding gene-target mining and studying cell metabolic characteristics.Therefore,there is increasing research interest in the construction of whole-cell computational models.Combining two classical languages of systems biology,this review expounds on the development and challenges of whole-cell computational modeling from the two classical methods of steady-state and dynamic modeling.Finally,we propose a new approach for constructing whole-cell computational models.

贝托司汀手性中间体(S)-CPMA整细胞催化合成工艺研究

针对不同条件对贝托司汀手性中间体(S)-CPMA的整细胞催化合成影响,进行单因素实验和正交设计优化研究.首先从耐热克鲁维酵母突变株SXBP-02预处理方式、不同催化反应介质、pH、底物质量浓度、温度、时间、催化剂用量、补料次数等8个方面进行考察,揭示其中pH、底物质量浓度、补料次数和催化剂用量4个因素对(S)-CPMA催化效率的影响较大;进一步对其进行正交优化,得出最佳工艺条件:在pH8的PEG4000双水相体系中,底物质量浓度为6g/L、补料次数为4次、催化剂用量为3g/L,温度为40℃条件下反应36h,可得到(S)-CPMA产率为88.1%,对映选择性在98.5%以上,4个因素对(S)-CPMA收率的影响主次顺序依次为补料次数>pH>催化剂用量>底物质量浓度,其中补料次数、pH为显著性影响因素,与优化前相比,底物质量浓度提高3倍,产率提高了6%.

奥西替尼联合全脑放疗对非小细胞肺癌的疗效分析

目的探讨奥西替尼联合全脑放疗对非小细胞肺癌的治疗效果。方法选取2019年6月至2020年6月确诊为非小细胞肺癌并伴随脑转移的86例患者,随机数字表法分为试验组和对照组,每组43例。对照组采取常规化疗方式联合全脑放疗进行治疗,试验组在常规化疗联合全脑放疗的基础上联合奥西替尼治疗,比较2组患者的近期疗效、血清肿瘤标志物含量、不良反应情况以及生存情况。结果试验组治疗有效率为88.4%高于对照组的62.8%(P<0.05)。试验组患者血清的CD4^(+)、CD8^(+)和CD4^(+)/CD8^(+)显著高于对照组(P<0.05);试验组的血清CYFRA21-1水平显著低于对照组(P<0.05)。试验组的患者的无病生存时间中位数为8个月,对照组为5个月,Log-rank检验显示,试验组的无病生存时间显著高于对照组(P<0.05)。结论奥西替尼联合全脑放疗作为治疗非小细胞肺癌脑转移患者的治疗方式,治疗效果较好,能延长患者的无病生存时间并且能提高患者的生存质量,值得临床推广应用。

小白链霉菌全细胞转化L-赖氨酸合成ε-聚赖氨酸的体系构建与优化

小白链霉菌(Streptomyces albulus)是天然抗菌肽ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)的主要生产菌株。为了提高小白链霉菌生产ε-PL效率,该文构建并优化了全细胞转化L-赖氨酸合成ε-PL体系:葡萄糖质量浓度80 g/L,菌龄12 h,反应温度30℃,L-赖氨酸质量浓度15 g/L,柠檬酸浓度15 g/L,初始反应p H 4.0,硫酸铵质量浓度6 g/L,湿菌体量为1900 g/L。基于该转化体系,实现小白链霉菌在96 h合成ε-PL产量和底物转化率达到13.80 g/L和38.9%,分别是常规摇瓶发酵的4.1、3.2倍。最后,在小白链霉菌中异源表达来自大肠杆菌的L-赖氨酸特异性通透蛋白基因lysp,获得的重组菌S.albulus OE-lysp实现L-赖氨酸利用能力和底物转化率较出发菌株分别提升26%和33%,ε-PL产量增加至17.21 g/L,约为常规摇瓶发酵ε-PL产量的6.4倍,这是文献报道的最高摇瓶规模ε-PL产量。该研究结果一方面说明了通过全细胞转化L-赖氨酸生产ε-PL的可行性,另一方面为S.albulus转化大宗氨基酸L-赖氨酸生产高值ε-PL奠定了坚实的技术基础,具有重要的理论意义和经济价值。

米根霉全细胞脂肪酶在化学-酶法环氧化反应体系中的稳定性

为了提高米根霉(Rhizopus oryzae CGMCC 3.5040)全细胞脂肪酶在化学-酶法环氧化反应体系中的稳定性,以α-蒎烯为模式底物,考察柠檬酸三钠用量、戊二醛交联细胞、过氧化氢(H_(2)O_(2))用量和回用方式对催化剂稳定性的影响。结果表明:过氧有机酸会影响酶稳定性,添加3.5 mmol柠檬酸三钠会与质量分数为30%H_(2)O_(2)水溶液形成高渗液,防止细胞涨破,同时能中和过量过氧酸,提高反应选择性与细胞的回用稳定性;经过戊二醛交联后,全细胞催化剂的热稳定性、储存稳定性和回用稳定性都显著提高;蒎烯环氧化的最适H_(2)O_(2)用量为5 mmol;可采用直接分离有机相,再加入新鲜有机相的方式进行回用。优化回用方式后,该全细胞催化剂第7次使用时,催化反应仍然有77.3%的转化率。