Effect of aerobically generated inoculum on xylose fermentation by an ethanologenic recombinantE. coli

Summary The anaerobic conversion of xylose to ethanol by a genetically engineredE. coli B (pLOI297) was investigated using anaerobically and aerobically grown cultures as inocula. Using anaerobically grown cells, an increase in the inoculation density from 50 to 340 mg dry wt. cells/L resulted in an increase in the overall volumetric productivity from 0.57 to 0.71 g/L/h. At the higher inoculation density, substitution of the anaerobic inoculum by aerobically grown cells resulted in a 15% reduction in volumetric productivity (0.61 g/L/h) that was caused by the introduction of a lag period during which the aerobic inoculum adapted to the anaerobic environment. In all cases, the ethanol yield from xylose approached the theoretical maximum and seemed unaffected by the physiological history of the inoculum with respect to aeration. It is concluded that aeration should be avoided in the production of high performance starter cultures.     

Optimization of high cell-density fermentation of Lactobacillus casei with function of removing microcystin

为提高一株具有藻毒素清除能力的干酪乳杆菌Lactobacillus casei BBE10-212单位体积的活菌数,针对其营养需求,研究了不同碳源、氮源、缓冲盐、微量元素及生长因子对该菌株生长情况及发酵特性的影响.通过响应面法对碳源、氮源、生长因子等进行优化,获得最佳培养基配方为:α-乳糖43.8 g/L,酵母膏79.5 g/L,无水乙酸钠13.12 g/L,冰醋酸9.17 mL/L,MnSO4 ·H2O 190mg/L,吐温-80 5.15 mL/L.经37℃培养18 h,菌体干重达到4.97 g/L,比在普通MRS培养基中(1.32 g/L)提高近4倍.基于乳酸菌发酵过程中的产酸特性,通过外源添加5 g/L谷氨酸,促使菌体浓度进一步提高15％,并提前1.2h进入生长稳定期.上述研究结果为食品行业重要生产菌株干酪乳杆菌的高密度培养技术提供了可借鉴的研究思路.     

一株具有藻毒素清除功能的干酪乳杆菌细胞高密度发酵条件优化

为提高一株具有藻毒素清除能力的干酪乳杆菌Lactobacillus casei BBEi0—212单位体积的活菌数,针对其营养需求,研究了不同碳源、氮源、缓冲盐、微量元素及生长因子对该菌株生长情况及发酵特性的影响。通过响应面法对碳源、氮源、生长因子等进行优化,获得最佳培养基配方为：α-乳糖43．8g/L,酵母膏79．5g/L,无水乙酸钠13．12g／L,冰醋酸9．17mL／L,MnSO4·H20190mg／L,吐温-805．15mL/L。经37℃培养18h,菌体干重达到4．97g/L,比在普通MRS培养基中（1．32g／L）提高近4倍。基于乳酸菌发酵过程中的产酸特性,通过外源添加5g／L谷氨酸,促使菌体浓度进一步提高15％,并提前1．2h进入生长稳定期。上述研究结果为食品行业重要生产菌株干酪乳杆菌的高密度培养技术提供了可借鉴的研究思路。     

Periplasmic production of native human proinsulin as a fusion to E. coli ecotin

Native proinsulin belongs to the class of the difficult-to-express proteins in Escherichia coli. Problems mainly arise due to its small size, a high proteolytic decay, and the necessity to form a native disulfide pattern. In the present study, human proinsulin was produced in the periplasm of E. coli as a fusion to ecotin, which is a small periplasmic protein of 16 kDa encoded by the host, containing one disulfide bond. The fusion protein was secreted to the periplasm and native proinsulin was determined by ELISA. Cultivation parameters were studied in parallel batch mode fermentations using E. coli BL21(DE3)Gold as a host. After improvement of fed-batch high density fermentation conditions, 153 mg fusion protein corresponding to 51.5mg native proinsulin was obtained per L. Proteins were extracted from the periplasm by osmotic shock treatment. The fusion protein was purified in one step by ecotin affinity chromatography on immobilized trypsinogen. After thrombin cleavage of the fusion protein, the products were separated by Ni-NTA chromatography. Proinsulin was quantified by ELISA and characterized by mass spectrometry. To evaluate the influence of periplasmic proteases, the amount of ecotin-proinsulin was determined in E. coli BL21(DE3)Gold and in a periplasmic protease deficient strain, E. coli SF120.     

High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

Continuous beer fermentation by high cell-density culture of bottom brewer's yeast

Continuous beer production was investigated in a high cell-density culture system which consisted of two stages for the fermentation and sedimentation of yeast cells. The continuous culture was carried out for a fermentation time of 5,500 h without contamination, at varying dilution rates and fermentation temperatures in the ranges of 0.017-0.033 h⁻¹ and 6.5–8.5°C, respectively. This process was found to be suitable for continuous and stable beer brewing. Under these conditions, the cell concentration in the first stage was about 80 times as high as that in the exit of the second stage. Concentrations of viable cells, sugar and ethanol were maintained at 1.3 × 10⁹ cells/ml, 25 and 36 g/l, respectively, and were hardly affected by fermentation temperature. Concentrations of ethyl acetate, isoamyl alcohol and isoamyl acetate were similar in the fermentation temperature ranges of 6.5–8.5°C, and the amounts at a fermentation temperature of 7°C were comparable to those of lager-type beer. Diacetyl flavor, which is known to be an effluent component that causes deterioration in the second stag e (young beer), was maintained at 1.2 ppm at a dilution rate and fermentation temperature of 0.022 h⁻¹ and 7°C, respectively. The diacetyl flavor was due to the accumulation of vicinal diketone, the precursor of which is acetohydroxy acid. The acetohydroxy acid was converted to vicinal diketone by pretreatment at 60°C for 30 min. The vicinal diketone was then consumed by the yeast during after-fermentation at a fermentation temperature of 3°C. Using this method, total vicinal diketone decreased below 0.3 ppm for an after-fermentation time of 6.8 h, which was 225 times as fast as that of after-fermentation without the pretreatment. This process may make it possible to achieve continuous beer fermentation from the fermentation stage to after-fermentation for diacetyl removal.     

产琥珀酸工程菌株的发酵工艺条件优化

对实验室构建的产琥珀酸大肠杆菌工程菌株(E.coliQZ1111)进行发酵工艺条件研究。以AM1低盐培养基为基础,研究不同C、N源及其质量浓度,培养基初始pH和发酵温度等因素对琥珀酸的影响,并在5L发酵罐中进行了补料-分批发酵实验。优化后的发酵条件为葡萄糖20g/L,玉米浆10g/L,pH6.4,发酵温度37℃。在5L发酵罐中培养,琥珀酸产量达到47.9g/L。     

重组大肠杆菌产尿素酶B高密度发酵条件的优化

探究重组大肠杆菌产尿素酶B（urease B subunit, UreB）的高密度发酵条件。通过实验室摇瓶和30 L发酵罐对UreB基因工程菌的发酵条件进行优化。结果表明：30 L发酵罐中以TB培养基为发酵培养基,接种量为5%,发酵温度为37 ℃,pH为6.8,溶氧量为30%左右,培养至2 h开始恒速流加50%甘油,4 h流加50%酵母提取物和50%胰蛋白胨,并加入终浓度为0.5 mmol/L的异丙基β-D-硫代半乳糖苷（isopropyl β-D-thiogalactoside,IPTG),诱导表达4 h,结束发酵,所得菌体干物质约为25.7 g/L,UreB表达量为31.4%。此工艺可以提高UreB的产量。     

Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA

The production of human proinsulin in its disulfide-intact, native form in Escherichia coli requires disulfide bond formation and the periplasmic space is the favourable compartment for oxidative folding. However, the secretory expression of proinsulin is limited by its high susceptibility to proteolysis and by disulfide bond formation, which is rate-limiting for proinsulin folding. In this report we describe a method for the production of high amounts of soluble, native human proinsulin in E. coli. We fused proinsulin to the C-terminus of the periplasmic disulfide oxidoreductase DsbA via a trypsin cleavage site. As DsbA is the main catalyst of disulfide bond formation in E. coli, we expected increased yields of proinsulin by intra- or intermolecular catalysis of disulfide bond formation. In the context of the fusion protein, proinsulin was found to be stabilised, probably due to an increased solubility and faster disulfide bond formation. To increase the yield of DsbA-proinsulin in the periplasm, several parameters were optimised, including host strains and cultivation conditions, and in particular growth medium composition and supplement of low molecular weight additives. We obtained a further, about three-fold increase in the amount of native DsbA-proinsulin by addition of L-arginine or ethanol to the culture medium. The maximum yield of native human proinsulin obtained from the soluble periplasmic fraction after specific cleavage of the fusion protein with trypsin was 9.2 mg g(-1), corresponding to 1.8% of the total cell protein.     

鲨肝刺激物质类似物在大肠杆菌中的高密度发酵

为建立重组鲨肝刺激物类似物(r-sHSA)的高密度发酵方法,本研究在利用单因素实验和均匀设计实验优化摇瓶发酵培养基的组成和浓度以及诱导剂(IPTG)浓度的基础上,利用5L发酵罐进行了放大试验,探讨了补料方式、补料培养基的组成和浓度、诱导剂加入时间和诱导后菌体的收获时间对工程菌生物量和r-sHSA产量的影响。结果表明:在改良LB培养基(0.97%甘油,0.91%酵母粉,0.72%胰蛋白胨,0.782%KH2PO4,0.267%K2HPO4·3H2O,0.062%MgSO4·7H2O,0.5%NaCl,pH7.0)中,当pH控制在7.0、溶氧浓度为25%～30%的前提下,采用指数补料方式加入优化后的补料培养基(620g/L甘油,94.8g/L胰蛋白胨,3.3mL/L微量元素,7.5g/LMgSO4·7H2O)进行培养,在工程菌的OD600达到23时,加入终浓度为0.5mmol/L的IPTG诱导6h后收获菌体,菌体的生物量可达(123.27±1.184)g/L,r-sHSA产量为(2.662±0.041)g/L,比优化前提高了13.7倍。     

Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K_m,app = 0.16 ± 0.02 mg/mL and k_cat,app = 79 ± 10 s^?1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k_cat,app/K_m,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with K_d of 27.2, 0.70, and 34.7 μM, respectively.     

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.     

碳源对重组大肠杆菌发酵生产GLP-1融合蛋白的影响

以一株表达人胰高血糖素样肽-1融合蛋白的重组大肠杆菌为研究对象,首先通过摇瓶实验对碳源种类进行了初步选择,发现葡萄糖和甘油对菌体生长以及GLP-1融合蛋白表达较为适宜。进一步在5 L反应器上对初始葡萄糖及甘油浓度进行了考察,发现高浓度碳源有利于菌体生长却抑制GLP-1融合蛋白表达,但能提高GLP-1融合蛋白的体积得率。在0.25%初始葡萄糖或甘油存在的条件下,在培养过程中流加葡萄糖或甘油维持其在发酵液中的浓度,比较了两者对菌体生长以及GLP-1融合蛋白表达的影响,结果发现,以甘油为碳源时,菌体生长以及GLP-1融合蛋白的表达量均高于以葡萄糖为碳源的结果,最终发酵液的菌浓(OD_(600))可达到25.4,较葡萄糖为碳源时19.1提高了33.0%,GLP-1融合蛋白表达水平和体积得率分别可达到22.4%和1.051 g/L,较葡萄糖为碳源的15.8%和0.504 g/L分别提高41.8%和108.5%。该结果对GLP-1融合蛋白表达菌株发酵条件的进一步优化提供了依据。     

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l(-1) (OD600 approximately 60) and the production of 700 mg l(-1) of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l(-1) of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l(-1) with E. coli as the production host.     

Effect of growing conditions of recombinantE.coli in carrageenan gel beads upon biomasse production and plasmid stability

Summary The biomass production and the plasmid stability of immobilizedE. coli cells in K-carrageenan gel beads were investigated in continuous cultures. Several factors, such as inoculum size, gel bead volume and gel concentration were examined in order to increase the cell concentration inside the immobilized cell reactor, and therefore to increase the overall productivity.     

Enhanced beta-galactosidase production by high cell-density culture of recombinant Bacillus subtilis with glucose concentration control

Cell concentration, recombinant protein (beta-galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed-batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed-batch culture. Glucose concentration of the culture broth was monitored by an automatic on-line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed-batch culture. (c) 1992 John Wiley & Sons, Inc.     

Optimization of phytase production by solid substrate fermentation

The production of phytase by three feed-grade filamentous fungi (Aspergillus ficuum NRRL 3135, Mucor racemosus NRRL 1994 and Rhizopus oligosporus NRRL 5905) on four commonly used natural feed ingredients (canola meal, cracked corn, soybean meal, wheat bran) was studied in solid substrate fermentation (SSF). A. ficuum NRRL 3135 had the highest yield [15 IU phytase activity/g dry matter (DM)] on wheat bran. By optimizing the supplementation of wheat bran with starch and (NH₄)₂SO₄, phytase production increased to 25 IU/g DM. Optimization was carried out by Plackett-Burman and central composite experimental designs. Using optimized medium, phytase, phosphatase, alpha-amylase and xylanase production by A. ficuum NRRL 3135 was studied in Erlenmeyer flask and tray SSF. By scaling up SSF from flasks to stationary trays, activities of 20 IU phytase activity/g DM were reproducibly obtained. Electronic Publication     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

重组大肠杆菌产疏绵状嗜热丝孢菌脂肪酶分批补料发酵工艺

为了获得低成本的疏绵状嗜热丝孢菌脂肪酶(TLL),在5L发酵罐发酵中对工程菌E.coliBL21(DE3)/pET28b-TLL的分批补料发酵工艺进行研究。结果表明:以葡萄糖为碳源的补料培养基,采用溶氧(DO)反馈补料策略进行补料,当OD600=60时降温至28℃,分2次加入终质量浓度为30g/L乳糖进行诱导表达。优化后TLL的表达量提高到798.5U/L,是优化前的2.4倍。本研究为规模化发酵重组菌生产TLL奠定了基础。     

Gene expression usingnar promoter under anaerobic condition with recombinantE. coli

Thenar promoter as an inducible promoter was characterized for the process development for the gene expression and the protein production under anaerobic condition. The LB medium was selected as a main culture medium showing the enzyme activity of 18,000 units/min/g cell in the flask cultivation. The optimum concentration of nitrate was 1%. Under anaerobic conditions, the gene expression was fully induced in the presence of nitrate.