常压室温等离子体诱变选育L-精氨酸生产菌及发酵条件优化

【目的】通过常压室温等离子体诱变技术选育L-精氨酸高产菌株,利用响应面设计探索突变菌株生产L-精氨酸的最佳发酵条件。【方法】采用常压室温等离子体生物诱变系统对实验室保藏的Corynebacterium glutamicum GUI089进行系列诱变,选育L-高精氨酸和8-氮鸟嘌呤抗性菌株。在单因子实验的基础上,应用Plackett-Burman设计从7个因素中筛选出对L-精氨酸合成具有显著效应的(NH4)2SO4、葡萄糖和尿素3个因素。基于上述结果,进一步采用响应面设计优化出主要影响因素的最佳参数水平。【结果】经过一系列的诱变和筛选,选育出一株L-高精氨酸(15 g/L)和8-氮鸟嘌呤(0.7 g/L)抗性菌株,并将此菌株命名为C.glutamicum ARG 3-16。此菌株的L-精氨酸产量比出发菌株提高了49.79%,且发酵液中杂酸的浓度明显降低,特别是L-脯氨酸、L-谷氨酸和L-缬氨酸。在经响应面优化后的最佳发酵条件下,L-精氨酸的产量达到39.72±0.75 g/L,比优化前提高了10.49%。【结论】通过常压室温等离子体诱变技术成功选育出一株L-精氨酸高产菌株,利用响应面法有效地优化了发酵条件,实验结果表明突变株ARG 3-16具有潜在的生产应用价值。     

双功能尿苷酰转移/去除酶GlnD在谷氨酸棒杆菌JNR中的功能

【目的】通过改造谷氨酸棒杆菌JNR中双功能尿苷酰转移/去除酶GlnD,减弱尿苷酰去除酶的活性,增强NH_4~+的转运和利用,提高L-精氨酸的合成。【方法】本文对来源于谷氨酸棒杆菌的突变菌株JNR中的双功能尿苷酰转移/去除酶GlnD进行整合突变,采用同源重组的方法将H_(414)和D_(415)位点突变为两个丙氨酸AA,在此菌株的基础上过量表达PII蛋白GlnK,并对其进行尿苷酰化研究,离子色谱检测摇瓶发酵过程中NH4+的浓度,并对最终的改造菌株进行连续流加发酵分析。【结果】该双功能尿苷酰转移/去除酶在谷氨酸棒杆菌中成功进行整合突变,有效减弱了尿苷酰去除酶的活性;同时过表达PII蛋白GlnK,其酰基化程度明显增强。摇瓶发酵结果表明菌株L4消耗NH_4~+增加,L-精氨酸产量为36.2±1.2 g/L,比对照菌株L3高出22.7%。5-L发酵罐实验结果显示改造菌株L4的L-精氨酸的产量为52.2 g/L,较野生型菌株L0提高了25.3%。【结论】谷氨酸棒杆菌合成L-精氨酸的过程中氮源是必不可少的。减弱GlnD尿苷酰去除酶的活性后,胞内尿苷酰化的GlnK-UMP增加,GlnK-UMP与氮转录调控因子AmtR结合,转运至胞内的NH_4~+浓度提高,促使L-精氨酸产量显著提高。     

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.     

Improvement of milbemycin-producing <Emphasis Type="Italic">Streptomyces bingchenggensis</Emphasis> by rational screening of ultraviolet- and chemically induced mutants

Milbemycin antibiotics are produced by Streptomyces hygroscopicus subsp. aureolacrimosus and a newly isolated Streptomyces bingchenggensis, respectively. According to the biosynthetic pathway of milbemycins generated by S. hygroscopicus subsp. aureolacrimosus, a rational screening procedure with UV irradiation and N-methyl-N′-nitroso-N-nitrosoguanidine (NTG) mutation was performed to obtain high milbemycin-producing S. bingchenggensis. Aminoacetic acid (Glycine)-resistant mutants (AA^r), propionate-resistant mutants (PRP^r), streptomycin-resistant mutants (STR^r) and 2-deoxy-d-glucose-resistant mutants (DOG^r) were selected successively. A strain S. bingchenggensis BC-109-6 with AA^r, PRPr, STR^r and DOG^r was obtained and its production of milbemycin A3 and A4 reached 1,450 μg/ml, which was 80% higher than that of the ancestral strain S. bingchenggensis BC-101-4. The subculture experiments indicated that the hereditary characteristic of high productivity of S. bingchenggensis BC-109-6 was stable. The production of milbemycin A3 and A4 by S. bingchenggensis BC-109-6 in a 50-l fermentor could reach 1,380 μg/ml after 360 h batch fermentation.     

A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

Fuel ethanol from hardwood hemicellulose hydrolysate by genetically engineeredEscherichia coli B carrying genes fromZymomonas mobilis

Summary Escherichia coli B (ATCC 11303) carrying the PET operon on plasmid pLOI 297 converted hemicellulose hydrolysate to ethanol at an efficiency of 94% theoretical maximum, which is 15% better than the highest efficiency reported for pentose utilizing yeasts in a comparable system. Aspen prehydrolysate (APH), that had been produced by theBio-Hol Process using a Wenger extruder with SO₂ as catalyst, was used as feedstock. The fermentation medium contained predominantly xylose (35g/L) with acetic acid present at about 6g/L. With the pH controlled at 7.0, this concentration of acetic acid was not inhibitory for growth or xylose fermentation. When the APH was fortified with nutrients (tryptone and yeast extract), the recombinant (inoculated at 0.5 g dry wt/L) converted 100% of the xylose to ethanol with a volumetric productivity of 0.29 g/L/hr. Overliming the APH with Ca(OH)₂, followed by neutralization to pH 7 with sulphuric acid and removal of the insolubles, resulted in a 2-fold increase in productivity. The max. productivity was 0.76 g/L/hr. The productivity in Ca(OH)₂-treated APH, fortified with only mineral salts, was 0.26 g/L/hr.     

离子转运蛋白调控钝齿棒杆菌离子和pH稳态促进L-精氨酸合成

L-精氨酸是一种半必需氨基酸,广泛应用于食品、制药、饲料等行业。【目的】当前对L-精氨酸生产菌株的研究,极少涉及离子转运领域。在本研究中,发现在发酵时适量添加外源K~+有利于促进钝齿棒杆菌(Corynebacterium crenatum) SYPA5-5合成L-精氨酸。【方法】在C. crenatum SYPA5-5发酵培养基外源添加0.5 g/L和2.5 g/L的K_3PO_4,取对数期发酵样品进行转录组数据分析,挖掘出K~+转运相关的阳离子转运ATP酶CTAP1以及单价阳离子/H~+逆转运蛋白Mrp1A,研究其在C. crenatum SYPA5-5快速合成L-精氨酸阶段,对菌株生长及L-精氨酸合成的影响。【结果】对基因ctap1和mrp1分别进行敲除和过表达,深入研究突变株对L-精氨酸合成的影响。研究发现同时过表达离子转运蛋白CTAP1和Mrp1A更有利于胞内离子、pH稳态和渗透压调节,最终提高L-精氨酸的产量。在补料分批发酵中分别过表达Mrp1A、CTAP1以及同时过表达Mrp1A和CTAP1的菌株L-精氨酸产量分别达到61.4 g/L、63.9 g/L和65.3 g/L,产率分别为0.383 g/g、0.392 g/g和0.395 g/g,比C. crenatum SYPA5-5分别提高了34.9%、38.0%和39.1%。【结论】CTAP1是特异性的K~+转运ATP酶,可以将培养基中的K~+运输到胞内。同时Mrp1A可将胞内K~+和Na~+等单价阳离子运输到胞外,将胞外H~+运输至胞内,中和胞内L-精氨酸所导致的碱性环境,从而维持胞内pH稳定。CTAP1和Mrp1A的研究为解析离子转运机制和L-精氨酸合成之间的联系奠定了基础。     

Development of food-grade expression system for d-allulose 3-epimerase preparation with tandem isoenzyme genes in Corynebacterium glutamicum and its application in conversion of cane molasses to D-allulose

D -Allulose 3-epimerase (DAE) has been applied to produce D -allulose, a low-calorie and functional sweetener. In this study, a new DAE from Paenibacillus senegalensis was characterized in Escherichia coli. Furthermore, we presented a tandem isoenzyme gene expression strategy to express multiple DAEs in one cell and construct food-grade expression systems based on Corynebacterium glutamicum. Seventeen expression cassettes based on three DAE genes from different organisms were constructed. Among all recombinant strains, DAE16 harboring three DAE genes in an expression vector exhibited the highest enzyme activity with 22.7 U/mg. Whole-cell transformation of DAE16 produced 225 g/L D -allulose with a volumetric productivity of 353 g·g ⁻¹·hr ⁻¹. The catalytic efficiency of strain C-DAE9 integrating total 11 DAE genes in chromosome was 16.4-fold higher than strains carrying one DAE. Fed-batch culture of C-DAE9 gave enzyme activity of 44,700 U/L. We also expressed a thermostable invertase in C. glutamicum and obtained enzyme activity of 29 U/mg. Immobilized cells expressing DAE or invertase exhibited 80% of retained activity after 30 cycles of catalytic reactions. Those immobilized cells were coupled to produce 61.2 g/L D -allulose from cane molasses in a two-step reaction process. This study provided an efficient approach for enzyme preparation and allowed access to produce D -allulose from other abundant and low-cost feedstock enriched with sucrose.     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

Pullulan production from synthetic medium by a new mutant of Aureobasidium pullulans

Pullulan with different molecular-weight could be applied in various fields. A UV-induced mutagenesis Aureobasidium pullulans UVMU6-1 was obtained from the strain A. pullulans CGMCC3.933 for the production of low-molecular-weight pullulan. First, the obtained polysaccharide from A. pullulans UVMU6-1 was purified and identified to be pullulan with thin-layer chromatography, Fourier transform infrared, and nuclear magnetic resonance. Then, culture medium and conditions for this strain were optimized by flask fermentation. Based on the optimized medium and culture conditions (pH 4, addition of 4?g/L Tween 80 for 96?hr of cultivation), continuously fermentation was performed. The highest pullulan production and dry biomass was 109 and 125?g/L after fermentation for 114?hr, respectively. The average productivity was about 1?g/L/hr, which was intensively higher than the previous reported. This study would lay foundations for the industrial production of pullulan.     

Improvement of pristinamycin-producing Streptomyces pristinaespiralis by rational screening

Summary According to the biosynthetic pathway of pristinamycin, a rational selection procedure with u.v. mutation was performed to obtain a high pristinamycin-producing strain. Aminoacetic acid-resistant mutants (AA^r), valine hydroxamate-resistant mutants (VH^r), kitasamycin-resistant mutants (KTM^r) and 2-deoxy-D-glucose-resistant mutants (DOG^r) were selected, successively. A strain Streptomyces pristinaespiralis 12–55 with AA^r, Val^r, KTM^r, and DOG^r was obtained, and its production of pristinamycin reached 3000 u/ml which is 100 times higher than that of the parent strain S. pristinaespiralis ATCC 25486. It is inferred that S. pristinaespiralis 12–55 can alleviate catabolite repression caused by carbon sources, provide more acetic acid and valine for pristinamycin biosynthesis and increase its resistance to pristinamycin produced by itself, all of which are favorable for pristinamycin production. The subculture experiments indicated that the hereditary character of high productivity of S. pristinaespiralis 12–55 is stable. The pristinamycin production of S. pristinaespiralis 12–55 in a 15-l fermentor could reach 3010 u/ml after a 56 h batch fermentation.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> under oxygen deprivation

In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l⁻¹) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h.     

l-Valine production with minimization of by-products’ synthesis in Corynebacterium glutamicum and Brevibacterium flavum

Corynebacterium glutamicum ATCC13032 and Brevibacterium flavum JV16 were engineered for l-valine production by over-expressing ilvEBN ^r C genes at 31?°C in 72?h fermentation. Different strategies were carried out to reduce the by-products’ accumulation in l-valine fermentation and also to increase the availability of precursor for l-valine biosynthesis. The native promoter of ilvA of C. glutamicum was replaced with a weak promoter MPilvA (P-ilvAM1CG) to reduce the biosynthetic rate of l-isoleucine. Effect of different relative dissolved oxygen on l-valine production and by-products’ formation was recorded, indicating that 15?% saturation may be the most appropriate relative dissolved oxygen for l-valine fermentation with almost no l-lactic acid and l-glutamate formed. To minimize l-alanine accumulation, alaT and/or avtA was inactivated in C. glutamicum and B. flavum, respectively. Compared to high concentration of l-alanine accumulated by alaT inactivated strains harboring ilvEBN ^r C genes, l-alanine concentration was reduced to 0.18?g/L by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, and 0.22?g/L by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. Meanwhile, l-valine production and conversion efficiency were enhanced to 31.15?g/L and 0.173?g/g by C. glutamicum ATCC13032MPilvA△avtA pDXW-8-ilvEBN ^r C, 38.82?g/L and 0.252?g/g by B. flavum JV16avtA::Cm pDXW-8-ilvEBN ^r C. This study provides combined strategies to improve l-valine yield by minimization of by-products’ production.     

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.Nomenclature S glucose concentration (g/L) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - Q_p volumetric ethanol productivity (g/L.h) - Q_X volumetric biomass productivity (g/L.h) - Y_X/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Osmotic stress restricts glycolytic flux, growth (rate and yield), d-lactate productivity, and d-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mm betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of d-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l⁻¹ h⁻¹; 0.8 to 2.3 g l⁻¹ h⁻¹). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during d-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol d-lactate (90 g) per 100 g sugar (glucose or sucrose). Revisions requested 17 January 2006; Revisions received 7 February 2006     

Continuous 2-Keto-l-gulonic acid fermentation by mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium or Xanthomonas maltophilia

The fermentation process of 2-keto-L-gulonic acid (2KGA) from L-sorbose was developed using a two-stage continuous fermentation system. The mixed culture of Ketogulonicigenium vulgare DSM 4025 and Bacillus megaterium DSM 4026 produced 90 g/L of 2KGA from 120 g/L of L-sorbose at the dilution rate of 0.01 h⁻¹ in a single-stage continuous fermentation process. But after the production period was beyond 150 h, the significant decrease of 2KGA productivity was observed. When the non-spore forming bacteria Xanthomonas maltophilia IFO 12692 was used instead of B. megaterium DSM 4026 as a partner strain for K. vulgare DSM 4025, the 2KGA productivity was significantly improved in a two-stage continuous culture mode, in which two fermentors of the same size and volume were connected in series. In this mode, with two sets of 3-L jar fermentors, the steady state could be continued to over 1,331.5 h at least, when the dilution rates were 0.0382 h⁻¹ and 0.0380 hour⁻¹, respectively, for the first and second fermentors. The overall productivity was calculated to be 2.15 g/L/h at 113.1 g/L and a molar conversion yield of 90.1%. In the up-scaling fermentation to 30-L jar fermentors, 118.5 g/L of 2KGA was produced when dilution rates in both stages were 0.0430 hour⁻¹, and the overall productivity was calculated to be 2.55 g/L/h.     

Production of acetone and butanol by Clostridium acetobutylicum in continuous culture with cell recycle

Summary To increase the solvent productivity of the acetone-butanol fermentation, a continuous culture of Clostridium acetobytylicum with cell recycling was used. At a dry cell mass concentration of 8 g l^-1 and a dilution rate of D=0.64 h^-1, a solvent productivity of 5.4 g l^-1 h^-1 was attained. To prevent degeneration of the culture, which occurs with high concentrations of solvents (acetone, butanol and ethanol), different reactor cascades were used. A two-stage cascade with cell recycling and turbidostatic cell concentration control turned out to be the best solution, the first stage of which was kept at relatively low cell and product concentrations. A solvent productivity of 3 and 2.3 g l^-1 h^-1, respectively, was achieved at solvent concentrations of 12 and 15 g l^-1.Symbols D Dilution rate (h^-1) - r _p solvent productivity (g l^-1 h^-1) - s residual glucose concentration (g l^-1) - V _R reactor volume (l) - V _O overall volume (l) - x (dry) cell mass concentration (g l^-1) - Y _P/S solvent yield (g g^-1)     

Tower fermentation using Zymomonas mobilis for ethanol production

Summary Flocculation was induced in a pure strain of the bacteria Zymomonas mobilis. When fermenting glucose to ethanol, cell densities of up to 40g/l were achieved and sustained in a 0.92 litre tower fermenter with dilution rates of up to 2.3 hr^-1. A maximum productivity of 100g EtOH/l/hr with 98% conversion of the 105g/l glucose feed was achieved. The limitation to performance with increase in throughput arose from incomplete fermentation of the feed glucose, rather than washout of the flocculated bacteria.