耐铵型产琥珀酸放线杆菌的选育

以Actinobacillus succinogenes NJ113为出发菌株,经硫酸二乙酯(DES)诱变,在含8～20 g/L硫酸铵平板中筛选到一株耐铵型突变株YZ25,该菌株在含8 g/L硫酸铵培养基中厌氧发酵,琥珀酸产量达32.68 g/L,比出发菌提高了180.5%,对葡萄糖收率达65.4%,副产物乙酸、甲酸产量分别下降3.5%、28.7%,琥珀酸/乙酸比值由0.63提高到2.5。在7.5 L发酵罐中,用氨水调节pH分批实验,发酵34 h琥珀酸产量达27.13 g/L,较出发菌株提高了85.3%。     

超高静压在琥珀酸生产菌株选育中的应用

采用超高静压对一株产琥珀酸放线杆菌A3进行诱变育种, 进一步提高其生产性能。考察了压力、变压速度、生长期对菌株致死率的影响。在压力为200 MPa, 50 MPa/min变压速度的诱变条件下对稳定期菌株进行诱变, 筛选到一株突变菌株产琥珀酸放线杆菌B19, 琥珀酸产量达到 32.2 g/L, 比出发菌株A3提高了17.9%, 代谢副产物乙酸的产量降低了11.5%, 经过6次传代表明突变菌株具有稳定的遗传特性。     

琥珀酸发酵高产菌株的选育

以富马酸钠为唯一碳源的选择性平板,从牛的瘤胃中筛选出一株产琥珀酸的菌株Actinobacillus succinogenes CGMCC1593。以该菌株为出发菌株进行NTG诱变,挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落,经过初筛和复筛,发现SF-9菌株产琥珀酸能力强且积累的乙酸较少。以50 g/L的葡萄糖为碳源,在培养瓶厌氧发酵条件下其琥珀酸产量(35.09 g/L)比出发菌株(28.91 g/L)提高了21.4%,琥珀酸/乙酸比率(w/w)从3.3∶1提高到7.5∶1。用SF-9菌株在5 L发酵罐上进行分批发酵,发酵36 h时琥珀酸积累量达到40.51 g/L,对葡萄糖的转化率为0.81(w/w),琥珀酸/乙酸比率为9∶1,副产物乙酸量比出发菌株降低了约50%。     

基因组改组技术选育耐酸性琥珀酸放线杆菌

以琥珀酸产生菌Actinobacillus succinogenes CGMCC 1593为出发菌,分别经过紫外线-甲基磺酸乙酯(UV-EMS)和紫外线-硫酸二乙酯(UV-DES)诱变处理,得到7株耐酸性有所提高的突变株.以此作为候选菌库,经3轮原生质体递进融合,筛选获得4株可以在pH 5.6下生长的改组菌株.其中改组菌株F3-21在pH 5.6的完全液体培养基中生长的OD值是原始菌的7倍,在pH 5.2条件下仍能生长;其摇瓶发酵48h琥珀酸产量较原始菌株提高48%.在5L发酵罐中进行分批发酵,当控制pH在较低值(5.6～6.0)时,F3-21厌氧发酵48h积累琥珀酸38.1g/L,较出发菌株提高了45%;当控制pH在6.5～7.0时,F3-21厌氧发酵32h积累琥珀酸40.7g/L.F3-21在5L发酵罐中进行补料分批发酵,厌氧发酵72h,产琥珀酸达67.4g/L.结果说明基因组改组技术能够改进琥珀酸放线菌的耐酸性能及其琥珀酸的产量.     

耐铵型产琥珀酸放线杆菌的选育及铵离子对其生长代谢的影响

经硫酸二乙酯(DES)诱变,在含61～242mmol/LNH4+梯度平板中,筛选到一株耐铵型突变株YZ25,该菌株在含121mmol/LNH4+发酵培养基中,琥珀酸产量达32.68g/L,转化率为65.4%,比出发菌提高了180.5%。进一步考察了不同形态铵盐对YZ25生长的影响,结果表明添加少量铵盐能够提高突变菌的生长速率,但当超过一定量后菌株生长受到抑制,不同铵盐对菌株的抑制程度不同,硫酸铵、碳酸氢铵、氯化铵和硝酸铵对突变株YZ25的半抑制浓度分别为:215mmol/L、265mmol/L、235mmol/L、210mmol/L。为了考察铵离子对YZ25发酵产琥珀酸的影响过程,在3.0L发酵罐以氨水作为pH的调控剂发酵,结果表明在稳定期前菌株生长基本不受铵离子抑制,生物量能够达到正常水平,但是进入稳定期后铵离子抑制作用越来越明显,导致菌株生长提前结束,耗糖不完全,产酸受阻。最后结合产琥珀酸放线杆菌Actinobacillus succinogenes代谢途径分析了铵离子对菌株抑制作用的机理。     

产琥珀酸放线杆菌发酵生产琥珀酸的研究进展

近年来,因瘤胃微生物产琥珀酸放线杆菌Actinobacillus succinogenes具有高的琥珀酸产量,并能够利用多种碳源进行发酵等优点,在利用发酵法生产琥珀酸领域具有广泛的应用前景和商业化价值,因而其代谢途径和发酵工艺等基础研究成为国内外研发的热点。近年来,人们在产琥珀酸放线杆菌的代谢途径、琥珀酸发酵动力学模型、新型经济培养基以及高产菌株选育等方面的研究取得了很大进展,对研发琥珀酸发酵工艺、降低生产成本和节能减耗等具有重要的理论意义。     

超高静压在琥珀酸生产菌株选育中的应用

采用超高静压对一株产琥珀酸放线杆菌A3进行诱变育种,进一步提高其生产性能.考察了压力、变压速度、生长期对菌株致死率的影响.在压力为200 MPa,50 MPa/min变压速度的诱变条件下对稳定期菌株进行诱变,筛选到一株突变菌株产琥珀酸放线杆菌B19,琥珀酸产量达到32.2 g/L,比出发菌株A3提高了17.9%,代谢副产物乙酸的产量降低了11.5%,经过6次传代表明突变菌株具有稳定的遗传特性.     

琥珀酸放线杆菌发酵培养基的优化

对琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC1593发酵产生琥珀酸培养基的主要成分,及其含量进行优化。通过单因素试验,得出发酵培养基中葡萄糖、酵母膏和玉米浆的含量对产生琥珀酸有显著影响;采用响应面法(RSM),得出多元二次回归方程拟合的三种因素与琥珀酸含量间的函数关系,并根据优化结果与实验,CGMCC1593产琥珀酸达到41.69g/L。     

基因组改组技术选育耐高温、耐高乙醇酿酒酵母菌株的研究

当以f4、f5、f6作为出发菌株,用酵母菌原生质体紫外诱变的方法,在不同温度下,用含有不同浓度乙醇的平板筛选,分别获得了在耐高温和耐乙醇性状有较大提高的f4.2、f5.1、f6.2、f4.5等正突变菌株。以这些菌株作为出发菌株,进一步用硫酸二乙酯诱变,获得了f5.1.1、f4.2.1两个乙醇耐受性能较高的菌株。在建立了上述不同突变株后,通过基因组改组(genome shuffling)的方法,将上述不同特性的菌株经过两轮genome shuffling,获得了耐高温性能和耐乙醇性能都较好的酵母菌株。经过摇瓶发酵后证明,R24株在35℃发酵过程中,发酵液中的最高乙醇浓度12.93%(W/V),比原始出发菌株f4在35℃的发酵液中最高乙醇浓度8.11%提高了近5%。     

产琥珀酸放线杆菌的原生质体制备与再生

基因组重组技术是一项重要的菌种改造技术,原生质体制备和再生是进行基因组重组的前提和基础。目前少有关于产琥珀酸放线杆菌(Actinobacillus succinogenes)CGMCC2650原生质体研究的报道。为了优化该菌的原生质体制备和再生条件,及利用基因组重组技术构建优良菌种提供参考,研究了甘氨酸预处理,菌龄,酶浓度,作用时间,温度对产琥珀酸放线杆菌原生质体制备和再生的影响,并考察了不同渗透压稳定剂对其再生的影响。结果表明,菌体在添加了0.6mg/ml甘氨酸的TSB培养基中培养5h后收集,用SMM稀释到OD660=1.0,用0.025mg/ml溶菌酶在37℃下酶解45min制备原生质体,将原生质体涂布于含0.3mol/L蔗糖的再生培养基中,再生率最大,达到40.9%。确定了产琥珀酸放线杆菌原生质体制备和再生的最佳条件,所用的原生质体制备的方法对琥珀酸的产生没有影响,这为进一步开展该菌的原生质体诱变及基因组重组等研究奠定了基础。     

Actinobacillus succinogenes抗氟乙酸突变株的选育及其代谢流量分析

琥珀酸是一种用于合成树脂、可降解塑料及许多化学中间体的重要绿色化工原料。为了提高琥珀酸的发酵产率, 基于Actinobacillus succinogenes的代谢流量分布情况对其育种机制进行了研究。以Actinobacillus succinogenes CGMCC1593为原始菌株进行NTG诱变, 挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落, 经过初筛和复筛, 发现SF-9菌株产生更多琥珀酸且积累乙酸较少。以50 g/L的葡萄糖为碳源, 在5 L发酵罐上进行分批发酵, 该菌株发酵32 h时琥珀酸产量(34.8 g/L)提高了23.4%, 琥珀酸/乙酸比率为9:1, 副产物乙酸量比原始菌株降低了约50%。代谢流量分析(MFA)结果表明, PEP是影响琥珀酸合成的关键节点, PYR是影响乙酸等杂酸生成比例的关键节点, 并且这两个节点均非刚性节点。通过氟乙酸抗性诱变, 成功地筛选出了流向乙酸、甲酸和乳酸等杂酸的流量相对减少, 而流向琥珀酸的流量明显增强的突变菌株SF-9。     

Actinobacillus succinogenes抗氟乙酸突变株的选育及其代谢流量分析

琥珀酸是一种用于合成树脂、可降解塑料及许多化学中间体的重要绿色化工原料。为了提高琥珀酸的发酵产率, 基于Actinobacillus succinogenes的代谢流量分布情况对其育种机制进行了研究。以Actinobacillus succinogenes CGMCC1593为原始菌株进行NTG诱变, 挑选在含有50~100 mmol/L氟乙酸平板生长较快的菌落, 经过初筛和复筛, 发现SF-9菌株产生更多琥珀酸且积累乙酸较少。以50 g/L的葡萄糖为碳源, 在5 L发酵罐上进行分批发酵, 该菌株发酵32 h时琥珀酸产量(34.8 g/L)提高了23.4%, 琥珀酸/乙酸比率为9:1, 副产物乙酸量比原始菌株降低了约50%。代谢流量分析(MFA)结果表明, PEP是影响琥珀酸合成的关键节点, PYR是影响乙酸等杂酸生成比例的关键节点, 并且这两个节点均非刚性节点。通过氟乙酸抗性诱变, 成功地筛选出了流向乙酸、甲酸和乳酸等杂酸的流量相对减少, 而流向琥珀酸的流量明显增强的突变菌株SF-9。     

Continuous succinic acid fermentation by Actinobacillus succinogenes

Fermentations were performed in an external recycle bioreactor using CO₂ and d-glucose at feed concentrations of 20 and 40 g L⁻¹. Severe biofilm formation prevented kinetic analysis of suspended cell (‘chemostat’) fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells. The highest productivity of 6.35 g L⁻¹ h⁻¹ was achieved at a dilution rate of 0.56 h⁻¹. A constant succinic acid yield of 0.69 ± 0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm build-up – approximately 56% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h⁻¹ when the product concentrations were low (succinic acid < 10 g L⁻¹), while minimal growth was observed at succinic acid concentrations above this threshold.     

Development of a Markerless Knockout Method for Actinobacillus succinogenes

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes''s auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.