基于同步辐射软X射线诱变的产L-乳酸米根霉育种研究
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摘要
L-乳酸是广泛应用于食品、医药等行业的重要有机酸，以其为原料制备的聚L-乳酸是一种生物相容性优良的新型可生物降解材料。米根霉发酵生产L-乳酸具有营养要求简单、产L-乳酸光学纯度高等优点。但目前用于发酵产L-乳酸的米根霉菌种普遍存在发酵强度低、发酵性能差等问题。辐射诱变是微生物菌种获得稳定、优良性状的重要方法，而同歩辐射软X射线覆盖了生命分子重要组成元素的碳、氮、氧的k吸收边波长，具有很好的生物学效应，是一种具有开发应用前景的微生物辐射诱变源。
本文基于同步辐射软X射线诱变，依据米根霉产L-乳酸的代谢控制原理，以提高L-乳酸产量、增大发酵强度、改善发酵条件适应性为目标，针对米根霉的诱变方法与定向选育技术开展研究，采用代谢分析、基因突变检测、蛋白质组表达量差异分析等技术对其突变的机理进行探讨，主要研究结论如下：
（1）在中国科技大学国家同步辐射实验室的软X射线辐照装置上，通过考察孢子吸附固定载体的材料、样品架载样量等因素，确定了适合米根霉同步辐射软X射线辐照的条件。研究获得米根霉孢子在Ck（4.4nm）、Nk（3.02nm）、Ok（2.3nm）波长软X射线辐照条件下的致死剂量曲线，并通过致死效应、能量沉积效应分析，选择Nk波长软X射线为米根霉主要的辐射诱变源。以乙醇脱氢酶（ADH）活力弱化突变率为指标，通过分析致死率与正突变率的关系，确定了同步辐射软X射线Nk辐射诱变米根霉突变最适剂量范围为0.4kGy-0.8kGy。
（2）以AS3.819为出发株，经同步辐射软X射线诱变选育获得ADH弱化突变株ADH-21。突变株的L-乳酸产量为94.01g/L，比出发株的L-乳酸产量82.49g/L提高了13.97％，而乙醇生产量较出发株降低了70.99%。突变株的乳酸脱氢酶（LDH）活力较出发株提高了34.7％，而ADH活性比出发株的降低了79.85％。可见下调ADH活性是减少米根霉副产物乙醇生成量，增加L-乳酸产量的有效方法。经分离、纯化得到出发株与突变株的ADH，发现pH值及[Zn2+]、[Mg2+]对二者的ADH活性影响规律基本相同，但突变株的ADH最适反应温度更低，会导致突变株ADH活力下降。另外，通过对出发株与突变株ADH基因的cDNA测序、分析，获得比对结果。
（3）利用弱化氧化磷酸化过程降低胞内能荷水平对糖酵解影响的调控机理，以AS3.819为出发株，经同步辐射软X射线诱变选育获得F0F1-ATPase活力明显降低、胞内能荷下降了29.5%的突变株F0F1-17。突变株的糖酵解途径关键酶磷酸果糖激酶（PFK）、丙酮酸激酶（PK）、磷酸甘油醛脱氢酶（GADPH）的活性分别比出发株增加了58.5%，24.8%和19.8%。突变株的葡萄糖消耗速率、平均比消耗速率（qs）、L-乳酸发酵强度及平均比生成速率（qp）较出发株分别提高17.5%，85.9%，24.1%及61.5%。突变株的最高产量达到91.54g/L，发酵周期较出发株缩短了约6h。说明胞内的能荷状态降低，糖酵解途径关键酶活性增强，导致米根霉发酵产L-乳酸的发酵性能提高。
（4）利用Mg2+激活己糖激酶（HK），研究[Mg2+]上调对F0F1-17及AS3.819的发酵性能影响。表明在Mg2+调节作用下，HK活性的微幅增加可导致米根霉菌株产L-乳酸发酵过程的发酵性能微幅增强，这种增强的幅度在F0F1-ATP合酶（F0F1-ATPase）活力弱化突变株中表现更明显。由此，以米根霉F0F1-17为出发株，经同步辐射软X射线诱变选育获得HK活力增加85.3%突变株HK-3。与出发株及AS3.819相比，HK-3的葡萄糖消耗速率、葡萄糖的平均比消耗速率分别增加了29.8%、141.4%及27.9%、50.3%。HK-3发酵L-乳酸产量达到97.53g/L，其发酵强度及产物平均比生成速率qp分别比出发株F0F1-17增加41%和71.8%、比AS3.819分别增加75%和177.5%。说明降低F0F1-ATPase活性、提高HK活性，米根霉菌株能获得较好的发酵性能。
（5）为提高米根霉在酸性环境中的发酵性能，以AS3.819为出发株，经同步辐射软X射线诱变选育获得耐酸突变株acid-18。中和剂碳酸钙添加量为30g/L时，突变株的L-乳酸产量为83.5g/L，比出发株提高26.53%，其LDH比出发株提高42.2％。另外，突变株的质膜H+-ATPase酶活高于出发株的原因可能与突变株耐酸性增强相关。通过二维双向电泳分离、PDQuest软件分析，发现耐酸突变株与出发株蛋白质组表达量差异点有475个，其中丰度差异显著的12个点，并确定这12个蛋白的分子量和等电点。经质谱分析、数据库检索鉴定出其中的5个蛋白。
本文利用同步辐射软X射线对真菌米根霉诱变进行了首次有益探索，在建立米根霉的软X射线诱变条件的基础上，研究通过ADH活力弱化、F0F1-ATPase活力弱化与HK活力增强、菌株耐酸性增强等途径提高L-乳酸发酵强度、改善发酵性能方法，综合分析突变株在代谢物、代谢关键酶、蛋白质组及cDNA水平的突变效应。研究结果为良好发酵性能的米根霉工业菌株选育提供了基础研究数据。
L-lactic acid is an important organic acid and widely applied in food,pharmaceutical and other industries. There is an increased interest inappling L-lactic acid as a starting material for the synthesis ofpolylacticacid, a biodegradable and good biocompatibility plastic.Rhizopus oryzae could be a high-performance strain to produce highoptical purity L-lactic acid, because it does not require complicatenutrition in fermentation. For all that, the primary limitation of theRhizopus fermentation for L-lactic acid is in the relatively poor yield oflactate per mole of glucose consumed and low performance in conditionsof higher temperature or lower pH. Radiation mutagenesis is an importanttechnology for microbiology to produce high-performance strains.Because synchronous radiation(SR) soft X-ray coveres K-absorption edgewavelength of fundamental life elements such as C, N and O, which hasstronger radiation biological effect, the radiobiological studies of it hadbeen payed more attentions in recent years.
Based on SR soft X-ray radiation, breeding of Rhizopus oryzae hasbeen developed towards the goal of increasing L-lactic acid yield, highvolumetric productivity, and high performance in conditions of low pH.And mutation effects were carefully investigated on metalbolism, cDNAand proteomics level.
Summary and main conclusions as follows:
(1) The soft X-ray mutagenesis was carried out in SynchronousRadiation Laboratory (SNRL) at University of Science and Technology ofChina (USTC). The soft X-ray radiation conditions for Rhizopus oryzaemutagenesis were established after investigated several main factors suchas sample holder for spores and sample carriers. The lethal curves of Ck(4.4nm), Nk (3.02nm), and Ok (2.3nm) of soft X-ray on the spores ofRhizopus oryzae were developed, respectively. Using lethal curves andenergy deposition of short wavelength radition analysis, Nk wavelengthsoft-X ray with moderate energy deposition was used for Rhizopus oryzaemutagenesis. Optimum radiation dose range,0.4-0.8kGy, for Rhizopusoryzae mutagenization was concluded by analysising relation betweenlethal rate and positive mutation rate of weakened alcohol dehydrogenase（ADH） activity mutation by Nk wavelength soft X-ray radiation.
(2) A mutant ADH-21, with high L-lactic acid yield and weakenedADH activity, was screened with yeast peptone dextrose（YPD） platecontaining allyl alcohol from Rhizopus oryzae mutants which weremutagenized by soft X-ray. L-lactic acid yield of ADH-21and paraentstrain were94.01g/L and82.49g/L, respectively. Compared with that ofparaental strain, L-lactic acid yield of ADH-21increased by13.97%,while ethanol yield decreased by70.99%. Furthermore, compared withthat of parental strain, lactate dehydrogenase(LDH) activity of ADH-21 increased by34.7％, while ADH activity of ADH-21decreased by79.85％. These results indicate that inhibition of the activity of ADH is aneffective way to reduce the production of ethanol while improve theproduction of L-lactic acid. ADH of Rhizopus oryzae AS3.819andADH-21were purified for further chartacterization. Effects of pH,concentration of Zn2+, and Mg2+on Rhizopus oryzae AS3.819andADH-21were similar, while optimum reaction temperature of ADH frommutant were lower than that from parental strain. In addition, cDNAsequence of adh gene from parant strain was compared with mutant.
(3) For increasing the glycolytic flux of Rhizopus oryzae by defectingoxidative phosphorylation, a mutant F0F1-17, with high L-lactic acid yieldand65%of F0F1-ATPase activity decrease, was screened with PDA platecontaining neomycin from Rhizopus oryzae mutants which weremutagenized by soft X-ray. As a consequence, the glycolytic pathway keyenzyme phosphofructokinase(PFK), pyrutate kinase(PK), glyceraldehydesphosphate dehydrogenase (GAPDH) activity in the mutant increased by58.5%,24.8%and19.8%, which resulted that rate of glucose consumed,average specific glucose consumption rate, L-lactic acid productivity andaverage specific L-lactic acid production rate of the mutant increased by17.5%,85.9%,24.1%and61.5%than those of the paraental strain,respectively. The maximum L-lactic acid yield (91.54g/L) by mutant wasobtained within about48h of fermentation which was about6h shorter than that by the paraental.
(4) The specific activity of hexokinase increased with the increase ofMg2+concentration in fermentation medium. Effects of positivlyregulating Mg2+concentration on the fermentation performance of F0F1-17and AS3.819were resulted that a slightly increase of the hexokinaseactivity resulted in a slightly increase of performances of the L-lactic acidfermentation, especially in more harsh effects on the low-energy chargeF0F1-17strains than those on the Rhizopus oryzae AS3.819. Consequently,for increasing the glycolytic flux of Rhizopus oryzae by increasing HKactivity, a mutant HK-3, with high L-lactic acid yield and85.3%ofhexokinase(HK) activity increase, was screened with medium containing2-DG as a sole carbon source from Rhizopus oryzae mutants which weremutagensised by soft X-ray. Compared with paraental strain, rate ofglucose consumed, average specific glucose consumption rate, L-lacticacid productivity and average specific L-lactic acid production rate of themutant increased by29.8%,27.9%,71.8%and41%, respectively.Compared with AS3.819, rate of glucose consumed, average specificglucose consumption rate, L-lactic acid productivity and average specificL-lactic acid production rate of the mutant increased by141.4%,50.3%,177.5%and75%, respectively. The maximum L-lactic acid yield (97.53g/L) by mutant was obtained within~36h of fermentation. It is provedthat a Rhizopus oryzae mutant with lower F0F1-ATPase activity and higher hexokinase activity could be a good performance industry strain.
(5) For increasing fermentation performance of Rhizopus oryzae inacid-resistant conditions, a mutant acid-18, with26.53%higher L-lacticacid yield (83.5g/L), was screened with accumunation in mediumcontaining L-lactic acid from Rhizopus oryzae mutants which weremutagenized by soft X-ray. Consistently, compared with that of parentalstrain, LDH activity of mutant increased by42.2％, and H+-ATPaseactivity increased. All proteins from mutant and parental strain wereseparated by two-dimensional electrophoresis. PDQuest software analysisto electrophoresis map showed that there were475proteins with differentabundance between the acid-resistant mutant and parental strain, and12points with significantly different abundance whose molecular weight,isoelectric point and other properties were analysised and5proteins of12were identified by Maldi-toff mass spectrometry and database search.
This thesis results have laid the experimental foundation for SR softX-ray applications to Rhizopus oryzae mutagenization. On the basis ofstudy to radiation mutagenesis conditions by SR soft X-ray with Rhizopusoryzae as a model object, methods aimed at increasing fermentationperformance, including breeding mutant with weakened ADH activity,breeding mutant with weakened F0F1-ATPase activity and increased HKactivity, breeding mutant with increased acid-resistant, were studied by SRsoft X-ray radiation mutagenesis. And mutation effects of above mutants were analysised in metabolism, key enzymes activity, proteome and cDNAlevels. This study presents underlying data for breeding a goodfermentation performance Rhizopus oryzae suitable for industryprocessing.
本文基于同步辐射软X射线诱变，依据米根霉产L-乳酸的代谢控制原理，以提高L-乳酸产量、增大发酵强度、改善发酵条件适应性为目标，针对米根霉的诱变方法与定向选育技术开展研究，采用代谢分析、基因突变检测、蛋白质组表达量差异分析等技术对其突变的机理进行探讨，主要研究结论如下：
（1）在中国科技大学国家同步辐射实验室的软X射线辐照装置上，通过考察孢子吸附固定载体的材料、样品架载样量等因素，确定了适合米根霉同步辐射软X射线辐照的条件。研究获得米根霉孢子在Ck（4.4nm）、Nk（3.02nm）、Ok（2.3nm）波长软X射线辐照条件下的致死剂量曲线，并通过致死效应、能量沉积效应分析，选择Nk波长软X射线为米根霉主要的辐射诱变源。以乙醇脱氢酶（ADH）活力弱化突变率为指标，通过分析致死率与正突变率的关系，确定了同步辐射软X射线Nk辐射诱变米根霉突变最适剂量范围为0.4kGy-0.8kGy。
（2）以AS3.819为出发株，经同步辐射软X射线诱变选育获得ADH弱化突变株ADH-21。突变株的L-乳酸产量为94.01g/L，比出发株的L-乳酸产量82.49g/L提高了13.97％，而乙醇生产量较出发株降低了70.99%。突变株的乳酸脱氢酶（LDH）活力较出发株提高了34.7％，而ADH活性比出发株的降低了79.85％。可见下调ADH活性是减少米根霉副产物乙醇生成量，增加L-乳酸产量的有效方法。经分离、纯化得到出发株与突变株的ADH，发现pH值及[Zn2+]、[Mg2+]对二者的ADH活性影响规律基本相同，但突变株的ADH最适反应温度更低，会导致突变株ADH活力下降。另外，通过对出发株与突变株ADH基因的cDNA测序、分析，获得比对结果。
（3）利用弱化氧化磷酸化过程降低胞内能荷水平对糖酵解影响的调控机理，以AS3.819为出发株，经同步辐射软X射线诱变选育获得F0F1-ATPase活力明显降低、胞内能荷下降了29.5%的突变株F0F1-17。突变株的糖酵解途径关键酶磷酸果糖激酶（PFK）、丙酮酸激酶（PK）、磷酸甘油醛脱氢酶（GADPH）的活性分别比出发株增加了58.5%，24.8%和19.8%。突变株的葡萄糖消耗速率、平均比消耗速率（qs）、L-乳酸发酵强度及平均比生成速率（qp）较出发株分别提高17.5%，85.9%，24.1%及61.5%。突变株的最高产量达到91.54g/L，发酵周期较出发株缩短了约6h。说明胞内的能荷状态降低，糖酵解途径关键酶活性增强，导致米根霉发酵产L-乳酸的发酵性能提高。
（4）利用Mg2+激活己糖激酶（HK），研究[Mg2+]上调对F0F1-17及AS3.819的发酵性能影响。表明在Mg2+调节作用下，HK活性的微幅增加可导致米根霉菌株产L-乳酸发酵过程的发酵性能微幅增强，这种增强的幅度在F0F1-ATP合酶（F0F1-ATPase）活力弱化突变株中表现更明显。由此，以米根霉F0F1-17为出发株，经同步辐射软X射线诱变选育获得HK活力增加85.3%突变株HK-3。与出发株及AS3.819相比，HK-3的葡萄糖消耗速率、葡萄糖的平均比消耗速率分别增加了29.8%、141.4%及27.9%、50.3%。HK-3发酵L-乳酸产量达到97.53g/L，其发酵强度及产物平均比生成速率qp分别比出发株F0F1-17增加41%和71.8%、比AS3.819分别增加75%和177.5%。说明降低F0F1-ATPase活性、提高HK活性，米根霉菌株能获得较好的发酵性能。
（5）为提高米根霉在酸性环境中的发酵性能，以AS3.819为出发株，经同步辐射软X射线诱变选育获得耐酸突变株acid-18。中和剂碳酸钙添加量为30g/L时，突变株的L-乳酸产量为83.5g/L，比出发株提高26.53%，其LDH比出发株提高42.2％。另外，突变株的质膜H+-ATPase酶活高于出发株的原因可能与突变株耐酸性增强相关。通过二维双向电泳分离、PDQuest软件分析，发现耐酸突变株与出发株蛋白质组表达量差异点有475个，其中丰度差异显著的12个点，并确定这12个蛋白的分子量和等电点。经质谱分析、数据库检索鉴定出其中的5个蛋白。
本文利用同步辐射软X射线对真菌米根霉诱变进行了首次有益探索，在建立米根霉的软X射线诱变条件的基础上，研究通过ADH活力弱化、F0F1-ATPase活力弱化与HK活力增强、菌株耐酸性增强等途径提高L-乳酸发酵强度、改善发酵性能方法，综合分析突变株在代谢物、代谢关键酶、蛋白质组及cDNA水平的突变效应。研究结果为良好发酵性能的米根霉工业菌株选育提供了基础研究数据。
L-lactic acid is an important organic acid and widely applied in food,pharmaceutical and other industries. There is an increased interest inappling L-lactic acid as a starting material for the synthesis ofpolylacticacid, a biodegradable and good biocompatibility plastic.Rhizopus oryzae could be a high-performance strain to produce highoptical purity L-lactic acid, because it does not require complicatenutrition in fermentation. For all that, the primary limitation of theRhizopus fermentation for L-lactic acid is in the relatively poor yield oflactate per mole of glucose consumed and low performance in conditionsof higher temperature or lower pH. Radiation mutagenesis is an importanttechnology for microbiology to produce high-performance strains.Because synchronous radiation(SR) soft X-ray coveres K-absorption edgewavelength of fundamental life elements such as C, N and O, which hasstronger radiation biological effect, the radiobiological studies of it hadbeen payed more attentions in recent years.
Based on SR soft X-ray radiation, breeding of Rhizopus oryzae hasbeen developed towards the goal of increasing L-lactic acid yield, highvolumetric productivity, and high performance in conditions of low pH.And mutation effects were carefully investigated on metalbolism, cDNAand proteomics level.
Summary and main conclusions as follows:
(1) The soft X-ray mutagenesis was carried out in SynchronousRadiation Laboratory (SNRL) at University of Science and Technology ofChina (USTC). The soft X-ray radiation conditions for Rhizopus oryzaemutagenesis were established after investigated several main factors suchas sample holder for spores and sample carriers. The lethal curves of Ck(4.4nm), Nk (3.02nm), and Ok (2.3nm) of soft X-ray on the spores ofRhizopus oryzae were developed, respectively. Using lethal curves andenergy deposition of short wavelength radition analysis, Nk wavelengthsoft-X ray with moderate energy deposition was used for Rhizopus oryzaemutagenesis. Optimum radiation dose range,0.4-0.8kGy, for Rhizopusoryzae mutagenization was concluded by analysising relation betweenlethal rate and positive mutation rate of weakened alcohol dehydrogenase（ADH） activity mutation by Nk wavelength soft X-ray radiation.
(2) A mutant ADH-21, with high L-lactic acid yield and weakenedADH activity, was screened with yeast peptone dextrose（YPD） platecontaining allyl alcohol from Rhizopus oryzae mutants which weremutagenized by soft X-ray. L-lactic acid yield of ADH-21and paraentstrain were94.01g/L and82.49g/L, respectively. Compared with that ofparaental strain, L-lactic acid yield of ADH-21increased by13.97%,while ethanol yield decreased by70.99%. Furthermore, compared withthat of parental strain, lactate dehydrogenase(LDH) activity of ADH-21 increased by34.7％, while ADH activity of ADH-21decreased by79.85％. These results indicate that inhibition of the activity of ADH is aneffective way to reduce the production of ethanol while improve theproduction of L-lactic acid. ADH of Rhizopus oryzae AS3.819andADH-21were purified for further chartacterization. Effects of pH,concentration of Zn2+, and Mg2+on Rhizopus oryzae AS3.819andADH-21were similar, while optimum reaction temperature of ADH frommutant were lower than that from parental strain. In addition, cDNAsequence of adh gene from parant strain was compared with mutant.
(3) For increasing the glycolytic flux of Rhizopus oryzae by defectingoxidative phosphorylation, a mutant F0F1-17, with high L-lactic acid yieldand65%of F0F1-ATPase activity decrease, was screened with PDA platecontaining neomycin from Rhizopus oryzae mutants which weremutagenized by soft X-ray. As a consequence, the glycolytic pathway keyenzyme phosphofructokinase(PFK), pyrutate kinase(PK), glyceraldehydesphosphate dehydrogenase (GAPDH) activity in the mutant increased by58.5%,24.8%and19.8%, which resulted that rate of glucose consumed,average specific glucose consumption rate, L-lactic acid productivity andaverage specific L-lactic acid production rate of the mutant increased by17.5%,85.9%,24.1%and61.5%than those of the paraental strain,respectively. The maximum L-lactic acid yield (91.54g/L) by mutant wasobtained within about48h of fermentation which was about6h shorter than that by the paraental.
(4) The specific activity of hexokinase increased with the increase ofMg2+concentration in fermentation medium. Effects of positivlyregulating Mg2+concentration on the fermentation performance of F0F1-17and AS3.819were resulted that a slightly increase of the hexokinaseactivity resulted in a slightly increase of performances of the L-lactic acidfermentation, especially in more harsh effects on the low-energy chargeF0F1-17strains than those on the Rhizopus oryzae AS3.819. Consequently,for increasing the glycolytic flux of Rhizopus oryzae by increasing HKactivity, a mutant HK-3, with high L-lactic acid yield and85.3%ofhexokinase(HK) activity increase, was screened with medium containing2-DG as a sole carbon source from Rhizopus oryzae mutants which weremutagensised by soft X-ray. Compared with paraental strain, rate ofglucose consumed, average specific glucose consumption rate, L-lacticacid productivity and average specific L-lactic acid production rate of themutant increased by29.8%,27.9%,71.8%and41%, respectively.Compared with AS3.819, rate of glucose consumed, average specificglucose consumption rate, L-lactic acid productivity and average specificL-lactic acid production rate of the mutant increased by141.4%,50.3%,177.5%and75%, respectively. The maximum L-lactic acid yield (97.53g/L) by mutant was obtained within~36h of fermentation. It is provedthat a Rhizopus oryzae mutant with lower F0F1-ATPase activity and higher hexokinase activity could be a good performance industry strain.
(5) For increasing fermentation performance of Rhizopus oryzae inacid-resistant conditions, a mutant acid-18, with26.53%higher L-lacticacid yield (83.5g/L), was screened with accumunation in mediumcontaining L-lactic acid from Rhizopus oryzae mutants which weremutagenized by soft X-ray. Consistently, compared with that of parentalstrain, LDH activity of mutant increased by42.2％, and H+-ATPaseactivity increased. All proteins from mutant and parental strain wereseparated by two-dimensional electrophoresis. PDQuest software analysisto electrophoresis map showed that there were475proteins with differentabundance between the acid-resistant mutant and parental strain, and12points with significantly different abundance whose molecular weight,isoelectric point and other properties were analysised and5proteins of12were identified by Maldi-toff mass spectrometry and database search.
This thesis results have laid the experimental foundation for SR softX-ray applications to Rhizopus oryzae mutagenization. On the basis ofstudy to radiation mutagenesis conditions by SR soft X-ray with Rhizopusoryzae as a model object, methods aimed at increasing fermentationperformance, including breeding mutant with weakened ADH activity,breeding mutant with weakened F0F1-ATPase activity and increased HKactivity, breeding mutant with increased acid-resistant, were studied by SRsoft X-ray radiation mutagenesis. And mutation effects of above mutants were analysised in metabolism, key enzymes activity, proteome and cDNAlevels. This study presents underlying data for breeding a goodfermentation performance Rhizopus oryzae suitable for industryprocessing.
引文
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