L-鸟氨酸发酵菌种的代谢工程研究进展

L-鸟氨酸是一种非蛋白类氨基酸参与尿素代谢及生物多胺类的合成,其对人体具有治疗肝脏疾病、增强免疫力等作用,被广泛应用于医疗、保健、食品等领域。工业上生产鸟氨酸主要有化学法、酶法及工业发酵法。其中,发酵法因其生产成本及环境保护等方面的优势而逐渐成为研究的焦点。本文归纳了近年来采用基因工程技术选育鸟氨酸高产菌种最新研究进展,重点讨论了产鸟氨酸谷氨酸棒杆菌的代谢工程改造策略,并对未来的研究方向进行了预测。     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

L-亮氨酸发酵的研究

钝齿棒状杆菌(Corynebacterlum crenatum)L-421是一株能产大量亮氨酸的突变株,它是从Corynebacterium crenatum AS1.1004菌株经NTG和快中子处理后获得的突变株。在含有葡萄糖、硫酸铵和尿素的培养基中,在2000升罐上,30℃发酵40小时,产亮氨酸可达20g/L。经强酸型阳离子交换树膳提取,收率可达40%以上,并得到符合美国药典85年21版标准的精品。发酵提取工艺简单,适于工业化生产。     

L-缬氨酸合成的代谢流量分析

分别测定谷氨酸棒杆菌（Corynebacterium glutamicum）AS1-495及其3个逐个叠加不同遗传标记的突变株AA361、AAT231和AATV341在特定培养时段（26～28h）L缬氨酸等代谢物的胞外浓度,由此计算这一时段这些代谢物在发酵液中积累（或消耗）的速率,分别做出这4株菌在拟稳态下的代谢流量分布图,进而研究育种过程中不同遗传标记的叠加对代谢网络中L-缬氨酸合成流量分布的影响。结果表明遗传标记的引入使流量分配发生了重大变化,节点处的流量分配朝着有利于L缬氨酸合成的方向改变。6-磷酸葡萄糖节点处流入EMP途径和HMP途径的流量分配由17.0∶83.0变为24.3∶75.7;丙酮酸节点处流入L-缬氨酸合成途径和其他途径的流量分配由15.8∶842变为76.7∶23.3/L-缬氨酸合成的分支途径上的流量由最初的5.37增大为37.3,乳酸合成途径的流量从11.1最后降为1.16,L-缬氨酸产量由4g/L提高到24.5 g/L。代谢流量分布的变化趋势与L缬氨酸产量的变化趋势是互相吻合的。以2-噻唑丙氨酸抗性突变（2TAr）和L天冬氨酸氧肟酸盐超敏性突变（LAAHss）有效地进行代谢流遗传导向的事实,在代谢流量分析的层面上,证明结构类似物抗性突变和结构类似物超敏性突变是代谢流导向和设计育种的十分有效的手段,代谢流量分析会成为设计育种的校正方法。     

L-亮氨酸发酵的研究

钝齿棒状杆菌(Corynebacterlum crenatum)L-421是一株能产大量亮氨酸的突变株,它是从Corynebacterium crenatum AS1.1004菌株经NTG和快中子处理后获得的突变株。在含有葡萄糖、硫酸铵和尿素的培养基中,在2000升罐上,30℃发酵40小时,产亮氨酸可达20g/L。经强酸型阳离子交换树膳提取,收率可达40%以上,并得到符合美国药典85年21版标准的精品。发酵提取工艺简单,适于工业化生产。     

L-亮氨酸发酵的研究

钝齿棒状杆菌(corynebacterium crenatum)L-421是一株能产大量亮氨酸的突变株,它是从Corynebactcrium crenatum Asl.1004菌株经NTG和快中子处理后获得的突变株。在含有葡萄糖、硫酸铵和尿素的培养基中,在2000升罐上,30℃发酵40小时,产亮氨酸可达20g/L。经强酸型阻离子交换树脂提取,收率可达40%以上,并得到符合美国药典85年21版标准的精品。发酵提取工艺简单,适于工业化生产。     

代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸

谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT (丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶 (ilvBN) 的调节亚基进行定点突变 (ilvBN1M13),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN1M13、乙酰羟酸异构酶 (ilvC)、二羟酸脱水酶 (ilvD)、支链氨基酸氨基转移酶 (ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。     

L-组氨酸高产菌株的选育

为得到L-组氨酸的高产菌株,以谷氨酸棒杆菌（Corynebacterium glutamicum）S9114为出发菌株,利用亚硝基胍（NTG）和硫酸二乙酯（DES）进行多次诱变,在D-组氨酸的抗性梯度平板上挑取正突变株,发酵检测,最终挑出一株S6（D—his＇）,可积累L-组氨酸327mg／L,比出发菌株提高47.3％。     

谷氨酸棒杆菌发酵过程中亮氨酸浓度近红外模型的建立

目的:建立谷氨酸棒杆菌发酵过程中亮氨酸浓度近红外模型,为实现谷氨酸棒杆菌发酵生产亮氨酸的发酵过程自动化控制提供理论基础和实践依据。方法:首先在5 L发酵罐中进行亮氨酸发酵,每隔一段时间采集发酵液样品,用高效液相色谱精确分析各样品中的亮氨酸实际浓度,再利用近红外分析仪和相关软件,对各样品进行近红外光谱扫描分析,并通过近红外光谱分析软件进行数据处理,建立谷氨酸棒杆菌发酵过程中亮氨酸浓度的近红外预测模型,最后通过外部检验方法检验模型的准确性。结果:结合偏最小二乘法,在波长为9043.3~7489.1 cm-1、减去一条直线作为光谱预处理的条件下,获得谷氨酸棒杆菌发酵过程中亮氨酸浓度最优近红外预测模型。该模型交叉验证误差均方根(RMSECV)、决定系数(R2)以及剩余预测偏差(RPD)分别为1.29 g/L、0.977和4.55。结论:经过验证,该模型的准确性和可靠性较强,实际值与预测值之间的误差较小,能够较好地检测发酵过程中的亮氨酸浓度。     

L-谷氨酸生产关键技术创新与产业化应用

L-谷氨酸是目前产量最大的氨基酸品种,也是我国生产规模最大的生物发酵产品。随着合成生物技术以及新型生产装备和技术的发展,国内L-谷氨酸菌种和生产技术近年来取得了明显的提升。本文从L-谷氨酸产业的现状分析和关键技术创新需求的角度出发,概述了L-谷氨酸菌种和生产技术的研究进展,介绍了近年来L-谷氨酸生产关键技术的创新开发和产业应用的进展。     

Glucose-controlled -isoleucine fed-batch production with recombinant strains of Corynebacterium glutamicum

-Isoleucine was produced in a fed-batch bioprocess with -leucine auxotrophic Corynebacterium glutamicum strains developed by genetic engineering. An efficient supply with nutrients was achieved by applying closed-loop control of glucose as the main carbon source, with a model-based, parameter-adaptive control strategy. This control strategy is based on an extended, semi-continuous Kalman filter for process identification and a minimum variance controller. The lab scale fed-batch process with C. glutamicum SM1 and C. glutamicum DR17 pECM3::ilvA38 was characterized with respect to biomass, product and by-product accumulation. A differential analysis of growth, specific productivities, and selectivities was performed to characterize the carbon flow over process time. Characterization of -isoleucine transport steps across the cell membrane resulted in a balance of -isoleucine transport over process time. Up to an extracellular -isoleucine concentration of 140 mM the cytosolic -isoleucine, provided by the biosynthesis, was quantitatively excreted into the medium via the export carrier system. Optimized feeding profiles for -leucine and phosphate in correlation with the on-line estimated glucose consumption were achieved up to the pilot scale (300-1 stirred tank reactor). The maximum -isoleucine concentration was 150 mM (21 g l⁻¹) with a space-time yield of 4.3 mmol l⁻¹ h⁻¹. With a 98% closed carbon balance the selectivity for isoleucine was 14%, for biomass 13%, and for CO₂ 68%.     

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO₂ via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础.30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力.因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开.研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时...     

Dynamics of glutamate synthesis and excretion fluxes in batch and continuous cultures of temperature-triggered Corynebacterium glutamicum

Corynebacterium glutamicum 2262 strain, when triggered for glutamate excretion, experiences a rapid decrease in growth rate and increase in glutamate efflux. In order to gain a better quantitative understanding of the factors controlling the metabolic transition, the fermentation dynamics was investigated for a temperature-sensitive strain cultivated in batch and glucose-limited continuous cultures. For non-excreting cells at 33°C, increasing the growth rate resulted in strong increases in the central metabolic fluxes, but the intracellular glutamate level, the oxoglutarate dehydrogenase complex (ODHC) activity and the flux distribution at the oxoglutarate node remained essentially constant. When subjected to a temperature rise to 39°C, at both high- and low-metabolic activities, the bacteria showed a rapid attenuation in ODHC activity and an increase from 28% to more than 90% of the isocitrate dehydrogenase flux split towards glutamate synthesis. Simultaneously to the reduction in growth rate, the cells activated a high capacity export system capable of expelling the surplus of synthesized glutamate.     

Temperature-sensitive cloning vector for Corynebacterium glutamicum

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.     

NADPH缺陷型Corynebacterium glutamicum重组菌株的构建及其性能分析

[目的]改造谷氨酸棒杆菌(Corynebacterium glutamicum)中NADPH合成途径,阻断胞内NADPH的合成,获得1株NADPH营养缺陷型菌株。[方法]通过失活L-赖氨酸高产菌C. glutamicum Lys-χ中葡萄糖-6-磷酸脱氢酶(Zwf)和苹果酸酶(MalE)并将NADP~+依赖型异柠檬酸脱氢酶(NADP~+-Icdcg)替换成变形链球菌(Streptococcus mutans)中的NAD~+-Icdsm,阻断胞内NADPH的合成。随后结合辅因子工程,引入大肠杆菌(Escherichia coli)中膜结合吡啶核苷酸转氢酶(PntAB)并通过不同强度启动子控制PntAB的表达水平。最后,分析不同重组菌中胞内氧化还原水平和L-赖氨酸生产强度的变化。[结果]重组菌C.glutamicum Lys-χΔZMI_(Cg)::I_(Sm)(即Lys-x1)胞内检测不到NADPH,为1株NADPH营养缺陷型菌株。该重组菌只在以葡萄糖酸为碳源的基础培养基中生长和积累L-赖氨酸,而以葡萄糖、丙酮酸、α-酮戊二酸和草酰乙酸为碳源时无法生长。此外,表达E.coli中的PntAB可回补重组菌Lys-χ1胞内NADPH的水平,但由于不同强度启动子控制PntAB表达水平不同,重组菌胞内NADPH水平也不同,并影响L-赖氨酸的生产强度。[结论]重组菌Lys-χ1可作为有效的底盘细胞,用于考察不同的NADPH再生策略,获得不同胞内NADPH水平的重组菌株,为进一步阐明NADPH调控微生物细胞生理代谢功能的机制提供研究基础。     

链霉菌中高效生产聚酮化合物的研究方法及进展北大核心CSCD

聚酮化合物是通过聚酮合成途径产生的一大类结构和生物活性多样的次级代谢产物,是链霉菌产生的主要次级代谢产物,具有重要的经济价值。为了在链霉菌中提高聚酮化合物产量,以满足工业生产需求,近年来,代谢工程的方法被广泛应用,例如,过表达合成途径中限速酶或途径特异性激活蛋白、强化前体供应、去除产物反馈抑制、合成基因簇异源表达等。本文将从代谢工程改造实例入手,全面综述链霉菌中聚酮化合物高效生物合成的研究方法及进展,并对利用合成生物学策略智能动态适配各个相关途径,进而提高该类化合物产量的研究思路进行展望。