稳定表达MVA途径基因提高番茄红素产量

萜类化合物的直接前体物质异戊烯焦磷酸(IPP)和二甲基烯丙基焦磷酸酯(DMAPP)可以由2-甲基-D-赤藻糖醇-4-磷酸途径(MEP途径)和甲羟戊酸途径(MVA途径)合成。在已经优化MEP合成途径、番茄红素合成途径关键基因表达的重组大肠杆菌LYC101中,引入MVA途径基因,进一步提高重组大肠杆菌合成萜类化合物的能力。质粒pALV23和pALV145是本实验室在研究MVA途径基因协调表达时,用核糖体结合位点(RBS)文库连接MVA途径各基因构建质粒文库,而筛选到的有效提高β-胡萝卜素产量的质粒。首先比较了两个质粒分别在低产和高产番茄红素的菌株中对番茄红素合成的影响。结果表明,两个质粒在高、低产番茄红素的菌株中都可以有效提高番茄红素产量。在高产菌LYC101中pALV23比pALV145使番茄红素产量更高。然后,用CRISPR-Cas9系统辅助同源重组的方法,将MVA途经基因和启动子一共6.7kb的条带整合到LYC101菌株的染色体上,得到遗传稳定的菌株LYC102。LYC102的番茄红素产率达40.9mg/g,是出发菌株LYC101产率的2.19倍,比用质粒表达MVA途径基因的菌株提高了20%。在重组大肠杆菌中同时表达MVA途径和MEP途径,可以有效提高萜类化合物产率;文中构建了不含质粒的、遗传稳定的高产番茄红素菌株,为产业化合成番茄红素提供基础;同时构建平台菌株,可以用于其他萜类化合物合成。     

代谢工程改造甘油代谢途径提高β-胡萝卜素产量

甘油是生物柴油的副产物,因其价格低廉和高还原性,成为生物发酵的重要碳源。为了进一步提高工程菌对甘油的利用能力,从而提高萜类化合物的合成能力,本研究从β-胡萝卜素高产菌CAR015出发,对其甘油代谢途径的多个基因进行了调控。首先敲除了编码3-磷酸甘油抑制子的glp R基因,然后分别用M1-37、M1-46和M1-93三个不同强度的人工调控元件对glp FK、glp D和tpi A三组基因进行单基因调控和多基因组合调控。研究发现用M1-46调控glp D基因后β-胡萝卜素产量达到了64.82 mg/L,是CAR015的4.86倍,甘油消耗速率也提高了100%;调控tpi A基因后β-胡萝卜素产量略有提高;调控glp FK基因后β-胡萝卜素产量略有降低。说明Glp D是甘油代谢途径中的关键限速步骤。Q-PCR结果表明,降低甘油代谢途径的glp D和glp FK基因转录水平,增加tpi A基因转录水平,可以增加细胞生长速度、提高β-胡萝卜素产量,可能是因为减少了丙酮醛毒性所致。组合调控glp D和tpi A基因,获得β-胡萝卜素产量最高菌株Gly003,其β-胡萝卜素产量达72.45 mg/L、产率达18.65 mg/g每克干细胞,分别是出发菌株CAR015的5.23倍和1.99倍。总之,Glp D是甘油代谢途径中的关键限速步骤,适当强度调控glp D,可以有效提高重组大肠杆菌的β-胡萝卜素产量。     

番茄红素基因工程菌多酶调控研究进展

作为类异戊二烯化合物中四萜的代表性产品番茄红素,在生物体中是典型的多酶参与催化的反应产物,在2-甲基-D-赤藓糖醇-4-磷酸盐(MEP)和甲羟戊酸(MVA)合成途径中起着至关重要的作用。从番茄红素在原核和真核中的多酶合成途径出发,针对番茄红素合成途径的各种优化策略,首先介绍了多酶合成中的常规调控方法,包括多基因共表达质粒构建、基因顺序调控、启动子与核糖体结合位点调控及基因敲除和替换等方法。之后介绍了一些新型的多酶调控方法,包括多片段组装技术、人工支架自组装方法等。最后重点介绍了这些多酶调控方法在番茄红素合成中的应用。这些多酶合成调控方法为构建高产番茄红素菌株提供了极大的启发和研究基础。     

提高大肠杆菌通过MVA途径合成异戊二烯

异戊二烯是橡胶合成的重要前体物质。为了提高菌株的异戊二烯产量,本实验室在研究中构建了一株异戊二烯产气的菌株BW-01,基于蛋白质预算理论的指导,理性设计通过改变质粒拷贝数、增加稀有密码子等合成生物学手段调控关键限速酶编码基因表达,从而提高大肠杆菌外源MVA代谢途径的异戊二烯产量。摇瓶发酵实验中我们构建的新产气菌株BW-07比原有的产气菌株BW-01的产量提高了73%,达到了761.1 mg/L。为后续菌株改造及进行发酵罐实验奠定了基础。     

高效合成番茄红素酿酒酵母菌株的构建

番茄红素作为一种高附加价值的萜类化合物已受到国内外研究者的广泛关注。首先对酿酒酵母Saccharomyces cerevisiae模式菌株S288c和YPH499合成番茄红素的能力进行分析比较,结果表明YPH499更适合作为底盘细胞用于番茄红素的合成。随后比较组成型启动子GPDpr、TEF1pr和诱导型启动子GAL1pr、GAL10pr对番茄红素合成的影响,结果发现以GPDpr、TEF1pr作为番茄红素合成途径基因crtE、crt B和crtI的启动子,摇瓶发酵60 h后,番茄红素产量为15.31 mg/L;以GAL1pr和GAL10pr为启动子时,其产量为123.89 mg/L,提高8.09倍。继续改造甲羟戊酸(MVA)途径,过量表达N-末端截短的关键酶基因t HMG1(3-羟基-3-甲基戊二酸单酰辅酶A还原酶),番茄红素产量为265.68 mg/L,单位菌体产量72.79 mg/g。文中所设计构建的异源表达番茄红素合成途径的酿酒酵母菌株单位细胞产量高,可以进一步改造和优化后用于番茄红素的工业化生产。     

大肠杆菌合成1,2,4-丁三醇的途径优化

1,2,4-丁三醇(BT)是一种在工业中有多种用途的重要的非天然化合物。文中通过将外源基因xdh和mdlC导入大肠杆菌BW25113表达,并敲除了xylA、xylB、yagE、yjhH、yiaE和ycdW等木糖和中间产物代谢旁路基因,构建了能够将D-木糖转化为BT的重组菌株。为优化BT合成途径,针对BT合成途径中的限速步骤——3-脱氧-D-甘油-戊酮糖酸的脱羧反应,进行了新酶的筛选和评价,获得了可显著提高反应效率的新的2-酮酸脱羧酶——KivD,并构建了表达该酶的重组菌株BW-025。在此基础上,通过初步条件优化,将BT产量提高至2.38g/L;进一步调节途径中各个酶的表达量,探究了它们对BW-025合成BT的影响,最终获得了BT产量较BW-025提高了48.62%的重组菌株BW-074。     

RBS文库调控重组大肠杆菌β-胡萝卜素合成途径关键基因提高β-胡萝卜素合成能力

β-胡萝卜素属于类胡萝卜素家族的一员,在药品、保健品、化妆品和食品行业有广泛的应用。本研究通过用RBS文库对重组大肠杆菌CAR005中β-胡萝卜素合成途径的关键基因dxs、idi和crt操纵子进行调控来提高β-胡萝卜素合成能力。研究发现3个基因分别用RBS文库调控后,与起始菌株相比β-胡萝卜素产量最高分别有7%、11%和17%的提高,表明使用RBS文库调控比使用多个固定强度启动子调控能筛选到更有利于目标产品合成的基因表达强度。三基因组合调控后,β-胡萝卜素产量相对于CAR005菌株提高了35%。同时发现,单基因文库筛选到的最优强度对于组合调控来说,未必是最优强度。本研究为利用基因表达调控优化目标产物合成途径提供了一种新的方案。     

挖掘与调控乙酸胁迫响应基因提高重组酿酒酵母合成番茄红素水平

【背景】乙酰辅酶A是酿酒酵母异源合成番茄红素的重要中间产物,胞质中乙酰辅酶A主要来自乙酰辅酶A合成酶催化乙酸合成。【目的】通过外源添加乙酸盐结合调控乙酸胁迫应答基因增加胞内乙酰辅酶A含量,改善细胞生长,促进番茄红素合成。【方法】在合成番茄红素的重组酵母菌中过表达乙酰辅酶A合成酶编码基因(acs2),在发酵过程中添加10g/L乙酸盐,结合转录组学分析挖掘乙酸胁迫响应基因,进行单一和组合调控。【结果】添加乙酸盐后,重组菌Y02中番茄红素含量增加了19.14%,但细胞生长受到抑制,转录组学结果表明adk2、fap7、hem13、elo3、pdc5、set5、pmt5、hst4、clb2和swe1表达水平增加,因此构建了单基因和双基因过表达菌株,其中Y02-set5-hst4菌在添加乙酸盐后细胞生长得到了显著改善,同时胞内乙酰辅酶A浓度提高了78.21%,番茄红素含量和产量达到12.62 mg/g-DCW和108.67 mg/L,与对照菌Y02相比分别提高了42.76%和67.13%。同时该菌中甲羟戊酸途径中关键基因erg12、erg20和hmg1的表达量与对照菌相比分别上调了1.70、1.4...     

甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。     

萜类生物合成的基因操作

萜类是一组结构迥异的化合物家族,其中很多具有较大的应用价值,如青蒿素和紫杉醇等,它们在多种微生物和植物中合成,但其天然产量低。萜类代谢工程通过DNA重组技术改造萜类合成细胞中的代谢途径,以提高萜类最终产量或在不含萜类的生物中合成萜类,为促进有用萜类合成提供了新的机会。以萜类化合物生物合成途径的基因转移与表达为切入点,综述了目前在微生物及植物中应用代谢工程提高萜类产量的研究进展。     

Enhanced lycopene production in Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate

To increase expression of lycopene synthetic genes crtE, crtB, crtI, and ipiHP1, the four exogenous genes were cloned into a high copy pTrc99A vector with a strong trc promoter. Recombinant Escherichia coli harboring pT-LYCm4 produced 17 mg/L of lycopene. The mevalonate lower pathway, composed of mvaK1, mvaK2, mvaD, and idi, was engineered to produce pSSN12Didi for an efficient supply of the lycopene building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Mevalonate was supplied as a substrate for the mevalonate lower pathway. Lycopene production in E. coli harboring pT-LYCm4 and pSSN12Didi with supplementation of 3.3 mM mevalonate was more than threefold greater than bacteria with pT-LYCm4 only. Lycopene production was dependent on mevalonate concentration supplied in the culture. Clump formation was observed as cells accumulated more lycopene. Further clumping was prevented by adding the surfactant Tween 80 0.5% (w/v), which also increased lycopene production and cell growth. When recombinant E. coli harboring pT-LYCm4 and pSSN12Didi was cultivated in 2YT medium containing 2% (w/v) glycerol as a carbon source, 6.6 mM mevalonate for the mevalonate lower pathway, and 0.5% (w/v) Tween 80 to prevent clump formation, lycopene production was 102 mg/L and 22 mg/g dry cell weight, and cell growth had an OD(600) value of 15 for 72 h.     

Production of lycopene by metabolically-engineered Escherichia coli

Escherichia coli strain CAR001 that produces β-carotene was genetically engineered to produce lycopene by deleting genes encoding zeaxanthin glucosyltransferase (crtX) and lycopene β-cyclase (crtY) from the crtEXYIB operon. The resulting strain, LYC001, produced 10.5 mg lycopene/l (6.5 mg/g dry cell weight, DCW). Modulating expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase B within central metabolic modules increased NADPH and ATP supplies, leading to a 76 % increase of lycopene yield. Ribosome binding site libraries were further used to modulate expression of genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) and the crt gene operon, which improved the lycopene yield by 32 %. The optimal strain LYC010 produced 3.52 g lycopene/l (50.6 mg/g DCW) in fed-batch fermentation.     

Screening for mevalonate biosynthetic pathway inhibitors using sensitized bacterial strains

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.     

Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways

Carotenoids are isoprenoid pigments of industrial and nutritional interest. Although they are produced in non-carotenogenic Escherichia coli engineered with the appropriate biosynthetic genes, only a limited pool of their metabolic precursors is available in these bacteria. We have compared the production of carotenoids (lycopene) in strains in which the supply of precursors was enhanced either by upregulating the endogenous pathway via overexpression of deoxyxylulose 5-phosphate synthase (DXS) or by incorporating an exogenous MVA+ operon. In strains expressing DXS under the control of a leaky IPTG-inducible promoter, lycopene accumulation was increased up to 8-fold in the absence of inducer. Addition of IPTG, however, negatively affected lycopene production. Although induction of too high levels of the MVA+ operon enzymes also appeared to cause interference with cell metabolism, supplementation with mevalonate (to be metabolized into carotenoid precursors) resulted in a 10-fold increase in lycopene levels in cells with a near wild-type background. An additional 2-fold increase (up to 228 mg/l) was obtained using an engineered BL21 strain. These results confirm that the MVA+ pathway is most convenient to upregulate the production of carotenoids (lycopene) production in E. coli but that factors other than precursor supply should be considered for high pigment accumulation levels.     

过表达乙酰辅酶A乙酰基转移酶2基因(RKAcat2)对红冬孢酵母产类胡萝卜素的影响

[背景] 乙酰辅酶A乙酰基转移酶（Acetyl Coenzyme A Acyltransferase,Acat）是硫解酶家族的一员,分为I型和II型,而II型作为甲羟戊酸（Mevalonate,MVA）途径的第一个限速酶,其表达水平和催化活性会影响萜类及其衍生物的合成量。[目的] 分析Acat II型基因的过表达对红冬孢酵母产类胡萝卜素的影响。[方法] 从红冬孢酵母YM25235菌株中克隆编码Acat II型的基因RKAcat2,将其回转到红冬孢酵母YM25235菌株中,构建一株RKAcat2基因过表达菌株进行分析。[结果] 与对照菌株相比,RKAcat2基因过表达使YM25235菌株中类胡萝卜素含量提高了50.53%,而菌株中油脂含量降低了22.80%,脂肪酸组成中油酸含量显著下降了17.78%,而且菌株中乙酰辅酶A （Coenzyme A,CoA）的含量也下降了13.64%。[结论] 过表达RKAcat2基因促进更多乙酰CoA进入MVA途径中,从而提高了类胡萝卜素的合成水平,这与部分MVA途径和类胡萝卜素合成途径中基因的转录分析结果一致。研究结果可为进一步通过代谢工程手段提高产油红酵母中类胡萝卜素及其特定组分含量的研究提供参考。     

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+/idi-::hph), crtE (crtE+/crtE-::hph), crtYB (crtYB+/crtYB-::hph), crtI (crtI+/crtI-::hph) and crtS (crtS+/crtS-::hph) and homozygote mutants crtYB (crtYB-::hph/crtYB-::hph), crtI (crtI-::hph/crtI-::hph) and crtS (crtS-::hph/crtS-::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.