微生物以5-氨基乙酰丙酸为唯一前体物合成血红素的研究进展*

随着人造肉热潮的兴起,血红素作为其呈色物质也愈发引起研究者的兴趣。血红素是一种含Fe²⁺的卟啉类化合物,以5-氨基乙酰丙酸为唯一前体物,在生物体中分别通过粪卟啉依赖性、原卟啉依赖性和西罗血红素依赖性三个途径合成,被认为是一种理想的补铁剂和着色剂。与化学合成法和生物提取法相比,微生物合成法具有操作方便、环境友好等优点,因此是一种非常有前景的血红素生产方法。介绍血红素的合成途径,总结微生物以5-氨基乙酰丙酸为唯一前体物合成血红素的最新进展,并简要分析其面临的挑战和前景。     

代谢改造重组谷氨酸棒杆菌C4途径高效合成5-氨基乙酰丙酸

5-氨基乙酰丙酸 (5-aminolevulinic acid,5-ALA) 在医药和农业等领域有着广泛作用,目前主要采用大肠杆菌或谷氨酸棒杆菌以微生物发酵法合成。为了进一步提高谷氨酸棒杆菌合成5-ALA的能力,对其C4代谢途径进行了系统代谢改造。首先分别在谷氨酸棒杆菌中异源表达荚膜红杆菌和沼泽红假单胞菌的5-氨基乙酰丙酸合成酶ALAS,选择酶活相对较高的沼泽红假单胞菌的RphemA基因作为关键合成酶基因,并筛选到能显著增强RphemA的酶活性的核糖体结合位点RBS5。重组菌株ALAS的比酶活可达 (221.87±3.10) U/mg,且5-ALA产量提高了14.3%;随后通过敲除α-酮戊二酸脱氢酶抑制蛋白基因 (odhI) 和琥珀酸脱氢酶基因 (sdhA),促进了前体琥珀酰CoA向5-ALA途径的流动;通过sRNA抑制hemB表达减少了5-ALA的降解;并且过表达半胱氨酸/O-乙酰丝氨酸转运蛋白eamA提高了5-ALA的输出效率;使用重组菌株C. glutamicum 13032/?odhI/?sdhA-sRNAhemB-RBS5RphemA-eamA摇瓶发酵,5-ALA最高产量达11.90 g/L,较出发菌株提高了57%。最后,在5 L发酵罐中进行补料分批发酵,48 h内5-ALA的产量达25.05 g/L,为目前以葡萄糖为碳源发酵的最高产量。本研究构建了高产5-ALA重组谷氨酸棒杆菌,具有良好的工业应用前景。     

提高类球红细菌5-氨基乙酰丙酸产率的措施

5-氨基乙酰丙酸是四吡咯化合物生物合成的必需前体物,它作为一种无公害的绿色除草剂、杀虫剂、植物生长促进剂以及治疗癌症的药物而受到广泛的关注。文章综述了类球红细菌5-氨基乙酰丙酸的发酵生产现状以及提高其发酵产率的策略与措施。     

功能性氨基酸实现大批量生产、肥料、生发剂等实用化领域多方面展开

在光合作用、电子传导等生物体内的重要反应中起关键作用的5-氨基乙酰丙酸，COSMO石油公司成功地开发出了批量生产的技术，并开始积极推动5-氨基乙酰丙酸在农业、医疗等多个领域的应用。在高浓度和低浓度下呈现相反作用这一特性的发现进一步加快了研究的进程。[编者按]     

代谢工程合成5-氨基乙酰丙酸的研究进展

5-氨基乙酰丙酸(5-ALA)是生物体内吡咯类物质生物合成的前体物质,在医药和农业领域显示出广阔的市场潜力。随着代谢工程的发展以及对5-ALA生物合成途径的研究,利用微生物合成5-ALA成为研究的新方向。与传统的化学法合成相比,利用微生物合成5-ALA具有低污染、高产率和低成本等优势。本文中,笔者介绍了5-ALA生物合成的最新研究进展,分析了5-ALA代谢途径的关键影响因素,并对未来的研究方向进行了展望。     

5-氨基乙酰丙酸的生物合成及其应用

5-氨基乙酰丙酸是一种新型农药,由于其在环境中易降解,无残留,对人蓄无毒性,所以是一种无公害的绿色农药而倍受关注,在农业领域应用非常广泛,主要应用于植物生长调节剂、绿色除草剂、杀虫剂等方面,还可以应用到医学、有机合成等方面。本文主要综述了生物合成五氨基乙酰丙酸的途径,同时还介绍了五氨基乙酰丙酸作为一种调节剂、新型农药、杀虫剂的研究进展及在医学领域的发展。以期为科研和生产提供指导。     

表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸(5-aminolevulinate,ALA)由5-氨基乙酰丙酸合酶(5-aminolevulinate synthase,ALAS)催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg/L培养基。纯化的ALA纯度达到90%。     

5-ALA在胶质瘤光动力治疗应用的进展

胶质瘤作为常见的颅内恶性肿瘤,传统的手术与放疗化疗联合的治疗方法难以取得令人满意的治疗效果。光动力治疗作为治疗恶性肿瘤有效的辅助方法,在胶质瘤治疗中得到广泛应用。5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)是在光动力治疗中应用最多的光敏剂前体物质。多年来针对5-氨基乙酰丙酸(5-aminolevulinic acid,5-ALA)在胶质瘤光动力治疗中的研究主要集中在如何增强光动力效应,这也是许多神经外科医生的兴趣所在。本文结合相关文献,对5-ALA在胶质瘤光动力治疗的研究进展及未来在此领域面临的挑战进行了综述。     

Biosynthesis and Application of 5-Aminolevulinic Acid

5-氨基乙酰丙酸是一种新型农药,由于其在环境中易降解,无残留,对人蓄无毒性,所以是一种无公害的绿色农药而倍受关注,在农业领域应用非常广泛,主要应用于植物生长调节剂、绿色除草剂、杀虫剂等方面,还可以应用到医学、有机合成等方面.本文主要综述了生物合成五氨基乙酰丙酸的途径,同时还介绍了五氨基乙酰丙酸作为一种调节剂、新型农药、杀虫剂的研究进展及在医学领域的发展.以期为科研和生产提供指导.     

产生5—氨基乙酰丙酸（ALA）光合细菌生物学研究进展

５－氨基乙酰丙酸（ＡＬＡ）是卟啉、血红素和维生素Ｂ１２４的类似物。ＡＬＡ作为一种无公害绿色除草剂、杀虫剂、抗微生物药剂、植物生长促进剂及治疗癌症与其它疾病等而备受国外研究者及产业界的关注。本文对ＡＬＡ光合细菌合成、调节途径、分子遗传学的研究作一简要综述。     

The common origins of the pigments of life—early steps of chlorophyll biosynthesis

The complex pathway of tetrapyrrole biosynthesis can be dissected into five sections: the pathways that produce 5-aminolevulinate (the C-4 and the C-5 pathways), the steps that transform ALA to uroporphyrinogen III, which are ubiquitous in the biosynthesis of all tetrapyrroles, and the three branches producing specialized end products. These end products include corrins and siroheme, chlorophylls and hemes and linear tetrapyrroles. These branches have been subjects of recent reviews. This review concentrates on the early steps leading up to uroporphyrinogen III formation which have been investigated intensively in recent years in animals, in plants, and in a wide range of bacteria.Abbreviations ALA 5-aminolevulinic acid - ALAS 5-aminolevulinic acid synthase - GR glutamyl-tRNA reductase - GSA glutamate-1-semialdehyde - GSAT glutamate-1-semialdehyde aminotransferase - HMB hydroxymethylbilane - PBG porphobilinogen - PBGD porphobilinogen deaminase - PBGS porphobilinogen synthase - URO uroporphyrin - URO'gen uroporphyrinogen - US uroporphyrinogen III synthase     

Erythromycin as a tool to investigate the tetrapyrrole biosynthetic pathways in habituated and normal sugarbeet calli

Erythromycin (ERT) has been shown to reduce the 5-aminolevulinic acid (ALA) synthesizing capacity of a normal (N) chlorophyllous sugarbeet callus, grown under light, in contrast to a habituated achlorophyllous non-organogenic (HNO) callus of the same species. Similar effects were obtained on total hemes and on catalase which is a hemoprotein used as marker. The effect of ERT, which is an inhibitor of plastid differentiation and of chlorophyll synthesis, was reversed in the N callus by a supply of glycine and succinate. The compounds are the precursors of ALA synthesized through 5-aminolevulinic acid synthase (ALAS) which is implied in the Shemin pathway. The involvement of ALAS appeared to be favoured when plastids were undifferentiated (HNO callus) or when plastids were inefficient (N callus under darkness or under light after ERT treatment).     

Pathway engineering of Escherichia coli for α-ketoglutaric acid production

α-Ketoglutaric acid (α-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for α-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for α-KG production in Escherichia coli. First, the TCA cycle was rewired for α-KG production starting from pyruvate, and the engineered strain E. coli W3110Δ4-PCAI produced 15.66 g/L α-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in α-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to α-KG production up to 28.54 g/L. Finally, the engineered strain E. coli W3110Δ4-P_(H)CAI_(H)A was able to produce 32.20 g/L α-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of α-KG.     

Control of the syntheses of bacteriochlorophyll,c- and b-type cytochromes,and coproporphyrin III in Rhodopseudomonas sphaeroides mutant H5

Rhodopseudomonas sphaerodes mutant H5 lacking 5-aminolevulinic acid synthase was grown phototrophically in chemostat cultures limited by malate. Tetrapyrrole formation was limited by 5-aminolevulinic acid. With variation of dilution rates the cultures exhibited two regions of almost constant cell protein, dry weight and bacteriochlorophyll levels suggesting the formation of two physiological modifications of the strain. These modifications were further characterized by differences in the rates of 5-aminolevulinic acid consumption, the production of reserve material, the stoichiometries of 5-aminolevulinic acid consumption and bacteriochlorophyll or cytochrome production, specific bacteriochlorophyll and cytochrome contents as well as the ratio of bacteriochlorophyll protein complexes. In contrast, cellular levels of coproporphyrin II stayed almost constant over the entire range of dilution rates employed. Bacteriochlorophyll and b-type cytochrome cellular levels exhibited hyperbolic dependencies on the specific rate of 5-aminolevulinic acid consumption, and c-type cytochrome levels a signmoidal dependency. Bacteriochlorophyll cellular levels showed a biphasic dependency with half maximal saturations at 2.6 and 15.4 nmol of 5-aminolevulinic acid consumed per mg of protein and h, and maximal levels of 15.2 and 21 nmol bacteriochlorophyll per mg of protein. Cellular levels of c- and b-type cytochromes were half maximally saturated at 19.5 and 14.5 nmol 5-aminolevulinic acid consumed per mg protein and h while maximal levels were reached at 0.5 and 0.17 nmol of c- and b-type cytochromes, respectively, per mg of protein.The data suggest that within the cell bacteriochlorophyll as well as c- and b-type cytochrome units are assembled according to a defined pattern of kinetics characteristic of each group of compounds. Under otherwise constant external conditions the expression of the pattern is controlled by the rate of 5-aminolevulinic acid supply.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Divergent pathways for δ-aminolevulinic acid synthesis in two species of Arthrobacter

Abstract Secretion of coproporphyrin III by suspensions of Arthrobacter photogonimos and A. globiformis facilitated analysis of the paths of synthesis of δ-aminolevulinic acid, the precursor of tetrapyrroles. Sensitivity of coproporphyrin accumulation to gabaculine and incorporation of ¹⁴C from [1-¹⁴C]glutamate indicated that suspensions of A. photogonimos synthesized δ-aminolevulinic acid from glutamate by the widespread C5 pathway. In contrast, A. globiformis cells switched from predominantly the C5 pathway for δ-aminolevulinic acid synthesis in early exponential phase cultures to δ-aminblevulinic acid synthase in stationary phase cultures.     

Effective 5-aminolevulinic acid production via T7 RNA polymerase and RuBisCO equipped Escherichia coli W3110

Chromosome-based engineering is a superior approach for gene integration generating a stable and robust chassis. Therefore, an effective amplifier, T7 RNA polymerase (T7RNAP) from bacteriophage, has been incorporated into Escherichia coli W3110 by site-specific integration. Herein, we performed the 5-aminolevulinic acid (5-ALA) production in four T7RNAP-equipped W3110 strains using recombinant 5-aminolevulinic synthase and further explored the metabolic difference in best strain. The fastest glucose consumption resulted in the highest biomass and the 5-ALA production reached to 5.5 g/L; thus, the least by-product of acetate was shown in RH strain in which T7RNAP was inserted at HK022 phage attack site. Overexpression of phosphoenolpyruvate (PEP) carboxylase would pull PEP to oxaloacetic acid in tricarboxylic acid cycle, leading to energy conservation and even no acetate production, thus, 6.53 g/L of 5-ALA was achieved. Amino acid utilization in RH deciphered the major metabolic flux in α-ketoglutaric acid dominating 5-ALA production. Finally, the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoribulokinase were expressed for carbon dioxide recycling; a robust and efficient chassis toward low-carbon assimilation and high-level of 5-ALA production up to 11.2 g/L in fed-batch fermentation was established.     

微生物发酵产二十二碳六烯酸代谢机理的研究进展

二十二碳六烯酸(简称DHA)是一种重要的长链多不饱和脂肪酸,对人体具有重要的生理功能。微生物发酵生产的DHA与鱼油来源的DHA相比,具有诸多优点,其发展前景广阔。以下从发酵菌株、代谢途径、关键酶以及油脂积累机制等方面进行了综述,为通过代谢工程等技术手段进一步提高DHA产量提供了参考。