光驱动二氧化碳转化系统的构建、优化与应用

利用光能驱动二氧化碳(carbon dioxide, CO₂)还原生产化学品对于缓解环境压力、解决能源危机具有重要意义。光捕获、光电转化和CO₂固定等作为影响光合作用效率的关键因素,同时也制约着CO₂的资源化利用效率。为了解决上述问题,本文从生物化学与代谢工程相结合的角度,系统总结了光驱动杂合系统的构建、优化与应用,并从酶杂合系统、生物杂合系统以及杂合系统应用3个方面分析了光驱动CO₂还原合成化学品的最新研究进展。在酶杂合系统方面,采用的策略主要有提升酶催化活性、增强酶稳定性等;在生物杂合系统方面,采用的方法主要包括增强生物捕光能力、优化还原力供应以及改善能量再生等;在杂合系统应用方面,主要阐述了光驱动CO₂还原生产一碳含能化合物、生物燃料以及生物食品等。最后,从纳米材料(包括有机材料和无机材料)和生物催化剂(包括酶和微生物)两个方面,展望了人工光合系统的进一步发展方向。     

“自我剪切”2A肽介导的Δ-12和ω-3脂肪酸脱氢酶以及过氧化氢酶在转基因小鼠肌肉表达研究

哺乳动物因为缺乏Δ-12和ω-3脂肪酸脱氢酶,不能自身合成必需的多不饱和脂肪酸．目前,通过转基因技术在哺乳动物体内表达ω-3脂肪酸脱氢酶,能将长链的n-6多不饱和脂肪酸转化成n-3多不饱和脂肪酸,造成体内长链的n-6多不饱和脂肪酸含量显著减低．本研究通过自我剪切2A肽介导Δ-12和ω-3脂肪酸脱氢酶(FAT-2和FAT-1)以及人过氧化氢酶(human catalase,hCAT)在小鼠的肌肉同时表达．结果表明,转基因小鼠肌肉中长链n-3多不饱和脂肪酸含量提高2.6倍,长链n-6多不饱和脂肪酸含量没有显著变化,而n-6/n-3比例显著降低(P < 0.01)．同时蛋白质印迹检测到人过氧化氢酶hCAT在小鼠的肌肉组织中表达,且过氧化氢酶活性比野生型小鼠显著提高(P < 0.01)．     

好氧氨氧化过程中的关键酶及N2O排放研究进展

氧化亚氮(nitrous oxide, N₂O)排放量的持续增加对全球生态平衡造成了严重的威胁。微生物N₂O排放占主要来源。其中,好氧氨氧化过程是氨在有氧的条件下氧化为亚硝酸盐,其直接或间接地影响着全球产生N₂O与释放量。氨氧化古菌(ammonia-oxidizing archaea, AOA)、氨氧化细菌(ammonia-oxidizing bacteria, AOB)、全程氨氧化菌(complete ammonia oxidization, Comammox)和异养氨氧化菌(heterotrophic ammonium oxidizing bacteria, HAOB)是氨氧化过程中主要的参与者,明确这四类微生物N₂O产生的机制对缓解全球N₂O排放是必要的。本文综述了AOA、AOB、Comammox和HAOB在好氧氨氧化过程中驱动的N₂O产生途径,并结合酶学分析了一些关键酶在N₂O产生途径中的作用。本文旨在为调控生物N₂O排放提供理论基础。     

氮素添加对贝加尔针茅草原土壤团聚体微生物群落的影响

大气氮沉降增加作为全球气候变化的重要因素,其对土壤生态系统影响的研究受到了生态学家的广泛关注。土壤微生物是有机物分解和养分循环的主要参与者,在维持土壤的功能多样性和可持续发展方面发挥着重要的作用。氮沉降的激增会引起土壤微生物群落结构和功能的改变。土壤中营养物质在不同团聚体组分中分布的不均匀,为微生物提供了空间异质微生境。为揭示草原土壤不同粒径团聚体中微生物群落分布及其对氮素添加响应特征。自2010年起,在内蒙古贝加尔针茅草原典型地段设置N₀（0 kg hm^-2 a^-1）、N₁₅（15 kg hm^-2 a^-1）、N₃₀（30 kg hm^-2 a^-1）、N₅₀（50 kg hm^-2 a^-1）、N₁₀₀（100 kg hm^-2 a^-1）、N₁₅₀（150 kg hm^-2 a^-1）6个氮素添加处理模拟氮沉降野外控制试验。采用磷脂脂肪酸（phospholipid fatty acid,PLFA）法测定>2 mm、0.25-2 mm和<0.25 mm 3个粒径土壤团聚体中微生物PLFA含量,探讨氮素添加对土壤团聚体微生物群落结构的影响。结果表明：氮素添加提高了土壤碳、氮含量,降低了土壤pH。氮素添加显著提高了0.25-2 mm土壤团聚体微生物群落磷脂脂肪酸总量、真菌磷脂脂肪酸含量和真菌/细菌（Fungi/bacteria,F/B）、革兰氏阳性菌/革兰氏阴性菌（Gram-positive bacteria/gram-negative bacteria,G⁺/G^-）的比值（P<0.05）,降低了土壤团聚体微生物Margalef丰富度指数（P<0.05）。相关性分析表明,土壤团聚体微生物总PLFAs、真菌PLFAs含量、G⁺/G^-、F/B与土壤有机碳、全氮含量呈显著正相关关系,与C/N值负相关。综合研究表明,连续8年氮素添加显著提高了土壤有机碳和全氮含量、降低了土壤pH;提高了0.25-2 mm土壤团聚体真菌群落,土壤有机碳、全氮的固持与真菌群落的增加有关。     

焦化废水O/H/O生物处理工艺二级好氧生物反应器的微生物结构和功能

【背景】焦化废水O/H/O生物处理工艺的二级好氧生物反应器O₂具有剩余污染物矿化和完全硝化功能,对废水的达标排放有重要作用。【目的】阐明O₂生物反应器的微生物结构和功能。【方法】利用16S rRNA基因测序,研究O₂生物反应器的微生物多样性和组成并进行功能预测,揭示其共现性特征和环境影响因子。【结果】O₂的优势菌门以变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、绿菌门(Chlorobi)为主。主要菌属中红游动菌属(Rhodoplanes)、溶杆菌属(Lysobacter)、硫杆菌属(Thiobacillus)等参与化学需氧量(chemical oxygen demand,COD)、酚类(phenols)和硫氰酸盐(thiocyanate,SCN^-)等剩余污染物的去除,亚硝化弧菌属(Nitrosovibrio)和硝化螺菌属(Nitrospira)分别作为氨氧化细菌(ammonia-oxidizing bacteria,AOB)和主要的亚硝酸盐氧化细菌(nitrite-oxidizing bacteria,NOB)。功能预测结果显示苯甲酸酯降解、氨基苯甲酸酯降解、氯烷烃和氯烯烃的降解、氟代苯甲酸酯降解和硝基甲苯降解是外源物质生物降解和代谢的前五大通路,广泛分布在主要菌属中,验证了微生物降解剩余污染物的作用。基因pmoA/B/C-amoA/B/C、hao和nxrA/B编码相关的酶,组成了完整的硝化途径。共现网络结果揭示溶杆菌属、Candidatus Solibacter和红游动菌属在O₂生态中的重要地位。通过冗余分析(redundancy analysis,RDA)表明COD和NH₃是影响O₂微生物群落的主要因素。【结论】红游动菌属和溶杆菌属是O₂中最核心的功能菌属,在污染物矿化和维持群落生态稳定上有重要作用。亚硝化弧菌属和硝化螺菌属是硝化作用的核心菌属。O₂中的代谢通路以剩余污染物矿化和完全硝化为主,微生物群落主要受COD和NH₃的影响。本研究阐明了O₂的微生物结构与功能,为焦化废水O/H/O生物处理工艺的改进提供了微生物学上的依据。     

地质封存二氧化碳与深地微生物相互作用研究进展

地质封存将工业和能源相关领域生产活动产生的二氧化碳（CO₂）进行捕集并注入到深部地下岩石构造中,以实现长期储存的目标,是降低温室气体排放、实现CO₂长期封存的重要可行性手段之一。向深部地下地质构造中注入大量CO₂会导致深地环境发生显著变化,进而引起原生微生物活性及群落结构发生明显改变。因此,地质封存CO₂能够直接或间接影响深地微生物驱动的生物地球化学过程。同时,微生物在短期和长期的超临界CO₂（scCO₂）胁迫作用下,也会通过不同的适应性进化方式影响CO₂在地下环境中的迁移、转化和赋存形态。本文介绍了国内外二氧化碳捕获与封存发展现状以及地质封存CO₂影响条件下的scCO₂-水-微生物-矿物的相互作用领域的最新科研进展,并展望了利用深地微生物强化CO₂固定以及将其转化为高附加值产物的潜力。     

应用聚合酶链式反应快速特异鉴定假单胞菌和伯克霍尔德氏菌（R）-3-羟基酯酰载酯蛋白-辅酶A转酰基酶基因

聚羟基脂肪酸酯 (PHA)是一类具有广泛应用前景的可降解生物塑料。因其可以以葡萄糖等廉价底物直接发酵生产PHA而日益受到重视。目前的研究表明在积累中长链PHA的假单胞菌中 ,由phaG基因编码的（R）3 羟基酯酰载酯蛋白 辅酶A转酰基酶 (PhaG)起关键作用 ,但目前为止对该蛋白还知之甚少。通过聚合酶链式反应 (PCR)建立了一种快速、特异鉴定phaG基因的方法 ,应用该方法成功地从两株积累不同PHA的假单胞菌Pseudomonasstutzeri 1317和Pseudomonasnitroreducens 080 2中分别克隆得到phaG基因 ,并在phaG基因突变株PseudomonasputidaPHAG_N-21中表达成功。同时 ,还首次报道了从非假单胞菌菌株Burkholderiacaryophylli AS 1.274 1中鉴定得到phaG基因 ,提示PhaG介导的中长链PHA合成途径作为一种通用的代谢模式在细菌中广泛存在 ,为进一步实现从廉价的非相关底物合成中长链PHA提供了必要的分子生物学基础。     

云贵高原喀斯特坡耕地土壤微生物量 C、N、P空间分布

土壤微生物是地球生物演化进程中的先锋种类,具有重要的生态修复功能,但空间分布格局是否存在的争议很大。以云贵高原典型喀斯特坡耕地为对象,基于网格法取样,用经典统计学和地统计学综合分析了土壤微生物生物量的空间变异特征。结果表明,云贵高原喀斯特坡耕地土壤微生物生物量碳(C_mic)、磷(P_mic)以及碳氮比(C_mic/N_mic)适宜,氮(N_mic)的含量较低,变异均很大,空间自相关性明显,最佳拟合模型均为指数模型。块金值C₀较小(0.0016-0.0087),C₀/(C₀+C)均<25%(2.6%-10.2%),变程a较短(22.2-51.0 m),其强烈的空间变异主要由结构性变异引起。Kriging等值线图表明,C_mic、N_mic和C_mic/N_mic的高值区分布在坡的中上部,P_mic的高值区则在坡的中下部和坡脚。云贵高原喀斯特坡耕地土壤微生物不仅存在着小尺度的空间分布格局,而且不同土壤微生物属性的空间分布不同。     

N-糖链加工影响里氏木霉形态发生并提高木质纤维素降解能力

[背景] 烟曲霉α-1,2-甘露糖苷酶MsdS在高尔基体中将N-糖链Man₈GlcNAc₂加工为成熟分泌糖蛋白的糖型Man₆GlcNAc₂,有研究表明MsdS与烟曲霉的形态发生、细胞壁合成及蛋白质分泌密切相关;与烟曲霉不同的是,里氏木霉的成熟分泌糖蛋白上的N-糖链结构为Man₈GlcNAc₂,细胞却能正常生长,说明丝状真菌N-糖链的加工具有物种特异性,但其生物学意义不明。[目的] 为研究N-糖链加工对里氏木霉细胞生长及蛋白质分泌的影响,本研究将烟曲霉MsdS转入里氏木霉中以改变其成熟分泌糖蛋白的糖型。[方法] 构建带有烟曲霉msdS基因的重组质粒并转入里氏木霉中,获得msdS表达菌株Tr-MsdS,分析Tr-MsdS菌株的生长表型、N-糖组、蛋白质分泌途径和纤维素酶活性的变化。[结果] 在里氏木霉msdS表达菌株Tr-MsdS中,分泌糖蛋白的主要糖型由出发株的Man₈GlcNAc₂转变为Man₆GlcNAc₂,细胞壁组分发生变化,但细胞壁完整性未受影响;与出发株相比,Tr-MsdS菌株产孢、出芽及分枝增多;另外,MsdS的表达还影响蛋白质分泌,在50℃时降解纤维素和β-葡聚糖的能力分别提高9.9%和32.2%。[结论] 研究结果表明,N-糖链的加工可影响里氏木霉蛋白质,尤其是纤维素酶的分泌,干扰N-糖链加工可能是提高里氏木酶纤维素酶产量的新策略。     

深黄被孢霉△6-脂肪酸脱氢酶基因的克隆及序列分析

γ－亚麻酸（ＧＬＡ,Ｃ１８：３△_{６,９,１２}）是由△_６－脂肪酸脱氢酶以亚油酸（ＬＡ,Ｃ１８：２△_９,１２）为底物,在Ｃ_６位脱氢形成的。由于在人体中,γ－亚麻酸是花生四烯酸、前列腺素类和白三烯类等生理活性物质的前体物,而深黄被孢霉是目前用于微生物发酵生产γ－亚麻酸的主要菌株。本文根据脂肪酸脱氢酶的保守区设计引物,利用反转录聚合酶链式反应从丝状真菌深黄被孢霉中克隆了编码△_６－脂肪酸脱氢酶的ｃＤＮＡ,全长为１３７４个核苷酸,编码４５７ 个氨基酸,但与其他位点的脂肪酸脱氢酶不同的是, △_６－脂肪酸脱氢酶在其序列的 Ｎ 端特有细胞色素 ｂ_５（Ｃｙｔｂ_５）区。这是国际上对深黄被孢霉△_６－脂肪酸脱氢酶基因的首次报道。     

Engineering Escherichia coli BL21 (DE3) for high-yield production of germacrene A,a precursor of β-elemene via combinatorial metabolic engineering strategies

β-elemene is one of the most commonly used antineoplastic drugs in cancer treatment. As a plant-derived natural chemical, biologically engineering microorganisms to produce germacrene A to be converted to β-elemene harbors great expectations since chemical synthesis and plant isolation methods come with their production deficiencies. In this study, we report the design of an Escherichia coli cell factory for the de novo production of germacrene A to be converted to β-elemene from a simple carbon source. A series of systematic approaches of engineering the isoprenoid and central carbon pathways, translational and protein engineering of the sesquiterpene synthase, and exporter engineering yielded high-efficient β-elemene production. Specifically, deleting competing pathways in the central carbon pathway ensured the availability of acetyl-coA, pyruvate, and glyceraldehyde-3-phosphate for the isoprenoid pathways. Adopting lycopene color as a high throughput screening method, an optimized NS^Y305N was obtained via error-prone polymerase chain reaction mutagenesis. Further overexpression of key pathway enzymes, exporter genes, and translational engineering produced 1161.09 mg/L of β-elemene in a shake flask. Finally, we detected the highest reported titer of 3.52 g/L of β-elemene and 2.13 g/L germacrene A produced by an E. coli cell factory in a 4-L fed-batch fermentation. The systematic engineering reported here generally applies to microbial production of a broader range of chemicals. This illustrates that rewiring E. coli central metabolism is viable for producing acetyl-coA-derived and pyruvate-derived molecules cost-effectively.     

酶的分子设计、改造与工程应用

酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。     

提高微生物油脂生产能力的研究进展

微生物油脂是生物柴油生产领域具有广阔前景的新油脂资源。然而, 利用产油微生物进行油脂的工业化生产仍存在限氮条件下油脂生产强度不够高、对廉价高氮生物质原料的利用效率低等瓶颈问题。随着近年来发酵工程、生物信息学及分子生物学技术的发展, 国内外研究者利用不同策略优化微生物油脂的生产条件, 并对其油脂积累代谢途径进行改造, 旨在获得适用于工业化生产的产油性能优良的油脂菌。本综述总结了国内外利用生化工程、基因工程以及新兴的转录因子工程策略提高产油微生物油脂生产强度和扩大产油微生物廉价底物利用范围方面的研究进展, 并展望了基于组学研究、模块途径工程以及反向代谢工程的综合策略在理性改造产油微生物以提高其油脂发酵性能中的应用。     

生态工程设计研究进展及特点——以黑龙江省肇东市玉米生态工程为例

生态工程设计是生态工程建设的核心。明确生态工程设计的特点,是进行良好的生态工程设计的根本。通过生态设计研究进展的综述,从１０个方面,以肇东市玉米生态工程设计为例,分析了生态工程设计的特点,并在此基础上,提出了今后进行生态工程设计应采取的策略。     

Rational design of thiolase substrate specificity for metabolic engineering applications

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3‐hydroxyacid (3HA) pathway, also known as the reverse β‐oxidation or coenzyme‐A‐dependent chain‐elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model‐guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3‐hydroxy‐hexanoic acid and 3‐hydroxybutyric acid. We identify thiolase mutants that result in nearly 10‐fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence–structure–function relationship for this important class of enzymes.     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

阿维菌素的生物合成与途径工程

阿维菌素是一种高效安全的大环内酯杀虫杀螨剂。本文介绍了阿维菌素生物合成的步骤及参与合成步骤的有关酶系统和基因簇。对阿维菌素 8个组分合成的遗传控制基因 ,特别是对其中B1a组分合成的遗传控制位点进行讨论分析 ,并介绍了利用途径工程改造阿维链霉菌生产合成单一高效组分B1a和提高活性组分产量的研究进展。     

Bioprocess engineering: now and beyond 2000

Abstract: Bioprocess engineering may be defined as the translation of life-science discoveries into practical products, processes, or systems capable of serving the needs of society. It is a critical link from discovery to commercialization. Current bioprocess engineering is primarily focused on biopharmaceutical products of high dollar value per gram such as erythropoietin or growth hormones. However, other products of current interest include ethanol, amino acids, organic acids, antibiotics, and specialty chemicals. Current challenges for increased use of bioprocesses for producing bulk and semi-bulk chemicals include both technical and infrastructural barriers. Technical barriers are easy to identify and at times can be overcome by engineering improvements or changes brought about radical developments in science (e.g. recombinant DNA). Infrastructural barriers, such as raw-material substitutions or educational limitations are more difficult to define and change. Recently the National Academy of Sciences examined barriers to bioprocess engineering and issued a report entitled: "Putting Biotechnology to Work: Bioprocess Engineering". A key recommendation was the establishment of a coordinated long-range plan of research, development, training and education in bioprocess engineering involving participation by industry, academe and the federal government. The report was the first national analysis devoted entirely to bioprocess engineering and covered new topics such as space bioprocess engineering. Other topics covered by the author include the current state of the US chemical industry and future directions in three promising areas of bioprocess engineering environmental bioprocess engineering, marine bioprocess engineering and microsystem bioprocess engineering.     

Concerns with computational protein engineering programmes IPRO and OptMAVEn and metabolic pathway engineering programme optStoic

It has become customary in engineering to require a modelling component in research endeavours. In addition, as the code for these models becomes more byzantine in complexity, it is difficult for reviewers and readers to discern their value and understand the underlying code. This opinion piece summarizes the negative experience of the author with the IPRO and OptMAVEn computational protein engineering models as well as problems with the optStoic metabolic pathway model. In our hands, these models often fail to predict reliable ways to engineer proteins and metabolic pathways.