甲醇的生物利用与转化*

甲醇作为一种来源广泛、价格低廉、还原度高的非粮原料有望成为下一代生物制造的关键原料。利用合成生物学技术构建能够高效利用甲醇的重组微生物以实现从甲醇到高值化学品的生物转化已成国内外研究热点,但由于甲醇代谢过程的特殊性及复杂性,目前人工设计的甲基营养菌还难以实现以甲醇为唯一碳源进行生长及产物合成。基于对天然甲基营养菌甲醇代谢过程的分析,从甲醇脱氢酶的筛选与改造、甲醛同化途径的重构与优化、甲醇到化学品的生物转化几个方面对合成型甲基营养菌的构建策略及面临的挑战进行总结与分析,以期为今后合成型甲基营养菌的人工设计和利用提供一定的借鉴。     

甲醇生物转化合成化学品的研究进展

甲醇来源丰富、价格低廉,已成为生物制造行业极具吸引力的底物之一。构建微生物细胞工厂实现甲醇到增值化学品的生物转化,具有过程绿色、条件温和、产品体系多样等优势,不仅能拓展基于甲醇的产品链,还能缓解当前生物制造“与民争粮、与粮争地”的问题,是实现绿色生物制造的重要手段。因此,阐明不同天然甲基营养菌中涉及甲醇氧化、甲醛同化和异化途径对于后续基因工程改造工作至关重要,也更有利于构建新型非天然甲基营养菌。本文讨论了甲基营养菌中甲醇代谢途径的研究现状,并结合近年来天然和人工合成甲基营养菌在甲醇生物转化中的应用进展及面临的挑战。     

一碳代谢关键酶——甲醇脱氢酶的研究进展与展望

甲醇和甲烷等一碳原料来源广泛,价格低廉,是生物制造的理想原料。甲醇脱氢酶(Methanol dehydrogenase,MDH)催化甲醇生成甲醛是一碳代谢的关键反应。目前已从天然甲基营养菌中发现了多种利用不同辅因子,具有不同酶学性质的MDH。其中,烟酰胺腺嘌呤双核苷酸(NAD)依赖型MDH被广泛应用于构建人工甲基营养菌。但是,NAD依赖型MDH的甲醇氧化活性较低,对甲醇的亲和力较差,导致甲醇氧化成为人工甲基营养菌代谢甲醇的限速步骤。为了提高甲醇氧化速率,进而提高人工甲基营养菌的甲醇利用效率,近年来大量研究集中于MDH的挖掘与改造研究。文中系统综述了不同类型MDH的发现、表征、改造以及在人工甲基营养菌中的应用进展,详细阐述了MDH的定向进化和多酶复合体的构建,并展望了通过细胞生长偶联的蛋白质进化和蛋白质理性设计获得高活性MDH的潜在策略。     

甲基营养菌代谢网络途径和代谢工程改造的研究进展

一碳化合物(甲醇或甲烷)高值化转化是当前生物工程领域的重要研究热点。甲基营养菌是一类以甲醇为碳源和能源生长的革兰氏阴性菌,随着基因组测序的开展和各类组学技术的发展,以扭脱甲基杆菌为代表的甲基营养菌的中心代谢网络途径和相关功能基因逐渐明晰。近年来,甲基营养菌中包括基因过表达、基因敲除整合等遗传操作工具的完善以及各种基因调控元件的开发,为甲基营养菌的底盘改造和异源途径优化表达提供了重要基础,国内外的研究推动了甲醇转化成多种高附加值产品。此外,人们渴望利用传统基因工程菌作为底盘构建合成型甲基菌,以期更高效实现甲醇催化转化。本文中,笔者综述了目前甲基营养菌,尤其是扭脱甲基杆菌的代谢网络特点、代谢工程改造策略与应用以及构建合成型甲基细菌这3个方面的研究进展,以期为甲基微生物催化转化的研究提供借鉴。     

甲烷生物利用及嗜甲烷菌的工程改造

作为来源广泛、储量丰富的有机碳一气体,甲烷被认为是下一代工业生物技术中最具潜力的碳原料之一。嗜甲烷菌能够利用其体内的甲烷单加氧化酶,将甲烷作为唯一的碳源和能源进行生长和代谢,这为温室气体减排及其开发利用提供了新的策略。目前,嗜甲烷菌生物催化体系的相关研究已开展多年,随着系统生物学和合成生物学的快速发展,利用代谢工程合理改造嗜甲烷菌代谢途径以提高甲烷转化效率,已经实现了生物转化甲烷制备多种大宗化学品和生物燃料。本文详细讨论并介绍了嗜甲烷菌催化氧化甲烷的相关代谢途径、高效细胞工厂构建及部分化学品生物合成的最新研究进展,并对甲烷生物转化未来的发展方向和面临的技术挑战进行了讨论和展望。     

化学品生物合成专刊序言

以酶及微生物细胞催化剂结合工程学方法将廉价、废弃原料进行高效生物转化可实现化学品的可持续生产。近年来,合成生物学、系统生物学及酶工程等技术的快速发展大大推动了化学品的可持续生物制造,既实现了多种新型化学品的生物合成,又显著提高化学品的生物合成效率。为展示化学品生物合成的最新进展并促进绿色生物制造的发展,《生物工程学报》特组织出版化学品生物合成专刊,从酶催化与生物合成机制、微生物细胞合成、一碳生物炼制以及关键核心技术等方面,介绍化学品生物合成的最新前沿、挑战以及潜在解决方案。     

1株甲基杆菌Methylobacterium sp.WGM16的鉴定及降解甲醇的最佳培养条件

从水稻根部土壤中筛选到1株粉红色、需氧的兼性甲基营养型菌株WGM16,该菌为革兰阴性杆菌。根据菌株16S rRNA基因序列比对分析及结合菌株常规形态特征、生理生化性状的鉴定,将该菌初步鉴定为Methylobacterium sp.PCR扩增到菌株WGM16编码甲醇脱氢酶α-亚基的mxaF基因,表明菌株WGM16中存在甲基营养代谢途径。在培养温度为32℃、以1%的甲醇作为碳源、pH值为8.0的培养条件下,其甲醇降解率可达75%。     

甲醇酵母代谢工程研究进展

甲醇酵母由于独特优点被认为是绿色生物制造的潜在宿主。特别是其天然甲醇利用性能有望建立甲醇生物转化路线,拓展生物炼制底物,具有重要经济价值和环保意义。文中综述了代谢工程改造甲醇酵母合成蛋白质和化学品的最新研究进展,并比较了其与模式生物酿酒酵母作为细胞工厂的优缺点。随后,分析了甲醇酵母代谢工程改造面临的挑战,并展望了潜在解决方案。随着基因操作工具开发和细胞代谢阐释,甲醇酵母将在未来绿色生物制造发挥越来越重要的作用。     

二氧化碳生物转化制甲烷技术研究进展

二氧化碳减量化与转化是当前业界关注及着手解决的重要问题,将二氧化碳作为资源转化为甲烷,有利于环境与社会的可持续发展。本文在分析二氧化碳转化为甲烷技术的基础上,重点介绍了国内外二氧化碳生物转化的研究与进展;总结了二氧化碳生物转化途径及其影响因素,分析了氢营养型、甲基营养型生物转化甲烷机理和生物转化能量来源;探讨了不同产甲烷菌微生物电合成产甲烷和氢气研究进展,总结了微生物电合成法、光合作用法和厌氧消化法等二氧化碳生物转化技术在反应器设计、电极材料选择、工艺条件优化及试验结果评估等方面取得的进展及存在的问题。重点就微生物电合成法的未来研究提出了增强微生物活性、提升氢气利用率、加快高效电极开发、提高能量效率、加强工业废气试验研究和强化光能转化等研究重点和发展方向,同时加强计算机模拟等交叉学科协同创新是促进二氧化碳生物转化技术进步的新方向。     

甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究

目的:研究甲醇脱氢酶基因mpq1818在甲基营养菌MP688生长代谢中的作用。方法:利用同源重组原理构建中间为庆大霉素抗性基因Gmr、两侧mpq1818基因上下游序列同源的敲除载体pAK0-up-Gmr-down,接合转移导入MP688,通过庆大霉素抗性和组合PCR方法筛选基因敲除菌,并检测其生长、甲醇脱氢酶活性、甲醇利用及吡咯喹啉醌(PQQ)生物合成能力等方面的差异。结果:抗性和PCR验证显示mpq1818缺失株构建成功;与野生菌相比,缺失株的甲醇脱氢酶活力及利用甲醇的能力降低,而且菌株的生长和PQQ产量也有显著下降。结论:基因mpq1818的缺失影响菌株前期生长与PQQ合成。     

Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methylotrophs have proved successful, autonomous methylotrophy, i.e. the ability to utilize methane or methanol as sole carbon and energy substrates, has not yet been realized. Here, we address an important limitation of autonomous methylotrophy in E. coli: the inability of the organism to synthesize several amino acids when grown on methanol. By activating the stringent/stress response via ppGpp overproduction, or DksA and RpoS overexpression, we demonstrate improved biosynthesis of proteinogenic amino acids via endogenous upregulation of amino acid synthesis pathway genes. Thus, we were able to achieve biosynthesis of several limiting amino acids from methanol-derived carbon, in contrast to the control methylotrophic E. coli strain. This study addresses a key limitation currently preventing autonomous methylotrophy in E. coli and possibly other synthetic methylotrophs and provides insight as to how this limitation can be alleviated via stringent/stress response activation.     

Recent advances toward the bioconversion of methane and methanol in synthetic methylotrophs

Abundant natural gas reserves, along with increased biogas production, have prompted recent interest in harnessing methane as an industrial feedstock for the production of liquid fuels and chemicals. Methane can either be used directly for fermentation or first oxidized to methanol via biological or chemical means. Methanol is advantageous due to its liquid state under normal conditions. Methylotrophy, defined as the ability of microorganisms to utilize reduced one-carbon compounds like methane and methanol as sole carbon and energy sources for growth, is widespread in bacterial communities. However, native methylotrophs lack the extensive and well-characterized synthetic biology toolbox of platform microorganisms like Escherichia coli, which results in slow and inefficient design-build-test cycles. If a heterologous production pathway can be engineered, the slow growth and uptake rates of native methylotrophs generally limit their industrial potential. Therefore, much focus has been placed on engineering synthetic methylotrophs, or non-methylotrophic platform microorganisms, like E. coli, that have been engineered with synthetic methanol utilization pathways. These platform hosts allow for rapid design-build-test cycles and are well-suited for industrial application at the current time. In this review, recent progress made toward synthetic methylotrophy (including methanotrophy) is discussed. Specifically, the importance of amino acid metabolism and alternative one-carbon assimilation pathways are detailed. A recent study that has achieved methane bioconversion to liquid chemicals in a synthetic E. coli methanotroph is also briefly discussed. We also discuss strategies for the way forward in order to realize the industrial potential of synthetic methanotrophs and methylotrophs.     

Chemical Production from Methanol Using Natural and Synthetic Methylotrophs

Methanol as a chemical feedstock is becoming increasingly important as it is derived from natural gas and is a feasible end‐product for captured carbon dioxide. Biological conversion of methanol through natural and synthetic methylotrophs increases the chemical repertoire and is an important direction for one carbon (C1) based chemical economy. Advances in the metabolic engineering and synthetic biology enable development of microbial cell factories for converting methanol into various platform chemicals. In this review, the current status of methanol utilizing microbial factory development is summarized. Also the development of synthetic methylotrophy and methanol‐augmented bioproductions is discussed.     

Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli

One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modelling, we designed a new route that “mixed and matched” two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput ¹³C-labeling experiments. The highest level of incorporation of methanol into central metabolism intermediates (e.g. 22% into the PEP), was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.     

Engineering Escherichia coli for methanol-dependent growth on glucose for metabolite production

Synthetic methylotrophy aims to engineer methane and methanol utilization pathways in platform hosts like Escherichia coli for industrial bioprocessing of natural gas and biogas. While recent attempts to engineer synthetic methanol auxotrophs have proved successful, these studies focused on scarce and expensive co-substrates. Here, we engineered E. coli for methanol-dependent growth on glucose, an abundant and inexpensive co-substrate, via deletion of glucose 6-phosphate isomerase (pgi), phosphogluconate dehydratase (edd), and ribose 5-phosphate isomerases (rpiAB). Since the parental strain did not exhibit methanol-dependent growth on glucose in minimal medium, we first achieved methanol-dependent growth via amino acid supplementation and used this medium to evolve the strain for methanol-dependent growth in glucose minimal medium. The evolved strain exhibited a maximum growth rate of 0.15 h⁻¹ in glucose minimal medium with methanol, which is comparable to that of other synthetic methanol auxotrophs. Whole genome sequencing and ¹³C-metabolic flux analysis revealed the causative mutations in the evolved strain. A mutation in the phosphotransferase system enzyme I gene (ptsI) resulted in a reduced glucose uptake rate to maintain a one-to-one molar ratio of substrate utilization. Deletion of the e14 prophage DNA region resulted in two non-synonymous mutations in the isocitrate dehydrogenase (icd) gene, which reduced TCA cycle carbon flux to maintain the internal redox state. In high cell density glucose fed-batch fermentation, methanol-dependent acetone production resulted in 22% average carbon labeling of acetone from ¹³C-methanol, which far surpasses that of the previous best (2.4%) found with methylotrophic E. coli Δpgi. This study addresses the need to identify appropriate co-substrates for engineering synthetic methanol auxotrophs and provides a basis for the next steps toward industrial one-carbon bioprocessing.     

Genome of Methylobacillus flagellatus, molecular basis for obligate methylotrophy, and polyphyletic origin of methylotrophy

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.     

Multiphyletic origins of methylotrophy in Alphaproteobacteria,exemplified by comparative genomics of Lake Washington isolates

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N‐methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl‐CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.     

Root nodule bacteria isolated from South African <Emphasis Type="Italic">Lotononis bainesii,L. listii</Emphasis> and <Emphasis Type="Italic">L. solitudinis</Emphasis> are species of <Emphasis Type="Italic">Methylobacterium</Emphasis> that are unable to utilize methanol

The South African legumes Lotononis bainesii, L. listii and L. solitudinis are specifically nodulated by highly effective, pink-pigmented bacteria that are most closely related to Methylobacterium nodulans on the basis of 16S rRNA gene homology. Methylobacterium spp. are characterized by their ability to utilize methanol and other C₁ compounds, but 11 Lotononis isolates neither grew on methanol as a sole carbon source nor were able to metabolize it. No product was obtained for PCR amplification of mxaF, the gene encoding the large subunit of methanol dehydrogenase. Searches for methylotrophy genes in the sequenced genome of Methylobacterium sp. 4-46, isolated from L. bainesii, indicate that the inability to utilize methanol may be due to the absence of the mxa operon. While methylotrophy appears to contribute to the effectiveness of the Crotalaria/M. nodulans symbiosis, our results indicate that the ability to utilize methanol is not a factor in the Lotononis/Methylobacterium symbiosis.