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1.
生物丁醇作为一种重要的化学品和石油基燃料的替代品引起了人们的广泛关注。大肠杆菌(Escherichia coli)是生物合成化学品的优良底盘菌株,已在其体内构建了丁醇的生物合成途径。但大肠杆菌合成丁醇存在:(1)代谢通量非最优;(2)辅因子和氧化还原不平衡;(3)丁醇产量和产率低等问题,为此,从高效酶选择、碳代谢流调控、辅因子调控、丁醇生产和工艺等方面已经对丁醇合成途径和丁醇发酵进行了优化。从该角度出发阐述近几年来大肠杆菌生物合成丁醇的研究进展,并展望了利用工程大肠杆菌生产丁醇的研究方向,旨在为应用其进行高效的丁醇生产提供参考。 相似文献
2.
生物丁醇作为一种重要的化学品和石油基燃料的替代品引起了人们的广泛关注。大肠杆菌(Escherichia coli)是生物合成化学品的优良底盘菌株,已在其体内构建了丁醇的生物合成途径。但大肠杆菌合成丁醇存在:(1)代谢通量非最优;(2)辅因子和氧化还原不平衡;(3)丁醇产量和产率低等问题,为此,从高效酶选择、碳代谢流调控、辅因子调控、丁醇生产和工艺等方面已经对丁醇合成途径和丁醇发酵进行了优化。从该角度出发阐述近几年来大肠杆菌生物合成丁醇的研究进展,并展望了利用工程大肠杆菌生产丁醇的研究方向,旨在为应用其进行高效的丁醇生产提供参考。 相似文献
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The potential advantages of biological production of chemicals or fuels from biomass at high temperatures include reduced enzyme loading for cellulose degradation, decreased chance of contamination, and lower product separation cost. In general, high temperature production of compounds that are not native to the thermophilic hosts is limited by enzyme stability and the lack of suitable expression systems. Further complications can arise when the pathway includes a volatile intermediate. Here we report the engineering of Geobacillus thermoglucosidasius to produce isobutanol at 50 °C. We prospected various enzymes in the isobutanol synthesis pathway and characterized their thermostabilities. We also constructed an expression system based on the lactate dehydrogenase promoter from Geobacillus thermodenitrificans. With the best enzyme combination and the expression system, 3.3 g/l of isobutanol was produced from glucose and 0.6 g/l of isobutanol from cellobiose in G. thermoglucosidasius within 48 h at 50 °C. This is the first demonstration of isobutanol production in recombinant bacteria at an elevated temperature. 相似文献
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Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C. acetobutylicum ATCC 824 Δcac15ΔuppΔbuk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridial species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate]int and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium. 相似文献
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Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement. 相似文献
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Microbial fatty acids are an attractive source of precursors for a variety of renewable commodity chemicals such as alkanes, alcohols, and biofuels. Rerouting lipid biosynthesis into free fatty acid production can be toxic, however, due to alterations of membrane lipid composition. Here we find that membrane lipid composition can be altered by the direct incorporation of medium-chain fatty acids into lipids via the Aas pathway in cells expressing the medium-chain thioesterase from Umbellularia californica (BTE). We find that deletion of the aas gene and sequestering exported fatty acids reduces medium-chain fatty acid toxicity, partially restores normal lipid composition, and improves medium-chain fatty acid yields. 相似文献
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The production of biofuels by recombinant Escherichia coli is restricted by the toxicity of the products. 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical with low toxicity to microbes, could be a promising alternative for biofuel production. However, the yield and productivity of 2,3-BD produced by recombinant E. coli strains are not sufficient for industrial scale fermentation. In this work, the production of 2,3-BD by recombinant E. coli strains was optimized by applying a systematic approach. 2,3-BD biosynthesis gene clusters were cloned from several native 2,3-BD producers, including Bacillus subtilis, Bacillus licheniformis, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae, inserted into the expression vector pET28a, and compared for 2,3-BD synthesis. The recombinant strain E. coli BL21/pETPT7-EcABC, carrying the 2,3-BD pathway gene cluster from Enterobacter cloacae, showed the best ability to synthesize 2,3-BD. Thereafter, expression of the most efficient gene cluster was optimized by using different promoters, including PT7, Ptac, Pc, and Pabc. E. coli BL21/pET-RABC with Pabc as promoter was superior in 2,3-BD synthesis. On the basis of the results of biomass and extracellular metabolite profiling analyses, fermentation conditions, including pH, agitation speed, and aeration rate, were optimized for the efficient production of 2,3-BD. After fed-batch fermentation under the optimized conditions, 73.8 g/L of 2,3-BD was produced by using E. coli BL21/pET-RABC within 62 h. The values of both yield and productivity of 2,3-BD obtained with the optimized biological system are the highest ever achieved with an engineered E. coli strain. In addition to the 2,3-BD production, the systematic approach might also be used in the production of other important chemicals through recombinant E. coli strains. 相似文献
8.
Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production. 相似文献
9.
As the demand for biofuels for transportation is increasing, it is necessary to develop technologies that will allow for low-cost production of biodiesel. Conventional biodiesel is mainly produced from vegetable oil by chemical transesterification. This production, however, has relatively low land-yield and is competing for agricultural land that can be used for food production. Therefore, there is an increasing interest in developing microbial fermentation processes for production of biodiesel as this will allow for the use of a wide range of raw-materials, including sugar cane, corn, and biomass. Production of biodiesel by microbial fermentation can be divided into two different approaches, (1) indirect biodiesel production from oleaginous microbes by in vitro transesterification, and (2) direct biodiesel production from redesigned cell factories. This work reviews both microbial approaches for renewable biodiesel production and evaluates the existing challenges in these two strategies. 相似文献
10.
Recent advances in metabolic engineering have enabled microbial factories to compete with conventional processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with synthetic and systematic tools play central roles in metabolic engineering to create and improve a selected microbial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting biological diversity and high-throughput screening have been demonstrated as more effective tools for improving various phenotypes of interest. In particular, identification of unprecedented targets to rewire metabolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review highlights general principles and the features of the combinatorial approaches using various libraries to implement desired phenotypes for strain improvement. In addition, recent applications that harnessed the combinatorial approaches to produce biofuels and biochemicals will be discussed. 相似文献
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Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σs-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production. 相似文献
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The fermentative production of l-threonine and l-isoleucine with Corynebacterium glutamicum is usually accompanied by the by-production of l-lysine, which shares partial biosynthesis pathway with l-threonine and l-isoleucine. Since the direct precursor for l-lysine synthesis, diaminopimelate, is a component of peptidoglycan and thus essential for cell wall synthesis, reducing l-lysine by-production could be troublesome. Here, a basal strain with eliminated l-lysine production was constructed from the wild type C. glutamicum ATCC13869 by deleting the chromosomal ddh and lysE. Furthermore, the basal strain as well as the ddh single mutant strain was engineered for l-threonine production by over-expressing lysC1, hom1 and thrB, and for l-isoleucine production by over-expressing lysC1, hom1, thrB and ilvA1. Fermentation experiments with the engineered strains showed that (i) deletion of ddh improved l-threonine production by 17%, and additional deletion of lysE further improved l-threonine production by 28%; (ii) deletion of ddh improved l-isoleucine production by 8% and improved cell growth by 21%, whereas additional deletion of lysE had no further influence on both l-isoleucine production and cell growth; (iii) l-lysine by-production was reduced by 95% and 86% in l-threonine and l-isoleucine production, respectively, by deletion of ddh and lysE. This is the first report on improving l-threonine and l-isoleucine production by deleting ddh and lysE in C. glutamicum. The results demonstrate deletion of ddh and lysE as an effective strategy to reduce l-lysine by-production without surrendering the cell growth of C. glutamicum. 相似文献
15.
生物法制备平台化合物乙偶姻的最新研究进展 总被引：1，自引：0，他引：1
乙偶姻(3-羟基-2-丁酮)作为一种应用广泛的食用香料和重要的平台化合物,具有广阔的工业应用前景。与传统的化学合成方法不同,高效、环保的乙偶姻生物制备方法,可以减轻资源和环境压力,促进我国低碳经济的发展。近来,生物法制备平台化学品乙偶姻取得了丰硕的研究成果。总结了最近几年国内外在该领域最新的研究热点及方向,简述了发酵法生产乙偶姻的优势菌株概况,重点综述了以糖类物质为底物生产乙偶姻的最新策略及研究成果、将微生物改造为生产手性乙偶姻的高效细胞炼制工厂以及将2,3-丁二醇或双乙酰作为发酵底物的研究趋势,并介绍了乙偶姻的分离纯化工艺。使用非致病性的安全菌株,高效率地利用廉价底物,并采用经济、简单、环保的分离纯化方式,从而生产具有高附加值的食品级或高手性纯度乙偶姻,是生物法制备乙偶姻产业化发展的可靠保障。 相似文献
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High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15 g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3 g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies. 相似文献
18.
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. 相似文献
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Hiroshi Hisano Rangaraj Nandakumar Zeng-Yu Wang 《In vitro cellular & developmental biology. Plant》2009,45(3):306-313
The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to
break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation
of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases
for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation
of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel
crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis
of cellulosic feedstocks, although some potential fitness issues should also be addressed. 相似文献
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