Metagenomics for new enzyme discovery and industrial biocatalysis

用于新酶发现和工业生物催化的宏基因组学

• 批准号: BB/L007444/1
• 负责人: John Ward
• 金额: $ 131.32万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FL007444%2F1
• 关键词: Metagenomics; new; enzyme; discovery; industrial

The aim of the proposed research is to find new enzymes that have potential uses in industry by searching for the genes for these enzymes in the DNA extracted directly from soil, compost or other environments. Enzymes are very useful in biocatalysis which is a sustainable method of making chemicals in industry. If enzymes are used the eventual industrial process can be cleaner and greener as it avoids the use of toxic reagents such as metals needed for many chemical catalysis steps, and often uses water-based systems. Biocatalysis can also replace several steps in a chemical process with one enzyme step due to their selectivity and this has a major effect of saving money and time in the overall process for making high value chemicals such as bioactive compounds in the fine chemical and pharmaceutical industry.We will use a technique called metagenomics to find new enzymes for biocatalysis. Many enzymes are derived from microbial sources and these would normally be found by growing bacteria on agar plates and analysing the enzymes they contain using special assays. However, several years ago scientists studying soil microorganisms found that there was a very large difference between the numbers of bacteria they could grow from a soil sample compared with the numbers they could identify by analysing the DNA from the same quantity of soil. These DNA techniques showed that there were over 1,000 times more bacteria in the soil than can be grown on agar plates. So by using plating and growth techniques to find bacteria for biocatalytic enzymes were are missing over 99.9% of the potential enzymes. A technique called metagenomics was developed by several researchers which started with the extraction of DNA directly from a soil sample and this DNA would potentially contain all the genes of the bacteria including the genes from bacteria that cannot be grown in the laboratory. We will use this metagenomic technique to isolate DNA from soils and other environmental samples. The metagenomic DNA will be sequenced and potential genes for biocatalysis will be searched for using computer based techniques to analyse the metagenome. When we find what could be useful genes we will amplify the gene from a sample of the metagenomic DNA and put the amplified gene into a laboratory bacterium that we can grow in large amounts and test the activity of the new biocatalytic enzyme. We call this overall method Functional Metagenomics.The new biocatalysts will be tested in collaboration with researchers at Almac who use enzymes and chemistry to make pharmaceutical compounds. We will test the range of reactions the new biocatalysts can perform and test the chemicals made.A new concept called enrichment metagenomics will also be investigated where we will enrich for bacteria able to use a specific compound before doing the metagenomics. This has the potential to increase the number of bacteria with the desired biocatalytic enzyme. Another new concept called cDNA metagenomics will be tested where we extract messenger RNA from the sample and convert this into what is known as cDNA. This technique will allow us to look for genes from the microorganisms such as soil fungi that have introns in their DNA. This could enable us to find a hitherto unaccessed pool of new enzymes for biocatalysis.

这项研究的目的是通过在直接从土壤、堆肥或其他环境中提取的DNA中寻找这些酶的基因来寻找在工业中有潜在用途的新酶。酶在生物催化中非常有用，生物催化是工业中制造化学品的可持续方法。如果使用酶，最终的工业过程可以更清洁，更环保，因为它避免使用有毒试剂，如许多化学催化步骤所需的金属，并且通常使用水基系统。生物催化还可以用一个酶步骤取代化学过程中的几个步骤，这是由于它们的选择性，这在整个过程中节省了资金和时间，在精细化工和制药工业中生产高价值的化学品，如生物活性化合物。我们将使用一种称为宏基因组学的技术来寻找新的生物催化酶。许多酶来源于微生物，通常通过在琼脂平板上培养细菌并使用特殊测定法分析它们所含的酶来发现。然而，几年前，研究土壤微生物的科学家发现，他们可以从土壤样本中培养出的细菌数量与他们通过分析相同数量土壤的DNA所能识别的数量之间存在很大差异。这些DNA技术表明，土壤中的细菌比琼脂平板上的细菌多1,000多倍。因此，通过平板培养和生长技术来寻找细菌中用于生物催化的酶被缺失了99.9%以上的潜在酶。几位研究人员开发了一种称为宏基因组学的技术，该技术从直接从土壤样本中提取DNA开始，这种DNA可能包含细菌的所有基因，包括无法在实验室中生长的细菌的基因。我们将使用这种宏基因组技术从土壤和其他环境样本中分离DNA。将对宏基因组DNA进行测序，并使用基于计算机的技术分析宏基因组来搜索生物催化的潜在基因。当我们发现可能有用的基因时，我们将从宏基因组DNA样本中扩增基因，并将扩增的基因放入实验室细菌中，我们可以大量生长并测试新生物催化酶的活性。我们称这种整体方法为功能性宏基因组学。新的生物催化剂将与Almac的研究人员合作进行测试，他们使用酶和化学来制造药物化合物。我们将测试新的生物催化剂可以执行的反应范围，并测试所产生的化学物质。还将研究一种称为富集宏基因组学的新概念，在进行宏基因组学之前，我们将富集能够使用特定化合物的细菌。这有可能增加具有所需生物催化酶的细菌数量。另一个新概念称为cDNA宏基因组学将进行测试，我们从样本中提取信使RNA，并将其转化为所谓的cDNA。这项技术将使我们能够从微生物中寻找基因，例如在DNA中含有内含子的土壤真菌。这可能使我们能够找到一个迄今为止尚未获得的生物催化新酶库。

项目成果

Metagenomic ene-reductases for the bioreduction of sterically challenging enones.

• DOI: 10.1039/c9ra06088j
• 发表时间: 2019-11-11
• 期刊: RSC ADVANCES
• 影响因子: 3.9
• 作者: Dobrijevic, Dragana;Benhamou, Laure;Aliev, Abil E.;Mendez-Sanchez, Daniel;Dawson, Natalie;Baud, Damien;Tappertzhofen, Nadine;Moody, Thomas S.;Orengo, Christine A.;Hailes, Helen C.;Ward, John M.
• 通讯作者: Ward, John M.

Direct Conversion of Hydrazones to Amines using Transaminases.

• DOI: 10.1002/cctc.202101008
• 发表时间: 2021-11-08
• 期刊: CHEMCATCHEM
• 影响因子: 4.5
• 作者: Carter, Eve M.;Subrizi, Fabiana;Ward, John M.;Sheppard, Tom D.;Hailes, Helen C.
• 通讯作者: Hailes, Helen C.

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis.

• DOI: 10.1039/d1ob01237a
• 发表时间: 2021-07-28
• 期刊: Organic & biomolecular chemistry
• 影响因子: 3.2
• 作者: Cárdenas-Fernández M;Subrizi F;Dobrijevic D;Hailes HC;Ward JM
• 通讯作者: Ward JM

Stereoselective Transaminase-Mediated Synthesis of Serotonin and Melatonin Receptor Agonists

立体选择性转氨酶介导的血清素和褪黑激素受体激动剂的合成

• DOI: 10.1002/adsc.202200146
• 发表时间: 2022
• 期刊: Advanced Synthesis & Catalysis
• 影响因子: 5.4
• 作者: Baud D

A metagenomics approach for new biocatalyst discovery: application to transaminases and the synthesis of allylic amines

• DOI: 10.1039/c6gc02769e
• 发表时间: 2017-02-21
• 期刊: GREEN CHEMISTRY
• 影响因子: 9.8
• 作者: Baud, Damien;Jeffries, Jack W. E.;Hailes, Helen C.
• 通讯作者: Hailes, Helen C.