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.

拟议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，找到在工业中具有潜在用途的新酶。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加清洁和绿色，因为它避免使用有毒试剂，例如许多化学催化步骤所需的金属，并且通常使用水基系统。由于其选择性，生物催化还可以用一个酶步骤代替化学过程中的多个步骤，这对于在精细化工和制药行业中制造生物活性化合物等高价值化学品的整个过程中具有节省金钱和时间的重大作用。我们将使用一种称为宏基因组学的技术来寻找用于生物催化的新酶。许多酶源自微生物来源，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这些酶。然而，几年前，研究土壤微生物的科学家发现，从土壤样本中生长的细菌数量与通过分析相同数量的土壤中的 DNA 识别出的细菌数量相比，存在很大差异。这些 DNA 技术表明，土壤中的细菌数量比琼脂平板上可以生长的细菌数量多 1,000 倍以上。因此，通过使用平板接种和生长技术来寻找生物催化酶的细菌，超过 99.9% 的潜在酶都缺失了。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潜在的生物催化基因。当我们找到可能有用的基因时，我们将从宏基因组 DNA 样本中扩增该基因，并将扩增的基因放入实验室细菌中，我们可以大量培养该细菌并测试新生物催化酶的活性。我们将这种总体方法称为功能宏基因组学。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。我们将测试新生物催化剂可以执行的反应范围并测试所制造的化学品。还将研究一种称为富集宏基因组学的新概念，我们将在进行宏基因组学之前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。另一个称为 cDNA 宏基因组学的新概念将得到测试，我们从样本中提取信使 RNA，并将其转化为 cDNA。这项技术将使我们能够从土壤真菌等微生物中寻找 DNA 中含有内含子的基因。这可以使我们找到迄今为止未曾接触过的用于生物催化的新酶库。

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.