Mining enzymes from extreme environments

熊 玉 翠 10708054

Current advances in metagenomics have revolutionized the research in fields of microbial ecology and biotechnology, enabling the high-throughput discovery of new enzymes for industrial bioconversions.

What is Metagenomics?

Metagenomics is a strategy, characterizing DNA extracted directly from environmental samples.

美國黃石公園的熱泉內棲息著地球上最原始的微生物

南极深处发现的活着的微生物

南极冰下湖存在着复杂的微生物群

Two obstacles :

First, most extremophiles cannot be cultured using traditional culturing technologies

Second, the very low biomass densities often do not yield enough DNA and reduces the effectiveness of cloning

Introduction ：

Currently, about 4% of all sequencing projects deal with extremophiles that have industrial applications.

The extremophiles thrive in the harshest environments.

Methanococcus jannaschii

however, if the final goal is the identification of extremozymes

we must also improve annotations through empirical work on

specific candidates

not all proteins from extreme environments are extremozymes

Second, in other environments(extremes of pH or salinity/osmolarity, the presence of organic solvents or high levels of radiation)

maintain intracellular conditions more typical of non-extremophiles

First, in some environments(extreme temperature orpressure)

both intracellular and extracellular biochemical machineries are therefore exquisitely adapted to such conditions.

they are not expected to be extremozymes

they are expected to be extremozymes

Estimates of microbial diversity in extreme and non-extreme microbial communities

The microbial diversity on our planet is mostly comprised by the microorganisms, in terms of both absolute numbers of cells and of the biomass, and the numbers of species.

Different culture-independent technologies

Recent data suggests that the community composition and diversity can shift greatly in human-perturbed areas, as well as within small vertical and horizontal distances, possibly in response to changes in the extreme physical and chemical conditions.

Protein engineering versus metagenomics

In the past two years, 45 extremozymes have been identified from pure cultures isolated from different extreme environments.

Although, many of these enzymes have been proven to be useful for chemical synthesis, one still might exhibit some unpredictable features with unnatural substrates and often exhibit low operational stability.

To overcome these problems…

using protein engineering and chemical modification screening it is currently possible to create ‘artificial extremozymes’ with altered protein topology, thermal stability and tolerance to organic solvents.

However, these strategies have a few hindrances, because the accumulation of subtle changes at genetic level do not always lead to the best enzymatic fitness.

Metagenomics constitutes a challenging researchdomain with great potential for both fundamental and industrial applications that range from the understanding of microbial adaptation and evolution to the discovery of new enzymes for their direct use.

challenges and bottlenecks

One of the main challenges for mining enzymes from extreme environments is not only to develop a robust screening or selection system to select for specific extremozymes, but also to overcome the problem of the low biomass yields and very low cell numbers that hinder high yields of DNA for cloning.

To solve this problem…

multiple displacement amplification (MDA) whole genome amplification(WGA)

high-throughput sequencing has revolutionized the study of microbial community metagenomes

certain limitations and difficulties

Future advances should focus on designing cell-free systems with a double aim:

to increase substrate bioavailability and to reduce inhibition and cross reactivity of cell components.

Prospect

Thanks for your attention!

DGGE 电泳设备是用于进行 DGGE 电泳的仪器，DGGE 技术是 DNA 指纹技术。

定量 PCR

FISH 荧光原位杂交

(1) The random hexamers (represented by a blue line) bind to the denatured DNA (represented by a green line); (2) The DNA polymerase (represented by a blue circle) extends the primers until it reaches newly synthesized double-stranded DNA (represented by an orange line); (3) The enzyme proceeds to displace the strand and continues the polymerization, while primers bind to the newly synthesized DNA; (4) Polymerization starts on the new strands, forming a hyperbranched structure.

Applied Biosystems Prism 3700 型基因分析仪为 96 道毛细管基因分析仪，是高通量基因分析的技术平台。

超高通量基因组测序系统 GS FLX 系统