Progress of Research on Origins of Life in China

ZHAO Yufen HUA Yuejin ZHANG Hongyu HE Yujian ZHU Ting LIU Yan WU Li

Progress of Research on Origins of Life in China

ZHAO Yufen1,2,3HUA Yuejin4ZHANG Hongyu5HE Yujian6ZHU Ting7LIU Yan1WU Li6

1 (361005) 2 (315221) 3 (100084) 4 (310029) 5 (430070) 6 (101408) 7 (100084)

The development of Chinese space science and technology plays a great role in promoting the researches in the field of the origin of life. With the multidisciplinary cooperation, there are fruitful achievements in this research field obtained over the past two years. This report summarizes the major progress of the basic researches about the origin of life in China during 2018–2020.

Origin of genetic code, Origin of metabolism, Evolution prototype of modern enzyme, Mirror- image biological system, Extremophiles

1 Introduction

Where did life come from? The origin and evolution of life are one of the fundamental science problems, which is always fascinating and widely concerned in the world today. So far there are two main theories. One is “meteoritic origin”, that means life on Earth must have originated in outer space and come to Earth through meteorites with organic matters, especially prebiotic materials as life seeds. Another is “the origin of living systems on the Earth” that life arose from lifeless matters under some extreme conditions on the Earth. However, no matter which theoretical system is, both follow the basic concept of the origin of life. That means modern biological functional macromolecules are originated from the original, simple inorganic or organic small molecules which go through the evolution of structure and function. It is the origin of chemical stage in the process of the origin of life.

Investigation of the internal nature of the origin of life involves in the multidisciplinary cooperation covers a wide range of researches. As one of the frontiers of science, related researches inevitably promote the development and innovation of the scientific and technological level in China. With the development of national economy and the construction of the Chinese manned space station and recoverable satellite science experimental platform, there are more and more opportunities to be created for exploring the origin of life in space with a broader perspective. In 2007, the Professional Committee of the Chinese Space Science Society on Space Life Ori­gin and Evolution was established at Xiamen University. Since then, the researchers with the common interest in exploring the origin of life have a platform to realize the extensive discussion and cooperation. Herein, we review and summarize the researches on the origin of life contributed by Chinese scientists in the past two years.

2 Regulation and Catalysis of Phosphorus in Prebiotic Chemistry Conditions

As the significant element for life, phosphorus inevitably plays a vital role in the process of the origin of life. In 2019, in view of phosphorus chemistry, Zhao.[1]collected their previous and systematic researches focusing on the origin of life in a book named as “Phosphorus Chemistry: The role of pho­sphorus in prebiotic chemistry”. The book mainly contents are as these follows: (i) penta-coordinate phosphorus activated intermediates have a vital regulation role in biological system; (ii) phosphorus selects the α-amino acids to be the structural unit of proteins; (iii)-phosphoryl amino acid is a delicate Co-evolution Model of Nucleic acids and Proteins; (iv) the discovery of Ser-His, the smallest functional dipeptide; (v) the chemical models for the investigation of the origin of chirality and the genetic code. The correlative researches will trigger people’s consideration about the effects of phosphorus in the origin of life. It will provide some novel clues and pivotal experiment supports to explain the eternal enigma during the evolution process of life.

3 Discovery of Analogs of 5’-aa-AMPs (aa-N-NMPs) in Prebiotic Chemistry System

The genetic code undoubtedly plays an essential role in life sciences; thus its origin, which remains ambiguous, is the most fundamental issue. Aminoacyladenylates (5’-aa-AMPs) are key intermediates in peptide synthesis of life system. In 2018, Zhao[2]reported analogs of 5’-aa-AMPs, namely nucleotide amidates (aa--NMPs), could be obtained under Hadean conditions (Figure 1). Significantly, dipeptides were detected from the above reactions and their yields varied with different nucleosides through the formation of different aa--NMPs. That means the nucleoside could regulate peptide formation through the intermediate aa--NMP. In addition, aa--NMP further reacts with the-terminal of peptide (p=) to form nucleotide-peptide (NMP- peptide,p=+1). Then, the peptide (p=+1) was formed by NMP-peptide hydrolysis (Figure 1). The related mechanism for producing the intermediate aa--NMP was double checked in the organic phase reaction system[3]. This chemical model provides the primordial version of the genetic code and reasonably explained the possible mechanism of prebiotic peptides synthesis.

Fig. 1 Mechanism influencing the yield of peptide by nucleosides

4 Seryl-histidine Dipeptide: Potential Evolution Prototype of Modern Enzyme

Seryl-histidine dipeptide (Ser-His) has been identified as the shortest functional peptide with multifarious hydrolysis cleavage activity, such as the hydrolysis of protein, DNA, and carboxyl ester. Ser-His (Figure 2) has the two critical functional amino acid residues of the Ser-His-Asp catalytic triad of serine proteases. Therefore, it is worth exploring if there are some evolutionary relationships between Ser-His and modern serine proteases.

To this end, four differently folded proteins were treated with Ser-His, such as bovine serum albumen, green fluorescent protein, cyclophilin A and myog­lobin. The resulting digestion products were evaluated with high-resolution mass spectrometry. The cleavage efficiency and cleavage propensity of Ser-His against these protein substrates were calculated at both the primary and secondary sequence levels. It was found that Ser-His cleaves a broad spectrum of substrate proteins of varying secondary structures. Ser-His has the original digestion function of serine proteases to cleave at all 20 amino acids with different efficiencies according to the protein. Besides, through comparing the catalytic sites and cleavage sites of 340 extant serine proteases derived from 17 representative organisms, a consensus motif Ser-[X]- His was identified as the major pattern at the catalytic sites of serine proteases from all of the organisms represented except, which uses Ser-Lys instead. This finding indicates that Ser-His is the core component of the serine protease catalytic site. Moreover, the above analysis revealed that the cleavage sites of modern serine proteases have become more specific over the evolutionary history of this family[4].

Fig. 2 Molecular structure of Ser-His dipeptide

5 Contributions of Phosphates to the Metabolism Origin

As mentioned before, phosphates are basic components of many biomolecules and essential for modern biochemical reactions. However, most of the phosphates are either insoluble in water or have low reactivity, which is considered to be problematic for primordial biological use. To avoid this phosphorus problem, Goldfordconstructed a phosphate- free metabolic network starting from a set of prebiotically abundant compounds excluding phosphates and proposed an unconventional new view: a phosphorus-independent metabolism could exist before the emergence of the phosphate-based genetic coding system. This new opinion stimulated researchers’ interest in the systematical exploration of the role that phosphorus played in the origin of metabolism.

In Zhang’s laboratory, first, phosphorus-depen­dent protometabolic networks were constructed using the method of Goldford. with seed sets containing different prebiotically available phosphates. The obtained phosphorus-dependent metabolic net­works were composed of the same reactions, implying that the network was robust to different phosphorus sources (Figure 3). The following bioinformatics and chemical informatics analysis showed that the phosphorus-dependent metabolic network exhibits several ancient features. The enrichment of the Last Universal Common Ancestor (LUCA)-related features observed in the phosphorus-dependent network implied that a great portion of the reactions in this network also existed in the earliest life. The origins of the enzymes participating in the networks were tra­ced by the protein-domain structure-based molecular clocks.

This analysis showed that phosphorus-dependent and independent metabolic networks originated at the same evolutionary stage. By analyzing the che­mical characteristics, it was found that the metabolites in both kinds of networks have higher water solubility, stronger molecular polarity, and smaller molecular weight than modern metabolites. These findings showed that the phosphorus-dependent me­tabolic network exhibits critical ancient characters, thus both phosphorus-dependent and -independent networks have an ancient origin.

Subsequently, the influence of phosphates on the protometabolic network was studied by the KEGG pathway enrichment analysis. It was found that phos­phates promote carbon metabolism as well as car­bohydrate metabolism. These metabolic pathways are extremely important for sustaining energy flow in modern life and for the formation of the RNA world. In addition, the presence of phosphorus also promotes the production of complex metabolites and enhanced the diversity of metabolites.

Fig. 3 Construction of phosphorus-dependent metabolic network. Network expansion simulation was executed using a set of defined seed compounds and all balanced reactions in the background metabolism pool derived from the updated KEGG reactions. The figure displays the obtained phosphorus-dependent network in which metabolites are linked if they have a reactant-product relationship during the expansion. The metabolites generated at different iteration steps during the network expansion process are represented by nodes in different colors. The size of node represents the degree of the node, i.e., the number of reactions added in the subsequent iteration

To explore the influence of phosphorus on the thermodynamic feasibility of ancient metabolic system, the thermodynamically constrained network expansion with various forms of phosphates was simulated. This study found that some phosphorous intermediates of the glycolytic pathway could dramatically alleviate the thermodynamic bottlenecks and promote the expansion of the network. Further study of scale-limiting reactions during the thermo­dynamically constrained network expansion showed that the expansion of ancient metabolic network might be feasible with the presence of phosphorous intermediates such as glucose 6-phosphate and gly­ceraldehyde 3-phosphate.

In summary, the phosphorous-dependent metabolism could originate in very early stage of biochemical processes. Phosphorus can promote reactions which are essential for life and some high- energy phosphates can ensure the prebiotic metabolism under feasible energetic constraints. Ta­ken together, phosphorus is indispensable for meta­bolism origin, as material and energy sources, which provides deeper insights into the origin of life[6].

3）注水系统是油田的关键设备，以后要加强对注水水质的监测以及注水设备的定期问题排查。特别是细过滤器的填料漏失情况，要定期通过观察孔进行观察，通过滤料高度的对比（添加填料后已经做了标记），及时分析查找滤料漏失问题。

6 Origin of Homochirality and Mirror-image Biological System

The essence of life is chirality. It is well known that the protein-helix is a right-handed helix, and the structural unit amino acids of protein are all in the L-configuration. DNA is a right-handed helix in which the ribose is in the D-configuration. Therefore, the origin of life is the origin of homochirality. Up to now, the origin of homochirality in life on the Earth is still a puzzle.

It is interesting to know what happens to the mirror-image molecular system containing L-nucleo­sides during their replication and transcription and whether it would bring about deleterious effects during the evolution of nucleic acids. He.[7]previously investigated that several general DNA poly­merases and T7 RNA polymerase catalyze poly­merization reactions of nucleotides directed by the DNA template containing an L-Thymidine (L-T). It was found that mirror-image thymidine discriminates against incorporation of deoxyribonucleotide trip­hosphate into DNA and repairs itself by normal DNA polymerases.

As we have known, protein, which consists of L-amino acid, is the direct performer of vital move­ment, and weed-out of D-amino acids within living cells or organism that are necessary for the maintenance of life. A small but moderate amount of D- amino acids is also essential for life. Excess D-amino acids cause DNA oxidative damage. Some diseases of the human body are positively correlated with the content of D-amino acids in the body. D-amino acid toxicology is very important for studying certain diseases and aging mechanisms. Stereospecific enzyme plays an important role in the metabolism, which controls the balance of L or D-amino acids. D-amino Acid Oxidase (DAAO) catalyzes the oxidative deamination of a majority of D-amino acids rather than L-amino acids. Once the enantioselectivity of DAAO is disturbed, the metabolic systems might be disordered, which might be found as precipitating factors of chiral disease. Enzymatic enan­tioselectivity of amino acids with DAAO could be controlled by adjusting the pH of aqueous solution and adding ionic liquid, which means that the external chemical environments are likely to affect the enzyme specificity[8]. Additionally, D-amino acids occur widely in food. The related reactive oxygen species accumulation and biochemical damage caused by D-amino acids are evaluated by usingas a model[9]or in beer[10].

The racemization of amino acids is liable to happen under extreme conditions, such as space ra­diation and strong acid or alkalinity, microbial fermentation. Therefore, the space response of chiral molecules, such as amino acids, chiral medicine and so on, is worth obtaining more attention, which is of great importance for space medicine and astronaut’s health.

The chiral selection rules above-mentioned are for the life system on the Earth. How about extraterrestrial life? Does the extraterrestrial life have the same rules with terrestrial life? The search for mirror-image biology systems in the universe may open the next frontier for biomedical technology development and discovery. Zhu’s group at Tsinghua University first demonstrated that two key steps in the central dogma of molecular biology, the template-directed polymerization of DNA and transcription into RNA, can be catalyzed by a chemically synthesized D-amino acid polymerase on an L-DNA template[11]. The two chirally mirrored polymerase systems can operate in a racemic mixture without obvious enantiomeric cross-inhibition to the activity of each other. Additionally, a mutant version of the thermostableP2 DNA polymerase IV (Dpo4) consisting of D-amino acids was designed and chemically synthesized, and used for realizing mirror-image PCR[12]. More recently, they further demonstrated the transcription of a mirror- image gene into L-RNA, and reverse transcription into L-DNA by synthetic D-polymerases based on designed mutants of the mirror-image Dpo4[13].

Furthermore, a practical method for sequencing mirror-image DNA by adopting the Maxam-Gilbert sequencing approach was developed through nucleobase-specific cleavage by achiral chemicals. The technique may facilitate the therapeutic application of nuclease-resistant L-aptamer drugs, and bring the vision of building an alternative, mirror-image self- replicating system closer to reality[14].

After the realization of mirror-image genetic replication, transcription, and reverse transcription, the big remaining challenge in establishing a mirror-image version of the central dogma of molecular biology is to build a mirror-image ribosomal translation apparatus. Zhu’s group reported the chemical synthesis of three ribosomal proteins (L5, L18, and L25) in the large subunit of theribosome with post-translational modifications, which can fold properly in vitro and assemble with enzymatically transcribed mirror-image 5S ribosomal RNA into stable ribonucleoprotein complexes through chiral- specific binding[15].

In summary, a mirror-image form of life based on D-amino acids and L-nucleic acids, is conceivable and is possible to exist on other planets. The above­mentioned ground-based research may provide experimental support for the exploration of extraterrestrial life.

7 Progress of Survival and Evolution of Earth-originating Extremophiles in Space Environment

How does the Earth life deal with and adapt to the extreme environment such as cosmic ray, anoxia and dryness of space is not only a key scientific problem to understand the origin and evolution of life on the Earth, but also a technical problem to be solved for human beings to realize “livable” in the extraterrestrial space such as Mars. Deep space exploration driven by the research on the origin, evolution, and livable environment of life has become the consensus of the international scientific community. At present, the research on the survival ability and stress response of related organisms in the space environment is mainly ground-based experiments. With the initiation of a series of National Science and Technology Major Projects, including the China Manned Space Station Project and Deep Space Exploration mission,., an unprecedented opportunity for space life sciences has been provided. Because of the stress- tolerance of various extremophiles on the Earth, it is of great scientific value using the extremophiles as model organisms to explore the survival and evolution of life in extraterrestrial space and even Mars. These researches will greatly help to develop the knowledge and techniques to enable life on the Earth to be livable in space.

Extremophiles refer to the microorganisms that can live in various extreme environments of the Earth, including irradiation and desiccation. They are unique biological resources and precious research materials left to human beings by this planet. For example,is one of the most radioresistant organisms on the Earth. It is not only tolerant of strong ionizing radiation and ultraviolet radiation, but also endure extreme environment such as oxidative stress and desiccation, and its radioresistant ability of ionizing radiation is 200 times of that ofand more than 1000 times of that of human beings.is a kind of strictly anaerobic archaea emerging in the early stage of life evolution on the Earth, which can withstand extreme environments such as hypoxia and low temperature. For example,isolated from Antarctica has a unique cold adaptation mechanism.

For many years, Hua[16,17]have been engaged in the research on the stress-resistant mechanism of. It was found that the extraordinary resistance ofwas attributed to its efficient DNA damage response and repair system. The global regulatory factor PprI (Inducer of Pleiotropic Proteins Promoting DNA Repair) and its DNA damage response regulatory pathway were identified. A series of important fin­dings are as follows: PprI is a new type of protease, which can specifically cleave the substrate protein DdrO (DNA damage response protein O). The novel DNA damage response regulatory pathway mediated by PprI-DdrO regulates the transcription of more than 20 genes involved in DNA damage response and repair related, and the pathway is different from the classical SOS pathway[18, 19]; the research on the new pathway has found many functional genes, and expanded a number of new functions of genes[20]. The connection of multiple functional modules in the cell was completed, and a new idea that the stress response of cells is closely related to DNA damage repair was proposed. In addition, a new technology was developed using the PprI as a regulatory element to enhance the resistance of some microorganisms and plants to various stresses[21]. PprI can improve the resistance of microorganisms (,) or plants (corn,.) to stresses such as osmotic pressure, oxi­dation, or high temperature, demonstrating a good application prospect. These findings and techniques laid a solid theoretical and application foundation for exploring the origin and evolution of life and the utilization of extreme microorganism resources.

8 Planned Experiments On-orbit and on Manned Space Experiment Platform

In 2019, we have planned to carry out projects including the experiment of returning space payload carrying the Earth’s extremophile and an application mission project in China’s manned space station. These projects take extremophiles of the Earth as the model organisms, and investigate the survival ability and the changing pattern of genome, transcriptome and proteome in the extreme environment of the space through space flight or space station in-situ experiments, so as to reveal the survival ability, stress response, evolution and diversity formation mechanism of organisms in the space environment. The implementation of the project will greatly pro­mote the research on the survival and evolution of life on the Earth in the extraterrestrial space. Meanwhile, it will provide key technologies for the Earth’s life and ecosystems suitable for survival in the Mars environment.

The experiment of returning space payload carr­ying the Earth’s extremophiles has cooperated with China Academy of Space Technology (CAST). The stress response mechanism of radiation-resistantin space environment will be studied by using unmanned spacecraft. The objective is to reveal the changing pattern and regulatory network of genetic material and proteins in the radioresistant bacterium under the extreme environment of space. The results are of great scientific significance to evaluate the living ability and stress response of organisms to the space environment, and understand the evolution and diversity formation of organisms in the early extreme environment of the Earth. The project is in the stage of launch preparation, and the samples have been sent to the launch site. It is expected to launch and complete the experiment in 2020.

The application mission project in China’s manned space station “The survival and evolution of the Earth or man-made anaerobic life in the Martian environment” is in the application stage. The project objective is to research the survival and evolution of the Earth life in the simulated Martian environment in China’s manned space station. The manned space experiment platform’s instruments support two wor­king modes: in-place operation experiment and remote control experiment. During the on orbit operation of the space station, the microorganism ana­lysis payload, life ecology experiment payload, space biology exposure experiment device, and the radiation effect analysis platform will be used to carry out the radiation biology and omics analysis of microorganisms. We planned to investigate on the extreme environmental adaptation mechanism and re-cons­truction of a variety of extreme microorganisms (,,and), including the following aspects.

(1) To explore the stress-response, adaptation and change of life phenotype at different levels of components (cells, organelle, biological macromolecules) after entering space, and explore the survival and evolution of life in extraterrestrial space and even Mars environment. Through interdisciplinary research and comprehensive research, the expected results will reveal the survival of life in the extreme environment of space, the evolution of its genome, transcriptome and proteome, and clarify the stress response mec­hanism of DNA repair, regulatory factors, metabolic synthase and other important proteins in space.

(2) To investigate the adaptability of artificial mo­dified anaerobic life in space. Through the fusion and transformation of the genome, new species that can survive in the space environment similar to Mars are selected and constructed, which provides the basis for the subsequent improvement of the soil ecosystem of Mars and the establishment of Mars agricultural base.

These studies are planned to set up ground- based experiments simulating the space environment, such as hypoxia and radiation, and carry out the parallel experimental study of biological samples.

9 Future Expectations

With the construction of Chinese manned space station and recoverable satellite science experimental platform, more and more space-based experiments focusing on the origin of life have opportunities to be carried out. In the future, at the molecular level, there are some key issues that should be further explored to well-understand the origin of homochirality and genetic code. For examples, which are critical factors to induce the chiral symmetry breaking? Is there the common genetic code between extraterrestrial and terrestrial life? What will be the potential biomarker for extraterrestrial life exploration besides water? How about oxygen isotope ratios of PO43? Additionally, the photochemical response behavior of chiral molecules, such as amino acids, chiral medicines, and so on, under space extreme environments should further get more attention.

To take extremophiles as the model organism, through the research on extremophiles in space environment including space on-orbit flight and China’s manned space station in-situ experiments, the expected results will reveal the survival of organisms in the extreme environment of space, the changes and evolution of their genomes, transcriptome and proteome,. The results will shed light on the stress response mechanism and key factors in the interaction between stresses such as cosmic rays and organisms. At the same time, by using synthetic bio­logy and other technologies, through genome fusion, modification, and transformation, the starting species will be constructed to adapt to the Martian environment. The important role of man-made organisms in the synthesis of energy sources, carbon sources and ecosystem construction will be investigated further, so as to provide technical support for the research and improvement of the space adaptability of the Earth’s organisms.

The relative researches on the origin and evolution of life should execute more sufficient and extensive interdisciplinary cooperation, involving as­trobiology, chemistry, biology, geology, space science and technology, and so on. If the special fund will be established to support these relevant studies, it will be better to promote the development in this research field.

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ZHAO Yufen, HUA Yuejin, ZHANG Hongyu, HE Yujian, ZHU Ting, LIU Yan, WU Li. Progress of Research on Origins of life in China., 2020, 40(5): 937-945. DOI:10.11728/cjss2020.05.937

March 21, 2020

E-mail: stacyliu@xmu.edu.cn