Difference between revisions of "Team:CAU China/Description"

In China, the theoretical amount of crop stalk is 1.04 billion tons per year, and the collectable amount is 900 million tons [1]. The lignocellulose in crop stalk is a good carbon source and has a high recycling value. However, the methods of stalk disposal in China are still relatively primitive - burning is still the main method of stalk disposal in a wide range of rural areas. According to the estimation of the proportion of open burning fire points monitored by satellite remote sensing of the Ministry of Environmental Protection, the amount of open burning stalk in China in 2015 is about 81.1 million tons, and the total carbon emission is about 34.5 million tons¹. Burning stalk not only fails to utilize the rich carbon source in it, but also brings burden to the environment. Therefore, it is urgent to find environmentally friendly, economical and reasonable stalk degradation methods.

At present, there are five comprehensive utilization methods of stalk in China, which are fertilizer, feed, substrate (used for edible fungi), fuel and raw material (used for paper making or board making). Nevertheless, these five utilization methods only deal with stalk simply and the added value of the products is relatively low, which can not make up for the manpower and technical cost in the process of stalk recycling. With stalk fuel into the case, 1 t stalk can produce about 50 kg (59 L) fuel². At present, biomass fuel produced in China can only reach the standard of 0# diesel oil, whose price is about 6.30 yuan/L. As a result, the biomass fuel output value of 1 t stalk is about 371 yuan. In spite of that, after visiting the farmers, it is known that the labor wage of recycling 1 t of corn stalk is about 400 yuan, not to mention the running cost of equipment in the factory. Therefore, most biomass energy enterprises can only rely on state subsidies to maintain operations³.

In order to make stalk treatment enterprises profitable without subsidies and fundamentally solve the problem of stalk incineration, it is necessary to carry out in-depth treatment of stalk and produce high value-added products. Astaxanthin is the most powerful antioxidant found in nature. It has a wide range of health care functions, including fighting high blood pressure by reducing oxidative stress and relaxing blood vessel walls and even inhibiting cancer metastasis. Astaxanthin has a promising market, with over 98% pure products sold at SIGMA for up to $200 /50 mg. Hence, we decided to construct an engineering Escherichia coli using cellulose to produce astaxanthin to deal with the dilemma of stalk treatment in China.

There are a variety of enzymes that degrade cellulose, such as β-1,4-glucanase, β-1,4-glucanase and β-glucosidase from Streptomyces azure. These three enzymes work together to degrade cellulose into glucose. Cellulose, however, has a high molecular weight, is insoluble in water, and cannot enter the cells of e. coli, which means we need to display the three cellulose degrading enzymes on the surface. Ice Crystal Nucleoprotein (INP) is a kind of secreted outer membrane protein, which is widely distributed in Pseudomonas Syringae, Pseudomonas fluorescens and other gram-negative bacteria. Compared with other surface technology carrier proteins, INP has the advantages of stable expression of foreign proteins and can display foreign proteins with larger molecular weight⁵.

The enzymes involved in each step of the astaxanthin synthesis pathway have been well understood, which lays a foundation for our construction of engineered strains. Astaxanthin is a terpenoid compound, which can be synthesized by FPP, the intermediate metabolite of e. coli. The synthesis of astaxanthin requires the catalysis of six key enzymes, and the detailed metabolic process is shown on the left. Due to the whole genome sequencing of a large number of valuable microorganisms, the genes related to astaxanthin biosynthesis can be found from a variety of microorganisms. It gives us the opportunity to select those microorganisms with high activity of astaxanin-related synthases that are easy to culture as our target gene source.

In order to realize the conversion from cellulose to astaxanthin, we need to transfer the cellulose degradation genes and astaxanthin synthesis genes into e. coli. Considering we need to transfer a large number of genes, we divided the project into two parts: degradation of cellulose and production of astaxanthin. After realizing the functions of the two parts, they will be integrated together. In the part of cellulose degradation, we need to link β-1,4-glucanase gene, β-1,4-glucanase gene and β-glucosidase gene from Streptomyces azure with INP respectively, anchor them on the outer membrane of e. coli, and then express them on the surface. In this way, e. coli can come into contact with cellulose in the environment and degrade it.

In the part of astaxanthin biosynthesis, we took advantage of the original DXP pathway from e. coli and transferred six foreign genes into it, therefore directed the metabolic flow from FPP to the astaxanthin biosynthesis pathway. In the artificially constructed astaxanin synthesis pathway, the first four genes CrtE, CrtB, CrtI and CrtY can be obtained directly from PCR amplification of bacterial genomic DNA, while the last two genes CrtZ and BKT need codon optimization and modification, so we decided to obtain these two genes by chemical synthesis. All six genes need to be integrated into one plasmid to avoid significant differences in protein expression due to plasmid copy number differences. We selected a low-copy inducible plasmid (pACYC184-M) as the vector of these six genes, it can make e. coli begin to synthesize astaxanthin after passing the logarithmic growth period. Besides, the lower copy number of plasmid can maintain the amount of astaxanthin at a more appropriate level, thus avoiding e. coli poisoning caused by excessive astaxanthin accumulation.

Our ultimate goal is to integrate cellulose degradation and astaxanthin synthesis into a single strain, which means that we need to co-transform plasmids carrying cellulose degradation and astaxanthin synthesis functions into e. coli, or integrate genes responsible for cellulose degradation into the genome of e. coli. Both of these schemes involve a lot of work, so we may first try to experiment with the conversion of cellulose to astaxanthin in a mixed culture.

1. Shi Zuliang; Jia Tao; Wang Yajing; Wang Jiuchen; Sun Renhua; Wang Fei; Li Xiang; Bi Yuyun.Comprehensive utilization of crop stalks and estimation of incineration carbon emissions in China [J] .Agricultural Resources and regionalization of China, 2017,38 (9).
2. Tianjin University Scientific Research Project. Stalk "Changes" into Gas Oil [J] .Resource Conservation and Environmental Protection, 2016 (02): 7.
3. ZHU Hao, HU Qichun, TANG Xiaoyu, LI Qian.Progress of fuel utilization and development of crop stalk resources in China [J] .China Biogas, 2017,35 (02): 115-120.
4. Wang Hongmei, Tu Yan, Zhang Naifeng, Si Bingwen, Ma Tao, Sa Zhula, Na Ya, Diao Qiyu.Current situation of crop stalk resources and utilization of "five materials" in China [J] .science and Technology Review, 2017,35 (21): 81-88.
5. Li Mingya, Lin Chenshui, Limingya, et al. Ice crystal nucleoprotein and its application in bacterial surface display [J] .amino acids and biological resources, 2016,38 (2): 7-11.
6. Park, S. Y., Binkley, R. M., Kim, W. J., Lee, M. H. & Lee, S. Y. Metabolic engineering of Escherichia coli for high-level astaxanthin production with high productivity. Metabolic engineering 49, 105-115, doi:10.1016/j.ymben.2018.08.002 (2018)