Project background
In China, the theoretical amount of crop stalks is 1.04 billion tons per year, and the collectable amount is about 900 million tons1. The lignocellulose in crop stalk is a good carbon source and has a high recycling value. However, the treatment of wasted stalk in China are still relatively primitive - burning is 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 stalks not only fails to utilize the rich carbon source in it, but also brings heavy burden to the environment. Therefore, it is urgent to find the environment-friendly, economical and effective stalk degradation methods.
At present, there are five comprehensive utilization methods of stalk in China: fertilizer, feed, substrate (used for edible fungi), fuel and raw material (used for paper making or board making). Nevertheless, these five utilization methods only treat stalks at the primitive level and the added value of the products is relatively low, which even cannot make up for the manpower and technical cost in the process of stalk recycling. Take the biofuel made by stalks as an example , 1 ton stalks can produce about 50 kg (59 L) fue2. At present, biomass fuel produced in China can only reach the standard of 0# diesel oil, whose price is about 6.30 RMB/L. As a result, the biomass fuel output value of 1 ton stalks is about 371 RMB. Additionally, after visiting the local villagers, we learned that the labor wage of recycling 1 ton of crop stalks is about 400 RMB, not to mention the running cost of equipment in the factory. Therefore, most biomass energy enterprises in China have to rely on state subsidies to maintain their operations3.
In order to make enterprises of stalks treatment profitable without subsidies and fundamentally solve the problem of stalk incineration, it is necessary to carry out in-depth treatment of stalks and produce products with higher additional values. Astaxanthin is the most powerful antioxidant found in nature so far. It has been applied to a wide range of health care products, in which it functions to fight against high blood pressure by reducing oxidative stress, relax blood vessel walls and even inhibit cancer metastasis. As a natural ingredient, astaxanthin has a promising market, products with over 98% purity sold at SIGMA can be up to $200 /50 mg. Hence, we intended to engineer Escherichia coli to consume cellulose and meanwhile produce astaxanthin to deal with the stalk degradation dilemma in China.
Research Foundation
There are a variety of enzymes that degrade cellulose, such as β-1, 4-endoglucanase, β-1,4-exoglucanase and β-glucosidase from Streptomyces Coelicolor. These three enzymes work together to degrade cellulose into glucose. Yet the large molecular weight of cellulose prohibit it to be dissolved in water and enter the E. coli cells. Ice-nucleation protein (INP) is a secreted outer membrane protein, which is widely distributed in Pseudomonas syringae, Pseudomonas fluorescens and other Gram-negative bacteria. Compared with other surface carrier proteins, ice-nucleation protein has the advantage of stably expressing heterogeneous proteins and displaying proteins with larger molecular weight5, which inspired us to employ the property of INP to display the three cellulases on the cell surfaces so that the E.coli cells are able to break cellulose outside the cell and intake the glucose as the energy resource. Moreover, the N terminus of INP(INP-N), which is much shorter than the intact INP or INP-NC but still functions as anchoring sequence, is employed in our project.
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(Farnesyl pyrophosphate), 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 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 astaxanthin-related synthases that are easy to culture as our target gene source.
Our project:
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 cells. Considering that we need to transfer a number of genes, we divided the project into two subsections: degradation of cellulose and production of astaxanthin. After realizing the functions of the two subsections, they will be integrated together. In the subsection of cellulose degradation, we plan to join β-1, 4-endoglucanase, β-1,4-exoglucanase and β-glucosidase with INP respectively. The INP-cellulases are induced by IPTG and anchored on the outer membrane surface of the cell, which allows E. coli cells to contact the cellulose on their cell surface and degrade it.
In the subsection of astaxanthin biosynthesis, we took the advantage of the original DXP pathway from E. coli and transferred six more heterogeneous genes into the cell, therefore directed the metabolic flow from FPP to the astaxanthin biosynthesis pathway. In the artificially constructed astaxanthin synthesis pathway, the first four genes CrtE, CrtB, CrtI and CrtY can be obtained from PCR amplification of bacterial genomic DNA, while the last two genes CrtZ and CrtBKT require codon optimization and modification before transferred, so we decided to obtain these two genes by direct 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 employ a low-copy inducible plasmid (pACYC184-M) as the backbone of these six genes, it allows E. coli to start to synthesize astaxanthin after passing the logarithmic growth period. Besides, the lower copy number of the plamids can maintain the amount of astaxanthin at a more appropriate level, thus avoiding poisonous effect caused by excessive astaxanthin accumulation.
Our ultimate goal is to integrate cellulose degradation and astaxanthin synthesis into a single strain, which may require co-transfer of plasmids carrying cellulases and astaxanthin synthesis enzymes.
References
- 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).
- Tianjin University Scientific Research Project. Stalk "Changes" into Gas Oil [J] .Resource Conservation and Environmental Protection, 2016 (02): 7.
- 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.
- 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.
- 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.
- 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)
