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.
Research Foundation
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.
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. 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.
The iGEM team CAU_China is in conformity with iGEM`s safety and security regulations and CAU laboratory safety regulations. All our experiments are conducted in the Innovation Laboratory for Undergraduates, which is in the charge of Professor Qinhong Cao. And our laboratory is classified as Biosafety Level 1(McLeod, 2010; NIH, 2019), hence no pathogenic or infectious organisms posing any hazard are allowed to be used.
All our team members have finished the course Safety Protection of Laboratory during the previous semester and have been trained to conduct specific procedures before they start experiments about our project. We follow the laboratory regulations according to safety and security regulations of the Innovation Laboratory for Undergraduates, the full form can be found here.
For the consideration of laboratory safety when different team members conducting various experimental procedures, apart from complying with the existing lab rules, we have made and implemented several new rules applied to our team.
Not all team members are experienced with molecular cloning and biochemical experiments at first, thus, we require all our team members, especially the sophomore members, not to conduct experiment alone, and each sophomore member is assigned to a junior member as a partner when conducting an experiment.
We should conduct experiments in our iGEM-specialized area and clean up the bench after work. Except the marked items in the iGEM Reagent List, all other reagents should be purchased by ourselves, and our member Ruogu Liu, as our purchasing agent, is in charge of purchasing and ordering matters.
When using autoclave, we should ask a graduate student to oversee the operation.
All re-usable equipment such as glassware containing experimental organisms waste should be washed with disinfecting water to eliminate all living organisms before washing with detergent.
Project safety
Our project of this year involves several different microorganisms. Apart from Escherichia coli BL21 cells as chassis, Rhodobacter sphaeroides, Rhodospirillum rubrum, Pantoea agglomerans, and Streptomyces coelicolor are used as gene donors. According to the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules(NIH, 2019) and ABSA International’s Risk Group Database("ABSA Risk Group Database,"), all the organisms mentioned belongs to Risk Group 1, which means they are generally safe and pose no hazard to the human body or environment. And since we have specific rules for our microbial waste, all living cells in the waste would be killed by chlorine bleach or autoclave to ensure no environmental harm occurs. Moreover, the genes from donors we use are naturally located in the genome of organisms with no artificial engineering, therefore, they would do no harm even if they escape from the laboratory conditions. Additionally，considering the impact of antibiotic resistance, our Escherichia coli strains were screened by chloramphenicol, ampicillin and kanamycin.
In addition, since the experiment involves various organic reagents such as acetone, we require the experimenter to wear a white coat, a mask and gloves to conduct the experiment under the specified environment. After use, we have a special waste bottle for recycling and collective treatment, thus will not cause environmental pollution.
As for the potentially dangerous experimental facilities such as ultrasonic crushing apparatus involved in the experiment, all operations were completed under the guidance and supervision of experienced graduate students, thus avoiding the risk that may be caused by improper operation of the experiment,
