Inspired by waste of straw resources and air pollution, we set out to develop a strain that aims to produce lycopene high yields through gene editing technology. Through biosynthetic methods, we researched, designed, created and tested potential solutions to this problem and now present a proof of concept. On this page, we will discuss the problem at hand, the solutions we propose, and the outlook for the future.
1.1 China is a large agricultural country, straw resources are huge, the average annual output is about 865 million tons, the resource utilization rate is 81.68% [1], China’s straw resources are mainly fertilizer utilization, the utilization rate is 53.93%, feed and fuel utilization are supplemented, the utilization rate is 23.42% and 14.27%, and the utilization rate of base material and raw material is low, 4.89% and 3.40%[2]. With the adjustment of industrial structure and the improvement of residents’ living conditions, there are regional, seasonal and structural excess problems in straw production, resulting in the phenomenon of piling up or concentrated burning of a large number of straw in the field. This not only seriously pollutes the environment, but also causes a lot of waste of resources. Therefore, improving the comprehensive utilization efficiency of straw resources has a promoting effect on the improvement of environmental quality and the comprehensive green transformation of economic and social development.
1.2 The impact of high temperature stress caused by global warming on vegetable production is becoming increasingly serious. In the past 100 years, the global average temperature has increased by 0.5°C, and the global average temperature will increase by 0.4°C after 20 years, and the global average temperature is expected to rise by 1.0~3.4°C by 2100. High temperature and drought stress seriously limit the yield of vegetables, bring huge losses to the agricultural economy, if traditional methods, the quality and yield of tomatoes are difficult to guarantee, and directly extract lycopene from tomatoes, but also waste a lot of food resources. Through our learning and understanding, we want to use the CRISPR-MAD7(Cas12a) gene editing system to engineer Corynebacterium glutamicum to achieve high levels of lycopene production. While meeting the demand for high value-added products on the market, these measures can make better use of straw resources, thereby creating favorable conditions for the wide application of lycopene and alleviating problems such as environmental pollution and waste of food resources.
Lycopene is a compound belonging to carotenoid pigments that are widely found in a variety of fruits and vegetables, especially in tomatoes. In addition to its use in natural colors, lycopene is increasingly recognized for its role in functional foods, pharmaceuticals and cosmetics. At the same time, lycopene is the main bioactive component in a variety of plants, with pharmacological effects such as anticancer, antioxidant, heart protection and blood pressure reduction.
The human body cannot make lycopene on its own and needs to be supplemented from food[3]. Given the potential application prospects of lycopene, its production has been studied. At present, there are three production methods of lycopene: plant extraction, chemical synthesis, and biosynthesis[4].
The plant extraction method mainly extracts and purifies lycopene from mature plant fruits such as tomatoes, but this method is unstable due to various factors such as region, season, tomato product and ripeness. In China, tomatoes planted in Xinjiang (long sunshine time) are mainly selected to extract lycopene, but the content of lycopene in tomatoes is very low, generally only 20 mg/kg, even in the local content of high tomato peel is less than 0.4 g/kg tomato peel[5],the extraction cost is high, the extract often contains other carotenoids, affecting the purity of the product, and due to the low content, the extraction process consumes a lot of organic solvents, and the environmental pollution is greater.
The chemical synthesis method is mainly composed of octatrienedialdehyde and triphenylphosphorus chloride or triphenylphosphonium sulfonide (Wittig reaction) to synthesize lycopene[6]. The chemical synthesis method has the characteristics of high yield (more than 65%), cheap and recoverable raw materials, and mild reaction conditions. Although the chemical synthesis method has high yield and low cost, isomers are prone to occur due to the large number of double bonds in the structure of lycopene, and the products of the chemical synthesis method have chemical reagents and intermediate product residues, resulting in certain toxicity and carcinogenicity, limiting product quality and scope of use[7], and there are safety risks.
To the extent that existing methods are dangerous, inefficient, and environmentally polluting, it is necessary to take a better approach to solve this problem.
The biosynthesis method allows microorganisms to ferment and produce lycopene using abundant and readily available raw materials such as sugars, corn syrup, and inorganic salts. The microbial fermentation method not only has the safety of plant extraction method (all natural biological metabolic sources, non-synthetic), but also has the advantages of low cost and large-scale production of chemical synthesis method, and is considered to be an ideal method for the production of lycopene in the future[4].
Corynebacterium glutamicum ATCC13032 is an important industrial microbial model strain and food-grade safe strain in current biotechnology, Corynebacterium glutamicum has the advantages of no endotoxin, greater metabolic flux and strong secretion ability. It has been widely used in the production of various amino acids, organic acids, alcohols, diamines, aromatic compounds and terpenes. As a chassis microorganism that can produce complex terpenoids, Corynebacterium glutamicum has advantages over other engineering strains in the production of lycopene, and was selected as an excellent engineering strain in this project.
| CRISPR/Cas9 | CRISPR/MAD7(Cas12a) | |
| Cas protein size | 1000-1600 aa | 1200-1300 aa |
| crRNA length | 100-200 nt | 42-44 nt |
| crRNA type | Double strand | Single strand |
| Original spacer sequence proximity motif (PAM) | 3’-GGN(SpCas9) | 5’-TTN(FnCas12a) |
| Endonuclease domains | RuvC and HNH | RuvC and Nuc |
| Processing pre-crRNA | RNaseIII | Cas12a |
| Type of incision | Flat end | Sticky ends |
| Multi-gene editing efficiency | low | high |
3.2.1 CRISPR/MAD7(Cas12a) crRNA is significantly shorter than other types of CRISPR systems, and the secondary structure is simpler, which means that designing, synthesizing, and transcribing crRNA will be more economical and convenient.
3.2.2 CRISPR/MAD7(Cas12a) can realize tandem multiple crRNAs for multigene targeted editing, which is almost the same as the efficiency of single gene editing[8].
3.2.3 Cas12a is a single RNA-guided endonuclease enzyme, and its complexes are sufficient to mediate DNA targeting. In contrast, Cas9 requires crRNA and tracrRNA double strands to mediate targeted DNA.
3.2.4 At the same time, the Cas12a nuclease catalytic site can process pre-crRNA by itself, without RNaseIII, which makes up for the defect of low RNase III. activity in some species and improves DNA cleavage efficiency[9].
3.2.5 Cas12a cutting DNA strand produces a prominent sticky end notch at the 5nt5’ end, and the cutting site is far from the PAM site, which will promote precise gene replacement of non-homologous end joining (NHEJ) and homology directed repair (HDR); Cas9, on the other hand, produces a flat-end gap and is inefficient in gene replacement[10].
Studies have shown that dilute acid pretreatment removes hemicellulose from lignofiber raw materials, so that more cellulose is exposed, which is conducive to cellulase interaction with it, thereby improving the enzymatic hydrolysis conversion rate[11][12].
So our project uses dilute sulfuric acid to pretreat wheat straw, and then uses cellulase to enzymatically hydrolyze saccharification, and finally obtain the glucose solution we need, compared with the standard glucose solution produced after straw pretreatment, straw sugar can provide a relatively stable carbon source for the growth of Corynebacterium glutamicum, and promote the production of lycopene by Corynebacterium glutamicum. This can not only solve the problem of waste of straw resources, but also reduce straw burning, thereby solving the problem of environmental pollution, and then promoting the comprehensive utilization of straw, which is of great significance and far-reaching impact for stabilizing agricultural ecological balance, alleviating resource constraints and reducing environmental pressure.
Returning straw to the field will harm agricultural production, and if the straw is burned, it will not only cause waste of resources, but also cause pollution to the environment. The development of green circular agriculture is an important way to achieve sustainable development. Biomass energy represented by crop straw resources is a recyclable, clean and pollution-free renewable energy, and the development and utilization of crop straw resources can effectively alleviate the contradiction of energy supply shortage and reduce the damage to the ecology and environment caused by original energy use[12].
While realizing the mass production of lycopene, we wanted to measure the physicochemical properties of lycopene and make lycopene complex products that prevent oxidation and extend shelf life on the basis of lycopene antioxidant. At the same time, the color change characteristics of lycopene composite products after oxidation can also be designed to detect the storage time and temperature and shelf life of articles[13].
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