World’s first CRISPR-edited sugarcane developed in Brazil

The Flex sugarcanes respond to challenges of the sector and facilitate the production of (first and second generation) ethanol and the extration of other bioproducts. In the photo, Flex II

Scientists from Embrapa Agroenergy developed the first gene-edited sugarcanes that are considered non-transgenic in the world (through DNA-free genome editing), according to the Normative Resolution 16 (RN 16) by the Brazilian National Technical Commission on Biosafety (CTNBio) published on Dec 9, 2021. They are the Flex Iand Flex II sugarcane varieties, which respectively offer higher cell wall digestibility and higher concentration of sucrose in plant tissues. They respond to one of the biggest challenges of the sector: increasing the access of enzymes to the sugars imprisoned in the cells, which facilitates the production of ethanol (first and second generation) and the extraction of other bioproducts.

The Flex I sugarcane is the result of silencing the gene responsible for the rigidity of the plant’s cell walls. This structure was modified and showed higher “digestibility”, that is, allowed more access to enzyme attacks during the stage of enzymatic hydrolysis, a chemical process that extracts compounds from plant biomass.

Flex II: more sucrose

Meanwhile the second variety was generated by silencing a gene in the plant tissue, which caused a considerable increment in the production of sucrose in the culms of the model plant, Setaria viridis.

Hugo Bruno Correa Molinari, Researcher of Embrapa Agroenergy

“Once this trait of sugar accumulation was identified in the model plant, we transferred this knowledge to sugarcane, the target of our research. Again an increment in sucrose of around 15% in the sugarcane culms was observed, alongside an increase in other sugars that are present in the plant like glucose and fructose, both in the plant and in the fresh plant tissue”, explains the Embrapa researcher Hugo Molinari.

The team also observed increments of around 200% in sugar in sugarcane leaves. “We also performed tests to see if the gene had action towards improving saccharification, which is the conversion of cellulose into industrial sugar, and we observed an increment of around 12%”, the researcher adds.

Molinari lists some of the advantages of the Flex II sugarcane: increased efficiency in bioethanol production, the discovery of a variety that is more suited to industrial processing, the obtainment of bagasse with higher digestibility for use in animal feed, and aggregating value to the sugarcane production chain as a whole.

“In 2020/2021, the estimated total sugar production in the world was 188 million tons, and Brazil was responsible for 39 million tons, the equivalent to 21% of the world production”, Molinari asserts.

Another point underscored by the researcher is sugarcane’s contribution to a cleaner energy mix. “Today we know that more than 45% of the Brazilian energy mix is renewable and that sugarcane contributes with a share of over 30% of such renewable sources,” he informs.

Flex I

Research used revolutionary genome editing technique

Embrapa Agroenergy had already been studying genes related to acyltransferases, enzymes responsible for the formation and modification in the structure of plant cell walls and that allow access to sugar. “Specifically in the case of the Flex II sugarcane, our group identified a candidate gene belonging to the family of acyltransferases that proved to be a very promising and viable biotechnological asset to increase the production of sugars in grasses”, the researcher explains.

Both studies used CRISPR (Clustered Regularly Interspaced Shorts Palindromic Repeats), a revolutionary technique to manipulate genes discovered in 2012. The technology uses the Cas9 enzyme to cut DNA in given points, modifying specific regions. The discovery yielded the 2020 Nobel Prize in Chemistry to the researchers who published the first paper on the subject: Emmanuelle Charpentier and Jennifer A. Doudna.

Developing the Flex I and II sugarcanes thus did not entail changing the DNA of the plant, it only involved the silencing of the genes. That is why, the National Technical Commission on Biosafety (CTNBio) classified the new varieties as non-transgenic or not genetically modified.

“The controversy around the use of transgenic plants in agriculture has made each country in the world create specific regulation on the subject, which escalated the cost of inserting genetically modified (GM) varieties in the market. Nowadays we have seen the rise of a new technology, genome editing, with which the introduction of exogenous sequences from other species into the genome of the target species is no longer necessary”, Molinari reports.

The technological development of sugarcane crops throughout time was the main factor responsible for the expansion of the sector. – Photo: Hugo Molinari

According to the scientist, although transgenics remains an important strategy to solve countless problems in agriculture and to add value to species, genome editing with techniques like CRISPR allow for more precise, expeditious and economical manipulation of DNA in comparison with transgenics.

“The CRISPR technology has allowed a democratization of the use of biotechnology in agriculture, not only from the point of view of more companies and institutions participating in the development of products that reach the market, but also by allowing more species of interest to benefit from it”, Molinari explains. According to him, the estimated cost for the development of a GM plant is of about US$ 136 million and between 30% and 60% of this amount is aimed at the deregulation stages.

Molinari recalls that the technological development of sugarcane crops throughout time was the main factor responsible for the expansion of the sector. For decades, different research groups in the world have dedicated basic research efforts towards a better understanding of plant sugar metabolism and control during plant development in model species. “Sugar metabolism is well-known today, as the integration of several enzymes and the metabolic processes of transportation and accumulation have been unveiled”, the researcher observes.

According to the deputy head of Research and Development at Embrapa Agroenergy, Bruno Laviola, othe development of new sugarcane cultivars throught the CRISPR technique is at the frontier of knowledge. “These cultivars are only the beginning and they pave the way for the development and delivery of other cultivars for the production sector with characteristics that will directly impact on the productivity of sugarcane and reduce production costs,” he announces.

Flex II: minimal return on investment of 10% a year

With the aid of the economist Rosana Guiducci, researcher of Embrapa Agroenergy, the Flex II sugarcane variety was analysed in different adoption scenarios and there was an assessment of economic impacts in the sugar and energy sector. The analysis was the subject of Molinari’s MBA thesis, and the economist was his co-advisor.

The work conducted in his MBA aimed at assessing the economic viability of this new variety regarding the increase in sugar content and better exploitation of bagasse and straw for the production of second generation ethanol (2GE).

To estimate economic gains with the adoption of the technology, the study assessed two possible scenarios, an optimist one and a conservative one. The first one would be the gradual expansion of the adoption of the Flex II sugarcane in 1% a year, reaching 10% of the production observed in the 2020/2021 sugarcane harvest in Brazil within ten years.

Flex II

In the second, more conservative scenario, the expansion rate would be of 0.5% a year, reaching 5% of the sugarcane yield observed in the 2020/2021 harvest within ten years. “In both scenarios, we considered that a standard mill would process such production, separating 50% of the sugarcane to produce sugar and 50% for first generation ethanol, and 60% of the straw and bagasse to produce 2GE in the mill”, Guiducci explains.

The economic viability analysis of the revenue stream considered the advantages expected from Flex II sugarcane, obtained in the production of sugar, and 1G and 2G ethanol, in comparison with conventional sugarcane.

To expand the mill’s infrastructure and processing capacity, it considered an investment of around R$ 2 billion (optimistic scene) and two disbursements of R$ 1 billion (conservative scenario), both with annual maintenance expenses at around R$100 million.

The final analysis indicated that the investment is viable, once the additional gains expected from Flex II recorded an internal rate of return (IRR) of 27% and 16% and a net present value (NPV) of R$ 4.19 million and R$ 982,700 in the optimistic and conservative scenarios, respectively.

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