Advances in the Synthesis of Three typical tetriterpenoids: beta-carotene, lycopene and astaxanthin

Research background

Carotinoide are typical tetriterpenoids, a class of important natural pigments commonly found in animals, plants, fungi and algae. At present, the common and widely used carotenoids are β-carotene, Lycopin und Astaxanthin. With the development of isolation and analysis technology, some new carotenoids (cyclococcinin, dandelaxanthin, etc.) have also been studied and applied. According to the chemical structure, carotenoids can be divided into two categories: one is only carbon and hydrogen without oxygen carotenoids, including beta-carotene and lycopene; One is carotenoids containing hydroxyl, ketone, carboxyl and other oxygen-containing functional groups, including lutein and astaxanthin. Research content

Carotenoids are commonly produced in the following ways. Organic solvent extraction method: common organic solvents are acetone, petroleum ether, dimethyl sulfoxide, ethyl acetate and so on. However, there are some problems such as low extraction rate, poor purity and safety risk. Chemical synthesis: This method is one of the main methods for large-scale production of carotenoids. Most carotenoids can be produced from α- and β-ionic ketones through a series of complex chemical reactions such as acetylation, acidification, hydrogenation, halogenation rearrangement and witting reaction. Biosynthesis: The biosynthesis pathway of carotenoids can be divided into two stages: the first stage is the synthesis of isoprene from carbon sources, which is a common precursor of carotenoids; The second is the synthesis of downstream carotenoids. The synthesis of different carotene is affected by different enzymes. In the first stage, the microbes convert the carbon source into intermediates such as CoA, pyruvate, and glyceraldehyde 3-phosphate, which are then converted to precursor isoprene via the mevalonate pathway (MVA) and the 4-phospho-methylerythritol (MEP) pathway or the artificial isopentenol (IUP) utilization pathway.

The chassis strains selected in the production model of carotenoid include Escherichia coli, Saccharomyces cerevisiae, etc. Escherichia coli is the most commonly used chassis microorganism in the field of industrial biotechnology because of its mature genetic tools and easy cultivation. For example, Escherichia coli has a natural MEP pathway in the body, and only the introduction of carotenoid genes can achieve heterologous carotenoid biosynthesis. For example, after overexpressing the carotene gene of Erwinia with synthetic operons, a form of E. coli that produces beta-carotene was genetically constructed. The recombinant bacteria eventually produced 390 mg/l of beta-carotene in 50 L fermenter 25. By introducing the whole MVA pathway to increase the synthesis of precursor IPP and feeding batch culture, the maximum yield of β-carotene in engineering Escherichia coli reached 663mg/L. In the engineered strain DH416, the lycopene level could reach 1.22 g/L, and the average yield was 61.0 mg/L·h-1. The synthetic pathway of mevalonate and lycopene has been established in Escherichia coli FA03-PM. Through fed-batch fermentation, the lycopene content reached 94mg/g, which is the highest level reported in metabolically engineered Escherichia coli to date. When producing astaxanthin from Escherichia coli CAR026 as the starting strain, the recombinant strain Gro-46 produced 1.18g/L astaxanthin under the feeding condition, representing the highest output of astaxanthin in engineered Escherichia coli so far. Since Saccharomyces cerevisiae lacks a complete carotenoid metabolic pathway, heterogeneous carotenoid genes are introduced into saccharomyces cerevisiae. For example, after introducing the CRT gene of red yeast into Saccharomyces cerevisiae INVSc1, overexpression of recombinant saccharomyces cerevisiae INVSc1 at 20℃ increased β-carotene level to 528.8μg/g. Unsaturated fatty acids can improve the fluidity and storage space of cell membranes, thereby increasing the production of carotenoids. Supplementation with 60mg/L oleic acid or palmitoleic acid increased beta-carotene content by 83.7% and 130.2%, respectively. In terms of lycopene production, by optimizing lycopene synthases from different sources, saccharomyces cerevisiae can produce up to 3.28g/L of lycopene. A Gal4 mutant with a temperature-sensitive (TS) phenotype of Saccharomyces cerevicae accumulated 44% more biomass and 177% more lycopene through two-stage fermentation than wild-type Gal4. At the same time, the temperature response regulation system was introduced into astaxanthin production transgenic saccharomyces cerevisiae, and astaxanthin production was separated from cell growth. Finally, 235mg/L astaxanthin was produced by two-stage high density fermentation.

With the rapid development of synthetic biology, protein engineering, metabolic engineering and fermentation engineering, synthetic microorganisms will undoubtedly provide a new choice for the large-scale production of natural products. At present, synthetic microbial co-culture systems have been fully designed to synthesize metabolites through division of labor, especially metabolites with long metabolic pathways. Therefore, the design of a reasonable microbial co-culture system and the modularization of carotenoid product synthesis pathway can effectively reduce the metabolic pressure of single cells and achieve metabolic energy balance.

Due to the widespread use of carotenoids in food and medicine, the demand for natural carotenoids will also increase. Extracting carotenoids directly from natural resources is far from satisfying the needs of consumers. Therefore, the synthesis of natural carotenoids by microbial fermentation paves the way for mass production.

author: Nanjing University of Technology, Xin Feng Xue, Zhang Wenming

Journal: Biotechnology Advances

Year: 2022

DOI:doi.org/10.1016/J.BIOTECHADV.2022.108033

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