On April 19, the international academic journal ACS Sustainable Chemistry & Engineering published a research paper entitled "Design and optimization for efficient production of (S)-canadine in Escherichia coli" completed by Wang Yong's research group from the Center for Excellence in Molecular Plant Science, Chinese Academy of Sciences. This study achieved the efficient synthesis of (S)-tetrahydroberberine in Escherichia coli for the first time, which provided a reference for the microbial synthesis of chiral drugs.
Background:
Research background
(S)-Tetrahydroberberine is a protoberberine compound with multiple activities such as hypoglycemic and lipid-lowering and antioxidant, and is also a key precursor for the synthesis of berberine (antimicrobial) and noscapine (antineoplastic drug), which has great potential in drug research and development. At present, (S)-tetrahydroberberine can be obtained by plant extraction or chemical semi-synthesis. However, the low content of (S)-tetrahydroberberine in the rhizomes of ranunculus plants and the poor stereoselectivity of chemical synthesis limit its large-scale production. Microbial manufacturing has unique advantages in the synthesis of chiral compounds, and is an important technical means for the synthesis of such high-value products. At present, the common precursor (S)-reticulosoperine of benzylisoquinoline alkaloids has been efficiently synthesized in the microbial system, but due to the non-functional expression of some membrane proteins, the high-efficiency de novo synthesis of (S)-tetrahydroberberine has not been realized.
Outline of Research
Research content
In this study, CRISPR-Cas9 technology was used to construct a high-tyrosine-producing E. coli chassis SQZB18, which could accumulate 2.11 g/L of tyrosine after 2 days of shake flask culture (Figure 1). Subsequently, the researchers found that the synthesis trend of (S)-reticuloannon in rat and Drosophila-derived tyrosine hydroxylase (TYH)-derived strains was reversed before and after the integration of the THPSO cycle pathway into the genome. Combined with the structural characteristics of tyramine and the results of in vivo feeding experiments, the inhibitory effect of tyramide on RnTYHWR was speculated and confirmed (Fig. 2). Subsequently, the researchers cleverly used EcHpaBC-D11 to convert tyramine into dopamine, relieved the inhibitory effect of tyramine, and obtained strain SQZ227 by overexpressing purine nucleic acid phosphorylase, which produced 100.13 mg/L (S)-reticululosoperine after two days of shake flask culture (Fig. 3). In order to overcome the problem of functional expression of berberine bridge enzyme (BBE) and tetrahydroberine synthase (CAS), the researchers carried out homology modeling and molecular docking of berberine bridgease (AmBBE1) derived from Argemone mexicana L., and obtained 98% In addition, on the basis of optimizing the soluble expression of CAS, with the help of protein-peptide interaction domain (SH3) and its ligand (SH3-lig), trCjCAS-AtCPR2 with scaffold structure was finally screened from a combination of different CAS sources, and 32% of the substrate was converted to berberine under in vitro conditions (Figure 4). On this basis, combined with the effector factor (RraA) and cytochrome b5 that improve the expression efficiency of membrane proteins, the researchers obtained the best-performing (S)-tetrahydroberberine-producing bacterium SQZ266, which achieved a yield of 16.81 mg/L under shake flask culture and 165.74 mg/L under reactor fermentation (Fig. 5), which is the highest level of de novo synthesis of proberberine by microorganisms. This study not only provides a design idea for an efficient microbial chassis for alkaloids, but also lays a foundation for the green and sustainable production of alkaloids.
Figure 1. (a) Schematic diagram of the construction of tyrosine-producing strains and (b) Tyrosine titer and biomass of engineered strains
Figure 2. (a) Effect of BH4 supply pathway and TYH source on (S)-reticulated soursonine production; (b) Effect of BH4 cycle pathway and DDC on TYH s activity; (c) Effect of tyramide on RnTYH activity
Figure 3. (a) Strategy diagram (b) Targets that improve the metabolic flow of dopamine and (c) affect (S)-reticululosophane production
Figure 4. (a) Molecular docking of AmBBE1 and in vitro activity test results of mutants (b) Effect of spatial distribution of CAS and CPR on CAS activity
Figure 5. (a) Effect of combinatorial optimization of elements and improved membrane protein efficiency on (S)-tetrahydroberberine yield and (b) Result of fed-batch fermentation of strain SQZ266
Related information
Related information
Zhang Qian, a doctoral student in Wang Yong's research group at the Center for Excellence in Molecular Plant Science of the Chinese Academy of Sciences, is the first author of the paper, and Wu Yuhan, a doctoral student in Wang Yong's research group, participated in the related work. The research was supported by the National Key R&D Program of China, the Shanghai Academic Leader Program, the National Natural Science Foundation of China, the Pilot Program of the Chinese Academy of Sciences, and the State Key Laboratory of Plant Molecular Genetics.
Wang Yong researches group photos
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