Introduction
To synthesize rosmarinic acid in a co-culture system, many different enzymes must be expressed. Each BioBrick was strategically designed to be optimally expressed and functional in either bacteria or yeast. The BioBricks express enzymes that will modify intermediate molecules starting from the precursor molecule in each organism.
BioBrick Expression for Pathway Synthesis
Endogenously, the engineered bacteria can convert glucose to 4-hydroxyphenylpyruvate through the L-tyrosine biosynthesis pathway. The 4-hydroxy phenylacetate 3-monooxygenase reductase complex, composed of HpaB and HpaC expression, converts 4-hydroxyphenylpyruvate into 4-hydroxyphenyllactate by adding a hydroxyl group to the benzene ring. The enzyme lactate dehydrogenase (D-LDH) will convert the molecule further by a reduction reaction, converting a carbon double-bonded oxygen to a carbon-bonded hydroxyl group to produce salvianic acid A [1].
In parallel, in the engineered yeast culture, the expression of tyrosine aminotransferase (TyrB) converts 4-hydroxyphenylpyruvate into L-tyrosine with the addition of an amine group and the formation of a double-bonded oxygen. Tyrosine ammonia lyase (TAL) then converts L-tyrosine to p-coumaric acid by removing the amine group. The expression of 4-hydroxy phenylacetate 3-monooxygenase reductase complex (HpaB and HpaC) adds a hydroxyl group to the benzene ring of p-coumaric acid, resulting in the formation of caffeic acid. This intermediate is then modified further by 4-coumaroyl CoA-ligase (4Cl) that attaches a CoA group to the end carbon, allowing the formation of caffeoyl CoA. The final expression of rosmarinic acid synthase (RAS) mediates a condensation reaction between salvianic acid A and caffeoyl CoA, resulting in rosmarinic acid [2-3].
BioBrick Design and Function
This composite BioBrick BBa_K4588034 expresses the enzyme lactate dehydrogenase (D-LDH) in Escherichia coli necessary to convert 4-dihydroxyphenyllactate into salvianic acid A [1]. The L-rhamnose inducible promoter BBa_K914003 allows for moderate upregulation of recombinant protein expression in the presence of rhamnose. Following the promoter is a strong ribosome binding site BBa_B0034 for optimal translation initiation. The coding region consists of the BioBrick basic part BBa_K4588022 that was codon optimized. Following the coding region, a 6x His Tag (BBa_K12230) was inserted for protein expression detection. The strong double terminator (BBa_B0015) was chosen to terminate gene transcription completely.
The expression of BioBricks BBa_K4588033 and BBa_K45880 results in the 4-hydroxyphenylacetate 3-monooxygenase reductase complex (HpaBC). This enzyme complex expressed in bacteria converts 4-hydroxyphenylpyruvate into 4-dihydroxyphenyllactate [1]. These BioBricks contain T7 promoters ( BBa_1712074) for strong expression and a strong ribosome binding site ( BBa_B0034) for optimal translation initiation. The composite BioBrick for HpaB consists of the coding region HpaB (BBa_K4588023) and the composite BioBrick HpaC (BBa_K4588024, both codon optimized for Escherichia coli expression. Following the coding regions, a 6x His Tag ( BBa_K1223006) was inserted for protein expression detection. The strong double terminator ( BBa_B0015) was chosen to terminate gene transcription completely. These BioBricks also contain unique restriction sites required for multigene assembly in a single vector.
The BioBrick BBa_K4588036 expresses tyrosine aminotransferase in Saccharomyces cerevisiae. This enzyme converts 4-hydroxyphenylpyruvate into L-tyrosine [3]. This BioBrick contains a GAL1 promoter (BBa_K2637059) that supports strong inducible expression in the presence of galactose. Following this promoter, the sequence contains a Kozak sequence (BBa_J63003) for optimal ribosome binding. The protein coding sequence, BBa_K4588025, was optimized for S. cerevisiae expression. The coding region also contains an HA tag (BBa_K1150016) for protein detection. The strong ADH1 terminator (BBa_K1486025) (BBa_K1486025) completely halts protein expression. This BioBrick was also designed with unique restriction sites for multigene vector assembly.
The BioBrick BBa_K4588037expresses tyrosine ammonia lyase in Saccharomyces cerevisiae. This enzyme converts L-tyrosine into p-coumaric acid [3]. This BioBrick contains a GAL1 promoter (BBa_K2637059)that supports strong inducible expression in the presence of galactose. Following this promoter, the sequence contains a Kozak sequence (BBa_J63003) for optimal ribosome binding. The protein coding sequence, BBa_K4588026, was optimized for S. cerevisiae expression. The coding region also contains a 6x His tag (BBa_K1223006) for protein detection. The strong ADH1 terminator (BBa_K1486025) efficiently stops protein expression. This BioBrick was also designed with unique restriction sites for multigene vector assembly.
The expression of BioBricks BBa_K4588038 and BBa_K4588039 results in the 4-hydroxyphenylacetate 3-monooxygenase reductase complex (HpaBC). This enzyme complex expressed in Saccharomyces cerevisiae converts p-coumaric acid to caffeic acid [3]. These BioBricks contain GAL1 promoters (BBa_K2637059) that support strong inducible expression in the presence of galactose. Following this promoter, the sequence contains a Kozak sequence (BBa_J63003) for optimal ribosome binding. The BioBrick for HpaB consists of the coding region HpaB (BBa_K4588027) and the BioBrick HpaC (BBa_K4588028) both codon optimized for S. cerevisiae expression. Following the coding regions, a 6x His Tag (BBa_K1223006) was inserted for protein expression detection. The strong ADH1 terminator (BBa_K1486025) was chosen to terminate gene transcription completely. Each of these BioBricks also contains unique restriction sites required for multigene assembly in a single vector.
The BioBrick BBa_K4588040 expresses 4-coumaroyl CoA-ligase in Saccharomyces cerevisiae. This enzyme converts caffeic acid to caffeoyl CoA [3]. This BioBrick contains a GAL1 promoter (BBa_K2637059) that supports strong inducible expression in the presence of galactose. Following this promoter, the sequence contains a Kozak sequence (BBa_J63003) for optimal ribosome binding. The protein coding sequence, BBa_K4588029, was optimized for S. cerevisiae expression. The coding region also contains a HA tag (BBa_K1150016) for protein detection. The strong ADH1 terminator (BBa_K1486025) completely stops protein expression. This BioBrick was also designed with unique restriction sites for multigene vector assembly.
The BioBrick BBa_K4588041 expresses rosmarinic acid synthase in Saccharomyces cerevisiae. This enzyme fuses caffeoyl CoA and salvianic acid A to produce rosmarinic acid [3]. This BioBrick contains a GAL1 promoter (BBa_K2637059)that supports strong inducible expression in the presence of galactose. Following this promoter, the sequence contains a Kozak sequence (BBa_J63003) for optimal ribosome binding. The protein coding sequence, BBa_K4588041, was optimized for S. cerevisiae expression. The coding region also contains a 6x His tag (BBa_K1223006) for protein detection. The strong ADH1 terminator (BBa_K1486025) completely stops protein expression. This BioBrick was also designed with unique restriction sites for multigene vector assembly.
Parts Collection
Basic Parts
Part Number | Part Name | Type | Description | Length | RFC Compatible |
---|---|---|---|---|---|
BBa_K4588023 | HpaB.Bacteria.Coding | Basic: Coding | Coding sequence for a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain B - E. coli codon optimized. | 1431 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588024 | HpaC.Bacteria.Coding | Basic: Coding | Coding sequence for a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain C - E. coli codon optimized. | 498 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588025 | TyrB.Coding | Basic: Coding | Coding sequence for tyrosine aminotransferase. | 1191 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588026 | TAL.Coding | Basic: Coding | Coding sequence for tyrosine ammonia lyase. | 1518 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588027 | HpaB.Yeast.Coding | Basic: Coding | Coding sequence for a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain B -S. cerevisiae codon optimized. | 1431 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588028 | HpaC.Yeast.Coding | Basic: Coding | Coding sequence for a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain C - S. cerevisiae codon optimized. | 504 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588029 | 4Cl.Coding | Basic: Coding | Coding sequence for 4-coumaroyl CoA-ligase. | 1683 bp | 10, 12, 23, 25, 1000 |
BBa_K4588030 | RAS.Coding | Basic: Coding | Coding sequence for rosmarinic acid synthase. | 1290 bp | 10, 12, 21, 23, 25, 1000 |
Composite Parts
Part Number | Part Name | Type | Description | Length | RFC Compatible |
---|---|---|---|---|---|
BBa_K4588034 | D-LDH | Composite | Expresses lactate dehydrogenase. This protein converts 4-dihydroxyphenyllactate into salvianic acid A. | 1277 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588033 | HpaB.Bacteria | Composite | Expresses a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain B. This protein in conjuntion with HpaC converts 4-hydroxyphenylpyruvate into 4-dihydroxyphenyllactate in E. coli. | 1645 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588035 | HpaC.Bacteria | Composite | Expresses a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain C. This protein in conjuntion with HpaB converts 4-hydroxyphenylpyruvate into 4-dihydroxyphenyllactate in E. coli. | 712 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588036 | TyrB | Composite | Expresses tyrosine ammonia lyase. This protein converts L-tyrosine into p-coumaric acid. | 1881 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588037 | TAL | Composite | Expresses tyrosine ammonia lyase. This protein converts L-tyrosine into p-coumaric acid. | 2208 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588038 | HpaB.Yeast | Composite | Expresses a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain B. This protein in conjuntion with HpaC converts p-coumaric acid to caffeic acid in S. cerevisiae. | 2121 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588039 | HpaC.Yeast | Composite | Expresses a 4-hydroxyphenylacetate 3-monooxygenase reductase complex domain C. This protein in conjuntion with HpaB converts p-coumaric acid to caffeic acid in S. cerevisiae. | 1194 bp | 10, 12, 21, 23, 25, 1000 |
BBa_K4588040 | 4Cl | Composite | Expresses 4-coumaroyl CoA-ligase. This protein converts caffeic acid to caffeoyl CoA. | 2373 bp | 10, 12, 23, 25, 1000 |
BBa_K4588041 | RAS | Composite | Expresses rosmarinic acid synthase. This protein fuses caffeoyl CoA and salvianic acid A to produce rosmarinic acid. | 1980 bp | 10, 12, 21, 23, 25, 1000 |
- Bloch, S. E., & Schmidt‐Dannert, C. (2014). Construction of a chimeric biosynthetic pathway for the de novo biosynthesis of rosmarinic acid in escherichia coli. ChemBioChem, 15(16), 2393–2401. https://doi.org/10.1002/cbic.201402275
- Petersen, M., Hausler, E., Karwatzki, B., & Meinhard, J. (1993). Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus Blumei Benth. Planta, 189(1), 10–14. https://doi.org/10.1007/bf00201337
- Babaei, M., Borja Zamfir, G. M., Chen, X., Christensen, H. B., Kristensen, M., Nielsen, J., & Borodina, I. (2020). Metabolic engineering of saccharomyces cerevisiae for rosmarinic acid production. ACS Synthetic Biology, 9(8), 1978–1988. https://doi.org/10.1021/acssynbio.0c00048
- Dym, O., Pratt, E. A., Ho, C., & Eisenberg, D. (2000). The crystal structure of D-lactate dehydrogenase, a peripheral membrane respiratory enzyme. Proceedings of the National Academy of Sciences of the United States of America, 97(17), 9413–9418. https://doi.org/10.1073/pnas.97.17.9413
- Kim, S. H., Hisano, T., Takeda, K., Iwasaki, W., Ebihara, A., & Miki, K. (2007). Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8. The Journal of biological chemistry, 282(45), 33107–33117. https://doi.org/10.1074/jbc.M703440200
- Kim, S. H., Hisano, T., Iwasaki, W., Ebihara, A., & Miki, K. (2008). Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity. Proteins, 70(3), 718–730. https://doi.org/10.1002/prot.21534
- Blankenfeldt, W., Nowicki, C., Montemartini-Kalisz, M., Kalisz, H. M., & Hecht, H. J. (1999). Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode. Protein science : a publication of the Protein Society, 8(11), 2406–2417. https://doi.org/10.1110/ps.8.11.2406
- Louie, G. V., Bowman, M. E., Moffitt, M. C., Baiga, T. J., Moore, B. S., & Noel, J. P. (2006). Structural determinants and modulation of substrate specificity in phenylalanine-tyrosine ammonia-lyases. Chemistry & biology, 13(12), 1327–1338. https://doi.org/10.1016/j.chembiol.2006.11.011
- Li, Z., & Nair, S. K. (2015). Structural Basis for Specificity and Flexibility in a Plant 4-Coumarate:CoA Ligase. Structure (London, England : 1993), 23(11), 2032–2042. https://doi.org/10.1016/j.str.2015.08.012
- Levsh, O., Pluskal, T., Carballo, V., Mitchell, A. J., & Weng, J. K. (2019). Independent evolution of rosmarinic acid biosynthesis in two sister families under the Lamiids clade of flowering plants. The Journal of biological chemistry, 294(42), 15193–15205. https://doi.org/10.1074/jbc.RA119.010454