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].


pathway
Figure 1. Synthesis pathway for rosmarinic acid and expressed enzymes for intermediate modification.

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.

DLDH
Figure 2. Protein structure of lactate dehydrogenase (D-LDH) [4]

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.

HpaBC
Figure 3. Pictured on the left is protein structure of 4-hydroxyphenylacetate 3-monooxygenase reductase component, HpaB [5]. Pictured on the right is protein structure of 4-hydroxyphenylacetate 3-monooxygenase reductase component, HpaC [6].

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.

TyrB
Figure 4. Protein structure of tyrosine aminotransferase (TyrB) [7].

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.

TAL
Figure 5. Protein structure of tyrosine ammonia lyase (TAL) [8].

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.

HpaBC
Figure 6. Pictured on the left is protein structure of 4-hydroxyphenylacetate 3-monooxygenase reductase component, HpaB [5]. Pictured on the right is protein structure of 4-hydroxyphenylacetate 3-monooxygenase reductase component, HpaC [6].

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.

4Cl
Figure 8. Protein structure of 4-coumaroyl CoA-ligase (4Cl) [9] .

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.

RAS
Figure 9. Protein structure of rosmarinic acid synthase (RAS) [10].


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


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