We have also developed yeast strains that produce BIA metabolites along two of the major branches of reticulin: the sanguinarine/berberine branch and the morphinan branch. Yeast strains were engineered to express one or more of the P heterologous BIA pathway enzymes. The fragmentation pattern of the 4'OMT reaction product shows no evidence of nonspecific methylation.
The CNMT variants are the most highly expressed and/or stable of the recombinant enzymes. We co-transformed yeast cells with all possible combinations of the 6OMT, CNMT and 4'OMT enzymes of T. All strains harboring different combinations of the three methyltransferase enzyme variants showed the production of reticulin from norlaudanosoline, as verified by LC- MS/MS ( Fig. 4.6a).
This difference in activity can be attributed to the differences in substrate affinities of the CNMT variants observed in studies of a single enzyme such as P. Although BIA metabolites to reticulin exist only in the (S)- conformation in plants, our studies showed that three methyltransferases accepted both stereoisomers as substrates equally in vivo to give a racemic mixture of (R,S)-reticuline from (R,S)-norlaudanosoline66, 67 (Fig. 4.6c). a) LC-MS/MS analysis of the growth medium of engineered yeast strains supplemented with 1 mM norlaudanosoline and grown for 48 h confirms reticulin production. Control experiments in which strains lacking any of the three required enzymes or grown in the absence of substrate did not accumulate the metabolite peak identified as reticulin.
Data shown are a 1:10 dilution of growth media (norlaudanosoline, red; reticulin, blue) and a ∼1:2 dilution of cell extract (norlaudanosoline, green; reticulin, magenta) of CSY288 supplemented with 1 mM norlaudanosoline and adult for 48 hours. Our titration assays indicated optimal expression levels for each of the BIA enzymes tested, with the possible exception of P. We examined metabolite synthesis along a major branch, the sanguinarine/berberine branch, by expressing additional enzymes from native plant pathways such as a demonstration of the various natural products and activities that can be produced in our engineered yeast strains.
The first conversion step in the sanguinarine/berberine branch of the BIA pathway is performed by the berberine bridge enzyme (BBE), which catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline to the berberine bridge carbon of (S ) )-scoulerine68 (Fig. 4.10a). LC-MS chromatograms of the growth media of (a) CSY288 supplemented with 2 mM norlaudanosoline and (b) CSY410 supplemented with 2 mM laudanosoline and grown for 48 h showing impurities and degradation products in the sample. a) The total ion chromatogram is shown in black and the extracted ion chromatogram for norlaudanosoline (MS 288) is shown in purple. All detectable BIA metabolites elute in this time frame with the rest of the spectra relatively flat.
Although the levels of the substrate (S)-tetrahydroberberine are relatively low in this extensive pathway, the correct reaction product was still detectable in the growth medium. Although expression of the full-length protein was confirmed (Figure 4.15b), no activity was observed under any of the conditions tested when this cDNA was expressed in yeast. However, several enzymes that carry out early conversion steps in the morphinan branch have not been cloned and characterized, precluding engineering efforts that rely on natural enzymes.
In the native plant pathway, only (S)-reticulin is produced and two additional enzymes are required to produce (R)-reticulin, which is used in the synthesis of the morphine alkaloids. b) LC-MS/MS analysis of the growth media of engineered yeast strains supplemented with 4 mM norlaudanosoline and grown for 48 h confirms salutaridin production.
Discussion
The main ions (m/z = 297 and 265) correspond to the expected fragments reported in the literature for salutaridin76. The x-axis shows the CYP2D6 and CPR expression constructs present in the CSY288 background strain. A third factor that may limit conversion is the lack of an active transport system to facilitate the passage of BIA metabolites across microbial cell membranes and allow for higher intracellular substrate concentrations.
As a heterologous production host for diverse BIA molecules, our engineered strains will be useful tools to advance the characterization of a ubiquitous plant secondary metabolic pathway. First, our strains can be used as tools to characterize enzymes and investigate regulatory strategies for the BIA pathway in a synthetic host. For example, we used our strains to investigate the relative activities of enzyme variants in the engineered pathway.
Second, our strains can be used as functional genomics tools to advance the elucidation of the BIA pathway and characterize novel enzyme activities from native plant hosts or non-native sources, as demonstrated here. The engineered strains can be used to screen cDNA libraries from BIA-producing plants in high-throughput assays for modifying activities. Such a tool is particularly powerful in light of the scarce availability of many BIA intermediates and challenges in plant metabolic engineering.
The microbial production levels of BIA reported here reached ∼150 mg l-1, similar to yields previously reported from initial engineering efforts on microbial strains to produce other plant natural products78. More importantly, engineered yeast strains offer the potential to produce an even broader spectrum of BIA metabolites through extensions of this synthetic pathway. For example, as novel enzyme activities are cloned from native plant hosts, the cDNAs can be expressed in our engineered strains to produce berberine and other BIA metabolites in the sanguinarine, morphinan, and bis-benzylisoquinoline branches.
As demonstrated here with a human P450 enzyme, there is also the exciting potential to express enzymes unrelated to the native BIA pathway, but which accept BIA metabolites as substrates in our synthetic hosts to access ' to obtain a larger pool of intermediate products and derivatives. Furthermore, strategies involving the recombination of native and novel enzyme activities and the feeding of alternative substrates can be used to produce non-natural BIA molecules, expanding molecular diversity. Finally, synthetic methods can be used in conjunction with in vivo biosynthesis to attach various functional groups to the molecular backbone, thereby.
Materials and Methods
We performed chromosomal integrations of DNA fragments by homologous recombination using a standard lithium acetate transformation protocol to construct strains stably expressing combinations of the BIA enzymes. The assembled DNA insert contained a multicloning site and was subcloned upstream of the selection marker in each construct. We gel purified integration cassettes using the Zymoclean Gel DNA Recovery Kit (Zymo Research) according to the manufacturer's instructions before transformation into the appropriate yeast strain.
Integration of the cassettes into the correct locus was confirmed by PCR analysis of the targeted region of the chromosome. To remove all selection markers from the final strains, we transformed cells with the plasmid pSH63, which expresses a GAL-inducible Cre recombinase81. We constructed template plasmids for chromosomal integrations for the enzyme titration studies by cloning the GAL1-10 promoter (amplified from pRS314-GAL38 using primers GAL.fwd and GAL.rev) in place of the TEF1 promoter in pUG-based plasmids.
We sampled aliquots of the growth medium for LC-MS/MS analysis 24 h after substrate addition. At appropriate times, aliquots of yeast cultures were centrifuged to recover cells as pellets and allow collection of the growth media. Quantification of metabolites was based on the integrated area of the extracted ion chromatogram peaks64 calculated using DataAnalysis for 6300 Series Ion Trap LC/MS Version 3.4 (Bruker Daltonik GmbH) and reported as the mean ± s.d.
A summary of the average yields for each of the synthesized BIA compounds is provided (Table 4.3). Yields are reported from the supernatant of growth cultures of the appropriate strains fed 5 mM norlaudonosoline. We constructed plasmids for Western blotting experiments by cloning the C-terminal epitope tag(s) of pYES-NT/A (Invitrogen) into a standard BIA expression vector, followed by subcloning of the enzyme of interest.
The membrane was incubated with Anti-V5 HRP antibody (Invitrogen) according to the manufacturer's instructions (Invitrogen) with 5% nonfat milk as a blocker. Proteins were detected with a West Pico Super Signal Detection kit (Pierce) and imaged on a ChemiDoc XRS system (Bio-Rad). The obtained data were analyzed with the iCycler iQ software according to the manufacturer's instructions.
We normalized transcript levels to the endogenous ACT1 gene83 using ACT1-specific primers. We tested buffer solutions from the chiral test kit (Beckman) with the substrates norlaudanosoline and laudanosoline and optimized conditions using the HS-β-CD separation buffer at 5% (w/v) according to the manufacturer's instructions.
