Selection of the optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli.

We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via L-DOPA, which is synthesized by hydroxylation of L-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for L-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 µM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes. Graphical abstract The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli KEY POINTS: • There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase. • Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli. • New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.

Microbial production of novel sulphated alkaloids for drug discovery.

Natural products from plants are useful as lead compounds in drug discovery. Plant benzylisoquinoline alkaloids (BIAs) exhibit various pharmaceutical activities. Although unidentified BIAs are expected to be of medicinal value, sufficient quantities of such BIAs, for biological assays, are sometimes difficult to obtain due to their low content in natural sources. Here, we showed that high productivity of BIAs in engineered Escherichia coli could be exploited for drug discovery. First, we improved upon the previous microbial production system producing (S)-reticuline, an important BIA intermediate, to obtain yields of around 160 mg/L, which was 4-fold higher than those of the previously reported highest production system. Subsequently, we synthesised non-natural BIAs (O-sulphated (S)-reticulines) by introducing human sulphotransferases into the improved (S)-reticuline production system. Analysis of human primary cells treated with these BIAs demonstrated that they affected a biomarker expression in a manner different from that by the parent compound (S)-reticuline, suggesting that simple side-chain modification altered the characteristic traits of BIA. These results indicated that highly productive microbial systems might facilitate the production of scarce or novel BIAs and enable subsequent evaluation of their biological activities. The system developed here could be applied to other rare natural products and might contribute to the drug-discovery process as a next-generation strategy.

Laboratory-scale production of (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids, using a bacterial-based method.

Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.

Total biosynthesis of opiates by stepwise fermentation using engineered Escherichia coli.

Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.

(R,S)-tetrahydropapaveroline production by stepwise fermentation using engineered Escherichia coli.

Tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) found in diverse pharmaceutical compounds, is used as a starting material for the production of BIA. THP also has various neurobiological properties but is difficult to synthesize. Therefore, a simple method for THP production is desired. Recent studies have shown that microbes, especially bacteria, can serve as platforms for synthesizing these complex compounds; however, because bacteria lack organelles, the designed synthetic pathway cannot be compartmentalized. Thus, the metabolic flow is frequently inhibited or disrupted by undesirable reactions. Indeed, in the first attempt to synthesize THP using a single strain of engineered Escherichia coli, the yield was quite low (<5 µM), mainly because of the oxidation of THP by tyrosinase, an essential enzyme in our production system. To circumvent these problems, we constructed a stepwise (R,S)-THP production system, in which the dopamine-producing step and the subsequent THP-producing step were separated. The yield of (R,S)-THP reached 1.0 mM (287 mg/L), the highest yielding BIA production method using a microbe reported to date. Furthermore, we demonstrated that (R,S)-THP produced by stepwise fermentation is useful for the production of reticuline, an important BIAs intermediate. Based on these observations, applying the stepwise fermentation method is discussed.

Improvement of reticuline productivity from dopamine by using engineered Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are pharmaceutically important compounds. We have previously devised a reticuline (BIA) production method from dopamine by using Escherichia coli; however, its productivity was relatively low (33 µM, 11 mg/L). We report here, by fine-tuning the method, higher reticuline productivity of 165 µM (54 mg/L), increasing the conversion efficiency by 8-fold. These results are important for developing an efficient route to fermentative reticuline production.

Constitutive expression of catABC genes in the aniline-assimilating bacterium Rhodococcus species AN-22: production, purification, characterization and gene analysis of CatA, CatB and CatC.

The aniline-assimilating bacterium Rhodococcus sp. AN-22 was found to constitutively synthesize CatB (cis,cis-muconate cycloisomerase) and CatC (muconolactone isomerase) in its cells growing on non-aromatic substrates, in addition to the previously reported CatA (catechol 1,2-dioxygenase). The bacterium maintained the specific activity of the three enzymes at an almost equal level during cultivation on succinate. CatB and CatC were purified to homogeneity and characterized. CatB was a monomer with a molecular mass of 44 kDa. The enzyme was activated by Mn2+, Co2+ and Mg2+. Native CatC was a homo-octamer with a molecular mass of 100 kDa. The enzyme was stable between pH 7.0 and 10.5 and was resistant to heating up to 90 degrees C. Genes coding for CatA, CatB and CatC were cloned and named catA, catB and catC respectively. The catABC genes were transcribed as one operon. The deduced amino acid sequences of CatA, CatB and CatC showed high identities with those from other Gram-positive micro-organisms. A regulator gene such as catR encoding a regulatory protein was not observed around the cat gene cluster of Rhodococcus sp. AN-22, but a possible relic of catR was found in the upstream region of catA. Reverse transcriptase-PCR and primer extension analyses showed that the transcriptional start site of the cat gene cluster was located 891 bp upstream of the catA initiation codon in the AN-22 strain growing on both aniline and succinate. Based on these data, we concluded that the bacterium constitutively transcribed the catABC genes and translated its mRNA into CatA, CatB and CatC.

Constitutive synthesis, purification, and characterization of catechol 1,2-dioxygenase from the aniline-assimilating bacterium Rhodococcus sp. AN-22.

A catechol 1,2-dioxygenase (CD) was found, which was synthesized constitutively in the aniline-assimilating bacterium Rhodococcus sp. AN-22 grown on a medium without aniline, as well as on aniline medium. The bacterium synthesized CD in its cells grown on all the 21 non-aromatic substrates examined, including four natural media such as meat and yeast extracts, one sugar, six organic acids, and 10 amino acids as carbon, energy, and nitrogen sources. When the bacterium was incubated on a medium with D-glucose, L-malate, isoleucine, leucine, etc., it synthesized more CD than that in cells grown on aniline. Two CDs, which were prepared from cells grown on aniline and L-malate, were purified separately to homogeneity and characterized. The two enzymes were apparently identical in molecular and catalytic properties including molecular mass, optimal pH, stability to heating, and substrate specificity for catechol analogues. However, they differed in the substrate specificity and resistance to sulfhydryl and chelating agents from the inducible CDs produced by other aniline-assimilating bacteria reported previously.