Enhanced Co-culture System Using Escherichia coli and Pichia pastoris (Komagataella phaffii) for Improved Microbial Production of Valuable Plant Alkaloids.

Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.

Gut bacterial aromatic amine production: aromatic amino acid decarboxylase and its effects on peripheral serotonin production.

Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.

Transport engineering using tobacco transporter NtJAT1 enhances alkaloid production in Escherichia coli.

Transporters have been used in the production of plant metabolites in microorganisms. This study introduced a tobacco multidrug and toxic compound extrusion transporter, NtJAT1, into alkaloid-producing Escherichia coli cells. NtJAT1 expression enhanced alkaloid production secretion into the medium by 14 folds. Our findings further demonstrate the usefulness of the transport-engineering approach.

Machine learning discovery of missing links that mediate alternative branches to plant alkaloids.

Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase.

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS-1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS-1 (PPIase KS-1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS-1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS-1. The recombinant E. coli cells expressing the PPIase KS-1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS-1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS-1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Establishment of a co-culture system using Escherichia coli and Pichia pastoris (Komagataella phaffii) for valuable alkaloid production.

BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli-E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.

Transport engineering for improving the production and secretion of valuable alkaloids in Escherichia coli.

Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.

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.

Author Correction: Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.

Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.

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.

Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles.

Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.

Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis.

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA-rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both in vitro and in vivo Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of ∼0.1 µm These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli.

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

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.

Tracing microbiota changes in yamahai-moto, the traditional Japanese sake starter.

Sake is made from steamed rice, malted rice, and water. Sake production begins with the preparation of a small-scale starter (moto); the quality of moto significantly influences the flavor and richness of sake. In the traditional starter, yamahai-moto, the growth of naturally occurring lactic acid bacteria represses the putrefactive micro-organisms, whereas in the modern starter, sokujo-moto, this is achieved by adding lactic acid. In this study, the successive change in bacterial flora of yamahai-moto was analyzed by pyrosequencing 16S ribosomal RNA genes. Lactobacillus was dominant throughout the process (93-98%). Nitrate-reducing bacteria that have been generally assumed to be the first colonizers of yamahai-moto were scarcely found in the early stage, but Lactobacillus acidipiscis dominated. Lactobacillus sakei drastically increased in the middle stage. This is the first report, though one case study, to show how the early stage microbiota in Japanese yamahai-moto is varyingly controlled without nitrate-reducing bacteria using next-generation sequencing.

(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.

Asymmetric synthesis of tetrahydroisoquinolines by enzymatic Pictet-Spengler reaction.

Norcoclaurine synthase (NCS) catalyzes the stereoselective Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde as the first step of benzylisoquinoline alkaloid synthesis in plants. Recent studies suggested that NCS shows relatively relaxed substrate specificity toward aldehydes, and thus, the enzyme can serve as a tool to synthesize unnatural, optically active tetrahydroisoquinolines. In this study, using an N-terminally truncated NCS from Coptis japonica expressed in Escherichia coli, we examined the aldehyde substrate specificity of the enzyme. Herein, we demonstrate the versatility of the enzyme by synthesizing 6,7-dihydroxy-1-phenethyl-1,2,3,4-tetrahydroisoquinoline and 6,7-dihydroxy-1-propyl-1,2,3,4-tetrahydroisoquinoline in molar yields of 86.0 and 99.6% and in enantiomer excess of 95.3 and 98.0%, respectively. The results revealed the enzyme is a promising catalyst that functions to stereoselectively produce various 1-substituted-1,2,3,4-tetrahydroisoquinolines.

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.