Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor.

BACKGROUND: Single-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides. RESULTS: The water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists. CONCLUSIONS: We established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Metabolome analysis of metabolic burden in Escherichia coli caused by overexpression of green fluorescent protein and delta-rhodopsin.

Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E. coli strains have distinct metabolic capacities. In this study, two proteins were overexpressed in four E. coli strains (MG1655(DE3), W3110(DE3), BL21star(DE3), and Rosetta(DE3)), and their effects on metabolic burden were investigated. Metabolomic analysis showed that E. coli strains overexpressing green fluorescent protein had decreased levels of several metabolites, with a positive correlation between the number of reduced metabolites and green fluorescent protein expression levels. Moreover, nucleic acid-related metabolites decreased, indicating a metabolic burden in the E. coli strains, and the growth rate and protein expression levels were improved by supplementation with the five nucleosides. In contrast, two strains overexpressing delta rhodopsin, a microbial membrane rhodopsin from Haloterrigena turkmenica, led to a metabolic burden and decrease in the amino acids Ala, Val, Leu, Ile, Thr, Phe, Asp, and Trp, which are the most frequent amino acids in the delta rhodopsin protein sequence. The metabolic burden caused by protein overexpression was influenced by the metabolic capacity of the host strains and the sequences of the overexpressed proteins. Detailed characterization of the effects of protein expression on the metabolic state of engineered cells using metabolomics will provide insights into improving the production of target compounds.

Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway.

Saccharomyces cerevisiae is a promising host for the bioproduction of higher alcohols, such as 2,3-butanediol (2,3-BDO). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO via glycolysis have been constructed. However, the specific 2,3-BDO production rates of engineered strains must be improved. To identify approaches to improving the 2,3-BDO production rate, we investigated the factors contributing to higher ethanol production rates in certain industrial strains of S. cerevisiae compared to laboratory strains. Sequence analysis of 11 industrial strains revealed the accumulation of many nonsynonymous substitutions in RIM15, a negative regulator of high fermentation capability. Comparative metabolome analysis suggested a positive correlation between the rate of ethanol production and the activity of the pyruvate-consuming pathway. Based on these findings, RIM15 was deleted, and the pyruvate-consuming pathway was activated in YHI030, a metabolically engineered S. cerevisiae strain that produces 2,3-BDO. The titer, specific production rate, and yield of 2,3-BDO in the test tube-scale culture using the YMS106 strain reached 66.4 ± 4.4 mM, 1.17 ± 0.017 mmol (g dry cell weight h)-1, and 0.70 ± 0.03 mol (mol glucose consumed)-1. These values were 2.14-, 2.92-, and 1.81-fold higher than those of the vector control, respectively. These results suggest that bioalcohol production via glycolysis can be enhanced in a metabolically engineered S. cerevisiae strain by deleting RIM15 and activating the pyruvate-consuming pathway.

Improvement of ethanol and 2,3-butanediol production in Saccharomyces cerevisiae by ATP wasting.

BACKGROUND: "ATP wasting" has been observed in 13C metabolic flux analyses of Saccharomyces cerevisiae, a yeast strain commonly used to produce ethanol. Some strains of S. cerevisiae, such as the sake strain Kyokai 7, consume approximately two-fold as much ATP as laboratory strains. Increased ATP consumption may be linked to the production of ethanol, which helps regenerate ATP. RESULTS: This study was conducted to enhance ethanol and 2,3-butanediol (2,3-BDO) production in the S. cerevisiae strains, ethanol-producing strain BY318 and 2,3-BDO-producing strain YHI030, by expressing the fructose-1,6-bisphosphatase (FBPase) and ATP synthase (ATPase) genes to induce ATP dissipation. The introduction of a futile cycle for ATP consumption in the pathway was achieved by expressing various FBPase and ATPase genes from Escherichia coli and S. cerevisiae in the yeast strains. The production of ethanol and 2,3-BDO was evaluated using high-performance liquid chromatography and gas chromatography, and fermentation tests were performed on synthetic media under aerobic conditions in batch culture. The results showed that in the BY318-opt_ecoFBPase (expressing opt_ecoFBPase) and BY318-ATPase (expressing ATPase) strains, specific glucose consumption was increased by 30% and 42%, respectively, and the ethanol production rate was increased by 24% and 45%, respectively. In contrast, the YHI030-opt_ecoFBPase (expressing opt_ecoFBPase) and YHI030-ATPase (expressing ATPase) strains showed increased 2,3-BDO yields of 26% and 18%, respectively, and the specific production rate of 2,3-BDO was increased by 36%. Metabolomic analysis confirmed the introduction of the futile cycle. CONCLUSION: ATP wasting may be an effective strategy for improving the fermentative biosynthetic capacity of S. cerevisiae, and increased ATP consumption may be a useful tool in some alcohol-producing strains.

Safety of laparoscopic liver resection for high-risk patients.

BACKGROUND/PURPOSE: To investigate the safety of laparoscopic liver resections (LLRs) for high-risk patients (HRs) with preoperative comorbidities affecting the heart, lungs, kidneys, glucose tolerance, and central nervous system. METHODS: This retrospective study included 585 patients who had undergone total hepatectomies from 2006 to 2020. Among them, 239 patients underwent LLRs, and 349 underwent open liver resections (OLRs). The safety and validity of LLRs were analyzed by comparing outcomes and preoperative records between HRs and nonhigh-risk patients (nHRs). HRs were defined as patients with any type of chronic heart disease rated New York Heart Association II or higher, chronic obstructive pulmonary disease rated stage III or higher, chronic kidney disease rated stage III or higher, insulin-dependent diabetes mellitus, or cerebrovascular disease with neurological sequelae. RESULTS: A total of 117 LLRs (49.0%) were performed in HRs, and there were more patients with ASA class III or higher than nHRs. Complications of Clavien-Dindo classification grade 3b or higher were not observed in HRs and in only one nHR. Furthermore, no postoperative exacerbations of the five HRs factors were observed in either group. CONCLUSIONS: Rigorous assessment of surgical indications and perioperative management can promote safe LLRs, even in HRs with comorbidities.

The Outcome of Conversion to Hand-Assisted Laparoscopic Surgery in Laparoscopic Liver Resection.

BACKGROUND: Hand-assisted laparoscopic surgery (HALS) is known as a useful option. However, the outcome and predictor of conversion to HALS in laparoscopic liver resection (LLR) are unclear. METHODS: Data from consecutive patients who planned pure LLR between 2011 and 2020 were retrospectively reviewed. Univariate and multivariate analyses were performed and compared pure LLR, HALS, and converted open liver resection (OLR). RESULTS: Among the 169 LLRs, conversion to HALS was performed in 19 (11.2%) and conversion to OLR in 16 (9.5%). The most frequent reasons for conversion to HALS were failure to progress (11 cases). Subsequently, bleeding (3 cases), severe adhesion (2 cases), and oncological factors (2 cases) were the reasons. In the multivariable analysis, the tumor located in segments 7 or 8 (p = 0.002) was evaluated as a predictor of conversion to HALS. Pure LLR and HALS were associated with less blood loss than conversion to OLR (p = 0.005 and p = 0.014, respectively). However, there was no significant difference in operation time, hospital stay, or severe complications. CONCLUSIONS: The predictor of conversion to HALS was a tumor located in segments 7 or 8. The outcome of conversion to HALS was not inferior to pure LLR in terms of bleeding, operation time, hospital stay, or severe complication.

Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source.

BACKGROUND: The filamentous fungus Trichoderma reesei has been used as a host organism for the production of lignocellulosic biomass-degrading enzymes. Although this microorganism has high potential for protein production, it has not yet been widely used for heterologous recombinant protein production. Transcriptional induction of the cellulase genes is essential for high-level protein production in T. reesei; however, glucose represses this transcriptional induction. Therefore, cellulose is commonly used as a carbon source for providing its degraded sugars such as cellobiose, which act as inducers to activate the strong promoters of the major cellulase (cellobiohydrolase 1 and 2 (cbh1 and cbh2) genes. However, replacement of cbh1 and/or cbh2 with a gene encoding the protein of interest (POI) for high productivity and occupancy of recombinant proteins remarkably impairs the ability to release soluble inducers from cellulose, consequently reducing the production of POI. To overcome this challenge, we first used an inducer-free biomass-degrading enzyme expression system, previously developed to produce cellulases and hemicellulases using glucose as the sole carbon source, for recombinant protein production using T. reesei. RESULTS: We chose endogenous secretory enzymes and heterologous camelid small antibodies (nanobody) as model proteins. By using the inducer-free strain as a parent, replacement of cbh1 with genes encoding two intrinsic enzymes (aspartic protease and glucoamylase) and three different nanobodies (1ZVH, caplacizumab, and ozoralizumab) resulted in their high secretory productions using glucose medium without inducers such as cellulose. Based on signal sequences (carrier polypeptides) and protease inhibitors, additional replacement of cbh2 with the nanobody gene increased the percentage of POI to about 20% of total secreted proteins in T. reesei. This allowed the production of caplacizumab, a bivalent nanobody, to be increased to 9.49-fold (508 mg/L) compared to the initial inducer-free strain. CONCLUSIONS: In general, whereas the replacement of major cellulase genes leads to extreme decrease in the degradation capacity of cellulose, our inducer-free system enabled it and achieved high secretory production of POI with increased occupancy in glucose medium. This system would be a novel platform for heterologous recombinant protein production in T. reesei.

[Surgical Resection as Local Therapy for Sister Mary Joseph's Nodule Due to Pancreatic Tail Cancer].

We experienced a case of resection of a metastatic umbilical tumor(Sister Mary Joseph's nodule: SMJN)derived from a pancreatic tail carcinoma. The patient was a 70-year-old woman. She visited her previous doctor with a chief complaint of lower abdominal pain and came to our hospital due to suspicion of pancreatic tail cancer. She was found to have metastases to multiple organs which was unresectable by surgery. After chemotherapy up to the second-line of treatment, she was diagnosed to have progressive disease. The decision was made to provide the best supportive care for the patient. Thereafter, the patient developed SMJN. She had hemorrhage from the tumor accompanied by body movement, and her activity of daily living became impaired. She had difficulty controlling the bleeding despite repeated hemostatic treatment at the outpatient clinic and at her home. However, she required frequent blood transfusions for her severe anemia. Therefore, we performed a resection of the SMJN to control bleeding and to relieve her symptoms. She had a good postoperative course and was discharged on the fifth postoperative day. Due to deterioration of her general condition, she expired on the 59th day after surgery. However, the patient was able to live at home without bleeding or pain by the umbilical tumor. The local resection was considered to be useful as a palliative surgical treatment for SMJN.

Online SFE-SFC-MS/MS colony screening: A high-throughput approach for optimizing (-)-limonene production.

Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.

Conversion of Mevalonate to Isoprenol Using Light Energy in Escherichia coli without Consuming Sugars for ATP Supply.

Bioconversion of key intermediate metabolites such as mevalonate into various useful chemicals is a promising strategy for microbial production. However, the conversion of mevalonate into isoprenoids requires a supply of adenosine triphosphate (ATP). Light-driven ATP regeneration using microbial rhodopsin is an attractive module for improving the intracellular ATP supply. In the present study, we demonstrated the ATP-consuming conversion of mevalonate to isoprenol using rhodopsin-expressing Escherichia coli cells as a whole-cell catalyst in a medium that does not contain energy cosubstrate, such as glucose. Heterologous genes for the synthesis of isoprenol from mevalonate, which requires three ATP molecules for the series of reactions, and a delta-rhodopsin gene derived from Haloterrigena turkmenica were cointroduced into E. coli. To evaluate the conversion efficiency of mevalonate to isoprenol, the cells were suspended in a synthetic medium containing mevalonate as the sole carbon source and incubated under dark or light illumination (100 µmol m-2 s-1). The specific isoprenol production rates were 10.0 ± 0.9 and 20.4 ± 0.7 µmol gDCW-1 h-1 for dark and light conditions, respectively. The conversion was successfully enhanced under the light condition. Furthermore, the conversion efficiency increased with increasing illumination intensity, suggesting that ATP regenerated by the proton motive force generated by rhodopsin using light energy can drive ATP-consuming reactions in the whole-cell catalyst.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli.

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established. In this study, we attempted to develop an evolutionary engineering method to improve the proton-pumping activity of rhodopsins. We first introduced random mutations into delta-rhodopsin (dR) from Haloterrigena turkmenica using error-prone PCR to generate approximately 7000 Escherichia coli strains carrying the mutant dR genes. Rhodopsin-expressing E. coli with enhanced proton pumping activity have significantly increased survival rates in prolonged saline water. Considering this, we enriched the mutant E. coli cells with higher proton pumping rates by selecting populations able to survive starvation under 50 µmol m-2 s-1 at 37 °C. As a result, we successfully identified two strains, in which proton pumping activity was enhanced two-fold by heterologous expression in E. coli in comparison to wild-type strains. The combined approach of survival testing using saline water and evolutionary engineering methods used in this study will contribute greatly to the discovery of a novel rhodopsin with improved proton pumping activity. This will facilitate the utilization of rhodopsin in industrial applications.

Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris.

In our previous study, we serendipitously discovered that protein secretion in the methylotrophic yeast Pichia pastoris is enhanced by a mutation (V50A) in the mating factor alpha (MFα) prepro-leader signal derived from Saccharomyces cerevisiae. In the present study, we investigated 20 single-amino-acid substitutions, including V50A, located within the MFα signal peptide, indicating that V50A and several single mutations alone provided significant increase in production of the secreted proteins. In addition to hydrophobicity index analysis, both an unfolded protein response (UPR) biosensor analysis and a microscopic observation showed a clear difference on the levels of UPR induction and mis-sorting of secretory protein into vacuoles among the wild-type and mutated MFα signal peptides. This work demonstrates the importance of avoiding entry of secretory proteins into the intracellular protein degradation pathways, an observation that is expected to contribute to the engineering of strains with increased production of recombinant secreted proteins.

A streamlined strain engineering workflow with genome-wide screening detects enhanced protein secretion in Komagataella phaffii.

Expression of secreted recombinant proteins burdens the protein secretion machinery, limiting production. Here, we describe an approach to improving protein production by the non-conventional yeast Komagataella phaffii comprised of genome-wide screening for effective gene disruptions, combining them in a single strain, and recovering growth reduction by adaptive evolution. For the screen, we designed a multiwell-formatted, streamlined workflow to high-throughput assay of secretion of a single-chain small antibody, which is cumbersome to detect but serves as a good model of proteins that are difficult to secrete. Using the consolidated screening system, we evaluated >19,000 mutant strains from a mutant library prepared by a modified random gene-disruption method, and identified six factors for which disruption led to increased antibody production. We then combined the disruptions, up to quadruple gene knockouts, which appeared to contribute independently, in a single strain and observed an additive effect. Target protein and promoter were basically interchangeable for the effects of knockout genes screened. We finally used adaptive evolution to recover reduced cell growth by multiple gene knockouts and examine the possibility for further enhancing protein secretion. Our successful, three-part approach holds promise as a method for improving protein production by non-conventional microorganisms.

Predictors of a difficult Pringle maneuver in laparoscopic liver resection and evaluation of alternative procedures to assist bleeding control.

PURPOSE: To evaluate the predictors of a difficult Pringle maneuver (PM) in laparoscopic liver resection (LLR) and to assess alternative procedures to PM. METHODS: Data from patients undergoing LLR between 2013 and 2020 were reviewed retrospectively. Univariate and multivariate analyses were performed and the outcomes of patients who underwent PM or alternative procedures were compared. RESULTS: Among 106 patients who underwent LLR, PM could not be performed in 18 (17.0%) because of abdominal adhesions in 14 (77.8%) and/or collateral flow around the hepatoduodenal ligament in 5 (27.8%). Multivariate analysis revealed that Child-Pugh classification B (p = 0.034) and previous liver resection (p < 0.001) were independently associated with difficulty in performing PM in LLR. We evaluated pre-coagulation of liver tissue using microwave tissue coagulators, saline irrigation monopolar, clamping of the hepatoduodenal ligament using an intestinal clip, and hand-assisted laparoscopic surgery as alternatives procedures to PM. There were no significant differences in blood loss (p = 0.391) or transfusion (p = 0.518) between the PM and alternative procedures. CONCLUSIONS: Child-Pugh classification B and previous liver resection were identified as predictors of a difficult PM in LLR. The alternative procedures were found to be effective.

LXRß Activation Inhibits the Proliferation of Small-cell Lung Cancer Cells by Depleting Cellular Cholesterol.

BACKGROUND/AIM: Liver X receptors (LXRs) are nuclear receptors with various functions, including the regulation of cholesterol metabolism, glucose homeostasis, and inflammation. We previously reported that LXR activation inhibits the growth of oral cancer cells by inducing cellular cholesterol efflux and that LXRß is expressed mainly in small-cell lung cancer (SCLC) tissues. SCLC is one of the most aggressive cancers, and identifying an effective therapeutic target molecule is desirable. Therefore, we investigated whether LXRß could be an effective target molecule for SCLC treatment through in vitro experiments. MATERIALS AND METHODS: We evaluated the influence of treatment with the LXR agonist T0901317 on cell proliferation and apoptosis in SCLC cell lines using cell viability, BrdU-ELISA, FACS, and western blot analyses. Moreover, the mechanism by which T0901317 inhibits SCLC cell proliferation was elucidated using qRT-PCR, western blot, a cholesterol quantification assay, and a genome editing technique. RESULTS: We showed that cultivated SCLC cells expressed LXRß and that an LXR agonist inhibited the proliferation of SCLC cells without toxicity to normal cells. Furthermore, the antitumoral effect of an LXR agonist on SCLC cells was attributed to the induction of ABCA1 by LXRß activation, resulting in an increase in cellular cholesterol efflux via ABCA1. CONCLUSION: The activation of LXRß up-regulates ABCA1 expression, causing cholesterol depletion in cancer cells. This mechanism could be a novel target strategy for SCLC.

Enhanced squalene production by modulation of pathways consuming squalene and its precursor.

Fermentative production of squalene in yeast as an alternative approach to extracting squalene from sharks or plants has attracted significant interest. However, squalene accumulation is limited due to its inevitable high-flux allocation toward ergosterol synthesis. In this study, we described expression control of squalene monooxygenase (Erg1p), the first-step enzyme of ergosterol synthesis from squalene, to significantly reduce squalene loss. We replaced the ERG1 promoter (PERG1) with three natural yeast promoters with different activities (PPCL2, PHCM1, and PTHI2). ERG1 controlled by PTHI2 showed 20 times higher squalene production compared with the wild-type strain, whereas the other two strains exhibited no significant difference. By combining the overexpression of rate-limiting enzyme and the deletion of non-essential competing pathway gene, the yeast Saccharomyces cerevisiae produced up to 379 mg/L of squalene.

REST Inactivation and Coexpression of ASCL1 and POU3F4 Are Necessary for the Complete Transformation of RB1/TP53-Inactivated Lung Adenocarcinoma into Neuroendocrine Carcinoma.

Although recent reports have revealed the importance of the inactivation of both RB1 and TP53 in the transformation from lung adenocarcinoma into neuroendocrine carcinoma (NEC), the requirements for complete transformation into NEC have not been elucidated. To investigate alterations in the characteristics associated with the inactivation of RB1/TP53 and define the requirements for transformation into NEC cells, RB1/TP53 double-knockout A549 lung adenocarcinoma cells were established, and additional knockout of REST and transfection of ASCL1 and POU class 3 homeobox transcription factors (TFs) was conducted. More than 60 genes that are abundantly expressed in neural cells and several genes associated with epithelial-to-mesenchymal transition were up-regulated in RB1/TP53 double-knockout A549 cells. Although the expression of chromogranin A and synaptophysin was induced by additional knockout of REST (which mimics the status of most NECs), the expression of another neuroendocrine marker, CD56, and proneural TFs was not induced. However, coexpression of ASCL1 and POU3F4 in RB1/TP53/REST triple-knockout A549 cells induced the expression of not only CD56 but also other proneural TFs (NEUROD1 and insulinoma-associated 1) and induced NEC-like morphology. These findings suggest that the inactivation of RB1 and TP53 induces a state necessary for the transformation of lung adenocarcinoma into NEC and that further inactivation of REST and coexpression of ASCL1 and POU3F4 are the triggers for complete transformation into NEC.

Engineering of Synthetic Transcriptional Switches in Yeast.

Transcriptional switches can be utilized for many purposes in synthetic biology, including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although to date such switches have mainly been developed in prokaryotes, those for eukaryotes are increasingly being reported as both rational and random engineering technologies mature. In this review, we describe yeast transcriptional switches with different modes of action and how to alter their properties. We also discuss directed evolution technologies for the rapid and robust construction of yeast transcriptional switches.

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.