Morphinan alkaloids are the most powerful narcotic analgesics currently used to treat moderate to severe and chronic pain. The feasibility of morphinan synthesis in recombinant Saccharomyces ...cerevisiae starting from the precursor (R,S)-norlaudanosoline was investigated. Chiral analysis of the reticuline produced by the expression of opium poppy methyltransferases showed strict enantioselectivity for (S)-reticuline starting from (R,S)-norlaudanosoline. In addition, the P. somniferum enzymes salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) were functionally co-expressed in S. cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Finally, we reconstituted a 7-gene pathway for the production of codeine and morphine from (R)-reticuline. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates showed that all enzymes were functionally co-expressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in S. cerevisiae and pave the way for their complete synthesis in recombinant microbes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
An upsurge in the bioeconomy drives the need for engineering microorganisms with increasingly complex phenotypes. Gains in productivity of industrial microbes depend on the development of improved ...strains. Classical strain improvement programmes for the generation, screening and isolation of such mutant strains have existed for several decades. An alternative to traditional strain improvement methods, genome shuffling, allows the directed evolution of whole organisms via recursive recombination at the genome level. This review deals chiefly with the technical aspects of genome shuffling. It first presents the diversity of organisms and phenotypes typically evolved using this technology and then reviews available sources of genetic diversity and recombination methodologies. Analysis of the literature reveals that genome shuffling has so far been restricted to microorganisms, both prokaryotes and eukaryotes, with an overepresentation of antibiotics- and biofuel-producing microbes. Mutagenesis is the main source of genetic diversity, with few studies adopting alternative strategies. Recombination is usually done by protoplast fusion or sexual recombination, again with few exceptions. For both diversity and recombination, prospective methods that have not yet been used are also presented. Finally, the potential of genome shuffling for gaining insight into the genetic basis of complex phenotypes is also discussed.
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CEKLJ, DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, ...including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (
S
)-reticuline to 4.6 g L
−1
, a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.
Plants display an immense diversity of specialized metabolites, many of which have been important to humanity as medicines, flavors, fragrances, pigments, insecticides and other fine chemicals. ...Apparently, much of the variation in plant specialized metabolism evolved through events of gene duplications followed by neo- or sub-functionalization. Most of the catalytic diversity of plant enzymes is unexplored since previous biochemical and genomics efforts have focused on a relatively small number of species. Interdisciplinary research in plant genomics, microbial engineering and synthetic biology provides an opportunity to accelerate the discovery of new enzymes. The massive identification, characterization and cataloguing of plant enzymes coupled with their deployment in metabolically optimized microbes provide a high-throughput functional genomics tool and a novel strain engineering pipeline.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Benzylisoquinoline alkaloids (BIAs) are a family of ∼2500 alkaloids with both potential and realized pharmaceutical value, including most notably the opiates such as codeine and morphine. Only a few ...BIAs accumulate readily in plants, which limits the pharmaceutical potential of the family. Shifting BIA production to microbial sources could provide a scalable and flexible source of these compounds in the future. This review details the current status of microbial BIA synthesis and derivatization, including rapid developments in the past 6 months culminating in the synthesis of opioids from glucose in a microbial host.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Organelles are intracellular compartments which are themselves compartmentalized. Biogenic and metabolic processes are localized to specialized domains or microcompartments to enhance their ...efficiency and suppress deleterious side reactions. An example of intra-organellar compartmentalization is the pyrenoid in the chloroplasts of algae and hornworts. This microcompartment enhances the photosynthetic CO2-fixing activity of the Calvin-Benson cycle enzyme Rubisco, suppresses an energetically wasteful oxygenase activity of Rubisco, and mitigates limiting CO2 availability in aquatic environments. Hence, the pyrenoid is functionally analogous to the carboxysomes in cyanobacteria. However, a comprehensive analysis of pyrenoid functions based on its protein composition is lacking. Here we report a proteomic characterization of the pyrenoid in the green alga Chlamydomonas reinhardtii. Pyrenoid-enriched fractions were analyzed by quantitative mass spectrometry. Contaminant proteins were identified by parallel analyses of pyrenoid-deficient mutants. This pyrenoid proteome contains 190 proteins, many of which function in processes that are known or proposed to occur in pyrenoids: e.g. the carbon concentrating mechanism, starch metabolism or RNA metabolism and translation. Using radioisotope pulse labeling experiments, we show that pyrenoid-associated ribosomes could be engaged in the localized synthesis of the large subunit of Rubisco. New pyrenoid functions are supported by proteins in tetrapyrrole and chlorophyll synthesis, carotenoid metabolism or amino acid metabolism. Hence, our results support the long-standing hypothesis that the pyrenoid is a hub for metabolism. The 81 proteins of unknown function reveal candidates for new participants in these processes. Our results provide biochemical evidence of pyrenoid functions and a resource for future research on pyrenoids and their use to enhance agricultural plant productivity. Data are available via ProteomeXchange with identifier PXD004509.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Saccharomyces cerevisiae
is a workhorse of industrial biotechnology owing to the organism’s prominence in alcohol fermentation and the suite of sophisticated genetic tools available to ...manipulate its metabolism. However,
S. cerevisiae
is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L
−1
adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select
Pichia occidentalis
for production of
cis
,
cis
-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for
P. occidentalis
, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L
−1
), yield (0.134 g g
−1
glucose) and productivity (0.511 g L
−1
h
−1
) that surpasses all metrics achieved using
S. cerevisiae
. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.
Biofoundries provide an integrated infrastructure to enable the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. Many ...biofoundries are being built and a Global Biofoundry Alliance has recently been established to coordinate activities worldwide.
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