Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae

Industrial biotechnology employs the controlled use of microorganisms for the production of synthetic chemicals or simple biomass that can further be used in a diverse array of applications that span the pharmaceutical, chemical and nutraceutical industries. Recent advances in metagenomics and in the incorporation of entire biosynthetic pathways into Saccharomyces cerevisiae have greatly expanded both the fitness and the repertoire of biochemicals that can be synthesized from this popular microorganism. Further, the availability of the S. cerevisiae entire genome sequence allows the application of systems biology approaches for improving its enormous biosynthetic potential. In this review, we will describe some of the efforts on using S. cerevisiae as a cell factory for the biosynthesis of high-value natural products that belong to the families of isoprenoids, flavonoids and long chain polyunsaturated fatty acids. As natural products are increasingly becoming the center of attention of the pharmaceutical and nutraceutical industries, the use of S. cerevisiae for their production is only expected to expand in the future, further allowing the biosynthesis of novel molecular structures with unique properties

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.Improved catalysis with engineered polyketide synthases: Pairing wild‐type polyketide synthases with non‐native substrates largely failed to produce the anticipated products. A series of hybrid modules bearing heterologous thioesterase domains were generated and employed to alleviate the observed catalytic bottleneck, resulting in the efficient processing of non‐native substrates and an unexpected path to product diversity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/3/anie202004991-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/2/anie202004991_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156208/1/anie202004991.pd

Probing Selectivity and Creating Structural Diversity Through Hybrid Polyketide Synthases

Engineering polyketide synthases (PKS) to produce new metabolites requires an understanding of catalytic points of failure during substrate processing. Growing evidence indicates the thioesterase (TE) domain as a significant bottleneck within engineered PKS systems. We created a series of hybrid PKS modules bearing exchanged TE domains from heterologous pathways and challenged them with both native and non‐native polyketide substrates. Reactions pairing wildtype PKS modules with non‐native substrates primarily resulted in poor conversions to anticipated macrolactones. Likewise, product formation with native substrates and hybrid PKS modules bearing non‐cognate TE domains was severely reduced. In contrast, non‐native substrates were converted by most hybrid modules containing a substrate compatible TE, directly implicating this domain as the major catalytic gatekeeper and highlighting its value as a target for protein engineering to improve analog production in PKS pathways.Improved catalysis with engineered polyketide synthases: Pairing wild‐type polyketide synthases with non‐native substrates largely failed to produce the anticipated products. A series of hybrid modules bearing heterologous thioesterase domains were generated and employed to alleviate the observed catalytic bottleneck, resulting in the efficient processing of non‐native substrates and an unexpected path to product diversity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/2/ange202004991-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/1/ange202004991.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/156161/3/ange202004991_am.pd

Synthesis of Enantiomerically Enriched Imidazolidin‐2‐Ones through Asymmetric Palladium‐Catalyzed Alkene Carboamination Reactions

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93726/1/ange_201205233_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/93726/2/10024_ftp.pd

Copper-Catalyzed Borylation of Cyclic Sulfamidates:Access to Enantiomerically Pure (β-and γ-Amino-alkyl)boronic Esters

Cyclic sulfamidates undergo borylation under copper‐catalyzed conditions using B(2)pin(2) to give enantiomerically (and diasteromerically) defined (aminoalkyl)boronic esters. External iodide is essential, but the intermediacy of simple alkyl iodides has been excluded; N‐sulfated intermediates are key in the borylation sequence. Based on stereochemical studies and trapping experiments, the involvement of carbon‐centered radicals under these copper‐catalyzed conditions appears likely

Advances in Pathway Engineering for Natural Product Biosynthesis

Biocatalysts provide an efficient, inexpensive and environmentally friendly alternative to traditional organic synthesis, especially for compounds with complex stereochemistries. The past decade has seen a significant rise in the use of biocatalysts for the synthesis of compounds in an industrial setting; however, the incorporation of single enzymatically catalysed steps into organic synthesis schemes can be problematic. The emerging field of synthetic biology has sparked interest in the development of whole-cell factories that can convert simple, common metabolites into complex, high-value molecules with a range of applications such as pharmaceuticals and biofuels. This Review summarises conventional methods and recent advances in metabolic engineering of pathways in microorganisms for the synthesis of natural products

Development of Non-Natural Flavanones as Antimicrobial Agents

With growing concerns over multidrug resistance microorganisms, particularly strains of bacteria and fungi, evolving to become resistant to the antimicrobial agents used against them, the identification of new molecular targets becomes paramount for novel treatment options. Recently, the use of new treatments containing multiple active ingredients has been shown to increase the effectiveness of existing molecules for some infections, often with these added compounds enabling the transport of a toxic molecule into the infecting species. Flavonoids are among the most abundant plant secondary metabolites and have been shown to have natural abilities as microbial deterrents and anti-infection agents in plants. Combining these ideas we first sought to investigate the potency of natural flavonoids in the presence of efflux pump inhibitors to limit Escherichia coli growth. Then we used the natural flavonoid scaffold to synthesize non-natural flavanone molecules and further evaluate their antimicrobial efficacy on Escherichia coli, Bacillus subtilis and the fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus. Of those screened, we identified the synthetic molecule 4-chloro-flavanone as the most potent antimicrobial compound with a MIC value of 70 µg/mL in E. coli when combined with the inhibitor Phe-Arg-ß-naphthylamide, and MICs of 30 µg/mL in S. cerevesiae and 30 µg/mL in C. neoformans when used alone. Through this study we have demonstrated that combinatorial synthesis of non-natural flavonones can identify novel antimicrobial agents with activity against bacteria and fungi but with minimal toxicity to human cells

Rhodium-Catalyzed Asymmetric Intramolecular Hydroamination of Unactivated Alkenes

One for the Rh(oad): The first rhodium-catalyzed asymmetric intramolecular hydroamination of unactivated olefins was developed by using dialkylbiaryl phosphine ligands (see scheme; cod=1,5-cyclooctadiene, Cy=cyclohexyl). A variety of 2-methylpyrrolidines have been synthesized with high enantioselectivities.National Institutes of Health (U.S.) (Grant GM46059)Merck & Co.Boehringer Ingelheim PharmaceuticalsNational Institutes of Health (U.S.) (GM 1S10RR13886-01

Co-production of hydrogen and ethanol from glucose in Escherichia coli by activation of pentose-phosphate pathway through deletion of phosphoglucose isomerase (pgi) and overexpression of glucose-6-phosphate dehydrogenase (zwf) and 6-phosphogluconate dehydrogenase (gnd)

Background: Biologically, hydrogen (H-2) can be produced through dark fermentation and photofermentation. Dark fermentation is fast in rate and simple in reactor design, but H-2 production yield is unsatisfactorily low as < 4 mol H-2/ mol glucose. To address this challenge, simultaneous production of H-2 and ethanol has been suggested. Co-production of ethanol andH(2) requires enhanced formation of NAD(P) H during catabolism of glucose, which can be accomplished by diversion of glycolytic flux from the Embden-Meyerh-of-Parnas (EMP) pathway to the pentose-phosphate (PP) pathway in Escherichia coli. However, the disruption of pgi (phosphoglucose isomerase) for complete diversion of carbon flux to the PP pathway made E. coli unable to grow on glucose under anaerobic condition. Results: Here, we demonstrate that, when glucose-6-phosphate dehydrogenase (Zwf) and 6-phosphogluconate dehydrogenase (Gnd), two major enzymes of the PP pathway, are homologously overexpressed, E. coli.pgi can recover its anaerobic growth capability on glucose. Further, with additional deletions of Delta hycA,Delta hyaAB,Delta hybBC,Delta ldhA, and Delta frdAB, the recombinant.pgi mutant could produce 1.69 mol H-2 and 1.50 mol ethanol from 1 mol glucose. However, acetate was produced at 0.18 mol mol(-1) glucose, indicating that some carbon is metabolized through the Entner-Doudoroff (ED) pathway. To further improve the flux via the PP pathway, heterologous zwf and gnd from Leuconostoc mesenteroides and Gluconobacter oxydans, respectively, which are less inhibited by NADPH, were overexpressed. The new recombinant produced more ethanol at 1.62 mol mol(-1) glucose along with 1.74 mol H-2 mol(-1) glucose, which are close to the theoretically maximal yields, 1.67 mol mol(-1) each for ethanol andH(2). However, the attempt to delete the ED pathway in the.pgi mutant to operate the PP pathway as the sole glycolytic route, was unsuccessful. Conclusions: By deletion of pgi and overexpression of heterologous zwf and gnd in E. coli Delta hycA Delta hyaAB Delta hybBC Delta ldhA Delta frdAB, two important biofuels, ethanol andH(2), could be successfully co-produced at high yields close to their theoretical maximums. The strains developed in this study should be applicable for the production of other biofuels and biochemicals, which requires supply of excessive reducing power under anaerobic conditions