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1. Engineered biosynthesis of plant heteroyohimbine and corynantheine alkaloids in Saccharomyces cerevisiae

   https://doi.org/10.1093/jimb/kuad047  

   Dror, Moriel J. ; Misa, Joshua ; Yee, Danielle A. ; Chu, Angela M. ; Yu, Rachel K. ; Chan, Bradley B. ; Aoyama, Lauren S. ; Chaparala, Anjali P. ; O'Connor, Sarah E. ; Tang, Yi ( December 2023 , Journal of Industrial Microbiology and Biotechnology)

   Monoterpene indole alkaloids (MIAs) are a class of natural products comprised of thousands of structurally unique bioactive compounds with significant therapeutic values. Due to difficulties associated with isolation from native plant species and organic synthesis of these structurally complex molecules, microbial production of MIAs using engineered hosts are highly desired. In this work, we report the engineering of fully integrated Saccharomyces cerevisiae strains that allow de novo access to strictosidine, the universal precursor to thousands of MIAs at 30–40 mg/L. The optimization efforts were based on a previously reported yeast strain that is engineered to produce high titers of the monoterpene precursor geraniol through compartmentalization of mevalonate pathway in the mitochondria. Our approaches here included the use of CRISPR-dCas9 interference to identify mitochondria diphosphate transporters that negatively impact the titer of the monoterpene, followed by genetic inactivation; the overexpression of transcriptional regulators that increase cellular respiration and mitochondria biogenesis. Strain construction included the strategic integration of genes encoding both MIA biosynthetic and accessory enzymes into the genome under a variety of constitutive and inducible promoters. Following successful de novo production of strictosidine, complex alkaloids belonging to heteroyohimbine and corynantheine families were reconstituted in the host with introduction of additional downstream enzymes. We demonstrate that the serpentine/alstonine pair can be produced at ∼5 mg/L titer, while corynantheidine, the precursor to mitragynine can be produced at ∼1 mg/L titer. Feeding of halogenated tryptamine led to the biosynthesis of analogs of alkaloids in both families. Collectively, our yeast strain represents an excellent starting point to further engineer biosynthetic bottlenecks in this pathway and to access additional MIAs and analogs through microbial fermentation.

   An Saccharomyces cerevisiae-based microbial platform was developed for the biosynthesis of monoterpene indole alkaloids, including the universal precursor strictosidine and further modified heteroyohimbine and corynantheidine alkaloids.

    

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2. Xylose and shikimate transporters facilitates microbial consortium as a chassis for benzylisoquinoline alkaloid production

   https://doi.org/10.1038/s41467-023-43049-w  

   Gao, Meirong ; Zhao, Yuxin ; Yao, Zhanyi ; Su, Qianhe ; Van Beek, Payton ; Shao, Zengyi ( November 2023 , Nature Communications)

   Plant-sourced aromatic amino acid (AAA) derivatives are a vast group of compounds with broad applications. Here, we present the development of a yeast consortium for efficient production of (S)-norcoclaurine, the key precursor for benzylisoquinoline alkaloid biosynthesis. A xylose transporter enables the concurrent mixed-sugar utilization inScheffersomyces stipitis, which plays a crucial role in enhancing the flux entering the highly regulated shikimate pathway located upstream of AAA biosynthesis. Two quinate permeases isolated fromAspergillus nigerfacilitates shikimate translocation to the co-culturedSaccharomyces cerevisiaethat converts shikimate to (S)-norcoclaurine, resulting in the maximal titer (11.5 mg/L), nearly 110-fold higher than the titer reported for anS. cerevisiaemonoculture. Our findings magnify the potential of microbial consortium platforms for the economical de novo synthesis of complex compounds, where pathway modularization and compartmentalization in distinct specialty strains enable effective fine-tuning of long biosynthetic pathways and diminish intermediate buildup, thereby leading to increases in production.

    

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3. Synthesis of polyketides from low cost substrates by the thermotolerant yeast Kluyveromyces marxianus

   https://doi.org/10.1002/bit.26976  

   McTaggart, Tami L. ; Bever, Danielle ; Bassett, Shane ; Da Silva, Nancy A. ( April 2019 , Biotechnology and Bioengineering)

   Kluyveromyces marxianusis a promising nonconventional yeast for biobased chemical production due to its rapid growth rate, high TCA cycle flux, and tolerance to low pH and high temperature. UnlikeSaccharomyces cerevisiae, K. marxianusgrows on low‐cost substrates to cell densities that equal or surpass densities in glucose, which can be beneficial for utilization of lignocellulosic biomass (xylose), biofuel production waste (glycerol), and whey (lactose). We have evaluatedK. marxianusfor the synthesis of polyketides, using triacetic acid lactone (TAL) as the product. The 2‐pyrone synthase (2‐PS) was expressed on a CEN/ARS plasmid in three different strains, and the effects of temperature, carbon source, and cultivation strategy on TAL levels were determined. The highest titer was obtained in defined 1% xylose medium at 37°C, with substantial titers at 41 and 43°C. The introduction of a high‐stability 2‐PS mutant and a promoter substitution increased titer four‐fold. 2‐PS expression from a multi‐copy pKD1‐based plasmid improved TAL titers a further five‐fold. Combining the best plasmid, promoter, and strain resulted in a TAL titer of 1.24 g/L and a yield of 0.0295 mol TAL/mol carbon for this otherwise unengineered strain in 3 ml tube culture. This is an excellent titer and yield (on xylose) before metabolic engineering or fed‐batch culture relative to other hosts (on glucose), and demonstrates the promise of this rapidly growing and thermotolerant yeast species for polyketide production.

    

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4. CRISPR-mediated multigene integration enables Shikimate pathway refactoring for enhanced 2-phenylethanol biosynthesis in Kluyveromyces marxianus

   https://doi.org/10.1186/s13068-020-01852-3  

   Li, Mengwan ; Lang, Xuye ; Moran Cabrera, Marcos ; De Keyser, Sawyer ; Sun, Xiyan ; Da Silva, Nancy ; Wheeldon, Ian ( December 2021 , Biotechnology for Biofuels)

   null (Ed.)

   Abstract Background 2-phenylethanol (2-PE) is a rose-scented flavor and fragrance compound that is used in food, beverages, and personal care products. Compatibility with gasoline also makes it a potential biofuel or fuel additive. A biochemical process converting glucose or other fermentable sugars to 2-PE can potentially provide a more sustainable and economical production route than current methods that use chemical synthesis and/or isolation from plant material. Results We work toward this goal by engineering the Shikimate and Ehrlich pathways in the stress-tolerant yeast Kluyveromyces marxianus . First, we develop a multigene integration tool that uses CRISPR-Cas9 induced breaks on the genome as a selection for the one-step integration of an insert that encodes one, two, or three gene expression cassettes. Integration of a 5-kbp insert containing three overexpression cassettes successfully occurs with an efficiency of 51 ± 9% at the ABZ1 locus and was used to create a library of K. marxianus CBS 6556 strains with refactored Shikimate pathway genes. The 3 3 -factorial library includes all combinations of KmARO4 , KmARO7 , and KmPHA2 , each driven by three different promoters that span a wide expression range. Analysis of the refactored pathway library reveals that high expression of the tyrosine-deregulated KmARO4 K221L and native KmPHA2 , with the medium expression of feedback insensitive KmARO7 G141S , results in the highest increase in 2-PE biosynthesis, producing 684 ± 73 mg/L. Ehrlich pathway engineering by overexpression of KmARO10 and disruption of KmEAT1 further increases 2-PE production to 766 ± 6 mg/L. The best strain achieves 1943 ± 63 mg/L 2-PE after 120 h fed-batch operation in shake flask cultures. Conclusions The CRISPR-mediated multigene integration system expands the genome-editing toolset for K. marxianus, a promising multi-stress tolerant host for the biosynthesis of 2-PE and other aromatic compounds derived from the Shikimate pathway. 

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5. Adaptive laboratory evolution of β-caryophyllene producing Saccharomyces cerevisiae

   https://doi.org/10.1186/s12934-021-01598-z  

   Godara, Avinash ; Kao, Katy C. ( May 2021 , Microbial Cell Factories)

   β-Caryophyllene is a plant terpenoid with therapeutic and biofuel properties. Production of terpenoids through microbial cells is a potentially sustainable alternative for production. Adaptive laboratory evolution is a complementary technique to metabolic engineering for strain improvement, if the product-of-interest is coupled with growth. Here we use a combination of pathway engineering and adaptive laboratory evolution to improve the production of β-caryophyllene, an extracellular product, by leveraging the antioxidant potential of the compound.

   Using oxidative stress as selective pressure, we developed an adaptive laboratory evolution that worked to evolve an engineered β-caryophyllene producing yeast strain for improved production within a few generations. This strategy resulted in fourfold increase in production in isolated mutants. Further increasing the flux to β-caryophyllene in the best evolved mutant achieved a titer of 104.7 ± 6.2 mg/L product. Genomic analysis revealed a gain-of-function mutation in the a-factor exporterSTE6was identified to be involved in significantly increased production, likely as a result of increased product export.

   An optimized selection strategy based on oxidative stress was developed to improve the production of the extracellular product β-caryophyllene in an engineered yeast strain. Application of the selection strategy in adaptive laboratory evolution resulted in mutants with significantly increased production and identification of novel responsible mutations.

    

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