Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs fromY. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica

Building Synthetic Yeast Factories to Produce Fat-soluble Antioxidants

Fat-soluble antioxidants play a vital role in protecting the body against oxidative stress and damage. The rapid advancements in metabolic engineering and synthetic biology have offered a promising avenue for economically producing fat-soluble antioxidants by engineering microbial chassis. This review provides an overview of the recent progress in engineering yeast microbial factories to produce three main groups of lipophilic antioxidants: carotenoids, vitamin E, and stilbenoids. In addition to discussing the classic strategies employed to improve precursor availability and alleviate carbon flux competition, this review delves deeper into the innovative approaches focusing on enzyme engineering, product sequestration, subcellular compartmentalization, multistage fermentation, and morphology engineering. We conclude the review by highlighting the prospects of microbial engineering for lipophilic antioxidant production.This is a manuscript of the article Published as Zhao, Yuxin, Zhanyi Yao, Vedika Desai, Dan Chen, and Zengyi Shao. "Building Synthetic Yeast Factories to Produce Fat-soluble Antioxidants." Current Opinion in Biotechnology 87 (2024): 103129. doi: https://doi.org/10.1016/j.copbio.2024.103129. Posted with Permission

Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs fromY. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.This is a pre-print of the article Lopez, Carmen, Mingfeng Cao, Zhanyi Yao, and Zengyi Shao. Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica. Authorea (2020). DOI: 10.22541/au.160691063.38058320/v1. Posted with permission.</p

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

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 in Scheffersomyces 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 from Aspergillus niger facilitates shikimate translocation to the co-cultured Saccharomyces cerevisiae that converts shikimate to (S)-norcoclaurine, resulting in the maximal titer (11.5 mg/L), nearly 110-fold higher than the titer reported for an S. cerevisiae monoculture. 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.This article is published as Gao, Meirong, Yuxin Zhao, Zhanyi Yao, Qianhe Su, Payton Van Beek, and Zengyi Shao. "Xylose and shikimate transporters facilitates microbial consortium as a chassis for benzylisoquinoline alkaloid production." Nature Communications 14, no. 1 (2023): 7797. doi: https://doi.org/10.1038/s41467-023-43049-w. © The Author(s) 2023. This is an Open Access article licensed under a Creative Commons Attribution 4.0 http://creativecommons.org/licenses/by/4.0/

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

Abstract 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 in Scheffersomyces 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 from Aspergillus niger facilitates shikimate translocation to the co-cultured Saccharomyces cerevisiae that converts shikimate to (S)-norcoclaurine, resulting in the maximal titer (11.5 mg/L), nearly 110-fold higher than the titer reported for an S. cerevisiae monoculture. 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

Effects of Warming and Precipitation on Soil CO₂ Flux and Its Stable Carbon Isotope Composition in the Temperate Desert Steppe

The stable carbon (C) isotope of soil CO2 efflux (δ13CO2e) is closely associated with soil C dynamics, which have a complex feedback relationship with climate. Three levels of warming (T0: ambient temperature (15.7 °C); T1: T0 + 2 °C; T2: T0 + 4 °C) were combined with three levels of increased precipitation (W0: ambient precipitation (245.2 mm); W1: W0 + 25%; W2: W0 + 50%) in order to quantify soil CO2 flux and its δ13CO2e values under nine treatment conditions (T0W0, T0W1, T0W2, T1W0, T1W1, T1W2, T2W0, T2W1, and T2W2) in desert steppe in an experimental beginning in 2015. A non-steady state chamber system relying on Keeling plots was used to estimate δ13CO2e. The temperature (ST) and moisture (SM) of soil as well as soil organic carbon content (SOC) and δ13C values (δ13Csoil) were tested in order to interpret variations in soil CO2 efflux and δ13CO2e. Sampling was carried out during the growing season in 2018 and 2019. During the experiment, the ST and SM correspondingly increased due to warming and increased precipitation. CO2 flux ranged from 37 to 1103 mg m−2·h−1, and emissions peaked in early August in the desert steppe. Warming of 2 °C to 4 °C stimulated a 14% to 30.9% increase in soil CO2 efflux and a 0.4‰ to 1.8‰ enrichment in δ13CO2e, respectively. Increased precipitation raised soil CO2 efflux by 14% to 19.3%, and decreased δ13CO2e by 0.5‰ to 0.9‰. There was a positive correlation between soil CO2 efflux and ST and SOC indicating that ST affected soil CO2 efflux by changing SOC content. Although the δ13CO2e was positively correlated with ST, it was negatively correlated to SM. The decline of δ13CO2e with soil moisture was predominantly due to intensified and increased diffusive fractionation. The mean δ13CO2e value (−20.2‰) was higher than that of the soil carbon isotope signature at 0–20 cm (δ13Csoil = −22.7‰). The difference between δ13CO2e and δ13Csoil (Δe-s) could be used to evaluate the likelihood of substrate utilization. 13C enriched stable C pools were more likely to be utilized below 20 cm under warming of 2 °C in the desert steppe. Moreover, the interaction of T × W neither altered the CO2 emitted by soil nor the δ13CO2e or Δe-s, indicating that warming combined with precipitation may alleviate the SOC oxidation of soil enriched in 13C in the desert steppe

Effects of Warming and Precipitation on Soil CO2 Flux and Its Stable Carbon Isotope Composition in the Temperate Desert Steppe

The stable carbon (C) isotope of soil CO2 efflux (&delta;13CO2e) is closely associated with soil C dynamics, which have a complex feedback relationship with climate. Three levels of warming (T0: ambient temperature (15.7 &deg;C); T1: T0 + 2 &deg;C; T2: T0 + 4 &deg;C) were combined with three levels of increased precipitation (W0: ambient precipitation (245.2 mm); W1: W0 + 25%; W2: W0 + 50%) in order to quantify soil CO2 flux and its &delta;13CO2e values under nine treatment conditions (T0W0, T0W1, T0W2, T1W0, T1W1, T1W2, T2W0, T2W1, and T2W2) in desert steppe in an experimental beginning in 2015. A non-steady state chamber system relying on Keeling plots was used to estimate &delta;13CO2e. The temperature (ST) and moisture (SM) of soil as well as soil organic carbon content (SOC) and &delta;13C values (&delta;13Csoil) were tested in order to interpret variations in soil CO2 efflux and &delta;13CO2e. Sampling was carried out during the growing season in 2018 and 2019. During the experiment, the ST and SM correspondingly increased due to warming and increased precipitation. CO2 flux ranged from 37 to 1103 mg m&minus;2&middot;h&minus;1, and emissions peaked in early August in the desert steppe. Warming of 2 &deg;C to 4 &deg;C stimulated a 14% to 30.9% increase in soil CO2 efflux and a 0.4&permil; to 1.8&permil; enrichment in &delta;13CO2e, respectively. Increased precipitation raised soil CO2 efflux by 14% to 19.3%, and decreased &delta;13CO2e by 0.5&permil; to 0.9&permil;. There was a positive correlation between soil CO2 efflux and ST and SOC indicating that ST affected soil CO2 efflux by changing SOC content. Although the &delta;13CO2e was positively correlated with ST, it was negatively correlated to SM. The decline of &delta;13CO2e with soil moisture was predominantly due to intensified and increased diffusive fractionation. The mean &delta;13CO2e value (&minus;20.2&permil;) was higher than that of the soil carbon isotope signature at 0&ndash;20 cm (&delta;13Csoil = &minus;22.7&permil;). The difference between &delta;13CO2e and &delta;13Csoil (&Delta;e-s) could be used to evaluate the likelihood of substrate utilization. 13C enriched stable C pools were more likely to be utilized below 20 cm under warming of 2 &deg;C in the desert steppe. Moreover, the interaction of T &times; W neither altered the CO2 emitted by soil nor the &delta;13CO2e or &Delta;e-s, indicating that warming combined with precipitation may alleviate the SOC oxidation of soil enriched in 13C in the desert steppe