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Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3p Syn4.1 ), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3p MC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3p MC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3p MC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3p MC -mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy., Competing Interests: Declaration of competing interest None., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Failure by RNA polymerase to break contacts with promoter DNA results in release of bound RNA and re-initiation of transcription. These abortive RNAs were assumed to be non-functional but have recently been shown to affect termination in bacteriophage T7. Little is known about the functional role of these RNA in other genetic models. Using a computational approach, we investigated whether abortive RNA could exert function in E. coli. Fragments generated from 3780 transcription units were used as query sequences within their respective transcription units to search for possible binding sites. Sites that fell within known regulatory features were then ranked based upon the free energy of hybridization to the abortive. We further hypothesize about mechanisms of regulatory action for a select number of likely matches. Future experimental validation of these putative abortive-mRNA pairs may confirm our findings and promote exploration of functional abortive RNAs (faRNAs) in natural and synthetic systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Saccharomyces cerevisiae has increasingly been engineered as a cell factory for efficient and economic production of fuels and chemicals from renewable resources. Notably, a wide variety of industrially important products are derived from the same precursor metabolite, acetyl-CoA. However, the limited supply of acetyl-CoA in the cytosol, where biosynthesis generally happens, often leads to low titer and yield of the desired products in yeast. In the present work, combined strategies of disrupting competing pathways and introducing heterologous biosynthetic pathways were carried out to increase acetyl-CoA levels by using the CoA-dependent n-butanol production as a reporter. By inactivating ADH1 and ADH4 for ethanol formation and GPD1 and GPD2 for glycerol production, the glycolytic flux was redirected towards acetyl-CoA, resulting in 4-fold improvement in n-butanol production. Subsequent introduction of heterologous acetyl-CoA biosynthetic pathways, including pyruvate dehydrogenase (PDH), ATP-dependent citrate lyase (ACL), and PDH-bypass, further increased n-butanol production. Recombinant PDHs localized in the cytosol (cytoPDHs) were found to be the most efficient, which increased n-butanol production by additional 3 fold. In total, n-butanol titer and acetyl-CoA concentration were increased more than 12 fold and 3 fold, respectively. By combining the most effective and complementary acetyl-CoA pathways, more than 100mg/L n-butanol could be produced using high cell density fermentation, which represents the highest titer ever reported in yeast using the clostridial CoA-dependent pathway., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The biocatalytic reduction of D-xylose to xylitol requires separation of the substrate from L-arabinose, another major component of hemicellulosic hydrolysate. This step is necessitated by the innate promiscuity of xylose reductases, which can efficiently reduce L-arabinose to L-arabinitol, an unwanted byproduct. Unfortunately,due to the epimeric nature of D-xylose and L-arabinose, separation can be difficult, leading to high production costs. To overcome this issue, we engineered an E. coli strain to efficiently produce xylitol from D-xylose with minimal production of L-arabinitol byproduct. By combining this strain with a previously engineered xylose reductase mutant, we were able to eliminate L-arabinitol formation and produce xylitol to near 100% purity from an equiweight mixture of D-xylose, L-arabinose, and D-glucose., (2010 Elsevier Inc. All rights reserved.)