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1. Enhancing Lipid Production in Yarrowia lipolytica Through Continuous Fermentation and Adaptive Laboratory Evolution in Bioreactors

   https://doi.org/10.1177/15509087251389709  

   Kurt, Elif; Qin, Jiansong; Sa, Lucas; Anjo, Evelyn; Xie, Dongming (January 2026, Industrial Biotechnology)

   Gross, Richard (Ed.)

   Yarrowia lipolyticaexcels in microbial lipid production, thriving across diverse conditions. Batch or fed-batch fermentation is the not only common practice to achieve higher lipid titer and yield but it is also subject to lower lipid productivity. Single-stage continuous fermentation (CF) provides a great potential for significantly higher productivity, but genetic instability is often seen and challenges strain performance over the long-period CF. This study harnesses single-stage CF to not only improve lipid productivity but also evolve high-lipid mutants from a previously engineeredY. lipolyticastrain E26 via adaptive laboratory evolution (ALE) in a continuous bioreactor, guided by a predictive kinetic model. The single-stage CF was run for 1128 hours (47 days) with key process parameters adjusted in a 1-L bioreactor to produce over 150 g/L yeast biomass, exceeding the targeted 113 g/L that is predicted by the model. Compared with the fed-batch fermentation process, the single-stage CF successfully improved lipid productivity from 0.3–0.5 g/L/h to about 1 g/L/h while maintaining the lipid yield at around 0.1 g/g. The CF sample at 1008 hours was used to isolate mutants with higher lipid production after ALE in the continuous bioreactor. A mutant E26E03 was identified, which demonstrated improvements in biomass, lipid content, and lipid yield by 43%, 30%, and 51%, respectively, over the original strain E26 in fed-batch fermentation. Our study indicated that using model-guided CF with ALE in a continuous bioreactor provides a great potential for significantly higher product titer, rate, and yield in biomanufacturing. 

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2. Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the β-oxidation reversal

   Lee, Seung_Hwan; Hu, Yang; Chou, Alexander; Chou; Chen, Jing; Gonzalez, Ramon (February 2024, Metabolic engineering)

   Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient syn thesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements. 

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3. Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants

   https://doi.org/10.1128/AEM.00487-21  

   Moxley, W. Chris; Eiteman, Mark A. (June 2021, Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli , pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase ( ldhA ) and pyruvate oxidase ( poxB ). The effect of deleting phosphoenolpyruvate synthase ( ppsA ) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h −1 , an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli . This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes. 

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4. 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)

   Abstract  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. One Sentence SummaryAn 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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5. Engineering of a Highly Efficient Escherichia coli Strain for Mevalonate Fermentation through Chromosomal Integration

   https://doi.org/10.1128/AEM.02178-16  

   Wang, Jilong; Niyompanich, Suthamat; Tai, Yi-Shu; Wang, Jingyu; Bai, Wenqin; Mahida, Prithviraj; Gao, Tuo; Zhang, Kechun; Vieille, C. (November 2016, Applied and Environmental Microbiology)

   ABSTRACT Chromosomal integration of heterologous metabolic pathways is optimal for industrially relevant fermentation, as plasmid-based fermentation causes extra metabolic burden and genetic instabilities. In this work, chromosomal integration was adapted for the production of mevalonate, which can be readily converted into β-methyl-δ-valerolactone, a monomer for the production of mechanically tunable polyesters. The mevalonate pathway, driven by a constitutive promoter, was integrated into the chromosome of Escherichia coli to replace the native fermentation gene adhE or ldhA . The engineered strains (CMEV-1 and CMEV-2) did not require inducer or antibiotic and showed slightly higher maximal productivities (0.38 to ∼0.43 g/liter/h) and yields (67.8 to ∼71.4% of the maximum theoretical yield) than those of the plasmid-based fermentation. Since the glycolysis pathway is the first module for mevalonate synthesis, atpFH deletion was employed to improve the glycolytic rate and the production rate of mevalonate. Shake flask fermentation results showed that the deletion of atpFH in CMEV-1 resulted in a 2.1-fold increase in the maximum productivity. Furthermore, enhancement of the downstream pathway by integrating two copies of the mevalonate pathway genes into the chromosome further improved the mevalonate yield. Finally, our fed-batch fermentation showed that, with deletion of the atpFH and sucA genes and integration of two copies of the mevalonate pathway genes into the chromosome, the engineered strain CMEV-7 exhibited both high maximal productivity (∼1.01 g/liter/h) and high yield (86.1% of the maximum theoretical yield, 30 g/liter mevalonate from 61 g/liter glucose after 48 h in a shake flask). IMPORTANCE Metabolic engineering has succeeded in producing various chemicals. However, few of these chemicals are commercially competitive with the conventional petroleum-derived materials. In this work, chromosomal integration of the heterologous pathway and subsequent optimization strategies ensure stable and efficient (i.e., high-titer, high-yield, and high-productivity) production of mevalonate, which demonstrates the potential for scale-up fermentation. Among the optimization strategies, we demonstrated that enhancement of the glycolytic flux significantly improved the productivity. This result provides an example of how to tune the carbon flux for the optimal production of exogenous chemicals. 

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