Enhancing the Co-utilization of Biomass-Derived Mixed Sugars by Yeasts

Plant biomass is a promising carbon source for producing value-added chemicals, including transportation biofuels, polymer precursors, and various additives. Most engineered microbial hosts and a select group of wild-type species can metabolize mixed sugars including oligosaccharides, hexoses, and pentoses that are hydrolyzed from plant biomass. However, most of these microorganisms consume glucose preferentially to non-glucose sugars through mechanisms generally defined as carbon catabolite repression. The current lack of simultaneous mixed-sugar utilization limits achievable titers, yields, and productivities. Therefore, the development of microbial platforms capable of fermenting mixed sugars simultaneously from biomass hydrolysates is essential for economical industry-scale production, particularly for compounds with marginal profits. This review aims to summarize recent discoveries and breakthroughs in the engineering of yeast cell factories for improved mixed-sugar co-utilization based on various metabolic engineering approaches. Emphasis is placed on enhanced non-glucose utilization, discovery of novel sugar transporters free from glucose repression, native xylose-utilizing microbes, consolidated bioprocessing (CBP), improved cellulase secretion, and creation of microbial consortia for improving mixed-sugar utilization. Perspectives on the future development of biorenewables industry are provided in the end

Enhancing the Co-utilization of Biomass-Derived Mixed Sugars by Yeasts

Plant biomass is a promising carbon source for producing value-added chemicals, including transportation biofuels, polymer precursors, and various additives. Most engineered microbial hosts and a select group of wild-type species can metabolize mixed sugars including oligosaccharides, hexoses, and pentoses that are hydrolyzed from plant biomass. However, most of these microorganisms consume glucose preferentially to non-glucose sugars through mechanisms generally defined as carbon catabolite repression. The current lack of simultaneous mixed-sugar utilization limits achievable titers, yields, and productivities. Therefore, the development of microbial platforms capable of fermenting mixed sugars simultaneously from biomass hydrolysates is essential for economical industry-scale production, particularly for compounds with marginal profits. This review aims to summarize recent discoveries and breakthroughs in the engineering of yeast cell factories for improved mixed-sugar co-utilization based on various metabolic engineering approaches. Emphasis is placed on enhanced non-glucose utilization, discovery of novel sugar transporters free from glucose repression, native xylose-utilizing microbes, consolidated bioprocessing (CBP), improved cellulase secretion, and creation of microbial consortia for improving mixed-sugar utilization. Perspectives on the future development of biorenewables industry are provided in the end

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

Anticancer Drugs

Plant‐derived anticancer drugs play a large role in anticancer pharmaceuticals. Through reviewing the four major types of plant anticancer drugs, namely vinca alkaloids, taxane diterpenoids, podophyllotoxin lignans, and camptothecin quinoline alkaloids, this article illustrates the development process, current status, existing challenges, and future perspective of the plant anticancer drug production. Moreover, this review explains how various biotechnologies, from the mature elicitation strategy to the “omics” techniques that are still undergoing development, can be applied to address the challenges in improving the production of the plant‐sourced anticancer drugs

Building microbial factories for the production of aromatic amino acid pathway derivatives: From commodity chemicals to plant-sourced natural products

The aromatic amino acid biosynthesis pathway, together with its downstream branches, represents one of the most commercially valuable biosynthetic pathways, producing a diverse range of complex molecules with many useful bioactive properties. Aromatic compounds are crucial components for major commercial segments, from polymers to foods, nutraceuticals, and pharmaceuticals, and the demand for such products has been projected to continue to increase at national and global levels. Compared to direct plant extraction and chemical synthesis, microbial production holds promise not only for much shorter cultivation periods and robustly higher yields, but also for enabling further derivatization to improve compound efficacy by tailoring new enzymatic steps. This review summarizes the biosynthetic pathways for a large repertoire of commercially valuable products that are derived from the aromatic amino acid biosynthesis pathway, and it highlights both generic strategies and specific solutions to overcome certain unique problems to enhance the productivities of microbial hosts

Electrochemical Conversion of Biologically Produced Muconic Acid: Key Considerations for Scale-Up and Corresponding Technoeconomic Analysis

Muconic acid, an unsaturated diacid that can be produced from cellulosic sugars and lignin monomers by fermentation, emerges as a promising intermediate for the sustainable manufacture of commodity polyamides and polyesters including Nylon-6,6 and polyethylene terephthalate (PET). Current conversion schemes consist in the biological production of cis,cis-muconic acid using metabolically engineered yeasts and bacteria, and the subsequent diversification to adipic acid, terephthalic acid, and their derivatives using chemical catalysts. In some instances, conventional precious metal catalysts can be advantageously replaced by base metal electrocatalysts. Here, we show the economic relevance of utilizing a hybrid biological−electrochemical conversion scheme to convert glucose to trans-3-hexenedioic acid (t3HDA), a monomer used for the synthesis of bioadvantaged Nylon-6,6. Potential roadblocks to biological and electrochemical integration in a single reactor, including electrocatalyst deactivation due to biogenic impurities and low faradaic efficiency inherent to side reactions in complex media, have been studied and addressed. In this study, t3HDA was produced with 94% yield and 100% faradaic efficiency. With consideration of the high t3HDA yield and faradaic efficiency, a technoeconomic analysis was developed on the basis of the current yield and titer achieved for muconic acid, the figures of merit defined for industrial electrochemical processes, and the separation of the desired product from the medium. On the basis of this analysis, t3HDA could be produced for approximately $2.00 kg−1. The low cost for t3HDA is a primary factor of the electrochemical route being able to cascade biological catalysis and electrocatalysis in one pot without separation of the muconic acid intermediate from the fermentation broth

A genetic toolbox for metabolic engineering of Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplex gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ∼100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis

Combining Metabolic Engineering and Electrocatalysis: Application to the Production of Polyamides from Sugar

Biorefineries aim to convert biomass to a spectrum of products ranging from biofuels to specialty chemicals. To achieve economically sustainable conversion it is crucial to streamline the catalytic and downstream processing steps. Here we report a route that integrates bio- and chemical catalysis to convert glucose into bio-based unsaturated nylon 6,6. An engineered strain of Saccharomyces cerevisiae, with the highest reported muconic acid titer of 559.5 mg L-1 in yeast, was used as the initial biocatalyst to convert glucose into muconic acid. Without any separation, muconic acid was further electrocatalytically hydrogenated to 3-hexenedioic acid with 94% yield, despite the presence of all the biogenic impurities. Bio-based unsaturated nylon 6,6 (unsaturated polyamide 6,6) was finally obtained by polymerization of 3-hexenedioic acid with hexamethylenediamine, demonstrating the integrated design of bio-based polyamides from glucose