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

Developing nuclear and mitochondrial DNA editing techniques for engineering yeasts as novel microbial factories and disease models

The repurposing of CRISPR/Cas9 as rapidly programmable and portable DNA nucleases revolutionized biotechnology in the 2010s, providing easy access to modify genomes at user-specified locations. Engineering of yeasts, whether as microbial factories for biochemical production or as simple models for human congenital disorders, represents one high-impact domain that benefitted from CRISPR/Cas9 technology. However, precisely integrating desired genetic information after Cas9 cleavage via homology-directed repair (HDR) still poses a major challenge, as most yeasts prefer to repair DNA double strand breaks (DDBs) via non-homologous end joining (NHEJ). Although the conventional yeast species S. cerevisiae prefers HDR, making it more amenable to genome editing, like any organism, its evolved traits are not optimal for all applications. Conversely, evolutionary diverse nonconventional yeasts provide phenotypes tailored for one’s application. To expedite the nonconventional yeast engineering, I developed Lowered Indel Nuclease system Enabling Accurate Repair, (LINEAR), which addressed shortcomings associated with conventional CRISPR platforms, in part by adopting a temporal control strategy employed in mammalian gene editing. LINEAR enabled precision nuclear gene editing capabilities in nonconventional yeasts without the need to inhibit their preferred NHEJ mode of DNA repair, maximizing their innate potentials as microbial factories. Casted in the shadow of the nuclear genome editing renaissance was the mitochondrial genome (mtDNA). Long overlooked and understudied, characterization of mtDNA mutations causal in disease has inspired development of TALE-based mtDNA editing tools. However, the presumed inability to deliver nucleic acid to mitochondria has stymied the adoption of more robust CRIPSR-based tools. The convoluted nature of mtDNA genotype-phenotype relationships in multicellular tissues necessitates a robust mtDNA tool in a simple organism that recapitulates congenital pathogenesis. I applied experience in nuclear genome editing to establish a first-generation CRISPR mtDNA base-editing platform in S. cerevisiae. The platform encodes a mitochondrial-localizing cytosine-deaminase:Cas9 nickase fusion and an engineered mitochondrial-localizing sgRNA on a conventional nuclear episomal plasmid. Although the first-generation platform caused growth defects and large-scale mtDNA deletions, deep sequencing of mtDNA revealed protection of the protospacer sequence, indicating Cas9:sgRNA-mediated protection of target mtDNA, and to the best of my knowledge, the first report of Cas9:sgRNA targeting of mtDNA in S. cerevisiae without biolistic transformation. The demonstrated potential merits future efforts to dampen toxicity and promote on-target base editing. Although the first-generation platform was developed in S. cerevisiae, the long-term vision is to leverage the nonconventional yeast Yarrowia lipolytica. The Warburg positive phenotype of S. cerevisiae, with the ability to survive without mtDNA, is not representative of human physiology. Conversely, Y. lipolytica is an obligate aerobe with a lipid accumulation phenotype, the latter indicating Y. lipolytica as a suitable adipocyte model. As the “powerhouse of the cell”, mitochondrion’s integral role in bioenergetics indicates its dysfunction is causal in obesity. Studying mtDNA mutagenesis and mitochondrial dysfunction in Y. lipolytica could elucidate deleterious mtDNA genotypes in adipocytes that potentiate obesity. Advancements in genome editing potentiates harnessing novel organisms with intrinsic abilities to execute desired tasks in parallel with traditional rewiring of conventional organisms to perform foreign functions. Insights gained from one approach can augment the other, expediting our fundamental understanding of the spatiotemporal nature of genomes and metabolic/regulatory networks. Deepening this knowledge will have long-reaching biotechnological and biomedical impac

Developing nuclear and mitochondrial DNA editing techniques for engineering yeasts as novel microbial factories and disease models

The repurposing of CRISPR/Cas9 as rapidly programmable and portable DNA nucleases revolutionized biotechnology in the 2010s, providing easy access to modify genomes at user-specified locations. Engineering of yeasts, whether as microbial factories for biochemical production or as simple models for human congenital disorders, represents one high-impact domain that benefitted from CRISPR/Cas9 technology. However, precisely integrating desired genetic information after Cas9 cleavage via homology-directed repair (HDR) still poses a major challenge, as most yeasts prefer to repair DNA double strand breaks (DDBs) via non-homologous end joining (NHEJ). Although the conventional yeast species S. cerevisiae prefers HDR, making it more amenable to genome editing, like any organism, its evolved traits are not optimal for all applications. Conversely, evolutionary diverse nonconventional yeasts provide phenotypes tailored for one’s application. To expedite the nonconventional yeast engineering, I developed Lowered Indel Nuclease system Enabling Accurate Repair, (LINEAR), which addressed shortcomings associated with conventional CRISPR platforms, in part by adopting a temporal control strategy employed in mammalian gene editing. LINEAR enabled precision nuclear gene editing capabilities in nonconventional yeasts without the need to inhibit their preferred NHEJ mode of DNA repair, maximizing their innate potentials as microbial factories. Casted in the shadow of the nuclear genome editing renaissance was the mitochondrial genome (mtDNA). Long overlooked and understudied, characterization of mtDNA mutations causal in disease has inspired development of TALE-based mtDNA editing tools. However, the presumed inability to deliver nucleic acid to mitochondria has stymied the adoption of more robust CRIPSR-based tools. The convoluted nature of mtDNA genotype-phenotype relationships in multicellular tissues necessitates a robust mtDNA tool in a simple organism that recapitulates congenital pathogenesis. I applied experience in nuclear genome editing to establish a first-generation CRISPR mtDNA base-editing platform in S. cerevisiae. The platform encodes a mitochondrial-localizing cytosine-deaminase:Cas9 nickase fusion and an engineered mitochondrial-localizing sgRNA on a conventional nuclear episomal plasmid. Although the first-generation platform caused growth defects and large-scale mtDNA deletions, deep sequencing of mtDNA revealed protection of the protospacer sequence, indicating Cas9:sgRNA-mediated protection of target mtDNA, and to the best of my knowledge, the first report of Cas9:sgRNA targeting of mtDNA in S. cerevisiae without biolistic transformation. The demonstrated potential merits future efforts to dampen toxicity and promote on-target base editing. Although the first-generation platform was developed in S. cerevisiae, the long-term vision is to leverage the nonconventional yeast Yarrowia lipolytica. The Warburg positive phenotype of S. cerevisiae, with the ability to survive without mtDNA, is not representative of human physiology. Conversely, Y. lipolytica is an obligate aerobe with a lipid accumulation phenotype, the latter indicating Y. lipolytica as a suitable adipocyte model. As the “powerhouse of the cell”, mitochondrion’s integral role in bioenergetics indicates its dysfunction is causal in obesity. Studying mtDNA mutagenesis and mitochondrial dysfunction in Y. lipolytica could elucidate deleterious mtDNA genotypes in adipocytes that potentiate obesity. Advancements in genome editing potentiates harnessing novel organisms with intrinsic abilities to execute desired tasks in parallel with traditional rewiring of conventional organisms to perform foreign functions. Insights gained from one approach can augment the other, expediting our fundamental understanding of the spatiotemporal nature of genomes and metabolic/regulatory networks. Deepening this knowledge will have long-reaching biotechnological and biomedical impac

Developing nuclear and mitochondrial DNA editing techniques for engineering yeasts as novel microbial factories and disease models

The repurposing of CRISPR/Cas9 as rapidly programmable and portable DNA nucleases revolutionized biotechnology in the 2010s, providing easy access to modify genomes at user-specified locations. Engineering of yeasts, whether as microbial factories for biochemical production or as simple models for human congenital disorders, represents one high-impact domain that benefitted from CRISPR/Cas9 technology. However, precisely integrating desired genetic information after Cas9 cleavage via homology-directed repair (HDR) still poses a major challenge, as most yeasts prefer to repair DNA double strand breaks (DDBs) via non-homologous end joining (NHEJ). Although the conventional yeast species S. cerevisiae prefers HDR, making it more amenable to genome editing, like any organism, its evolved traits are not optimal for all applications. Conversely, evolutionary diverse nonconventional yeasts provide phenotypes tailored for one’s application. To expedite the nonconventional yeast engineering, I developed Lowered Indel Nuclease system Enabling Accurate Repair, (LINEAR), which addressed shortcomings associated with conventional CRISPR platforms, in part by adopting a temporal control strategy employed in mammalian gene editing. LINEAR enabled precision nuclear gene editing capabilities in nonconventional yeasts without the need to inhibit their preferred NHEJ mode of DNA repair, maximizing their innate potentials as microbial factories. Casted in the shadow of the nuclear genome editing renaissance was the mitochondrial genome (mtDNA). Long overlooked and understudied, characterization of mtDNA mutations causal in disease has inspired development of TALE-based mtDNA editing tools. However, the presumed inability to deliver nucleic acid to mitochondria has stymied the adoption of more robust CRIPSR-based tools. The convoluted nature of mtDNA genotype-phenotype relationships in multicellular tissues necessitates a robust mtDNA tool in a simple organism that recapitulates congenital pathogenesis. I applied experience in nuclear genome editing to establish a first-generation CRISPR mtDNA base-editing platform in S. cerevisiae. The platform encodes a mitochondrial-localizing cytosine-deaminase:Cas9 nickase fusion and an engineered mitochondrial-localizing sgRNA on a conventional nuclear episomal plasmid. Although the first-generation platform caused growth defects and large-scale mtDNA deletions, deep sequencing of mtDNA revealed protection of the protospacer sequence, indicating Cas9:sgRNA-mediated protection of target mtDNA, and to the best of my knowledge, the first report of Cas9:sgRNA targeting of mtDNA in S. cerevisiae without biolistic transformation. The demonstrated potential merits future efforts to dampen toxicity and promote on-target base editing. Although the first-generation platform was developed in S. cerevisiae, the long-term vision is to leverage the nonconventional yeast Yarrowia lipolytica. The Warburg positive phenotype of S. cerevisiae, with the ability to survive without mtDNA, is not representative of human physiology. Conversely, Y. lipolytica is an obligate aerobe with a lipid accumulation phenotype, the latter indicating Y. lipolytica as a suitable adipocyte model. As the “powerhouse of the cell”, mitochondrion’s integral role in bioenergetics indicates its dysfunction is causal in obesity. Studying mtDNA mutagenesis and mitochondrial dysfunction in Y. lipolytica could elucidate deleterious mtDNA genotypes in adipocytes that potentiate obesity. Advancements in genome editing potentiates harnessing novel organisms with intrinsic abilities to execute desired tasks in parallel with traditional rewiring of conventional organisms to perform foreign functions. Insights gained from one approach can augment the other, expediting our fundamental understanding of the spatiotemporal nature of genomes and metabolic/regulatory networks. Deepening this knowledge will have long-reaching biotechnological and biomedical impac

Engineering of non-model eukaryotes for bioenergy and biochemical production

The prospect of leveraging naturally occurring phenotypes to overcome bottlenecks constraining the bioeconomy has marshalled increased exploration of nonconventional organisms. This review discusses the status of non-model eukaryotic species in bioproduction, the evaluation criteria for effectively matching a candidate host to a biosynthetic process, and the genetic engineering tools needed for host domestication. We present breakthroughs in genome editing and heterologous pathway design, delving into innovative spatiotemporal modulation strategies that potentiate more refined engineering capabilities. We cover current understanding of genetic instability and its ramifications for industrial scale-up, highlighting key factors and possible remedies. Finally, we propose future opportunities to expand the current collection of available hosts and provide guidance to benefit the broader bioeconomy.This is a manuscript of an article published as Ploessl, Deon, Yuxin Zhao, and Zengyi Shao. "Engineering of non-model eukaryotes for bioenergy and biochemical production." Current Opinion in Biotechnology 79 (2023): 102869. DOI: 10.1016/j.copbio.2022.102869. Copyright 2022 Elsevier Ltd. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Posted with permission

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.</p

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.This article is published as Gao, Meirong, Deon Ploessl, and Zengyi Shao, "Enhancing the Co-utilization of Biomass-derived Mixed Sugars by Yeasts." Frontiers in Microbiology 9 (2019): 3264. doi: 10.3389/fmicb.2018.03264. Posted with permission.</p