Biology subjects

Mooiman, C.

Publications and source records attributed to Mooiman, C..

4 recordsLinked to original sources

bioRxiv2021

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

bioengineering↗

bioRxiv2021

Re-oxidation of cytosolic NADH is a major contributor to the high oxygen requirements of the thermotolerant yeast Ogataea parapolymorpha in oxygen-limited cultures

Thermotolerance is an attractive feature for yeast-based industrial ethanol production. However, incompletely understood oxygen requirements of known thermotolerant yeasts are incompatible with process requirements. To study the magnitude and molecular basis of these oxygen requirements in the facultatively fermentative, thermotolerant yeast Ogataea parapolymorpha, chemostat studies were performed under defined oxygen-sufficient and oxygen-limited cultivation regimes. The minimum oxygen requirements of O. parapolymorpha were found to be at least an order of magnitude larger than those of the thermotolerant yeast Kluyveromyces marxianus. This high oxygen requirement coincided with absence of glycerol formation, which plays a key role in NADH reoxidation in oxygen-limited cultures of other facultatively fermentative yeasts. Co-feeding of acetoin, whose reduction to 2,3-butanediol can reoxidize cytosolic NADH, supported a 2.5-fold higher biomass concentration in oxygen-limited cultures. The apparent inability of O. parapolymorpha to produce glycerol correlated with absence of orthologs of the S. cerevisiae genes encoding glycerol-3P phosphatase (ScGPP1, ScGPP2). Glycerol production was observed in aerobic batch cultures of a strain in which genes including key enzymes in mitochondrial reoxidation of NADH were deleted. However, transcriptome analysis did not identify a clear candidate for the responsible phosphatase. Expression of ScGPD2, encoding NAD+-dependent glycerol-3P dehydrogenase, and ScGPP1 in O. parapolymorpha resulted in increased glycerol production in oxygen-limited chemostats, but glycerol production rates remained substantially lower than observed in S. cerevisiae and K. marxianus. These results identify a dependency on aerobic respiration for reoxidation of NADH generated in biosynthesis as a key factor in the unexpectedly high oxygen requirements of O. parapolymorpha. ImportanceThermotolerant yeasts hold great potential for anaerobic fermentation processes but their application is so far hampered by incompletely understood oxygen requirements. Based on quantitative physiological studies in oxygen-limited chemostat cultures, this study shows that the thermotolerant yeast Ogataea parapolymorpha has a much higher oxygen requirement than other, previously investigated facultatively fermentative yeasts. The large oxygen requirement of O. parapolymorpha was linked to an inability of oxygen-limited cultures to efficiently reoxidize NADH formed in biosynthetic processes by glycerol formation. These results provide a basis for reducing the oxygen requirements of O. parapolymorpha by targeted metabolic engineering. In addition, this study shows that diversity of oxygen requirements should be taken into account in selecting yeast species for application in anaerobic or oxygen-limited industrial processes.

physiology↗

bioRxiv2021

Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the absence of oxygen. Response of K. marxianus and S. cerevisiae to different oxygen-limitation regimes were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 {degrees}C. These results open up new directions in the development of thermotolerant yeast strains for anaerobic industrial applications.

biochemistry↗

bioRxiv2020

Identification of oxygen-independent pathways for pyridine-nucleotide and Coenzyme-A synthesis in anaerobic fungi by expression of candidate genes in yeast

Neocallimastigomycetes are rare examples of strictly anaerobic eukaryotes. This study investigates how these anaerobic fungi bypass reactions involved in synthesis of pyridine nucleotide cofactors and coenzyme A that, in canonical fungal pathways, require molecular oxygen. Analysis of Neocallimastigomycete proteomes identified a candidate L-aspartate-decarboxylase (AdcA), and L-aspartate oxidase (NadB) and quinolinate synthase (NadA), constituting putative oxygen-independent bypasses for coenzyme A synthesis and pyridine nucleotide cofactor synthesis, respectively. The corresponding gene sequences indicated acquisition by ancient horizontal gene transfer event involving bacterial donors. To test whether these enzymes suffice to bypass corresponding oxygen-requiring reactions, they were introduced into fms1Δ and bna2Δ Sacharomyces cerevisiae strains. Expression of nadA and nadB, and adcA from the Neocallimastigomycetes Piromyces finnis and Neocallimastix californiae, respectively, conferred cofactor prototrophy under aerobic and anaerobic conditions. This study simulates how horizontal gene transfer can drive eukaryotic adaptation to anaerobiosis, and provides a basis for elimination of auxotrophic requirements in anaerobic industrial applications of yeasts and fungi.Competing Interest StatementThe authors have declared no competing interest.View Full Text

microbiology↗