Biology subjects

Hasunuma, T.

Publications and source records attributed to Hasunuma, T..

5 recordsLinked to original sources

bioRxiv2024

Engineering Saccharomyces cerevisiae for growth on xylose using an oxidative pathway

The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth on xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast. Key pointsO_LI1.5 g/L ethylene glycol was produced via Dahms pathway in S. cerevisiae. C_LIO_LIEnzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways. C_LIO_LITested enzymes in this study may expand application of xylose oxidative pathway. C_LI

bioengineering↗

bioRxiv2024

Genome-scale prediction of gene ontology from mass fingerprints reveals new metabolic gene functions

Mass-based fingerprinting can characterize unknown strains, however expansion of these methods to predict specific gene functions is lacking. Therefore, rapid mass fingerprinting was developed to functionally profile a comprehensive yeast knockout library. Matrix assisted laser desorption ionization (MALDI)-time of flight (TOF) mass fingerprints of 3,238 Saccharomyces cerevisiae knockouts were digitized for correlation with gene ontology (GO) annotations. Random forests and support vector machine (SVM) algorithms precisely assigned GO accessions with AUC scores all above 0.83. SVM was the best predictor with average true positive and true negative rates of 0.975 and 0.991, respectively. The SVM model suggested new functions for 28 uncharacterized yeast genes. Metabolomics analysis of two knockouts (YDR215C and YLR122C) of uncharacterized genes predicted to be involved in methylation-related metabolism, showed altered intracellular contents of methionine-related metabolites. Increased S-adenosylmethionine in YDR215C highlights potential for enhancement of methylation pathways. These results demonstrate that MALDI-TOF fingerprints can be rapidly digitized, resulting in datasets that enable prediction of microbial genotypes and even the function of specific genes. This fingerprinting method can inform optimal bioproduction chassis selection.

systems biology↗

bioRxiv2023

Minimized dark cataplerosis of the Calvin cycle ensures prompt photosynthetic initiation in cyanobacteria

As primary contributors to oxygenic photosynthesis, cyanobacteria intricately regulate their metabolic pathways during the diurnal cycle to ensure survival and growth. Under dark conditions, breakdown of stored energy reserves of glycogen replenishes the intermediates, especially the downstream glycolytic metabolites necessary for photosynthetic initiation upon light irradiation. The intracellular level of the intermediates is maintained throughout the dark period. However, it remains unclear how their accumulation is maintained in the dark despite the limited availability of glycogen. Here, we showed that the metabolite accumulation stability is ensured by the low activities of phosphoenolpyruvate (PEP) converting enzymes, namely PEP carboxylase and pyruvate kinase, during the dark period. Overexpression of these enzymes significantly decreased the accumulation of glycolytic intermediates after dark incubation. The oxygen evolution ability simultaneously decreased in the overexpressing strains, indicating that the dark limitation of the PEP-consuming pathway facilitates photosynthetic initiation through the maintenance of glycolytic intermediates. This finding shed light on the importance of controlling cataplerotic flux during the dark for maintaining stable operation of the Calvin cycle.

plant biology↗

bioRxiv2022

Accumulated 3-phosphoglycerate in darkness is a primary starting substrate for stepwise photosynthetic induction process in cyanobacteria

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies showed that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid (on a time scale of seconds) dynamic changes in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enables estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicates that downstream glycolytic intermediates including phosphoglycerate and phosphoenolpyruvate accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant delays in the initiation of oxygen evolution are observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathway ({Delta}zwf, {Delta}gnd, and{Delta} glgP). Accordingly, the degree of delay in the oxygen evolution initiation is proportional to the accumulated pool size of the glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

plant biology↗

bioRxiv2020

Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial Saccharomyces cerevisiae as efficient whole cell biocatalysts

Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which is mainly composed of xylose and may comprise up to 40 % of the total biomass. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae – the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 gram per gram of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗