Quantitative Physiology of Non-Energy-Limited Retentostat Cultures of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

ABSTRACT So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyzes the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h−1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80% and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolically active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed aerobic fermentation and a partial uncoupling of catabolism and anabolism. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen or phosphorus limitation offers interesting prospects for high-yield production of bio-based chemicals. IMPORTANCE The yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various biochemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate therefore could have a wide range of industrial applications but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely, the nitrogen or phosphorus source, and carried out a quantitative physiological study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

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Quantitative physiology of non-energy-limited retentostat cultures ofSaccharomyces cerevisiaeat near-zero specific growth rates

10.1101/653816 ◽

2019 ◽

Author(s):

Yaya Liu ◽

Anissa el Masoudi ◽

Jack T. Pronk ◽

Walter M. van Gulik

Keyword(s):

Saccharomyces Cerevisiae ◽

Growth Rates ◽

Phosphate Limitation ◽

High Yield ◽

Fermentation Processes ◽

Yield Production ◽

Zero Growth ◽

Limited Supply ◽

Quantitative Physiology ◽

High Yield Production

AbstractSo far, the physiology ofSaccharomyces cerevisiaeat near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology ofS. cerevisiaeunder conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyses the physiology ofS. cerevisiaein aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h-1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80 % and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolic active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed a partial uncoupling of catabolism and anabolism and aerobic fermentation. The possibility to achieve stable, near-zero growth cultures ofS. cerevisiaeunder nitrogen- or phosphorus-limitation offers interesting prospects for high-yield production of bio-based chemicals.ImportanceThe yeastSaccharomyces cerevisiaeis a commonly used microbial host for production of various bio-chemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. EstablishingS. cerevisiaecultures in which growth is restricted by the limited supply of a non-energy substrate could therefore have a wide range of industrial applications, but remains largely unexplored. In this work we accomplished near-zero growth ofS. cerevisiaethrough limited supply of a non-energy nutrient, namely the nitrogen or phosphorus source and carried out a quantitative physiology study of the cells under these conditions. The possibility to achieve near-zero-growthS. cerevisiaecultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

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High-Yield Production of Herbicidal Thaxtomins and Thaxtomin Analogs in a NonpathogenicStreptomycesStrain

Applied and Environmental Microbiology ◽

10.1128/aem.00164-18 ◽

2018 ◽

Vol 84 (11) ◽

Cited By ~ 14

Author(s):

Guangde Jiang ◽

Yucheng Zhang ◽

Magan M. Powell ◽

Peilan Zhang ◽

Ran Zuo ◽

...

Keyword(s):

Natural Products ◽

Gene Cluster ◽

Herbicidal Activity ◽

High Yield ◽

Content Type ◽

Thaxtomin A ◽

Yield Production ◽

Agricultural Applications ◽

Synthetic Routes ◽

High Yield Production

ABSTRACTThaxtomins are virulence factors of most plant-pathogenicStreptomycesstrains. Due to their potent herbicidal activity, attractive environmental compatibility, and inherent biodegradability, thaxtomins are key active ingredients of bioherbicides approved by the U.S. Environmental Protection Agency. However, the low yield of thaxtomins in nativeStreptomycesproducers limits their wide agricultural applications. Here, we describe the high-yield production of thaxtomins in a heterologous host. The thaxtomin gene cluster fromS. scabiei87.22 was cloned and expressed inS. albusJ1074 after chromosomal integration. The production of thaxtomins and nitrotryptophan analogs was observed using liquid chromatography-mass spectrometry (LC-MS) analysis. When the engineeredS. albusJ1074 was cultured in the minimal medium Thx defined medium supplemented with 1% cellobiose (TDMc), the yield of the most abundant and herbicidal analog, thaxtomin A, was 10 times higher than that inS. scabiei87.22, and optimization of the medium resulted in the highest yield of thaxtomin analogs at about 222 mg/liter. Further engineering of the thaxtomin biosynthetic gene cluster through gene deletion led to the production of multiple biosynthetic intermediates important to the chemical synthesis of new analogs. Additionally, the versatility of the thaxtomin biosynthetic system inS. albusJ1074 was capitalized on to produce one unnatural fluorinated analog, 5-fluoro-thaxtomin A (5-F-thaxtomin A), whose structure was elucidated by a combination of MS and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) analyses. Natural and unnatural thaxtomins demonstrated potent herbicidal activity in radish seedling assays. These results indicated thatS. albusJ1074 has the potential to produce thaxtomins and analogs thereof with high yield, fostering their agricultural applications.IMPORTANCEThaxtomins are agriculturally valuable herbicidal natural products, but the productivity of native producers is limiting. Heterologous expression of the thaxtomin gene cluster inS. albusJ1074 resulted in the highest yield of thaxtomins ever reported, representing a significant leap forward in its wide agricultural use. Furthermore, current synthetic routes to thaxtomins and analogs are lengthy, and two thaxtomin biosynthetic intermediates produced at high yields in this work can provide precursors and building blocks to advanced synthetic routes. Importantly, the production of 5-F-thaxtomin A in engineeredS. albusJ1074 demonstrated a viable alternative to chemical methods in the synthesis of new thaxtomin analogs. Moreover, our work presents an attractive synthetic biology strategy to improve the supply of herbicidal thaxtomins, likely finding general applications in the discovery and production of many other bioactive natural products.

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Disruption of the Oxidative Pentose Phosphate Pathway Stimulates High-Yield Production Using Resting Corynebacterium glutamicum in the Absence of External Electron Acceptors

Applied and Environmental Microbiology ◽

10.1128/aem.02114-20 ◽

2020 ◽

Vol 86 (24) ◽

Author(s):

Jing Shen ◽

Jun Chen ◽

Christian Solem ◽

Peter Ruhdal Jensen ◽

Jian-Ming Liu

Keyword(s):

Pentose Phosphate Pathway ◽

Reducing Power ◽

Pentose Phosphate ◽

High Yield ◽

Glycolytic Flux ◽

Resting Cells ◽

Content Type ◽

Dairy Waste ◽

Yield Production ◽

High Yield Production

ABSTRACT Identifying and overcoming the limitations preventing efficient high-yield production of chemicals remain important tasks in metabolic engineering. In an attempt to rewire Corynebacterium glutamicum to produce ethanol, we attained a low yield (63% of the theoretical) when using resting cells on glucose, and large amounts of succinate and acetate were formed. To prevent the by-product formation, we knocked out the malate dehydrogenase and replaced the native E3 subunit of the pyruvate dehydrogenase complex (PDHc) with that from Escherichia coli, which is active only under aerobic conditions. However, this tampering resulted in a 10-times-reduced glycolytic flux as well as a greatly increased NADH/NAD+ ratio. When we replaced glucose with fructose, we found that the glycolytic flux was greatly enhanced, which led us to speculate whether the source of reducing power could be the pentose phosphate pathway (PPP) that is bypassed when fructose is metabolized. Indeed, after shutting down the PPP by deleting the zwf gene, encoding glucose-6-phosphate dehydrogenase, the ethanol yield on glucose increased significantly, to 92% of the theoretical. Based on that, we managed to rechannel the metabolism of C. glutamicum into d-lactate with high yield, 98%, which is the highest that has been reported. It is further demonstrated that the PPP-inactivated platform strain can offer high-yield production of valuable chemicals using lactose contained in dairy waste as feedstock, which paves a promising way for potentially turning dairy waste into a valuable product. IMPORTANCE The widely used industrial workhorse C. glutamicum possesses a complex anaerobic metabolism under nongrowing conditions, and we demonstrate here that the PPP in resting C. glutamicum is a source of reducing power that can interfere with otherwise redox-balanced metabolic pathways and reduce yields of desired products. By harnessing this physiological insight, we employed the PPP-inactivated platform strains to produce ethanol, d-lactate, and alanine using the dairy waste whey permeate as the feedstock. The production yield was high, and our results show that inactivation of the PPP flux in resting cells is a promising strategy when the aim is to use nongrowing C. glutamicum cells for producing valuable compounds. Overall, we describe the benefits of disrupting the oxidative PPP in nongrowing C. glutamicum and provide a feasible approach toward waste valorization.

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