RW
Ruud A. Weusthuis
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4 records found
1
Review
(2024)
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Enrico Orsi, Javier M. Hernández-Sancho, Maaike S. Remeijer, Aleksander J. Kruis, Daniel C. Volke, Nico J. Claassens, Caroline E. Paul, Frank J. Bruggeman, Ruud A. Weusthuis, Pablo I. Nikel
One-carbon (C1) feedstocks, such as carbon monoxide (CO), formate (HCO2H), methanol (CH3OH), and methane (CH4), can be obtained either through stepwise electrochemical reduction of CO2 with renewable electricity or via processing of organic side streams. These C1 substrates are increasingly investigated in biotechnology as they can contribute to a circular carbon economy. In recent years, noncanonical redox cofactors (NCRCs) emerged as a tool to generate synthetic electron circuits in cell factories to maximize electron transfer within a pathway of interest. Here, we argue that expanding the use of NCRCs in the context of C1-driven bioprocesses will boost product yields and facilitate challenging redox transactions that are typically out of the scope of natural cofactors due to inherent thermodynamic constraints.
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One-carbon (C1) feedstocks, such as carbon monoxide (CO), formate (HCO2H), methanol (CH3OH), and methane (CH4), can be obtained either through stepwise electrochemical reduction of CO2 with renewable electricity or via processing of organic side streams. These C1 substrates are increasingly investigated in biotechnology as they can contribute to a circular carbon economy. In recent years, noncanonical redox cofactors (NCRCs) emerged as a tool to generate synthetic electron circuits in cell factories to maximize electron transfer within a pathway of interest. Here, we argue that expanding the use of NCRCs in the context of C1-driven bioprocesses will boost product yields and facilitate challenging redox transactions that are typically out of the scope of natural cofactors due to inherent thermodynamic constraints.
Fermentation processes are used to sustainably produce chemicals and as such contribute to the transition to a circular economy. The maximum theoretical yield of a conversion can only be approached if all electrons present in the substrate end up in the product. Control over the electrons is therefore crucial. However, electron transfer via redox cofactors results in a diffuse distribution of electrons over metabolism. To overcome this challenge, we propose to apply non-canonical redox cofactors (NRCs) in metabolic networks: cofactors that channel electrons exclusively from substrate to product, forming orthogonal circuits for electron transfer.
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Fermentation processes are used to sustainably produce chemicals and as such contribute to the transition to a circular economy. The maximum theoretical yield of a conversion can only be approached if all electrons present in the substrate end up in the product. Control over the electrons is therefore crucial. However, electron transfer via redox cofactors results in a diffuse distribution of electrons over metabolism. To overcome this challenge, we propose to apply non-canonical redox cofactors (NRCs) in metabolic networks: cofactors that channel electrons exclusively from substrate to product, forming orthogonal circuits for electron transfer.
Journal article
(1994)
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R. A. Weusthuis, M. A.H. Luttik, W. A. Scheffers, J. P. Van Dijken, J. T. Pronk
Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0.1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the culture's respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol. With both yeasts diauxic growth was indeed observed on mixtures of ethanol and a disaccharide that gives rise to the Kluyver effect, with ethanol being the preferred substrate. In contrast, sugars which could be fermented were either utilized simultaneously with ethanol or preferred over this substrate.
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Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0.1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the culture's respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol. With both yeasts diauxic growth was indeed observed on mixtures of ethanol and a disaccharide that gives rise to the Kluyver effect, with ethanol being the preferred substrate. In contrast, sugars which could be fermented were either utilized simultaneously with ethanol or preferred over this substrate.
Effects of oxygen limitation on sugar metabolism in yeasts
A continuous-culture study of the Kluyver effect
Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1. With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol 1-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0.1 h-1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.
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Growth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0.1 h-1. With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol 1-1 h-1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0.1 h-1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.