The type of carbon source not the growth rate it supports can determine diauxie

  1. Yu Huo
  2. Weronika Danecka
  3. Iseabail Farquhar
  4. Kim Mailliet
  5. Tessa Moses
  6. Edward W. J. Wallace
  7. Peter S. Swain

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Abstract

How cells choose between potential carbon sources is a classic example of cellular decision-making, and we know that many organisms prioritise glucose. Yet there has been little investigation of whether other sugars are also preferred, blinkering our view of carbon sensing. Here we study eukaryotic budding yeast and its growth on mixtures of palatinose, an isomer of sucrose, with other sugars. We find that yeast prioritise galactose over palatinose, but not sucrose or fructose, despite all three of these sugars being able to support faster growth than palatinose. Our results therefore disfavour carbon flux-sensing as the sole mechanism. By using genetic perturbations and transcriptomics, we show that repression is active and through Gal4, the master regulator of the GAL regulon. Cells enforce their preference for galactose over palatinose by preventing runaway positive feedback in the MAL regulon, whose genes enable palatinose catabolism. They do so both by repressing MAL11, the gene encoding the palatinose transporter, and by first expressing the isomaltases, IMA1 and IMA5, which cleave palatinose and so prevent its intracellular concentration becoming enough to induce further MAL expression. Our results demonstrate that budding yeast actively maintain a preference for carbon sources other than glucose and that such preferences have been selected by more than differences in growth rates. They imply that carbon-sensing strategies even in unicellular organisms are more complex than previously thought.

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    Reply to the reviewers

    We thank the reviewers for their reading of our manuscript, which we believe has led to substantial improvements.

    To aid clarity, we have split Fig. 1 into three separate figures.

    For convenience, we have put all major changes in the text in blue.

    Reviewer #1

    Evidence, reproducibility and clarity

    Summary: Hui et al. tackle a crucial question in biology: what factors influence the preference for carbon sources in yeasts?

    They reveal that the growth rate on palatinose exceeds that on glucose,

    The above statement is incorrect --- we think the reviewer may have confused sugars.

    despite palatinose utilization being repressed in the presence of glucose. Consequently, the favored carbon source does not necessarily align with the one supporting the fastest growth rate. The study also delves into potential regulatory mechanisms governing carbon source preference and dismisses certain existing theories, such as the general carbon flux sensing mechanism proposed by Okano et al. [25].

    Major comments: None

    Minor comments:

    The authors suggest that a higher growth rate implies a higher glycolytic flux (l63), a crucial assumption underpinning their interpretation of the absence of a ``general carbon flux sensing mechanism' (l65). To substantiate this significant conclusion, they could calculate the extracellular uptake fluxes (based on the time-course concentrations of biomass and substrates).

    This suggestion is a good one, but unfortunately the number of data points in the new Fig. 3 are insufficient to estimate the uptake flux reliably.

    To address whether glycolytic flux increases, we have added a new paragraph to the introduction explaining how all the sugars we consider feed upper glycolysis, providing either its first or second metabolite. We therefore think it highly likely that any differences in growth rate are generated by differences in glycolytic flux. Indeed, Hackett et al., 2016, showed that the glycolytic flux increases with growth rate when they changed extracellular glucose concentrations. We now include this reference in the Discussion.

    The accumulation of certain by-products is known to be toxic, reducing cellular growth rate (e.g., acetate DOI: 10.1038/srep42135, ethanol DOI: 10.1016/B978-0-12-040308-0.50006-9, etc.), while they can also enhance growth under specific conditions (e.g., acetate DOI: 10.15252/embj.2022113079). Considering this is crucial to rule out certain hypotheses, such as the possibility that a by-product produced during growth on the first carbon source would not modulate growth on the second carbon source, potentially influencing the growth rate differentially in each phase. Although the authors use mutant strains to eliminate the role of some C2 compounds (acetate and ethanol), alternative pathways could be implicated in the (co-)utilization of these by-products. This aspect should be discussed, and ideally, the authors could quantify the time-course concentrations of by-products to assess their potential role.

    We agree with the reviewer that extracellular acetate and ethanol may inhibit growth, although budding yeast might be less sensitive than E. coli, the subject of most of the studies provided.

    Nevertheless, we think it unlikely that these chemicals modify the decision-making we see. First, the icl1Δ mutant we tested is unable to consume ethanol (Fernandez et al., 1992) or acetate (Lee et al., 2011) --- we now include these references in the SI --- and yet has wild-type behaviour (Fig. S2D). Second, we observe that isomaltase expression strongly decreases in the presence of galactose when we grow cells in a microfluidic device (Fig. S4), just like it does in batch culture (Fig. 3A), even though the constant flow of medium through the device removes any chemicals the cells excrete.

    The general flux-sensing regulatory mechanism proposed by Okano et al. [25], which has been dismissed by this study, has recently been questioned, as discussed in DOI: 10.15252/embj.2022113079. This aspect should be included in the discussion.

    Okano et al. studied E. coli while we study budding yeast. We therefore have shown that the understanding for that organism does not transfer to our eukaryotic example. We suspect that control in budding yeast combines both flux-sensing and specific regulation, as we say in the discussion, and so we consider our results to build on those of Okano et al.

    Significance

    Strengths & limitations: The work is robust, and the experiments in the study have been appropriately designed and conducted. The primary question of this study has been tackled using a combination of experimental and computational methods to thoroughly assess various regulatory and functional aspects. However, there are gaps in the data that could enhance key conclusions, notably the absence of glycolytic flux measurements. Moreover, further evidence is needed to substantiate the assertion that by-products do not play a role in carbon source preference.

    Advance: This study represents a significant step forward in comprehending the nutritional strategy of microbes. The authors demonstrate that the preferred carbon source may not necessarily be the one supporting the fastest growth rate. Furthermore, they dismiss certain theories that have been proposed to explain the growth strategy of microbes on mixed carbon sources.

    Audience: By addressing a fundamental question in life science, this work is important in the field of biology in general and of particular interest in systems biology, biotechnology, synthetic biology, and health. Consequently, it will be of interest to a broad audience.

    Reviewer #2

    Evidence, reproducibility and clarity

    Summary: The authors have used microtiter plates to produce growth profiles on combinations of different sugars. From this data they have evaluated whether the sugars are co-consumed or if there is a preference for either sugar, seen as a diauxic shift. They found diauxie between galactose and the disaccharide palatinose, but co-consumption between palatinose and fructose. They further used strains with perturbations in their GAL regulon to attempt to explain this discrepency.

    Major comments:

    I unfortunately found a large portion of the present manuscript unintelligable.

    Firstly, figures were incorrect to the point I could not dechiffre them: Figure 2A-C have black solid and dashed lines in the legend that are not found in the graph, instead there are orange and blue dashed lines in the graph with no legends. Figure 4C has no description of the y-axis. The growth rates in Figure 1C are very hard to follow, and there are definitely local maxima in both the blue and green profiles that are not being discussed (at 15-20 h). I cannot evaluate the conclusions drawn from the data until these issues have been resolved.

    We apologise for the difficulties experienced by this reviewer.

    The black lines in the old Fig. 2's legend, now Fig. 4, explain the different styles used: dashed lines are for single sugars regardless of their concentration and full lines are for mixtures regardless of their concentration. We now explicitly say this in the caption.

    We have fixed the missing label in what is now Fig. 6C and have moved the statement that we are showing two biological replicates for each set of concentrations earlier in Fig. 2's caption.

    We now explore the meaning of the shoulder for the fructose-palatinose mixture in Fig. 2B in the Discussion. This point is not a local maximum, unlike the case for diauxie, because the growth rate always decreases. The shoulder for the glucose-palatinose mixture was likely an artefact generated by measurement noise at low ODs because it was not present when we repeated the experiment. We now use that data for Fig. 2A & B. We also include a new Fig. S5 showing that there are sucrose-palatinose concentrations too that have a similar shoulder.

    Secondly, the language in the Results and Discussion sections is confusing. Alternating between present and imperfect tense as well as active and passive form makes it hard to distinguish the authors own results from literature findings (Results are usually written in passive, imperfect tense). Examples are found on lines 24, 29, 37-38, 59, 84, 131, and 165.

    We have made both sections flow more smoothly with substantial re-writing. As before, we cite all results that are not our own.

    The authors also do not consider the differences and similarities in catabolic pathways for assimilation of galactose, fructose and palatinose. Even if they do not see a reason to continue that as a possible explanation for the co-consumption between fructose and palatinose a discussion of why it is disregarded would not be out of place here.

    A good point, and we now state in the Introduction that all the sugars we study feed upper glycolysis.

    Significance

    There is some novelty to the authors findings, but I would argue it is being overstated in the present manuscript. Some examples of studies looking at catabolite repression, the main cause of diauxie, of sugars other than glucose can be found in: Simpson-Lavy and Kupiec (2019), Gancedo (1998), Prasad and Venkatesh (2008) and Borgstrom et al (2022).

    We strongly disagree with this statement. The papers cited do not address, as we do, the co-consumption between two sugars neither of which is glucose. Where they study two sugars, they always study glucose.

    Simpson-Lavy and Kupiec, 2019, investigate the interaction between acetate and ethanol, neither of which are sugars. Further, they are not independent carbon sources because cells convert ethanol into acetate when catabolising ethanol.

    Gancedo, 1998, is a review of glucose repression and describes how glucose represses the expression of genes for other sugars. Although Gancedo mentions ``galactose repression', this repression is of genes encoding enzymes for gluconeogenesis and the TCA and glyoxylate cycles, not of other sugar regulons, our subject.

    Prasad and Venkatesh, 2008, also focus on glucose and the well studied diauxie between glucose and galactose.

    Borgstrom et al., 2022, focus too on glucose and growth on glucose and xylose in recombinant strains. The standard laboratory strains we study have not be artificially engineered to consume xylose. They do mention that galactose causes repression of TPS1, which encodes an enzyme that synthesises the storage carbohydrate trehalose. This repression is again not of a sugar catabolic regulon, our subject.

    I would not say that the field would be significantly advanced by the publication of this manuscript, and the authors have themselves not explained the application of futhering the understanding palatinose metabolism in yeast. As mentioned above, the catabolite repression potential of galactose is already known, it just hasn't been shown for palatinose specifically before.

    We again strongly disagree. Our findings are novel. The reviewer did not provide any evidence for galactose repression of other sugar regulons, which is not widely recognised as we emphasised in the Discussion. We believe that the reviewer has confused the known "galactose repression' of gluconeogenic or TCA-cycle genes with our new report of repression of other sugar regulons in the presence of the sugar catabolised by the regulon.

    I would recommend a complete rewrite of the manuscript as presented, with a lower stated novelty, clearer language and comprehensible figures.

    Reviewer #3

    Evidence, reproducibility and clarity

    Summary: Microbes grow at different growth rates in different carbon sources. When more than one carbon sources are present in the media microbes often show a preference over certain carbon sources, and 'non-preferred' carbons sources are used only when the preferred carbon source is exhausted in the media, this process called diauxic shift.

    Why microbes exhibit such utilization preference over certain carbon sources, is an interesting question in microbiology and evolutionary biology, and the molecular mechanisms that enable microbes to preferentially use one carbon over another is worth investigating. It is intuitive to think that microbes will prefer to use a carbon source that confers maximum growth rate, but when tested experimentally it has been often observed that a carbon source in which microbes grow at sub optimal growth rate is actually preferentially used.

    Although the reviewer states that "it has been often observed that a carbon source in which microbes grow at sub optimal growth rate is actually preferentially used“, we are unaware of this work and would appreciate references, particularly for budding yeast. The most systematic study we know, in E. coli by Aidelberg et al., 2014 --- reference 13, concludes that "the faster the growth rate, the higher the sugar on the hierarchy“, the opposite behaviour.

    In this study authors demonstrate that budding yeast prefer to use galactose over palatinose, but not over sucrose or fructose where all three sugars can support faster growth rate compared to palatinose. Authors presented data where preferential galactose use and diauxic shift is observed in the growth curve when galactose and palatinose or glucose and palatinose combinations were used.

    No diauxic shift was observed in the growth curve when fructose-palatinose, or sucrose-palatinose combination were used. In fructose-palatinose and sucrose-palatinose combinations growth curves agree more with co-utilization strategies. Authors used transcriptomics and genetic perturbations to decipher the molecular mechanism of such preferential carbon use, and reports preference of galactose over palatinose is achieved by preventing positive feedback of MAL regulon, which encodes the genes for palatinose catabolism. We found this observation is interesting and the molecular mechanism of such preferential carbon use is nicely described in this paper. We also find claims authors made are well supported by experiments. Although catabolite repression and diauxic transitions are known in yeast, and authors also pointed out such previous references, but preferential use of a slower carbon source, i.e. galactose over at least one other fast-growing carbon is interesting enough for publication. We would like to support the publication of this article, but we have major concerns about the data analysis and data presentation. Authors must address our concerns which are mentioned below.

    Major comments:

    1. This study mainly hinges on growth rate measurements, but we found growth rates are not properly represented in the figures. Growth curves are always shown in linear scale, which makes it almost impossible to compare fast and slow growth when presented in same plot. All growth curves must be shown on log scale.

    We have changed all growth curves to log2 scale, following New et al., 2014, rather than Monod's choice of linear scale that we had originally.

    Our conclusions are unaffected.

    1. Growth rates of the Yeast strain growing individual single carbon sources (galactose, palatinose, sucrose and fructose) should be shown as a figure panel and t-test should be performed to conclude if the individual growth rates are significantly different or not.

    We already showed these growth rates in their own panel in Fig. 1B. Following the reviewer's suggestion, we have now added their statistical significance to the caption.

    1. Growth phase, lag phase, diauxic shift and post shift growth should be clearly shown in figure 2 and 4, each phase should be clearly marked, carbons used in each phase should be mentioned on the plot. Also, the growth curve must be plotted using log scale.

    Although we have changed all growth curves to log scale, we decided against include this additional labelling for two reasons. First, we are presenting evidence that some of the growth we observe is diauxic and labelling the curves as diauxic before we discuss this evidence undermines that discussion. Second, any further labels would clutter the figures, and we believe would hinder rather than help the reader.

    Instead we changed the colour scheme and the boldness of the diauxic growth curves in Fig. 2, which we hope the reviewer agrees adds the clarity they felt was missing.

    1. Authors has taken in account that MAL12 gene overexpression causes long lag when cells need to switch to maltose from glucose, and shown deletion of IMA1 decreases the lag with subsequent 2% growth rate increase in palatinose. How significant is this increase?

    We have confirmed the statistical significance through a t-test and added the results to the caption of Fig. 6C.

    1. Authors have an interesting observation that in sucrose-palatinose and fructose palatinose combinations, most probably co utilization of the carbons is taking place. Authors should discuss this in more details. In galactose-palatinose scenario intracellular galactose-based repression of gal80 and subsequent lack of feed forward of the Mal regulon is expected to stop co-utilization of palatinose. As authors have RNA seq data, can they make predictions for other carbon pairs, where sequential utilization can occur based on their model?

    We agree and have added more discussion of the fructose- and sucrose-palatinose mixtures to the Discussion and a new figure, Fig. S5.

    Our RNAseq data reveals the difference in gene expression caused by an active versus an inactive GAL regulon. In Fig. S11, we show that the hexose transporters HXT2 and HXT7 are down regulated in 0.1% fructose when the GAL regulon is active, perhaps implying that cells are able to prioritise galactose over other hexoses. Nevertheless, to predict if particular carbon sources are therefore favoured, we would need to know whether cells use specific hexose transporters to drive growth on different carbon sources, which has been little investigated.

    Minor comments:

    1. In figure 5, authors attempted to summarize the model, which is informative, but it will be more useful for non-specific reader if a cell-based cartoon, with transports on surface and catabolic enzymes inside is also added.

    We have re-designed Fig. 5, now Fig. 7, following this suggestion and agree it improves clarity.

    In this schematic diagram, switch from galactose (blue line) to red line (palatinose) shows a mixed color zone, it's a bit confusing, as this represents a bi-stable state. Authors should clearly comment on possibility of biostability while discussing their proposed mechanism.

    In the new figure, this part has been removed.

    1. The author may want to put their work in the context of other recent observations that bacteria do not try to maximize their growth rates in many conditions. Fast growth is often associated with expansive tradeoffs, and a carbon source which confers fast growth rate may confer selective disadvantage. Thus, there are evolutionary benefits of sub-optimal growth, which could be discussed in the manuscript. In this regard a recent study (bioRxiv (2023) doi:10.1101/2023.08.22.554312.) has established the link between resource allocation strategies, growth rates and tradeoffs, which may be taken in account while discussing. Are there any known tradeoffs, when galactose is used over palatinose and which is not the case sucrose or fructose?

    This is an interesting reference looking at growth on a single carbon source. We are unaware of similar tradeoffs relevant to our study. For example, we see little evidence for a constraint on the proteome because in a strain with a constitutively active GAL regulon there is no change in phenotype if we delete the genes for the three highly expressed GAL enzymes (Fig. S6B). Nevertheless and as we state in the penultimate paragraph of the Discussion, we agree that such a constraint must exist, although perhaps this constraint is ecological.

    Referees cross-commenting

    As other reviewers pointed out, this study has merit and addressed interesting questions, but needed to be written well in a more understandable form, we agree with this assessment. Also figures must be made much clearer, as all of the reviewers pointed out. In summary, this is an interesting study, but needs some work before publication.

    Significance

    General assessment: Strength and limitations:

    This study addressed an interesting question regarding resource preference and growth rate optimization in microbes. This is an important question in the field. Study is well designed and claims are backed up with experimental results. One of the limitations of the study is lack of predictability. Authors explained the mechanism for one pair of carbon sources, but how applicable that will be in general is not clear.

    We would argue that one of our important findings is to demonstrate that the scientific community is missing the information needed to make such predictions. We provide a counter example to the generally accepted belief that accurate predictions can be made using growth rates. Our work poses the question: what then are the physiological variables required to predict how a cell will consume a pair of carbon sources?

    Advance: This study helps to advance our knowledge. Their observation regarding preferential utilization of a carbon source which supports slower growth over a carbon source which can support faster growth, and the molecular mechanism provided will help researchers to understand resource allocation strategies better.

    Audience: Microbiology, systems biology, evolutionary biology, fermentation and bio process engineering research.

    Reviewer expertise: Biochemistry, systems biology, metabolic strategies and tradeoffs in microbes, microbial ecology.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Review of the paper by Yu Huo et al.

    Summary:

    Microbes grow at different growth rates in different carbon sources. When more than one carbon sources are present in the media microbes often show a preference over certain carbon sources, and 'non-preferred' carbons sources are used only when the preferred carbon source is exhausted in the media, this process called diauxic shift. Why microbes exhibit such utilization preference over certain carbon sources, is an interesting question in microbiology and evolutionary biology, and the molecular mechanisms that enable microbes to preferentially use one carbon over another is worth investigating. It is intuitive to think that microbes will prefer to use a carbon source that confers maximum growth rate, but when tested experimentally it has been often observed that a carbon source in which microbes grow at sub optimal growth rate is actually preferentially used. In this study authors demonstrate that budding yeast prefer to use galactose over palatinose, but not over sucrose or fructose where all three sugars can support faster growth rate compared to palatinose. Authors presented data where preferential galactose use and diauxic shift is observed in the growth curve when galactose and palatinose or glucose and palatinose combinations were used.

    No diauxic shift was observed in the growth curve when fructose-palatinose, or sucrose-palatinose combination were used. In fructose-palatinose and sucrose-palatinose combinations growth curves agree more with co-utilization strategies. Authors used transcriptomics and genetic perturbations to decipher the molecular mechanism of such preferential carbon use, and reports preference of galactose over palatinose is achieved by preventing positive feedback of MAL regulon, which encodes the genes for palatinose catabolism. We found this observation is interesting and the molecular mechanism of such preferential carbon use is nicely described in this paper. We also find claims authors made are well supported by experiments. Although catabolite repression and diauxic transitions are known in yeast, and authors also pointed out such previous references, but preferential use of a slower carbon source, i.e. galactose over at least one other fast-growing carbon is interesting enough for publication. We would like to support the publication of this article, but we have major concerns about the data analysis and data presentation. Authors must address our concerns which are mentioned below.

    Major comments:

    1. This study mainly hinges on growth rate measurements, but we found growth rates are not properly represented in the figures. Growth curves are always shown in linear scale, which makes it almost impossible to compare fast and slow growth when presented in same plot. All growth curves must be shown on log scale.
    2. Growth rates of the Yeast strain growing individual single carbon sources (galactose, palatinose, sucrose and fructose) should be shown as a figure panel and t-test should be performed to conclude if the individual growth rates are significantly different or not.
    3. Growth phase, lag phase, diauxic shift and post shift growth should be clearly shown in figure 2 and 4, each phase should be clearly marked, carbons used in each phase should be mentioned on the plot. Also, the growth curve must be plotted using log scale.
    4. Authors has taken in account that MAL12 gene overexpression causes long lag when cells need to switch to maltose from glucose, and shown deletion of IMA1 decreases the lag with subsequent 2% growth rate increase in palatinose. How significant is this increase?
    5. Authors have an interesting observation that in sucrose-palatinose and fructose palatinose combinations, most probably co utilization of the carbons is taking place. Authors should discuss this in more details. In galactose-palatinose scenario intracellular galactose-based repression of gal80 and subsequent lack of feed forward of the Mal regulon is expected to stop co-utilization of palatinose. As authors have RNA seq data, can they make predictions for other carbon pairs, where sequential utilization can occur based on their model?

    Minor comments

    1. In figure 5, authors attempted to summarize the model, which is informative, but it will be more useful for non-specific reader if a cell-based cartoon, with transports on surface and catabolic enzymes inside is also added.

    In this schematic diagram, switch from galactose (blue line) to red line (palatinose) shows a mixed color zone, it's a bit confusing, as this represents a bi-stable state. Authors should clearly comment on possibility of biostability while discussing their proposed mechanism.

    1. The author may want to put their work in the context of other recent observations that bacteria do not try to maximize their growth rates in many conditions. Fast growth is often associated with expansive tradeoffs, and a carbon source which confers fast growth rate may confer selective disadvantage. Thus, there are evolutionary benefits of sub-optimal growth, which could be discussed in the manuscript. In this regard a recent study (bioRxiv (2023) doi:10.1101/2023.08.22.554312.) has established the link between resource allocation strategies, growth rates and tradeoffs, which may be taken in account while discussing. Are there any known tradeoffs, when galactose is used over palatinose and which is not the case sucrose or fructose?

    Referees cross-commenting

    As other reviewers pointed out, this study has merit and addressed interesting questions, but needed to be written well in a more understandable form, we agree with this assessment. Also figures must be made much clearer, as all of the reviewers pointed out. In summary, this is an interesting study, but needs some work before publication.

    Significance

    General assessment: Strength and limitations: This study addressed an interesting question regarding resource preference and growth rate optimization in microbes. This is an important question in the field. Study is well designed and claims are backed up with experimental results. One of the limitations of the study is lack of predictability. Authors explained the mechanism for one pair of carbon sources, but how applicable that will be in general is not clear.

    Advance: This study helps to advance our knowledge. Their observation regarding preferential utilization of a carbon source which supports slower growth over a carbon source which can support faster growth, and the molecular mechanism provided will help researchers to understand resource allocation strategies better.

    Audience: Microbiology, systems biology, evolutionary biology, fermentation and bio process engineering research.

    Reviewer expertise: Biochemistry, systems biology, metabolic strategies and tradeoffs in microbes, microbial ecology.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary: The authors have used microtiter plates to produce growth profiles on combinations of different sugars. From this data they have evaluated whether the sugars are co-consumed or if there is a preference for either sugar, seen as a diauxic shift. They found diauxie between galactose and the disaccharide palatinose, but co-consumption between palatinose and fructose. They further used strains with perturbations in their GAL regulon to attempt to explain this discrepency.

    Major comments: I unfortunately found a large portion of the present manuscript unintelligable.

    Firstly, figures were incorrect to the point I could not dechiffre them: Figure 2A-C have black solid and dashed lines in the legend that are not found in the graph, instead there are orange and blue dashed lines in the graph with no legends. Figure 4C has no description of the y-axis. The growth rates in Figure 1C are very hard to follow, and there are definitely local maxima in both the blue and green profiles that are not being discussed (at 15-20 h). I cannot evaluate the conclusions drawn from the data until these issues have been resolved.

    Secondly, the language in the Results and Discussion sections is confusing. Alternating between present and imperfect tense as well as active and passive form makes it hard to distinguish the authors own results from literature findings (Results are usually written in passive, imperfect tense). Examples are found on lines 24, 29, 37-38, 59, 84, 131, and 165.

    The authors also do not consider the differences and similarities in catabolic pathways for assimilation of galactose, fructose and palatinose. Even if they do not see a reason to continue that as a possible explanation for the co-consumption between fructose and palatinose a discussion of why it is disregarded would not be out of place here.

    Significance

    There is some novelty to the authors findings, but I would argue it is being overstated in the present manuscript. Some examples of studies looking at catabolite repression, the main cause of diauxie, of sugars other than glucose can be found in: Simpson-Lavy and Kupiec (2019), Gancedo (1998), Prasad and Venkatesh (2008) and Borgstrom et al (2022).

    I would not say that the field would be significantly advanced by the publication of this manuscript, and the authors have themselves not explained the application of futhering the understanding palatinose metabolism in yeast. As mentioned above, the catabolite repression potential of galactose is already known, it just hasn't been shown for palatinose specifically before.

    I would recommend a complete rewrite of the manuscript as presented, with a lower stated novelty, clearer language and comprehensible figures.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary: Hui et al. tackle a crucial question in biology: what factors influence the preference for carbon sources in yeasts? They reveal that the growth rate on palatinose exceeds that on glucose, despite palatinose utilization being repressed in the presence of glucose. Consequently, the favored carbon source does not necessarily align with the one supporting the fastest growth rate. The study also delves into potential regulatory mechanisms governing carbon source preference and dismisses certain existing theories, such as the general carbon flux sensing mechanism proposed by Okano et al. [25].

    Major comments: None

    Minor comments:

    • The authors suggest that a higher growth rate implies a higher glycolytic flux (l63), a crucial assumption underpinning their interpretation of the absence of a "general carbon flux sensing mechanism" (l65). To substantiate this significant conclusion, they could calculate the extracellular uptake fluxes (based on the time-course concentrations of biomass and substrates).
    • The accumulation of certain by-products is known to be toxic, reducing cellular growth rate (e.g., acetate DOI: 10.1038/srep42135, ethanol DOI: 10.1016/B978-0-12-040308-0.50006-9, etc.), while they can also enhance growth under specific conditions (e.g., acetate DOI: 10.15252/embj.2022113079). Considering this is crucial to rule out certain hypotheses, such as the possibility that a by-product produced during growth on the first carbon source would not modulate growth on the second carbon source, potentially influencing the growth rate differentially in each phase. Although the authors use mutant strains to eliminate the role of some C2 compounds (acetate and ethanol), alternative pathways could be implicated in the (co-)utilization of these by-products. This aspect should be discussed, and ideally, the authors could quantify the time-course concentrations of by-products to assess their potential role.
    • The general flux-sensing regulatory mechanism proposed by Okano et al. [25], which has been dismissed by this study, has recently been questioned, as discussed in DOI: 10.15252/embj.2022113079. This aspect should be included in the discussion.

    Significance

    Strengths & limitations: The work is robust, and the experiments in the study have been appropriately designed and conducted. The primary question of this study has been tackled using a combination of experimental and computational methods to thoroughly assess various regulatory and functional aspects. However, there are gaps in the data that could enhance key conclusions, notably the absence of glycolytic flux measurements. Moreover, further evidence is needed to substantiate the assertion that by-products do not play a role in carbon source preference.

    Advance: This study represents a significant step forward in comprehending the nutritional strategy of microbes. The authors demonstrate that the preferred carbon source may not necessarily be the one supporting the fastest growth rate. Furthermore, they dismiss certain theories that have been proposed to explain the growth strategy of microbes on mixed carbon sources.

    Audience: By addressing a fundamental question in life science, this work is important in the field of biology in general and of particular interest in systems biology, biotechnology, synthetic biology, and health. Consequently, it will be of interest to a broad audience.

  5. Version published to 10.1101/2023.10.18.562896v1 on bioRxiv