MANIPULATION OF BACTERIA TO OPTIMIZE SYNTHESIS OF BIOPRODUCTS FROM GLYCEROL

1. MANIPULATION OF BACTERIA TO OPTIMIZE SYNTHESIS OF BIOPRODUCTS FROM GLYCEROL M. Julia Pettinari What to do with glycerol? How much glycerin soap can we use? We will most probably need multiple strategies to use it if we take into account the huge amounts that will be produced during biodiesel synthesis as a consequence of the biofuel laws that our countries are making these last years. As microbiologists, our thought is naturally directed towards the possibility of feeding it to our microorganisms and figuring out which kinds of useful products we can obtain from them. We know that many organisms can use glycerol as a C source to synthesize a variety of products, but it is a substrate that will normally not be fermented, so there are a number of industrial products that we are used to get from microbes that are not easily obtained when using glycerol as a substrate, such as ethanol. As a consequence, the challenge posed these days to microbiologists regarding glycerol is to convince our faithful microorganisms to produce what we need from the substrate we need to get rid of. In this talk I will tell you about two kinds of biotechnologically important products that we can obtain from glycerol using E. coli:: biodegradable thermoplastics and ethanol. What to do with glycerol? How much glycerin soap can we use? We will most probably need multiple strategies to use it if we take into account the huge amounts that will be produced during biodiesel synthesis as a consequence of the biofuel laws that our countries are making these last years. As microbiologists, our thought is naturally directed towards the possibility of feeding it to our microorganisms and figuring out which kinds of useful products we can obtain from them. We know that many organisms can use glycerol as a C source to synthesize a variety of products, but it is a substrate that will normally not be fermented, so there are a number of industrial products that we are used to get from microbes that are not easily obtained when using glycerol as a substrate, such as ethanol. As a consequence, the challenge posed these days to microbiologists regarding glycerol is to convince our faithful microorganisms to produce what we need from the substrate we need to get rid of. In this talk I will tell you about two kinds of biotechnologically important products that we can obtain from glycerol using E. coli:: biodegradable thermoplastics and ethanol.

2. What to do with glycerol? How much glycerin soap can we use? We will most probably need multiple strategies to use it if we take into account the huge amounts that will be produced during biodiesel synthesis as a consequence of the biofuel laws that our countries are making these last years. As microbiologists, our thought is naturally directed towards the possibility of feeding it to our microorganisms and figuring out which kinds of useful products we can obtain from them. We know that many organisms can use glycerol as a C source to synthesize a variety of products, but it is a substrate that will normally not be fermented, so there are a number of industrial products that we are used to get from microbes that are not easily obtained when using glycerol as a substrate, such as ethanol. As a consequence, the challenge posed these days to microbiologists regarding glycerol is to convince our faithful microorganisms to produce what we need from the substrate we need to get rid of. In this talk I will tell you about two kinds of biotechnologically important products that we can obtain from glycerol using E. coli:: biodegradable thermoplastics and ethanol. What to do with glycerol? How much glycerin soap can we use? We will most probably need multiple strategies to use it if we take into account the huge amounts that will be produced during biodiesel synthesis as a consequence of the biofuel laws that our countries are making these last years. As microbiologists, our thought is naturally directed towards the possibility of feeding it to our microorganisms and figuring out which kinds of useful products we can obtain from them. We know that many organisms can use glycerol as a C source to synthesize a variety of products, but it is a substrate that will normally not be fermented, so there are a number of industrial products that we are used to get from microbes that are not easily obtained when using glycerol as a substrate, such as ethanol. As a consequence, the challenge posed these days to microbiologists regarding glycerol is to convince our faithful microorganisms to produce what we need from the substrate we need to get rid of. In this talk I will tell you about two kinds of biotechnologically important products that we can obtain from glycerol using E. coli:: biodegradable thermoplastics and ethanol.

3. Metabolic Engeneering of microbial processes Strategies: Substrate manipulation Use of inhibitors (Genetic) Manipulation of metabolic pathways As microbiologists, our thought is naturally directed towards the possibility of feeding it to our microorganisms and figuring out which kinds of useful products we can obtain from them. We know that many organisms can use glycerol as a C source to synthesize a variety of products, but it is a substrate that will normally not be fermented, so there are a number of industrial products that we are used to get from microbes that are not easily obtained when using glycerol as a substrate, such as ethanol. As a consequence, the challenge posed these days to microbiologists regarding glycerol is to convince our faithful microorganisms to produce what we need from the substrate we need to get rid of. In this talk I will tell you about two kinds of biotechnologically important products that we can obtain from glycerol using E. coli:: biodegradable thermoplastics and ethanol. Among the different strategies used for metabolic engineering of microbial processes, classis techniques such as substrate manipulation, the use of inhibitors or manipulating other growth conditions can result in process improvements, but there are limitations imposed by the microorganism�s metabolic capacities. These limitations can be overcome by modifying the organisms metabolic pathways. This can be done by introducing mutations in genes that result in increasing the activity of some enzymes, or reducing, or directly blocking, others. Whole new pathways can also be introduced, and then the whole metabolism of the organism must be taken into account in order to predict what the results will be. For any kind of manipulation of genetic pathways, a deep knowledge of the organism�s metabolism and genes is necessary. Because of this, E coli, the best characterized microorganism, is ideal, not only because all kinds of information and tools are available to modify this organism, but also because it is very versatile and flexible. As microbiologists, our thought is naturally directed towards the possibility of feeding it to our microorganisms and figuring out which kinds of useful products we can obtain from them. We know that many organisms can use glycerol as a C source to synthesize a variety of products, but it is a substrate that will normally not be fermented, so there are a number of industrial products that we are used to get from microbes that are not easily obtained when using glycerol as a substrate, such as ethanol. As a consequence, the challenge posed these days to microbiologists regarding glycerol is to convince our faithful microorganisms to produce what we need from the substrate we need to get rid of. In this talk I will tell you about two kinds of biotechnologically important products that we can obtain from glycerol using E. coli:: biodegradable thermoplastics and ethanol. Among the different strategies used for metabolic engineering of microbial processes, classis techniques such as substrate manipulation, the use of inhibitors or manipulating other growth conditions can result in process improvements, but there are limitations imposed by the microorganism�s metabolic capacities. These limitations can be overcome by modifying the organisms metabolic pathways. This can be done by introducing mutations in genes that result in increasing the activity of some enzymes, or reducing, or directly blocking, others. Whole new pathways can also be introduced, and then the whole metabolism of the organism must be taken into account in order to predict what the results will be. For any kind of manipulation of genetic pathways, a deep knowledge of the organism�s metabolism and genes is necessary. Because of this, E coli, the best characterized microorganism, is ideal, not only because all kinds of information and tools are available to modify this organism, but also because it is very versatile and flexible.

4. In our lab we have been working for many years on polyhydroxyalkanoates. These biopolymers are linear polymers constituted by hydroxyesther units, and bacteria accumulate them as aresponse to environmental stress when there is a carbon source available but other nutrients are limiting. They have thremoplastic and elastomeric properties, so they can be used to replace petroleum derived plastics. They have two main advantages over traditional plastics: they can be obtained from renewable sources and they are completely biodegradble. They have one disadvantage: their production price is still high. One way to lower their price is to use low cost carbon substrates, and this is where glycerol enter the picture. In our lab we have been working for many years on polyhydroxyalkanoates. These biopolymers are linear polymers constituted by hydroxyesther units, and bacteria accumulate them as aresponse to environmental stress when there is a carbon source available but other nutrients are limiting. They have thremoplastic and elastomeric properties, so they can be used to replace petroleum derived plastics. They have two main advantages over traditional plastics: they can be obtained from renewable sources and they are completely biodegradble. They have one disadvantage: their production price is still high. One way to lower their price is to use low cost carbon substrates, and this is where glycerol enter the picture.

5. PHA synthesis and degradation are cyclic. THERE are many microorganisms that produce PHAs naturally. The most common PHA is PHB, with a 4 carbon monomer. This polymer can be produced in several ways , but the most common and extended pathway involves the condensation of two molecules of Acetyl-CoA, and then a NADPH consuming reduction to give 3-hydroxybutyryl-CoA, the monomer, that is then polymerized by a special enzyme. IN all natural producers the polymer is degraded to use it as a source of carbon and reducing power, completing the cycle.THERE are many microorganisms that produce PHAs naturally. The most common PHA is PHB, with a 4 carbon monomer. This polymer can be produced in several ways , but the most common and extended pathway involves the condensation of two molecules of Acetyl-CoA, and then a NADPH consuming reduction to give 3-hydroxybutyryl-CoA, the monomer, that is then polymerized by a special enzyme. IN all natural producers the polymer is degraded to use it as a source of carbon and reducing power, completing the cycle.

6. We have constructed a recombinant E coli capable of synthesizing PHB by introducing the three genes responsible for the synthesis of the polymer from an Azotobacter strain. The substrates for PHB synthesis in Ecoli are Acetyl-CoA and reducing power, so strategies to improve the synthesis of the polymer must maximize the avaliability of these compounds. Several strategies have been tested to increase reducing power. Some of these have focused on directing carbon flow through the Entner doudoroff pathway, that produces more reducing power from sugars than glycolisis. Mutations that inactivate the pgi enzyme and others that increase the activity of G6PDH have been tested, but although these mutations increased the production of PHB, they sometimes had a negative effect on growth. Other strategies, that we have tested in our lab, involve the inactivation of competing pathways, such as the synthesis of acetate and lactate, but these also sometimes have negative effects on growth, specially the inactivation of the acetate synthesising enzymes. On the other hand, the degradation of glycerol yields more reducing power than other substrates like glucose, so using glycerol should favor the production of reduced compounds such as PHB We have constructed a recombinant E coli capable of synthesizing PHB by introducing the three genes responsible for the synthesis of the polymer from an Azotobacter strain. The substrates for PHB synthesis in Ecoli are Acetyl-CoA and reducing power, so strategies to improve the synthesis of the polymer must maximize the avaliability of these compounds. Several strategies have been tested to increase reducing power. Some of these have focused on directing carbon flow through the Entner doudoroff pathway, that produces more reducing power from sugars than glycolisis. Mutations that inactivate the pgi enzyme and others that increase the activity of G6PDH have been tested, but although these mutations increased the production of PHB, they sometimes had a negative effect on growth. Other strategies, that we have tested in our lab, involve the inactivation of competing pathways, such as the synthesis of acetate and lactate, but these also sometimes have negative effects on growth, specially the inactivation of the acetate synthesising enzymes. On the other hand, the degradation of glycerol yields more reducing power than other substrates like glucose, so using glycerol should favor the production of reduced compounds such as PHB

7. The E. coli regulatory network The strategies I just mentioned involve changes in certain enzymes that produce changes in carbon flow. There are other ways to manipulate metabolism producing many more subtle changes, that can be accomplished using mutations in regulator genes. If we take into account the regulatory network in E.coli, we will see that there are several levels of regulators, some of them regulating only a small number of genes, and other regulating entire pathways There are 7 global regulators, that control all metabolism. Among these regulators, arCA is a central regulator that is in charge of redox control, and we focused our interest in it. The strategies I just mentioned involve changes in certain enzymes that produce changes in carbon flow. There are other ways to manipulate metabolism producing many more subtle changes, that can be accomplished using mutations in regulator genes. If we take into account the regulatory network in E.coli, we will see that there are several levels of regulators, some of them regulating only a small number of genes, and other regulating entire pathways There are 7 global regulators, that control all metabolism. Among these regulators, arCA is a central regulator that is in charge of redox control, and we focused our interest in it.

8. Regulation of aerobic and anaerobic metabolism in E. coli Redox control in E coli, that as a facultative aerobe must adapt to oxygen availability adjusting its metabolism is achieved by means of two global regulators: ArcA and Fnr. Arc is a Global regulator system composed by a cytosolic response regulator (ArcA) and a trans-membrane histidine kinase sensor (ArcB). This system exerts control over ca. 51 operons. ArcB is activated during the transition from aerobic to microaerobic growth and phos-phorylates ArcA, which represses the synthesis of many aerobic enzymes (e.g., those involved in TCA cycle).Redox control in E coli, that as a facultative aerobe must adapt to oxygen availability adjusting its metabolism is achieved by means of two global regulators: ArcA and Fnr. Arc is a Global regulator system composed by a cytosolic response regulator (ArcA) and a trans-membrane histidine kinase sensor (ArcB). This system exerts control over ca. 51 operons. ArcB is activated during the transition from aerobic to microaerobic growth and phos-phorylates ArcA, which represses the synthesis of many aerobic enzymes (e.g., those involved in TCA cycle).

9. Hypothesis Our working hypothesis was that... in conditions of low O2 availability, normally encountered by microorganisms in industrial fermentations, The E. coli arcA mutants are unregulated for aerobic pathways and the pool of reducing equivalents as NADH or NADPH is increased. We hypothesize that the accumulation of PHB will be favored in an arcA genetic background due to the generation of an excess reducing power that could be funneled into an electron sink. Our working hypothesis was that... in conditions of low O2 availability, normally encountered by microorganisms in industrial fermentations, The E. coli arcA mutants are unregulated for aerobic pathways and the pool of reducing equivalents as NADH or NADPH is increased. We hypothesize that the accumulation of PHB will be favored in an arcA genetic background due to the generation of an excess reducing power that could be funneled into an electron sink.

10. To start the analysis of the relationship between arc and PHB we first analyzed the effect of PHB accumulation on a phenotype characteristic of arc mutants, sensitivity to the redox dye toluidine blue. This sensitivity is related to the higher respiratory rate and O2 consumption of arc mutants, that produces high amounts of toxic ROS. We observed that the PHB accumulating arc strains were more resistant to AT, had lower O2 consumption rates and produced less ROS. In a second approach we used an essay that estimates NADH intracellular availability. Diamide causes an oxidative stress by inducing disulfide formation in proteins and low MW thiols in the arc strain. This effect is neutralized by the thioredoxin reductase, that uses NADH to reduce the S-S. The PHB accumulating strains had greater sensitivity, indicating a lower availability of reducing power, preseumably due to its utilization in PHB synthesis.To start the analysis of the relationship between arc and PHB we first analyzed the effect of PHB accumulation on a phenotype characteristic of arc mutants, sensitivity to the redox dye toluidine blue. This sensitivity is related to the higher respiratory rate and O2 consumption of arc mutants, that produces high amounts of toxic ROS. We observed that the PHB accumulating arc strains were more resistant to AT, had lower O2 consumption rates and produced less ROS. In a second approach we used an essay that estimates NADH intracellular availability. Diamide causes an oxidative stress by inducing disulfide formation in proteins and low MW thiols in the arc strain. This effect is neutralized by the thioredoxin reductase, that uses NADH to reduce the S-S. The PHB accumulating strains had greater sensitivity, indicating a lower availability of reducing power, preseumably due to its utilization in PHB synthesis.

11. Preliminary characterization of arcA mutants for PHB accumulation in microaerobiosis The experiments we just saw suggested that PHB synthesis consumes part of the excess reducing equivalents produced in the arc mutant strains. Now we wanted to find out wether this resulted in an enhanced polymer production in arc mutants. Arc regulation is active at low O2 availability conditions, so we analysed polymer production in these conditions, in which we expected to find differences between wt and arc strains. When we grew the strains we saw that both accumulated the polymer in aerobiosis, but only the arc mutant did it at low O2 availability conditions We analysed different aereation conditions, and observed that the relative accumulation (% CDW) of PHB in the mutant was inversely proportional to O2 availability. This was consistent with our hypothesis, as in low O2 availability the mutant does not reduce its respiration rate and reducing power generation. This reducing power is the funnelled into PHB synthesis, resulting in higher synthesis in lower O2 conc. The experiments we just saw suggested that PHB synthesis consumes part of the excess reducing equivalents produced in the arc mutant strains. Now we wanted to find out wether this resulted in an enhanced polymer production in arc mutants. Arc regulation is active at low O2 availability conditions, so we analysed polymer production in these conditions, in which we expected to find differences between wt and arc strains. When we grew the strains we saw that both accumulated the polymer in aerobiosis, but only the arc mutant did it at low O2 availability conditions We analysed different aereation conditions, and observed that the relative accumulation (% CDW) of PHB in the mutant was inversely proportional to O2 availability. This was consistent with our hypothesis, as in low O2 availability the mutant does not reduce its respiration rate and reducing power generation. This reducing power is the funnelled into PHB synthesis, resulting in higher synthesis in lower O2 conc.

12. After the preliminary tests run in erlenmeyer flasks we made some bioreactor cultures with the strains We observed that the arcA mutant accumulated more than an order of magnitude more polymer than the WT (see scale) , that probably did not produce enough reducing power to allow PHB synthesis. However, we observed that arc mutants had lower growth, probably because arc is a global regulator with pleiotropic effects that affect growth. For instance, arc mutants require growth supplements for growth.After the preliminary tests run in erlenmeyer flasks we made some bioreactor cultures with the strains We observed that the arcA mutant accumulated more than an order of magnitude more polymer than the WT (see scale) , that probably did not produce enough reducing power to allow PHB synthesis. However, we observed that arc mutants had lower growth, probably because arc is a global regulator with pleiotropic effects that affect growth. For instance, arc mutants require growth supplements for growth.

13. PHB accumulation in CT1061/pJP24 (arcA2) growing on glycerol in microaerobiosis We had another arc mutant that had a slightly different behavior. This strain had an even higher respiration rate than the deletion strain, but had less growth requirements and less sensitivity to the redox dye TB. When a strain carrying this mutation and the phb genes was tested for growth and PHb synthesis, it grew more and produced more polymer than the deletion strain, even in a poorer medium.We had another arc mutant that had a slightly different behavior. This strain had an even higher respiration rate than the deletion strain, but had less growth requirements and less sensitivity to the redox dye TB. When a strain carrying this mutation and the phb genes was tested for growth and PHb synthesis, it grew more and produced more polymer than the deletion strain, even in a poorer medium.

14. Effect of arcA on PHB producing E. coli Conclusions CONCLUSIONS The mutant strains allowed PHB accumulation in microaerobic conditions. The wild type strain did not produce detectable amounts of PHB in these conditions Higher biomass and PHB concentrations (resulting in an higher PHB yield) were reached in minimal medium in the strain bearing mutation arcA2 compared with the deletion mutant. This behaviour could be due to an arcA leaky phenotype. CONCLUSIONS The mutant strains allowed PHB accumulation in microaerobic conditions. The wild type strain did not produce detectable amounts of PHB in these conditions Higher biomass and PHB concentrations (resulting in an higher PHB yield) were reached in minimal medium in the strain bearing mutation arcA2 compared with the deletion mutant. This behaviour could be due to an arcA leaky phenotype.

15. Ethanol production from glycerol in arcA mutants Up to now we have discussed PHB production in recombinant E coli carrying the pha genes from an Azotobacter strain and the effect of mutations in arcA genes in this context. If the arcA mutants had increased reducing equivalents supply, then this conditiosn should also favor the synthesis of other reduced products, such as ethanol. Following this hypothesis, we measured teh amounts of ethanol produced by the wild type and the arcA mutants both in aerobiosis and microaerobiosis, and observed... Up to now we have discussed PHB production in recombinant E coli carrying the pha genes from an Azotobacter strain and the effect of mutations in arcA genes in this context. If the arcA mutants had increased reducing equivalents supply, then this conditiosn should also favor the synthesis of other reduced products, such as ethanol. Following this hypothesis, we measured teh amounts of ethanol produced by the wild type and the arcA mutants both in aerobiosis and microaerobiosis, and observed...

16. Metabolic characterization of arcA strains growing on glycerol To continue studying the metabolic effect of the mutations we measured the major fermentative metabolic producs in microaerobiosis in order to better understand the flow of carbon in the cells. We observed that arcA mutants had increased levels of reduced compounds such as ethanol, and lower levels of more oxidized products such as acetate. In a similar way as what we had seen in the PHB producing starins, the effect was much more pronounced in the arcA2 mutants.To continue studying the metabolic effect of the mutations we measured the major fermentative metabolic producs in microaerobiosis in order to better understand the flow of carbon in the cells. We observed that arcA mutants had increased levels of reduced compounds such as ethanol, and lower levels of more oxidized products such as acetate. In a similar way as what we had seen in the PHB producing starins, the effect was much more pronounced in the arcA2 mutants.

17. Facultative aerobes like E coli regulate the fermentation products according to their redox state. Because of this, the relative amount of acetate and ethanol can be used to estimate the intracellular redox state. A high ethanol to acetate ratio indicates a highly reduced state. We observed that the deletion mutant had a ratio 2 times higher that the wild type, and that the arcA2 mutant had a dramatically higher ratio, almost 14 times higher than the wild type and 6 times higher than the deletion mutant. In view of these results we measured the intracellular amounts of NAD and NADH. We observed that the relative amounts of reduced vs oxidised cofactor correlated well with the ethanol/acetate ratios, and that both arcA mutants had increasec proportions of reduced cofactor, almost equalling the amount of oxidized cofactor in the arcA2 mutant.Facultative aerobes like E coli regulate the fermentation products according to their redox state. Because of this, the relative amount of acetate and ethanol can be used to estimate the intracellular redox state. A high ethanol to acetate ratio indicates a highly reduced state. We observed that the deletion mutant had a ratio 2 times higher that the wild type, and that the arcA2 mutant had a dramatically higher ratio, almost 14 times higher than the wild type and 6 times higher than the deletion mutant. In view of these results we measured the intracellular amounts of NAD and NADH. We observed that the relative amounts of reduced vs oxidised cofactor correlated well with the ethanol/acetate ratios, and that both arcA mutants had increasec proportions of reduced cofactor, almost equalling the amount of oxidized cofactor in the arcA2 mutant.

18. Throughout this presentation we have analyzed two different mutants in the arcA gene, and we have seen that the strain bearing the arcA2 mutation, strain CT1061, grows more than................... We have observed many different traits in which the CT1061 strain presents advantages from the biotechnological point of view over its sister strain CT1062. The question is what exactly are the differences bewteen them? Do they only differ in their arcA allelle? Throughout this presentation we have analyzed two different mutants in the arcA gene, and we have seen that the strain bearing the arcA2 mutation, strain CT1061, grows more than................... We have observed many different traits in which the CT1061 strain presents advantages from the biotechnological point of view over its sister strain CT1062. The question is what exactly are the differences bewteen them? Do they only differ in their arcA allelle?

19. Detection of ArcA in Western blot What we first did was to characterize the arcA2 mutant at the molecular level. We thought that the mutation could result in a regulatory protein with special properties In the bibliography describing the mutant it was described as a null mutant due to a Tn10 insertion, this means that it either didn�t express the protein or that the protein produced had no activity. We cloned and sequenced the region, and found an IS10 insertion interrupting the protein�s coding sequence. On the other hand, using specific antibodies we could detect in Western Blot experiments the presence of a smaller protein in the mutant. The size of this mutant protein corresponded to the size predicted by an in silico analysis. This short protein did not contain the DNA binding sites of the regulator, so it was not expected to interact with DNA, and thus should not function as a regulator. The question was then why this mutant strain did not behave as the deletion mutant.What we first did was to characterize the arcA2 mutant at the molecular level. We thought that the mutation could result in a regulatory protein with special properties In the bibliography describing the mutant it was described as a null mutant due to a Tn10 insertion, this means that it either didn�t express the protein or that the protein produced had no activity. We cloned and sequenced the region, and found an IS10 insertion interrupting the protein�s coding sequence. On the other hand, using specific antibodies we could detect in Western Blot experiments the presence of a smaller protein in the mutant. The size of this mutant protein corresponded to the size predicted by an in silico analysis. This short protein did not contain the DNA binding sites of the regulator, so it was not expected to interact with DNA, and thus should not function as a regulator. The question was then why this mutant strain did not behave as the deletion mutant.

20. E. coli CT1061 construction We had constructed strain CT1061 using phage P1 to transfer the mutation by transduction from strain ECL618,so there was a possibility that other genes could have been transferred along with the arcA allelle and could be responsible for the phenotype. We analyzed the region in serach of other genes that could contribute to the phenotypic traits observed, and we noticed the cre operon just next to arcA We had constructed strain CT1061 using phage P1 to transfer the mutation by transduction from strain ECL618,so there was a possibility that other genes could have been transferred along with the arcA allelle and could be responsible for the phenotype. We analyzed the region in serach of other genes that could contribute to the phenotypic traits observed, and we noticed the cre operon just next to arcA

21. Some CreBC regulated genes The cre regulating system is a two compenent system analogous to arc. It has a sensor protein, creB, and a regulator, creC, that activates some geness, like ack/pta, and represses others, like mal. This system is induced in conditions of low nutrient availability. Many of the operons that the cre system controls play an important role in carbon flow, so it was reasonable to assume that the phenotypic differences observed between the strains could be related to differences in the cre genes. A bibliographic search revealed that there are many common E coli lab strains carrying a mutation in one of the cre genes, creC510. It is a constitutive mutation in the creC gene, originally described in strain HfrH, from which the donor strain for the arcA2 allelle descends. Because of this, it was possible that our arcA2 mutants carried this mutationThe cre regulating system is a two compenent system analogous to arc. It has a sensor protein, creB, and a regulator, creC, that activates some geness, like ack/pta, and represses others, like mal. This system is induced in conditions of low nutrient availability. Many of the operons that the cre system controls play an important role in carbon flow, so it was reasonable to assume that the phenotypic differences observed between the strains could be related to differences in the cre genes. A bibliographic search revealed that there are many common E coli lab strains carrying a mutation in one of the cre genes, creC510. It is a constitutive mutation in the creC gene, originally described in strain HfrH, from which the donor strain for the arcA2 allelle descends. Because of this, it was possible that our arcA2 mutants carried this mutation

22. Analysis of creC in E.coli strains Because of this we decided to analyze the creC gene both molecularly and phenotypically to see if this mutation was present in our strains. The creC510 mutant contains a sustitution of an Arg residue in position 77 by a proline. We amplified by PCR and sequenced a region of the creC gene, and found that�. We also tested the presence of the creC510 allelle phenotypically by testing growth of the strains on maltose. The constitutive mutant would always repress maltose utilization, so it would not be able to grow on maltose. The results indicated that strains�had the creC510 allelle, so we concluded that CT1061 contains BOTH an arcA mutation AND a creC mutationBecause of this we decided to analyze the creC gene both molecularly and phenotypically to see if this mutation was present in our strains. The creC510 mutant contains a sustitution of an Arg residue in position 77 by a proline. We amplified by PCR and sequenced a region of the creC gene, and found that�. We also tested the presence of the creC510 allelle phenotypically by testing growth of the strains on maltose. The constitutive mutant would always repress maltose utilization, so it would not be able to grow on maltose. The results indicated that strains�had the creC510 allelle, so we concluded that CT1061 contains BOTH an arcA mutation AND a creC mutation

23. In this table we can see several parameters measured in the mutants. We can notice the enhanced acetate kinase activity of strain CT1061, the only exhibiting the creCconst phenotype,that had the highest respiratory rate, and was the only strain that consumed O2 at high speed in acetate. We can also see that the creC mutant has a very high glucose consumption rate, that could explain its higher production Por ultimo se midio el contenido de los citocromos en las mutantes. En ecoli existen dos oxidasas terminales responsables de entregar los e al O2, la cit d oxidasa, que tiene alta afinidad por el O2 y se activa cuando hay baja disponibilidad, y la cit o oxidasa. El citoc o tiene menor afinidad por el O2, y se activa cuando este se encuentra en alta conc. Arc activa la expresion del cit d y reprime la del o. La distribuci�n de citocromos en CT1061 y CT1062 es compatible con el patr�n esperado para mutantes arc, ya que ArcA activa la expresi�n del citocromo bd y reprime la expresi�n del citocromo bo. Para analizar si la mutacion en cre afetaba la expresion de estos citocromos se midieron en las mutantes, y se observo que la mutacion en cre no esta involucrada en la regulacion de las transferasas que transfieren los electr al O2. La respuesta al efecto observado parece deberse a la mayor actividad metabolica de las cepas cre, ya que estas cepas presentan mayor consumo de glucosa, independientemente de la mutacion arc.In this table we can see several parameters measured in the mutants. We can notice the enhanced acetate kinase activity of strain CT1061, the only exhibiting the creCconst phenotype,that had the highest respiratory rate, and was the only strain that consumed O2 at high speed in acetate. We can also see that the creC mutant has a very high glucose consumption rate, that could explain its higher production Por ultimo se midio el contenido de los citocromos en las mutantes. En ecoli existen dos oxidasas terminales responsables de entregar los e al O2, la cit d oxidasa, que tiene alta afinidad por el O2 y se activa cuando hay baja disponibilidad, y la cit o oxidasa. El citoc o tiene menor afinidad por el O2, y se activa cuando este se encuentra en alta conc. Arc activa la expresion del cit d y reprime la del o. La distribuci�n de citocromos en CT1061 y CT1062 es compatible con el patr�n esperado para mutantes arc, ya que ArcA activa la expresi�n del citocromo bd y reprime la expresi�n del citocromo bo. Para analizar si la mutacion en cre afetaba la expresion de estos citocromos se midieron en las mutantes, y se observo que la mutacion en cre no esta involucrada en la regulacion de las transferasas que transfieren los electr al O2. La respuesta al efecto observado parece deberse a la mayor actividad metabolica de las cepas cre, ya que estas cepas presentan mayor consumo de glucosa, independientemente de la mutacion arc.

24. General conclusions C and redox balance mechanisms can be manipulated in order to enhance the use of the substrate for the production of highly reduced products, even when using a reduced substrate such as glycerol, that will be mostly used in an oxidative catabolism, which does not lead to the production of fermentation products.