When metabolic prowess is too much of a good thing: how carbon catabolite repression and metabolic versatility impede production of esterified α,ω-diols in Pseudomonas putida KT2440

Abstract Background Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols. Results AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain. Conclusions This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes.

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Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in Pseudomonas putida KT2440

Metabolic Engineering Communications ◽

10.1016/j.meteno.2017.05.002 ◽

2017 ◽

Vol 5 ◽

pp. 19-25 ◽

Cited By ~ 42

Author(s):

Christopher W. Johnson ◽

Paul E. Abraham ◽

Jeffrey G. Linger ◽

Payal Khanna ◽

Robert L. Hettich ◽

...

Keyword(s):

Pseudomonas Putida ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Global Regulator ◽

Pseudomonas Putida Kt2440

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Carbon catabolite repression correlates with the maintenance of near invariant molecular crowding in proliferating E. coli cells

BMC Systems Biology ◽

10.1186/1752-0509-7-138 ◽

2013 ◽

Vol 7 (1) ◽

pp. 138 ◽

Cited By ~ 10

Author(s):

Yi Zhou ◽

Alexei Vazquez ◽

Aaron Wise ◽

Tomoko Warita ◽

Katsuhiko Warita ◽

...

Keyword(s):

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Molecular Crowding ◽

E Coli

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Effect of deregulation of repressor-specific carbon catabolite repression on carbon source consumption in Escherichia coli

Applied Biological Chemistry ◽

10.1186/s13765-021-00627-0 ◽

2021 ◽

Vol 64 (1) ◽

Author(s):

Hyeon Jeong Seong ◽

Yu-Sin Jang

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Catabolite Repression ◽

Sole Carbon Source ◽

Carbon Catabolite Repression ◽

Cell Factory ◽

Essential Information ◽

E Coli ◽

Marine Biomass ◽

Galactose Utilization

AbstractEscherichia coli has been used as a host to construct the cell factory for biobased production of chemicals from renewable feedstocks. Because galactose is found in marine biomass as a major component, the strategy for galactose utilization in E. coli has been gained more attention. Although galactose and glucose co-fermentation has been reported using the engineered E. coli strain, few reports have covered fermentation supplemented with galactose as a sole carbon source in the mutant lacking the repressor-specific carbon catabolite repression (CCR). Here, we report the effects of the deregulation of the repressor-specific CCR (galR− and galS−) in fermentation supplemented with galactose as a sole carbon source, using the engineered E. coli strains. In the fermentation using the galR− and galS− double mutant (GR2 strain), an increase of rates in sugar consumption and cell growth was observed compared to the parent strain. In the glucose fermentation, wild-type W3110 and its mutant GR2 and GR2PZ (galR−, galS−, pfkA−, and zwf−) consumed sugar at a higher rate than those values obtained from galactose fermentation. However, the GR2P strain (galR−, galS−, and pfkA−) showed no difference between fermentations using glucose and galactose as a sole carbon source. This study provides essential information for galactose fermentation using the CCR-deregulated E. coli strains.

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Regulation of Arabinose and Xylose Metabolism in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01970-09 ◽

2009 ◽

Vol 76 (5) ◽

pp. 1524-1532 ◽

Cited By ~ 94

Author(s):

Tasha A. Desai ◽

Christopher V. Rao

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Plant Biomass ◽

Xylose Metabolism ◽

Sugar Utilization ◽

E Coli ◽

Metabolic Genes ◽

Pentose Sugars

ABSTRACT Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.

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Carbon catabolite repression inPseudomonas: optimizing metabolic versatility and interactions with the environment

FEMS Microbiology Reviews ◽

10.1111/j.1574-6976.2010.00218.x ◽

2010 ◽

Vol 34 (5) ◽

pp. 658-684 ◽

Cited By ~ 260

Author(s):

Fernando Rojo

Keyword(s):

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Metabolic Versatility

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Intracellular 2-keto-3-deoxy-6-phosphogluconate is the signal for carbon catabolite repression of phenylacetic acid metabolism in Pseudomonas putida KT2440

Microbiology ◽

10.1099/mic.0.027060-0 ◽

2009 ◽

Vol 155 (7) ◽

pp. 2420-2428 ◽

Cited By ~ 13

Author(s):

Juhyun Kim ◽

Jinki Yeom ◽

Che Ok Jeon ◽

Woojun Park

Keyword(s):

Pseudomonas Putida ◽

Transcriptional Repression ◽

Catabolite Repression ◽

Phenylacetic Acid ◽

Carbon Catabolite Repression ◽

Transcriptional Level ◽

Pseudomonas Putida Kt2440 ◽

Coa Ligase ◽

Additional Support

The growth pattern of Pseudomonas putida KT2440 in the presence of glucose and phenylacetic acid (PAA), where the sugar is used in preference to the aromatic compound, suggests that there is carbon catabolite repression (CCR) of PAA metabolism by glucose or gluconate. Furthermore, CCR is regulated at the transcriptional level. However, this CCR phenomenon does not occur in PAA-amended minimal medium containing fructose, pyruvate or succinate. We previously identified 2-keto-3-deoxy-6-phosphogluconate (KDPG) as an inducer of glucose metabolism, and this has led to this investigation into the role of KDPG as a signal compound for CCR. Two mutant strains, the edd mutant (non-KDPG producer) and the eda mutant (KDPG overproducer), grew in the presence of PAA but not in the presence of glucose. The edd mutant utilized PAA even in the presence of glucose, indicating that CCR had been abolished. This observation has additional support from the finding that there is high phenylacetyl-CoA ligase activity in the edd mutant, even in the presence of glucose+PAA, but not in wild-type cells under the same conditions. Unlike the edd mutant, the eda mutant did not grow in the presence of glucose+PAA. Interestingly, there was no uptake and/or metabolism of PAA in the eda mutant cells under the same conditions. Targeted disruption of PaaX, a repressor of the PAA operon, had no effect on CCR of PAA metabolism in the presence of glucose, suggesting that there is another transcriptional repression system associated with the KDPG signal. This is the first study to demonstrate that KDPG is the true CCR signal of PAA metabolism in P. putida KT2440.

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The PalkBFGHJKL Promoter Is under Carbon Catabolite Repression Control in Pseudomonas oleovorans but Not in Escherichia coli alk+Recombinants

Journal of Bacteriology ◽

10.1128/jb.181.5.1610-1616.1999 ◽

1999 ◽

Vol 181 (5) ◽

pp. 1610-1616 ◽

Cited By ~ 40

Author(s):

Ivo E. Staijen ◽

Rosanna Marcionelli ◽

Bernard Witholt

Keyword(s):

Escherichia Coli ◽

Catabolite Repression ◽

Carbon Catabolite Repression ◽

Carbon Sources ◽

Transcriptional Level ◽

Pseudomonas Oleovorans ◽

Hydroxylase Activity ◽

Alkane Hydroxylase ◽

Alkane Degradation ◽

E Coli

ABSTRACT The alk genes are located on the OCT plasmid ofPseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans andEscherichia coli alk + recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads.Pseudomonas putida GPo12 is a P. oleovoransderivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently inPseudomonas and in E. coli strains. These results also indicate that P alkBFGHJKL , the P alk promoter, might be useful in attaining high expression levels of heterologous genes in E. coligrown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.

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Cold-Active β-Galactosidases: Insight into Cold Adaption Mechanisms and Biotechnological Exploitation

Marine Drugs ◽

10.3390/md19010043 ◽

2021 ◽

Vol 19 (1) ◽

pp. 43

Author(s):

Marco Mangiagalli ◽

Marina Lotti

Keyword(s):

Low Temperature ◽

Molecular Mechanisms ◽

Building Blocks ◽

Cold Active ◽

Cold Adaption ◽

Glycosyl Donor ◽

Pharmaceutical Industries ◽

Biotechnological Processes ◽

Hydrolysis Of ◽

Insight Into

β-galactosidases (EC 3.2.1.23) catalyze the hydrolysis of β-galactosidic bonds in oligosaccharides and, under certain conditions, transfer a sugar moiety from a glycosyl donor to an acceptor. Cold-active β-galactosidases are identified in microorganisms endemic to permanently low-temperature environments. While mesophilic β-galactosidases are broadly studied and employed for biotechnological purposes, the cold-active enzymes are still scarcely explored, although they may prove very useful in biotechnological processes at low temperature. This review covers several issues related to cold-active β-galactosidases, including their classification, structure and molecular mechanisms of cold adaptation. Moreover, their applications are discussed, focusing on the production of lactose-free dairy products as well as on the valorization of cheese whey and the synthesis of glycosyl building blocks for the food, cosmetic and pharmaceutical industries.

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