Engineering of Primary Carbohydrate Metabolism for Increased Production of Actinorhodin in Streptomyces coelicolor

ABSTRACT The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.

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Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans

mBio ◽

10.1128/mbio.00805-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 4

Author(s):

Katharina Kremer ◽

Muriel C. F. van Teeseling ◽

Lennart Schada von Borzyskowski ◽

Iria Bernhardsgrütter ◽

Rob J. M. van Spanning ◽

...

Keyword(s):

Growth Rates ◽

Carbon Metabolism ◽

Paracoccus Denitrificans ◽

High Growth ◽

Central Carbon Metabolism ◽

High Biomass ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Content Type ◽

Central Carbon

ABSTRACT During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism’s central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism. IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable “operating system” of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

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Lycopene production in recombinant strains of Escherichia coli is improved by knockout of the central carbon metabolism gene coding for glucose-6-phosphate dehydrogenase

Biotechnology Letters ◽

10.1007/s10529-013-1317-0 ◽

2013 ◽

Vol 35 (12) ◽

pp. 2137-2145 ◽

Cited By ~ 27

Author(s):

Yan Zhou ◽

Komi Nambou ◽

Liujing Wei ◽

Jingjing Cao ◽

Tadayuki Imanaka ◽

...

Keyword(s):

Escherichia Coli ◽

Carbon Metabolism ◽

Central Carbon Metabolism ◽

Recombinant Strains ◽

Gene Coding ◽

Lycopene Production ◽

Central Carbon ◽

Glucose 6 Phosphate Dehydrogenase ◽

Metabolism Gene

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Cofactor Specificity of Glucose-6-Phosphate Dehydrogenase Isozymes in Pseudomonas putida Reveals a General Principle Underlying Glycolytic Strategies in Bacteria

mSystems ◽

10.1128/msystems.00014-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Daniel Christoph Volke ◽

Karel Olavarría ◽

Pablo Iván Nikel

Keyword(s):

Gene Duplication ◽

Pseudomonas Putida ◽

Carbon Metabolism ◽

Redox Balance ◽

Central Carbon Metabolism ◽

Evolutionary Strategy ◽

Content Type ◽

Cofactor Specificity ◽

Central Carbon ◽

Glucose 6 Phosphate Dehydrogenase

ABSTRACT Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity toward NADP+, some variants accept NAD+ similarly or even preferentially. Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from genomic, genetic, and biochemical perspectives. P. putida represents an ideal model to tackle this endeavor, as its genome harbors gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic “gatekeepers” for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. The abundance of G6PDH isoforms in these species goes hand in hand with low NADP+ affinity, at least in one isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy toward balancing the relative production of NADPH and NADH. IMPORTANCE Protein families have likely arisen during evolution by gene duplication and divergence followed by neofunctionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the coexistence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of coexisting glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+ or NADP+ specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy toward achieving redox balance according to the growth conditions.

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Cofactor specificity of glucose-6-phosphate dehydrogenase isozymes in Pseudomonas putida reveals a general principle underlying glycolytic strategies in bacteria

10.1101/2021.01.08.426012 ◽

2021 ◽

Author(s):

Daniel C. Volke ◽

Karel Olavarria ◽

Pablo Ivan Nikel

Keyword(s):

Pseudomonas Putida ◽

Carbon Metabolism ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Central Carbon Metabolism ◽

Pentose Phosphate ◽

Biochemical Network ◽

Evolutionary Strategy ◽

Cofactor Specificity ◽

Central Carbon ◽

Glucose 6 Phosphate Dehydrogenase

Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase also provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity towards nicotinamide adenine dinucleotide phosphate (NADP+), some variants accept nicotinamide NAD+ similarly (or even preferentially). Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from a genomic, genetic and biochemical perspective. P. putida represents an ideal model to tackle this endeavor, as its genome encodes numerous gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities, and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic 'gatekeepers' for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. Multiplication of G6PDH isoforms in these species goes hand-in-hand with low NADP+ affinity at least in one G6PDH isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy towards balancing the relative production of NADPH and NADH.

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