Engineering NADH metabolism in<i>Saccharomyces cerevisiae</i>: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

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Production of ␤-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol ⅐ mol ؊1 , an increasing rate of formate oxidation via a cytosolic NAD ؉ -dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol ⅐ mol ؊1 , the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as ␤-lactams.The filamentous fungus Penicillium chrysogenum is applied on a large scale (Ͼ60,000 tons year Ϫ1 ) (8, 36) for the industrial production of ␤-lactam antibiotics, such as penicillin G and penicillin V, and for the production of the cephalosporin precursor adipoyl-7-ADCA. ␤-Lactam antibiotics are formed in a multistep process in which the first two steps are common for penicillins and cephalosporins. The three amino acids cysteine, valine, and ␣-aminoadipic acid, derived from central metabolism, are condensed to form the tripeptide ACV (␣-aminoadipyl-cysteinyl-valine). The next step is a ring closure that leads to the characteristic penam structure of isopenicillin N, the branch point intermediate at which penicillin biosynthesis diverges from cephalosporin biosynthesis. Penicillin G is formed from isopenicillin N by exchanging its ␣-aminoadipic acid side chain for phenylacetic acid, using phenylacetyl-coenzyme A as a side chain donor.Overproduction of secondary metabolites can have a large impact on central metabolism if it requires significant amounts of carbon precursors, reducing equivalents (NADH and NADPH), and free energy equivalents (ATP). Previous studies on penicillin G production in a high-producing industrial strain of P. chrysogenum have shown that constraints in central metabolism may reside in the supply and regeneration of the cofactor NADPH rather than in the supply of the carbon precursors, ␣-aminoadipic aci...

Section: Discussionmentioning

confidence: 99%

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum : Impact on Penicillin G Production and Biomass Yield

Harris

Krogt

Gulik

et al. 2007

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“…These results are likely to be a combination of effects from alleviating the feedback inhibition of the TCA cycle by mitochondrial NADH and increasing respiratory capacity due to improved efficiency of oxidative phosphorylation, as quantified by the P/O ratio (15). There are no reports that describe the effect of increasing NADH in S. cerevisiae, although formate has been used previously as a source of additional reducing power in S. cerevisiae (2,4,11,23,24,27). Formate (HCOO Ϫ ) is efficiently oxidized to CO 2 by NAD ϩ -dependent formate dehydrogenase (27) and, therefore, cannot be used as a carbon source for biomass synthesis.…”

mentioning

confidence: 91%

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Hou

Scalcinati

Oldiges

et al. 2010

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD ؉ -dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.The traditional use of baker's yeast, Saccharomyces cerevisiae, for ethanol production has resulted in the accumulation of substantial information about its genetics, metabolism, and process development. Consequently, the collection of compounds that are produced using S. cerevisiae has expanded to include organic acids and even secondary metabolites (1,25,28). Unlike ethanol, many of these products are not redox neutral relative to commonly used substrates such as glucose. Therefore, in addition to stoichiometry, redox constraints play an important role in the formation of the products. Additional reducing power has to be supplied to produce compounds whose degree of reduction is higher than that of the substrate. On the other hand, producing compounds with a degree of reduction lower than that of the substrate will force the synthesis of other compounds with higher degrees of reduction to compensate for excess reducing power generated from substrate oxidation. These constraints may decrease the product yield substantially.The catabolic currency that balances the degree of reduction between the substrate and the products is usually NADH. In S. cerevisiae, NADH is produced in the cytosol by mainly glyceraldehyde-3-phosphate dehydrogenase and other assimilatory reaction enzymes (35). In the mitochondria, NADH is formed in the tricarboxylic acid (TCA) cycle and the reaction of the pyruvate dehydrogenase complex. Cytosolic NADH is oxidized by the glycerol-3-phosphate shuttle or the external cytosolic NADH dehydrogenases, which are part of the electron transp...

“…The working volume was kept at 1 liter by a Masterflex peristaltic effluent pump (Barrington, IL) coupled to an electric level sensor. The pH was kept at 5.0 by using an Applikon ADI 1030 biocontroller via the automatic addition of 2.0 M potassium hydroxide (15). Cultures were sparged with air at a flow rate of 0.5 liter min Ϫ1 using a Brooks model 5876 mass flow controller.…”

Section: Methodsmentioning

confidence: 99%

“…Biomass dry mass determination, substrate and metabolite analyses, off-gas analysis, preparation of cell extract, and determination of chemostat culture protein samples were performed as described previously (15). Formate concentrations were confirmed by a colorimetric method (24).…”

Section: Vol 74 2008 Engineered S Cerevisiae Coconsuming Formaldehmentioning

confidence: 99%

Engineering and Analysis of a Saccharomyces cerevisiae Strain That Uses Formaldehyde as an Auxiliary Substrate

Baerends

Hulster

Geertman

et al. 2008

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We demonstrated that formaldehyde can be efficiently coutilized by an engineered Saccharomyces cerevisiae strain that expresses Hansenula polymorpha genes encoding formaldehyde dehydrogenase (FLD1) and formate dehydrogenase (FMD), in contrast to wild-type strains. Initial chemostat experiments showed that the engineered strain coutilized formaldehyde with glucose, but these mixed-substrate cultures failed to reach steadystate conditions and did not exhibit an increased biomass yield on glucose. Subsequent transcriptome analyses of chemostat cultures of the engineered strain, grown on glucose-formaldehyde mixtures, indicated that the presence of formaldehyde in the feed caused biotin limitations. Further transcriptome analysis demonstrated that this biotin inactivation was prevented by using separate formaldehyde and vitamin feeds. Using this approach, steady-state glucose-limited chemostat cultures were obtained that coutilized glucose and formaldehyde. Coutilization of formaldehyde under these conditions resulted in an enhanced biomass yield of the glucose-limited cultures. The biomass yield was quantitatively consistent with the use of formaldehyde as an auxiliary substrate that generates NADH and subsequently, via oxidative phosphorylation, ATP. On an electron pair basis, the biomass yield increase observed with formaldehyde was larger than that observed previously for formate, which is tentatively explained by different modes of formate and formaldehyde transport in S. cerevisiae.In industrial biotechnology, the carbon feedstock is usually a sugar or sugar-rich substrate such as molasses, which often represents an important cost factor. In many processes, a large portion of the sugar feed is not converted into product but is instead dissimilated to provide free energy (e.g., in the form of ATP equivalents or proton motive force) and/or reducing power [in the form of NAD(P)H]. The use of mixed substrates consisting of a sugar and an additional, inexpensive source of reducing power and/or free energy represents an interesting approach to reduce the costs of industrial fermentation. Ideally, this practice should lead to a situation in which the sugar is used exclusively as a carbon source for biomass and/or product formation. The validity of this "auxiliary substrate" concept has been demonstrated in many academic studies using compounds such as formate or thiosulfate, whose oxidation can provide free energy but which cannot be used as carbon sources (2,7,26).Methanol is a low-cost chemical which can be derived from fossil sources or from biomass (via syngas, a CO-and H 2 -containing gas mixture generated by gasification of organic matter) (9, 17) and is an interesting candidate for the role of "auxiliary substrate" in industrial biotechnology. However, only a few industrial microorganisms are capable of utilizing methanol, including several yeast species that can grow on methanol as the sole carbon and energy source (35,42,46). In these methylotrophic yeasts, like Hansenula polymorpha and Pichia pastoris, meth...