Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

Methanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacterium Corynebacterium glutamicum toward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase from Bacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway of Bacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinant C. glutamicum strain showed an average methanol consumption rate of 1.7 ؎ 0.3 mM/h (mean ؎ standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13 C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m ؉ 1 mass isotopomers of various intracellular metabolites. In the background of a C. glutamicum ⌬ald ⌬adhE mutant being strongly impaired in its ability to oxidize formaldehyde to CO 2 , the m ؉ 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineered C. glutamicum strains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

Section: Discussionmentioning

confidence: 62%

Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

Witthoff

Schmitz

Niedenführ

et al. 2015

“…DM, dry mass. (5,20). The effect of the zwf1 deletion alone on anaerobic D-xylose fermentation in S. cerevisiae was previously described by Jeppsson et al (8).…”

Section: Discussionmentioning

confidence: 95%

Engineering Redox Cofactor Regeneration for Improved Pentose Fermentation in Saccharomyces cerevisiae

Verho

Londesborough

Penttilä

et al. 2003

193

127

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD ؉ , which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP ؉ -dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO 2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO 2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO 2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD ؉ . The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO 2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.

“…Since part of the glucose has to be used for the generation of ATP equivalents in dissimilation, it can be called an energy-deficient substrate (4). Due to this intrinsic free energy deficiency, experimentally obtained biomass yields on glucose are generally lower than the maximum biomass yield that could theoretically be reached when assimilatory reactions are fully optimized (5,9).…”

mentioning

confidence: 99%

“…Auxiliary substrates are compounds that can be dissimilated to provide free energy requirements but cannot be used as a carbon source for growth. An increase of the carbon source-to-biomass conversion efficiency has been demonstrated for many combinations of carbon sources and auxiliary substrates, including acetate-thiosulfate (14), acetate-formate (11), and glucose-formate (9,13,16). Formate is a suitable model auxiliary energy substrate for yeasts and fungi, as many of these organisms contain an NAD ϩ -linked formate dehydrogenase (EC 1.2.1.1; FDH) that catalyzes the oxidation of formate to CO 2 but are unable to assimilate formate (3,4,10,16,28,33,38).…”

mentioning

confidence: 99%

“…The stoichiometry of this process (P/O ratio) depends on the subcellular localization of FDH. In most eukaryotes, FDH appears to be a cytosolic enzyme (9,28), with the notable exception of plants, where it is located in the mitochondria and chloroplasts (18,25,26). At saturating rates of formate consumption, glucose-limited growth should switch from an energy-limited to an energy-excess or carbon-limited situation.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Self Cite

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...