A continuous culture study of methanol and formate utilization by the yeast Pichia pastoris

Hazeu, W.; Donker, R. A.

doi:10.1007/bf00131280

Cited by 35 publications

(12 citation statements)

References 25 publications

(18 reference statements)

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“…While formate is a useful model substrate for laboratory experiments, it does not meet this cost requirement. For industrial applications, the oxidation of methanol (via a linear oxidation pathway involving NAD ϩ -dependent methanol, formaldehyde, and FDHs) would be much more interesting, as it can yield up to 3 mol NADH per mol methanol (16,32,43). Such an approach would require metabolic engineering, as NAD ϩ -linked methanol dehydrogenases have been characterized for gram-positive bacteria (2, 17) but not for fungi.…”

Section: Discussionmentioning

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).…”

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confidence: 99%

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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

Appl Environ Microbiol

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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...

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Section: Discussionmentioning

confidence: 99%

mentioning

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

Appl Environ Microbiol

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“…In aerobic cultures of yeasts, formate cannot serve as a carbon source, but it can provide redox equivalents for respiratory dissimilation (van Dijken et al, 1981;Hazeu & Donker, 1983;Babel et al, 1993). In aerobic cultures of yeasts, formate cannot serve as a carbon source, but it can provide redox equivalents for respiratory dissimilation (van Dijken et al, 1981;Hazeu & Donker, 1983;Babel et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

“…Co-utilization of formic acid and sugars by yeasts represents an interesting case of mixed substrate utilization. In aerobic cultures of yeasts, formate cannot serve as a carbon source, but it can provide redox equivalents for respiratory dissimilation (van Dijken et al, 1981;Hazeu & Donker, 1983;Babel et al, 1993). In S. cerevisiae CEN.PK113-7D, two isozymes of NAD 1 -linked formate dehydrogenase (FDH) (EC 1.2.1.2), encoded by the FDH1 and FDH2 genes, are induced on cultivation in the presence of formate (Overkamp et al, 2002b).…”

Section: Introductionmentioning

confidence: 99%

Engineering NADH metabolism inSaccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Geertman

Dijken

Pronk

2006

FEMS Yeast Research

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Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.

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“…pastoris Mut + strains result in higher productivity and faster accumulation of methanol oxidation by-products (formaldehyde and hydrogen peroxide) inside the cell (Brierley, Bussineau, Kosson, Melton, & Siegel, 1990;Chiruvolu et al, 1997;Hazeu & Donker, 1983). Excess of these by-products can lead to cellular stress and ultimately to cell death (Couderc & Baratti, 1980;Cregg & Madden, 1988;Kern et al, 2007; Van der Klei, Bystrykh, & Harder, 1990;Zhang, Bevins, Plantz, Smith, & Meagher, 2000).…”

Section: Discussionmentioning

confidence: 99%

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

Bredell

Smith

Görgens

et al. 2018

Yeast

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Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Mut ) and GS115 (Mut ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Mut were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Mut strains than in the Mut strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L culture while P. pastoris Mut only produced 23.61 mg L . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high-level producer of heterologous proteins.

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A continuous culture study of methanol and formate utilization by the yeast Pichia pastoris

Cited by 35 publications

References 25 publications

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

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

Engineering NADH metabolism inSaccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Expression of unique chimeric human papilloma virus type 16 (HPV‐16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha

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