Effect of nitrogen availability on the poly-3-d-hydroxybutyrate accumulation by engineered Saccharomyces cerevisiae

Abstract

Poly-3-d-hydroxybutyrate (or PHB) is a polyester which can be used in the production of biodegradable plastics from renewable resources. It is naturally produced by several bacteria as a response to nutrient starvation in the excess of a carbon source. The yeast Saccharomyces cerevisiae could be an alternative production host as it offers good inhibitor tolerance towards weak acids and phenolic compounds and does not depolymerize the produced PHB. As nitrogen limitation is known to boost the accumulation of PHB in bacteria, the present study aimed at investigating the effect of nitrogen availability on PHB accumulation in two recombinant S. cerevisiae strains harboring different xylose consuming and PHB producing pathways: TMB4443 expressing an NADPH-dependent acetoacetyl-CoA reductase and a wild-type S. stipitis XR with preferential use of NADPH and TMB4425 which expresses an NADH-dependent acetoacetyl-CoA reductase and a mutated XR with a balanced affinity for NADPH/NADH. TMB4443 accumulated most PHB under aerobic conditions and with glucose as sole carbon source, whereas the highest PHB concentrations were obtained with TMB4425 under anaerobic conditions and xylose as carbon source. In both cases, the highest PHB contents were obtained with high availability of nitrogen. The major impact of nitrogen availability was observed in TMB4425, where a 2.7-fold increase in PHB content was obtained. In contrast to what was observed in natural PHB-producing bacteria, nitrogen deficiency did not improve PHB accumulation in S. cerevisiae. Instead the excess available carbon from xylose was shunted into glycogen, indicating a significant gluconeogenic activity on xylose.

Engineering NADH/NAD+ ratio in Halomonas bluephagenesis for enhanced production of polyhydroxyalkanoates (PHA)

“…The limitations on essential nutrients (such as N, P, S, Fe and Mg) and presence of sufficient carbon source are critical for most known NADPH dependent species to accumulate PHA (Steinbüchel, 1991). While oxygen limitation has been proven functional for inducing PHA synthesis only in A. beijerinckii and recombinant Saccharomyces cerevisiae harboring phaCAB genes from A. vinosum, as both contain NADH consumable PhaB (Portugal-Nunes et al, 2017;de las Heras et al, 2016;Jackson and Dawes, 1976;Senior and Dawes, 1971;Senior and Dawes, 1973).…”

Growth kinetics, fatty acid composition and metabolic activity changes of Crypthecodinium cohnii under different nitrogen source and concentration

“…In resent researches, the effect of N has been positively exploited in Monoraphidium sp. (Dhup and Dhawan 2014 ), Scenedesmus abundans (González-Garcinuño et al 2014 ), Saccharomyces cerevisiae (Portugal-Nunes et al 2017 ), S. rubescens (Lin and Lin 2011 ) and Stigeoclonium sp. (Liu et al 2016a , b ) for the production of commercially important lipids.…”

Current Challenges and Opportunities in Non-native Chemical Production by Engineered Yeasts

“…To strengthen the availability of cytosolic acetyl-CoA, acetate, an essential precursor for PHB production, was fed as a sole carbon source, resulting in a PHB content of 10.2% with a high titer of 7.35 g/L during fed-batch fermentation in a bioreactor ( Li et al, 2017 ). In S. cerevisiae , high PHB content was achieved during xylose fermentation ( Portugal-Nunes et al, 2017 ). An engineered xylose-utilizing strain of S. cerevisiae with a heterologously expressed PHB pathway based on PhaA and PhaC1 from C. necator and NADH-preferred PhaB1 from Allochromatium vinosum ( Figure 5A ) produced 730 mg/L PHB with a content of 16.4% during xylose fermentation with additional nitrogen supply under an anaerobic shake-flask culture condition ( Portugal-Nunes et al, 2017 ).…”

“…In S. cerevisiae , high PHB content was achieved during xylose fermentation ( Portugal-Nunes et al, 2017 ). An engineered xylose-utilizing strain of S. cerevisiae with a heterologously expressed PHB pathway based on PhaA and PhaC1 from C. necator and NADH-preferred PhaB1 from Allochromatium vinosum ( Figure 5A ) produced 730 mg/L PHB with a content of 16.4% during xylose fermentation with additional nitrogen supply under an anaerobic shake-flask culture condition ( Portugal-Nunes et al, 2017 ). PHB production in E. coli has been shown to be more promising than that in yeast strains.…”

“…Therefore, the construction of a non-native chemical production pathway involving numerous heterologous genes would facilitate the development of yeast cell factories. Interestingly, unconventional carbon sources other than glucose, such as xylose, acetate, and propionic acid, have been shown to better support metabolic flux through the biosynthesis pathway for non-native chemicals ( Wu and San, 2014 ; Li et al, 2017 ; Portugal-Nunes et al, 2017 ; Wu et al, 2019 ). Thus, non-native chemical production by the engineered yeast strains with non-native carbon metabolism could offer better promises for industrial-scale production of non-native chemicals.…”