Abstract
Carbon-conserving pathways have the potential of increasing product yields in biotechnological processes. The aim of this project was to investigate the functionality of a novel carbon-conserving pathway that produces 3 mol of acetyl-CoA from fructose-6-phosphate without carbon loss in the yeast Saccharomyces cerevisiae. This cyclic pathway relies on a generalist phosphoketolase (Xfspk), which can convert xylulose-5-phosphate, fructose-6-phosphate and sedoheptulose-7-phosphate (S7P) to acetyl phosphate. This cycle is proposed to overcome bottlenecks from the previously reported non-oxidative glycolysis (NOG) cycle. Here, in silico simulations showed accumulation of S7P in the NOG cycle, which was resolved by blocking the non-oxidative pentose phosphate pathway and introducing Xfspk and part of the riboneogenesis pathway. To implement this, a transketolase and transaldolase deficient S. cerevisiae was generated and a cyclic pathway, the Glycolysis AlTernative High Carbon Yield Cycle (GATHCYC), was enabled through xfspk expression and sedoheptulose bisphosphatase (SHB17) overexpression. Flux through the GATHCYC was demonstrated in vitro with a phosphoketolase assay on crude cell free extracts, and in vivo by constructing a strain that was dependent on a functional pathway to survive. Finally, we showed that introducing the GATHCYC as a carbon-conserving route for 3-hydroxypropionic acid (3-HP) production resulted in a 109% increase in 3-HP titers when the glucose was exhausted compared to the phosphoketolase route only.
Highlights
The native routes for carbon conversion in microorganisms have been optimized through evolution, but not always in a way that is optimal for bioproduction of valuable products
The kinetic parameters were extracted from published data (Messiha et al, 2014; Smallbone et al, 2013; Teusink et al, 2000) and from data deposited on the Biomodels website
The steady state simulation of non-oxidative glycolysis (NOG) on fructose with a specific (Fig. S1) or generalist phosphoketolase (Fig. 2A) shows accumulation of intermediates, mostly S7P, that in vivo would normally block the use of the NOG pathway
Summary
The native routes for carbon conversion in microorganisms have been optimized through evolution, but not always in a way that is optimal for bioproduction of valuable products. In metabolic engineer ing, the metabolic network of a cell is optimized to increase the fluxes towards the product of interest, with the goal of improving the Titer, Rate and Yield (TRY) metrices (Nielsen and Keasling, 2016). The yeast Saccharomyces cerevisiae is an attractive industrial production host due to the robustness of the organism, ease of genetic manipulation and a vast knowledgebase being available (Nielsen and Jewett, 2008). S. cerevisiae is a Crabtree positive yeast (De Deken, 1966), which means that it will exhibit fermentative growth when glucose is in excess, even in aerobic conditions, with ethanol as the main product. When the glucose is consumed (marking the end of the glucose phase), the diauxic shift occurs, where S. cerevisiae reprograms its metabolism from fermentative growth to respiratory growth on ethanol. The growth on the produced ethanol is usually referred to as the ethanol phase
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