Combined engineering of disaccharide transport and phosphorolysis for enhanced ATP yield from sucrose fermentation in Saccharomyces cerevisiae

Highlights

• Functional replacement of native S. cerevisiae sucrose hydrolysis and uptake.

• Replacement of sucrose hydrolysis by phosphorolysis increased biomass yield by 31%.

• Replacement of sucrose/H⁺-symport (Mal11) by PvSUF1 further increased yield by 8%.

• Overexpression of PGM2 increased the growth rate of LmSPase expressing strains.

Abstract

Anaerobic industrial fermentation processes do not require aeration and intensive mixing and the accompanying cost savings are beneficial for production of chemicals and fuels. However, the free-energy conservation of fermentative pathways is often insufficient for the production and export of the desired compounds and/or for cellular growth and maintenance. To increase free-energy conservation during fermentation of the industrially relevant disaccharide sucrose by Saccharomyces cerevisiae, we first replaced the native yeast α-glucosidases by an intracellular sucrose phosphorylase from Leuconostoc mesenteroides (LmSPase). Subsequently, we replaced the native proton-coupled sucrose uptake system by a putative sucrose facilitator from Phaseolus vulgaris (PvSUF1). The resulting strains grew anaerobically on sucrose at specific growth rates of 0.09 ± 0.02 h⁻¹ (LmSPase) and 0.06 ± 0.01 h⁻¹ (PvSUF1, LmSPase). Overexpression of the yeast PGM2 gene, which encodes phosphoglucomutase, increased anaerobic growth rates on sucrose of these strains to 0.23 ± 0.01 h⁻¹ and 0.08 ± 0.00 h⁻¹, respectively. Determination of the biomass yield in anaerobic sucrose-limited chemostat cultures was used to assess the free-energy conservation of the engineered strains. Replacement of intracellular hydrolase with a phosphorylase increased the biomass yield on sucrose by 31%. Additional replacement of the native proton-coupled sucrose uptake system by PvSUF1 increased the anaerobic biomass yield by a further 8%, resulting in an overall increase of 41%. By experimentally demonstrating an energetic benefit of the combined engineering of disaccharide uptake and cleavage, this study represents a first step towards anaerobic production of compounds whose metabolic pathways currently do not conserve sufficient free-energy.

Introduction

Microbial conversion of sugars from renewable feedstocks into chemicals and fuels offers a sustainable alternative to conventional petroleum-based production processes (Nielsen et al., 2013). In microbial processes for production of commodity chemicals, the cost of the sugar substrate can be up to 70% of the variable cost price. This impact of substrate costs on process economics necessitates high yield of product on substrate (Borodina and Nielsen, 2014, De Kok et al., 2012). The efficiency of free-energy conservation in central metabolism, expressed as conversion of ADP and phosphate to ATP, has a big impact on the product yield. For products whose synthesis from sugar requires a net input of ATP and therefore are produced in aerobic bioreactors, an increased efficiency of energy conservation would imply that less substrate has to be respired to provide the ATP required for product formation. As a result, more substrate carbon can be channelled towards the desired product. Additionally, the product yield on oxygen increases, which improves volumetric productivity (often limited by oxygen transfer (Meadows et al., 2016)) and/or decreases the cost of aeration and cooling (Luong and Volesky, 1980). Where possible, anaerobic conversion of sugars into fuels and chemicals would be even more beneficial (Cueto-Rojas et al., 2015, De Kok et al., 2012, Weusthuis et al., 2011).

Although many conversions of sugars into industrially relevant products are feasible based on thermodynamics and mass conservation, ATP formation by substrate-level phosphorylation in central metabolism can be insufficient to provide the energy required for product-formation pathways, product export, cellular growth and/or maintenance (Cueto-Rojas et al., 2015, De Kok et al., 2012). For example, in the conversion of glucose into lactic acid by Saccharomyces cerevisiae, all ATP formed by substrate-level phosphorylation in glycolysis is required for export of product (Abbott et al., 2009a, Abbott et al., 2009b, Van Maris et al., 2004). In this specific example, increased free-energy (ATP) conservation could enable homofermentative, anaerobic lactate production.

A negative Gibbs free-energy change for the conversion of substrate into product can either be conserved in the form of ATP, or used to drive the reaction. Therefore, a trade-off often exists between high energetic efficiency and high reaction rates (Pfeiffer et al., 2001). In nature, competition for resources is often more important than optimal free-energy conservation. Consequently, microbial evolution has in many cases yielded pathways with high turnover rates that facilitate fast substrate utilization at the expense of energetic efficiency (Pfeiffer et al., 2001). This evolutionary trade-off between yield and rate creates metabolic engineering opportunities for increasing free-energy conservation in industrial microorganisms.

The conversion of cheap and abundant substrates such as the disaccharide sucrose, which is mainly derived from sugar cane and sugar beet, is especially interesting for industrial applications (Marques et al., 2015). The yeast S. cerevisiae is very well suited for large-scale industrial fermentation processes due to its robustness and tolerance towards industrial conditions (Abbott et al., 2009a, Abbott et al., 2009b, Hong and Nielsen, 2012). S. cerevisiae can metabolize sucrose in two ways: extracellular hydrolysis followed by facilitated diffusion of the monosaccharides glucose and fructose (Fig. 1A) or uptake of sucrose by a proton-symport mechanism followed by intracellular hydrolysis (Fig. 1B) (Batista et al., 2005, Santos et al., 1982, Stambuk et al., 2000).

S. cerevisiae does not conserve the free energy of sucrose hydrolysis (∆G₀′ = −29 kJ/mol). In some anaerobic microorganisms sucrose is cleaved by phosphorolysis instead of hydrolysis. In the latter cleavage process, sucrose phosphorylase (SPase) converts inorganic phosphate and sucrose into glucose-1-phosphate and fructose. Glucose-1-phosphate can subsequently be converted into glucose-6-phosphate by phosphoglucomutase. As this phosphorolytic cleavage circumvents the ATP-requiring hexokinase reaction, it enables higher overall free-energy conservation than sucrose hydrolysis (Fig. 1C). Genes encoding SPase are known from various bacterial species (Kawasaki et al., 1996). Other disaccharide phosphorylases, such as maltose- and cellobiose phosphorylase, have previously been functionally expressed in S. cerevisiae (De Kok et al., 2011, Sadie et al., 2011).

While intracellular phosphorolysis theoretically enables a higher free-energy conservation (gain of 1 ATP per sucrose molecule), it requires transport of extracellular sucrose to the cytosol. However, in wild-type S. cerevisiae, uptake of sucrose via a proton-symporter (e.g. Mal11 (Stambuk et al., 1999)) and subsequent export of the proton via the H⁺-ATPase results in a net expense of 1 ATP (Weusthuis et al., 1993). Therefore, an improved free-energy conservation can be achieved when the proton-symport system is replaced by transport via facilitated diffusion (SUF, Fig. 1D). Sucrose transporters from Phaseolus vulgaris and Pisum sativum have been functionally expressed in S. cerevisiae and were described as probable sucrose facilitators (SUFs) (Zhou et al., 2007). Additionally, sucrose transporters from the SWEET family, e.g. from Arabidopsis thaliana and Oryza sativa, have also been proposed to catalyse facilitated diffusion (Chen et al., 2012, Chen et al., 2010, Lin et al., 2014).

The goal of this study was to explore whether free-energy conservation from sucrose fermentation by S. cerevisiae can be improved by replacing the first two steps of the native sucrose metabolism by facilitated uptake of the disaccharide and subsequent phosphorolytic cleavage. A previously constructed S. cerevisiae strain lacking all native sucrose proton-symporters and hydrolases, which remained sucrose-negative upon strong selective pressures (Marques et al., 2017), was used as a platform to avoid interference by native sucrose metabolising enzymes. For the phosphorolytic cleavage reaction, SPase from Leuconostoc mesenteroides ATTC 12291 was chosen in view of the compatibility of its temperature and pH optima with expression in yeast (Aerts et al., 2011, Goedl et al., 2010, Goedl et al., 2007, Kawasaki et al., 1996, Lee et al., 2008). Several proposed sucrose facilitators from plant origins were screened for their ability to support growth of the platform strain on sucrose: Phaseolus vulgaris SUF1 (PvSUF1), Pisum sativum SUF1 and SUF4 (PsSUF1 and PsSUF4), Arabidopsis thaliana SWEET12 (AtSWEET12) and Oryza sativa SWEET11 (OsSWEET11). The impact of these modifications on free-energy conservation was studied by analysis of biomass yields of engineered S. cerevisiae strains in anaerobic, sucrose-limited chemostat cultures.