Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe

Abstract

BackgroundEconomical production of value-added chemicals from renewable biomass is a promising path to sustainability. 3-Hydroxypropionic acid (3-HP) is an important chemical for building a bio-sustainable society. Establishment of 3-HP production from renewable resources such as glucose would provide a bio-sustainable alternative to the production of acrylic acid from fossil resources.ResultsHere, we describe metabolic engineering of the fission yeast Schizosaccharomyces pombe to enhance 3-HP production from glucose and cellobiose via the malonyl-CoA pathway. The mcr gene, encoding the malonyl-CoA reductase of Chloroflexus aurantiacus, was dissected into two functionally distinct fragments, and the activities of the encoded protein were balanced. To increase the cellular supply of malonyl-CoA and acetyl-CoA, we introduced genes encoding endogenous aldehyde dehydrogenase, acetyl-CoA synthase from Salmonella enterica, and endogenous pantothenate kinase. The resulting strain produced 3-HP at 1.0 g/L from a culture starting at a glucose concentration of 50 g/L. We also engineered the sugar supply by displaying beta-glucosidase (BGL) on the yeast cell surface. When grown on 50 g/L cellobiose, the beta-glucosidase-displaying strain consumed cellobiose efficiently and produced 3-HP at 3.5 g/L. Under fed-batch conditions starting from cellobiose, this strain produced 3-HP at up to 11.4 g/L, corresponding to a yield of 11.2% (g-3-HP/g-glucose; given that 1 g cellobiose corresponds to 1.1 g glucose upon digestion).ConclusionsIn this study, we constructed a series of S. pombe strains that produced 3-HP via the malonyl-CoA pathway. Our study also demonstrated that BGL display using cellobiose and/or cello-oligosaccharides as a carbon source has the potential to improve the titer and yield of malonyl-CoA- and acetyl-CoA-derived compounds.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1025-5) contains supplementary material, which is available to authorized users.

Benchmarking recombinant Pichiapastoris for 3‐hydroxypropionic acid production from glycerol

“…Such 3‐HP titre is considerably higher than those achieved in S . pombe (Takayama et al ., 2018) and S. cerevisiae (Chen et al ., 2014) harbouring a similar genetic construction and using glucose as a substrate (0.016 g l −1 and 0.093 g l −1 respectively). Furthermore, the C‐yield (Cmol of 3‐HP per Cmol of substrate, i.e.…”

“…The same outcome has been demonstrated in S . pombe , where the expression of the N‐terminal and the improved version of the C‐terminal domain of MCR using two independent expression cassettes led to a 30‐fold improvement in the final 3‐HP titre, compared to the starting strain expressing the original sequence of MCR Ca (Takayama et al ., 2018). Therefore, we introduced two expression cassettes into P. pastoris allowing the independent expression of the coding DNA sequences for the N‐terminal and the C‐terminal domains of MCR (including the wild‐type and the mutated version of the latter) under the control of the GAP promoter.…”

“…The overexpression of such enzyme has already been performed in E. coli (Rathnasingh et al ., 2012), S. cerevisiae (Chen et al ., 2014; Kildegaard et al ., 2016) and S . pombe (Takayama et al ., 2018) to increase the production of 3‐HP. The enzyme cPos5 produces NADPH by means of NADH and ATP consumption, and the overexpression of its gene leads to an increase in the NADPH/NADP ratio (Tomàs‐Gamisans et al ., 2020).…”

Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus

“…Production of 3HP in fungi has been explored in a number of species including Pichia pastoris ( Zhou et al, 2011 ), Schizosaccharomyces pombe ( Takayama et al, 2018 ), and via a variety of pathways in S. cerevisiae ( Borodina et al, 2015 ; Kildegaard et al, 2016 ). However, to our knowledge 3HP production has not been explored in filamentous fungi.…”

A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae

“…CRISPR/Cas9 gene editing is extensively used in metabolic pathway engineering in Saccharomyces cerevisiae and other yeasts, which is particularly relevant to industrial applications (Bracher et al, ; Löbs, Schwartz, Thorwall, & Wheeldon, ; Takayama et al, ). In addition to rationale design, industrial strain improvement is often achieved by directed evolution experiments (reviewed in Steensels et al, ).…”