Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway

Abstract

BackgroundIn the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs.ResultsHere we describe the metabolic engineering of baker’s yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella entericaSEacsL641P. Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L−1 3HP with a yield of 13 % C-mol C-mol−1 glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by 13C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed.ConclusionsIn this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0451-5) contains supplementary material, which is available to authorized users.

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

“…As discussed above, the fatty alcohol pathway produces excess NADH and requires additional NADPH. To remediate this redox cofactor imbalance in our fatty alcohol pathway, first we considered replacing native NADH-producing GapDH with an NADPH-producing variant as has been pursued for other bioproducts Kildegaard et al, 2016;Zhang et al, 2011). From our proteomics results, we found that of the three GapDH isoenzymes in yeast, Tdh3p was the most highly expressed.…”

The sole introduction of two single-point mutations establishes glycerol utilization in Saccharomyces cerevisiae CEN.PK derivatives

“…In particular, strains of the CEN.PK family have become a popular platform in both fundamental research and industrial applications as they offer a good compromise between physiological properties (e.g., the growth characteristics in shake-flask cultures) and genetic properties (e.g., transformation efficiency) [7]. CEN.PK strains have been applied in numerous metabolic and evolutionary engineering studies, such as for the production of lactate and pyruvate [8, 9], isoprenoids [10, 11], C 4 -dicarboxylic acids [12], ornithine [13], n-butanol [14], and 3-hydroxypropionic acid [15], and for the fermentation of pentose sugars [16–18]. Although some of these processes could profit from utilizing glycerol as a carbon source due to its high reducing power, wild-type strains of the CEN.PK family cannot utilize glycerol at all in synthetic medium, particularly when complex medium supplements such as yeast extract or peptone are omitted [19, 20].…”

Biological Production of 3-Hydroxypropionic Acid: An Update on the Current Status

“…Fed-batch bioreactor 1 L, 0.5 L, pH 5, 30 • C, 2 vvm, 800 rpm 9.8 0.1 [59] yhxA from B. cereus, ydfG from E. coli, panD from T. castaneum, overexpression of aat2, pyc1, and pyc2…”

“…Another positive characteristic of this yeast is its high tolerance of low pH values, which can result from accumulation of 3HP [58,59]. To permit conversion of glucose to 3HP, the malonyl-CoA pathway was also constructed in S. cerevisiae cells [59].…”