Development of an industrial yeast strain for efficient production of 2,3-butanediol

Abstract

As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation.

Improved 2,3-Butanediol Production Rate of Metabolically Engineered Saccharomyces cerevisiae by Deletion of RIM15 and Activation of Pyruvate Consumption Pathway

“…Moreover, the YHI030 strain produced 2,3-BDO with a yield of 0.41 g g −1 glucose in fed-batch cultivation using synthetic dextrose (SD) medium [13]. This level is similar to those of other strains reported previously in fed-batch cultivation using a rich medium (0.41-0.48 g g −1 glucose) [7,9]. First, the open reading frame (ORF) of RIM15 was deleted from YHI030 to construct the YMS101 strain.…”

“…Fed-batch 2 of 14 cultivation of the most advanced strains achieved 2,3-BDO production with very high titers [8,9]. However, the 2,3-BDO production rates of these engineered strains were less than one-tenth of the specific ethanol production rates of non-engineered S. cerevisiae [7,9,13].…”

“…The Ehrlich pathway converts pyruvate to ethanol via pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO were constructed by introducing an artificial threestep pathway from pyruvate to 2,3-BDO [6][7][8][9][10][11][12][13][14]. In addition, 2,3-BDO production is elevated by disrupting competing pathways and solving the cofactor imbalance [6][7][8][9][10][11][12][13][14][15][16][17].…”

“…Metabolically engineered S. cerevisiae strains that produce 2,3-BDO were constructed by introducing an artificial threestep pathway from pyruvate to 2,3-BDO [6][7][8][9][10][11][12][13][14]. In addition, 2,3-BDO production is elevated by disrupting competing pathways and solving the cofactor imbalance [6][7][8][9][10][11][12][13][14][15][16][17]. Fed-batch 2 of 14 cultivation of the most advanced strains achieved 2,3-BDO production with very high titers [8,9].…”

Inducible Synthetic Growth Regulation Using the ClpXP Proteasome Enhances cis,cis-Muconic Acid and Glycolic Acid Yields in Saccharomyces cerevisiae

“…For diverting sufficient resources to a synthetic pathway in order to reach industrially attractive production, the native pathways commonly need substantial optimization. , However, the synthetic pathways are usually in resource competition with cell growth or processes linked to that (e.g., growth-essential byproduct formation), and simply knocking out such competing native pathways is not feasible. Therefore, the resource share of desired compound production has been increased by reducing competing activities, − forcing biomass synthesis via alternative lower-yield routes, − or by using product sensors to control growth-essential gene expression . While shown to be successful in demonstration cases, the product sensor-driven control options are limited to available sensors, , and forcing growth to occur via alternative routes tends to require a high number of deletions .…”