Toward “homolactic” fermentation of glucose and xylose by engineered Saccharomyces cerevisiae harboring a kinetically efficient l‐lactate dehydrogenase within pdc1‐pdc5 deletion background

Abstract

l-Lactic acid is an important platform chemical and its production from the lignocellulosic sugars glucose and xylose is, therefore, of high interest. Tolerance to low pH and a generally high robustness make Saccharomyces cerevisiae a promising host for l-lactic acid fermentation but strain development for effective utilization of both sugars is an unsolved problem. The herein used S. cerevisiae strain IBB10B05 incorporates a NADH-dependent pathway for oxidoreductive xylose assimilation within CEN.PK113-7D background and was additionally evolved for accelerated xylose-to-ethanol fermentation. Selecting the Plasmodium falciparum l-lactate dehydrogenase (pfLDH) for its high kinetic efficiency, strain IBB14LA1 was derived from IBB10B05 by placing the pfldh gene at the pdc1 locus under control of the pdc1 promotor. Strain IBB14LA1_5 additionally had the pdc5 gene disrupted. With both strains, continued l-lactic acid formation from glucose or xylose, each at 50 g/L, necessitated stabilization of pH. Using calcium carbonate (11 g/L), anaerobic shaken bottle fermentations at pH ≥ 5 resulted in l-lactic acid yields (Y ) of 0.67 g/g glucose and 0.80 g/g xylose for strain IBB14LA1_5. Only little xylitol was formed (≤0.08 g/g) and no ethanol. In pH stabilized aerobic conversions of glucose, strain IBB14LA1_5 further showed excellent l-lactic acid productivities (1.8 g/L/h) without losses in Y (0.69 g/g glucose). In strain IBB14LA1, the Y was lower (≤0.18 g/g glucose; ≤0.27 g/g xylose) due to ethanol as well as xylitol formation. Therefore, this study shows that a S. cerevisiae strain originally optimized for xylose-to-ethanol fermentation was useful to implement l-lactic acid production from glucose and xylose; and with the metabolic engineering strategy applied, advance toward homolactic fermentation of both sugars was made. Biotechnol. Bioeng. 2017;114: 163-171. © 2016 Wiley Periodicals, Inc.

l-Lactic acid production from glucose and xylose with engineered strains of Saccharomyces cerevisiae: aeration and carbon source influence yields and productivities

“…A dependency of both Y LA and lactic acid productivity (Q LA ) on oxygen supply by aeration was also noted in a recent study from the current authors, in which two strains of S. cerevisiae , IBB14LA1 and IBB14LA1_5, producing lactic acid from xylose were presented [ 20 ]. Both these strains are descendants of strain IBB10B05, which harbors a XR/XDH pathway engineered for redox-neutral assimilation of xylose and has furthermore undergone evolution for accelerated xylose-to-ethanol fermentation [ 28 , 29 ].…”

“…To channel the metabolic flux away from ethanol towards lactic acid production, further engineering strategies were applied. These included (a) the alleviation or disruption of the ethanol pathway by gene deletion of pdc1 , pdc5 , pdc6 , adh1 , or a combination thereof [ 9 , 10 , 12 , 16 ]; (b) the optimization of ldh gene expression [ 11 , 17 , 18 ], (c) the application of LDHs with high catalytic activities [ 19 , 20 ]; (d) the perturbation of the intracellular redox balance to increase the availability of NADH for the LDH-catalyzed reaction [ 18 ]; (e) the reduction of by-product formation by disruption of the glycerol pathway [ 21 ], and (f) the reduction of ATP consumption by replacing the native with an ATP-independent pathway for acetyl-CoA production from acetaldehyde [ 15 , 22 ]. Recent studies by us [ 20 ] and others [ 23 ] further showed that xylose-to-lactic acid conversion in S. cerevisiae is possible, representing an important step to advance lignocellulose-to-lactic acid processes.…”

“…Thus, homolactate fermentation on glucose was so far only achieved with PDC-deficient strains of S. cerevisiae [ 10 , 12 , 16 ]. These strains, however, showed severe distortion of biomass growth and cell viability [ 20 , 24 – 26 ]. Further, under anaerobic conditions, the preferred set-up for larger scale applications, only low productivities were obtained and these typically declined rapidly over fermentation time [ 2 , 10 , 20 , 25 ].…”

“…These strains, however, showed severe distortion of biomass growth and cell viability [ 20 , 24 – 26 ]. Further, under anaerobic conditions, the preferred set-up for larger scale applications, only low productivities were obtained and these typically declined rapidly over fermentation time [ 2 , 10 , 20 , 25 ]. Previous studies suggested that lactic acid production in recombinant S. cerevisiae does not yield net ATP, likely because the generated ATP is consumed largely to manage export of lactic acid out of the cell [ 25 , 27 ].…”

Harnessing the Periplasm of Bacterial Cells To Develop Biocatalysts for the Biosynthesis of Highly Pure Chemicals

“…S ynthetic biology and metabolic engineering approaches have been used successfully to produce a wide range of value-added products (1)(2)(3)(4)(5)(6)(7)(8). Most efforts in biosynthesis have focused on the establishment and optimization of metabolic fluxes toward the targeted product in the cytoplasm, before excretion or extraction (9)(10)(11)(12). The product line of available biocatalysts is limited by current strategies, especially when the targeted compound is structurally similar to other metabolites, which complicates the downstream processes for purification.…”