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

BACKGROUND: Saccharomyces cerevisiae, engineered for L-lactic acid production from glucose and xylose, is a promising production host for lignocellulose-to-lactic acid processes. However, the two principal engineering strategies-pyruvate-to-lactic acid conversion with and without disruption of the competing pyruvate-to-ethanol pathway-have not yet resulted in strains that combine high lactic acid yields (YLA) and productivities (QLA) on both sugar substrates. Limitations seemingly arise from a dependency on the carbon source and the aeration conditions, but the underlying effects are poorly understood. We have recently presented two xylose-to-lactic acid converting strains, IBB14LA1 and IBB14LA1_5, which have the L-lactic acid dehydrogenase from Plasmodium falciparum (pfLDH) integrated at the pdc1 (pyruvate decarboxylase) locus. IBB14LA1_5 additionally has its pdc5 gene knocked out. In this study, the influence of carbon source and oxygen on YLA and QLA in IBB14LA1 and IBB14LA1_5 was investigated. RESULTS: In anaerobic fermentation IBB14LA1 showed a higher YLA on xylose (0.27 g g Xyl-1 ) than on glucose (0.18 g g Glc-1 ). The ethanol yields (YEtOH, 0.15 g g Xyl-1 and 0.32 g g Glc-1 ) followed an opposite trend. In IBB14LA1_5, the effect of the carbon source on YLA was less pronounced (~ 0.80 g g Xyl-1 , and 0.67 g g Glc-1 ). Supply of oxygen accelerated glucose conversions significantly in IBB14LA1 (QLA from 0.38 to 0.81 g L-1 h-1) and IBB14LA1_5 (QLA from 0.05 to 1.77 g L-1 h-1) at constant YLA (IBB14LA1 ~ 0.18 g g Glc-1 ; IBB14LA1_5 ~ 0.68 g g Glc-1 ). In aerobic xylose conversions, however, lactic acid production ceased completely in IBB14LA1 and decreased drastically in IBB14LA1_5 (YLA aerobic ≤ 0.25 g g Xyl-1 and anaerobic ~ 0.80 g g Xyl-1 ) at similar QLA (~ 0.04 g L-1 h-1). Switching from aerobic to microaerophilic conditions (pO2 ~ 2%) prevented lactic acid metabolization, observed for fully aerobic conditions, and increased QLA and YLA up to 0.11 g L-1 h-1 and 0.38 g g Xyl-1 , respectively. The pfLDH and PDC activities in IBB14LA1 were measured and shown to change drastically dependent on carbon source and oxygen. CONCLUSION: Evidence from conversion time courses together with results of activity measurements for pfLDH and PDC show that in IBB14LA1 the distribution of fluxes at the pyruvate branching point is carbon source and oxygen dependent. Comparison of the performance of strain IBB14LA1 and IBB14LA1_5 in conversions under different aeration conditions (aerobic, anaerobic, and microaerophilic) further suggest that xylose, unlike glucose, does not repress the respiratory response in both strains. This study proposes new genetic engineering targets for rendering genetically engineering S. cerevisiae better suited for lactic acid biorefineries.

Toward "homolactic" fermentation of glucose and xylose by engineered Saccharomyces cerevisiae harboring a kinetically efficient l-lactate dehydrogenase within pdc1-pdc5 deletion background.

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 (YLA ) 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 YLA (0.69 g/g glucose). In strain IBB14LA1, the YLA 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.