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.

Enhancement of l-lactic acid production via synergism in open co-fermentation of Sophora flavescens residues and food waste.

In this study, Sophora flavescens residues (SFR) were used for l-lactic acid production and were mixed with food waste (FW) to assess the effects of different compositions of SFR and FW. Positive synergistic effects of mixed substrates were achieved with co-fermentation. Co-fermentation increased the proportion of l-lactic acid by decreasing the co-products of ethanol and other organic acids. A maximum l-lactic acid concentration of 48.4g/L and l-lactic acid conversion rate of 0.904g/g total sugar were obtained through co-fermentation of SFR and FW at the optimal ratio of 1:1.5. These results were approximately 6-fold those obtained during mono-fermentation of SFR. Co-fermentation of SFR and FW provides a suitable C/N ratio and pH for effective open fermentative production of l-lactic acid.

Novel pH control strategy for efficient production of optically active l-lactic acid from kitchen refuse using a mixed culture system.

Uninvestigated control factors of meta-fermentation, the fermentative production of pure chemicals and fuels in a mixed culture system, were examined for production of optically pure l-lactic acid (LA) from food waste. In meta-fermentations by pH swing control, l-LA production with 100% optical purity (OPl-LA) was achieved even using unsterilized model kitchen refuse medium with preferential proliferation of l-LA-producing Bacillus coagulans, a minor member in the seed, whereas agitation decreased OPl-LA drastically. pH constant control shortened the fermentation time but decreased OPl-LA and LA selectivity (SLA) by stimulating growth of heterofermentative Bacillus thermoamylovorans. Deliberately switching from pH swing control to constant control exhibited the best performance for l-LA production: maximum accumulation, 39.2gL(-1); OPl-LA, 100%; SLA, 96.6%; productivity, 1.09gL(-1)h(-1). These results present a novel pH control strategy for efficient l-LA production in meta-fermentation based on a concept different from that of pure culture systems.

Fed-batch fermentation for enhanced lactic acid production from glucose/xylose mixture without carbon catabolite repression.

There has been tremendous growth in the production of optically pure l-lactic acid from lignocellulose-derived sugars. In this study, Enterococcus mundtii QU 25 was used to ferment a glucose/xylose mixture to l-lactic acid. Maintenance of the xylose concentration at greater than 10 g/L achieved homo-lactic acid fermentation and reduced the formation of byproducts. Furthermore, carbon catabolite repression (CCR) was avoided by maintaining the glucose concentration below 25 g/L; therefore, initial concentrations of 25 g/L glucose and 50 g/L xylose were selected. Supplementation with 5 g/L yeast extract enhanced the maximum xylose consumption rate and consequently increased lactic acid production and productivity. Finally, a 129 g/L lactic acid without byproducts was obtained with a maximum lactic acid productivity of 5.60 g/(L·h) in fed-batch fermentation with feeding a glucose/xylose mixture using ammonium hydroxide as the neutralizing agent. These results indicate a potential for lactic acid production from glucose and xylose as the main components of lignocellulosic biomasses.

Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803.

BACKGROUND: Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by "designer microbes." The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make "designer microbes" includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made. RESULTS: To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme's affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization. CONCLUSION: An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH.

L-lactic acid production by Bacillus subtilis MUR1 in continuous culture.

A novel UV-induced mutant strain of recombinant Bacillus subtilis MUR1 was used for the production of L-LA in continuous cultures with a variety of culture conditions. The maximal productivity of 17.6g/L/h was obtained with a L-LA concentration of 44.1g/L at the dilution rate of 0.4h(-1). The highest concentration of L-LA (77.1g/L) was produced at the dilution rate of 0.05 h(-1). This study showed that the maximum L-LA productivity of B. subtilis MUR1 which can only last for a very short period of time during the exponential phase in fed-batch cultures, can be extended indefinitely at steady state in continuous cultures. L-LA production increased with the increase of yeast extract concentrations in the medium. Moreover, temperature, agitation rate and various glucose concentrations in the feed were compared in continuous cultures. Different nitrogen sources (lysine, glutamine, ammonium sulphate and corn steep liquor) were studied to partly or completely replace yeast extract in the medium, most of them showed positive effects on L-LA production and cell growth. The L-LA productivities from continuous cultures in this study are higher than the productivity of current microbial industrial processes which use Lactobacillus to produce L-LA.

The Mutagenesis of Lactobacillus Thermophilus for Enhanced L-(+)-Lactic Acid Accumulation Induced by Heavy Ion Irradiation

ABSTRACT Screening promising L. thermophiles with high productivity, high efficiency and strong adaptability are very important in lactic acid industry. For this purpose, 80MeV/u carbon ions were applied to irradiate L. thermophiles. After high-throughput screening, a mutant, named SRZ50, was obtained. Different carbon sources or nitrogen sources were provided to investigate carbon or nitrogen source utilization between mutant SRZ50 and wild type, and different fermentation periods were also chose to study fermentation characteristic between mutant SRZ50 and wild type. The results showed that mutant SRZ50 exhibited the enhanced L-(+)-lactic acid production from wild type. When glucose or fructose was the sole carbon source, the L(+)-lactic acid production by mutant SRZ50 was both the highest, respectively, 23.16 ± 0.72 g/L or 23.24 ± 0.66 g/L, which had a significant increase from that of wild type (P<0.01), following obvious increase in biomass (P<0.05). When yeast powder was the sole nitrogen source, it can promote mutant SRZ50 to accumulate the highest L-(+)-lactic acid accumulation, which also had a significant increase from that of wild type (P<0.01). Under different fermentation periods, it was obtained that mutant SRZ50 all exhibited significant increase in L-(+)-lactic acid accumulation from wild type. In conclusion, a mutant strain with improved production profiles for L-(+)-lactic acid, was obtained, indicating that heavy ions can be an efficient tool to improve metabolic product accumulations in microbes.