Erin Garza

Homofermentative production of optically pure L-lactic acid from xylose by genetically engineered Escherichia coli B

BackgroundPolylactic acid (PLA), a biodegradable polymer, has the potential to replace (at least partially) traditional petroleum-based plastics, minimizing “white pollution”. However, cost-effective production of optically pure L-lactic acid is needed to achieve the full potential of PLA. Currently, starch-based glucose is used for L-lactic acid fermentation by lactic acid bacteria. Due to its competition with food resources, an alternative non-food substrate such as cellulosic biomass is needed for L-lactic acid fermentation. Nevertheless, the substrate (sugar stream) derived from cellulosic biomass contains significant amounts of xylose, which is unfermentable by most lactic acid bacteria. However, the microorganisms that do ferment xylose usually carry out heterolactic acid fermentation. As a result, an alternative strain should be developed for homofermentative production of optically pure L-lactic acid using cellulosic biomass.ResultsIn this study, an ethanologenic Escherichia coli strain, SZ470 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR ::pflBp6-acEF-lpd ΔmgsA), was reengineered for homofermentative production of L-lactic acid from xylose (1.2 mole xylose = > 2 mole L-lactic acid), by deleting the alcohol dehydrogenase gene (adhE) and integrating the L-lactate dehydrogenase gene (ldhL) of Pediococcus acidilactici. The resulting strain, WL203, was metabolically evolved further through serial transfers in screw-cap tubes containing xylose, resulting in the strain WL204 with improved anaerobic cell growth. When tested in 70 g L-1 xylose fermentation (complex medium), WL204 produced 62 g L-1 L-lactic acid, with a maximum production rate of 1.631 g L-1 h-1 and a yield of 97% based on xylose metabolized. HPLC analysis using a chiral column showed that an L-lactic acid optical purity of 99.5% was achieved by WL204.ConclusionsThese results demonstrated that WL204 has the potential for homofermentative production of L-lactic acid using cellulosic biomass derived substrates, which contain a significant amount of xylose.

Engineering and adaptive evolution of Escherichia coli W for L-lactic acid fermentation from molasses and corn steep liquor without additional nutrients.

The D-lactic acid producing strain, Escherichia coli HBUT-D, was reengineered for L(+)-lactic acid fermentation by replacing the D-lactate dehydrogenase gene (ldhA) with an L(+)-lactate dehydrogenase gene (ldhL) from Pedicoccus acidilactici, followed by adaptive evolution in sucrose. The resulting strain, WYZ-L, has enhanced expression of the sucrose operon (cscA and cscKB). In 100 g L(-1) of sucrose fermentation using mineral salt medium, WYZ-L produced 97 g L(-1) of l(+)-lactic acid, with a yield of 90%, a maximum productivity of 3.17 g L(-1)h(-1) and an optical purity of greater than 99%. In fermentations using sugarcane molasses and corn steep liquor without additional nutrients, WYZ-L produced 75 g L(-1) of l(+)-lactic acid, with a yield of 85%, a maximum productivity of 1.18 g L(-1)h(-1), and greater than 99% optical purity. These results demonstrated that WYZ-L has the potential to use waste molasses and corn steep liquor as a resource for L(+)-lactic acid fermentation.

Pilot scale demonstration of D-lactic acid fermentation facilitated by Ca(OH)2 using a metabolically engineered Escherichia coli.

In this study, a genetically engineered Escherichia coli strain, HBUT-D (ΔpflB Δpta ΔfrdABCD ΔadhE Δald ΔcscR), was initially evaluated on a laboratory scale (7 L) in a glucose (130 g L(-1)) mineral salts medium for d-lactic acid fermentation using 6N KOH, Ca(OH)2 or NH4OH as the neutralizing agent. Fermentations neutralized by Ca(OH) 2 achieved a volumetric productivity of 6.35 g L(-1) h(-1), tripling that achieved by KOH (1.71 g L(-1) h(-1)) and NH4OH (1.5 g L(-1) h(-1)). The facilitative effect of Ca(OH)2 neutralization was then demonstrated on a pilot scale (6 ton vessel, 130 kg glucose ton(-1)), resulting in a volumetric productivity of 6 kg ton(-1) h(-1), a titer of 126 kg ton(-1), a yield of 97%, and an optical purity of 99.5%. These results demonstrated that E. coli HBUT-D is a promising strain for large scale d-lactic acid fermentation using mineral salts medium and Ca(OH)2 for neutralization.