Development of ethanologenic bacteria.

The utilization of lignocellulosic biomass as a petroleum alternative faces many challenges. This work reviews recent progress in the engineering of Escherichia coli and Klebsiella oxytoca to produce ethanol from biomass with minimal nutritional supplementation. A combination of directed engineering and metabolic evolution has resulted in microbial biocatalysts that produce up to 45 g L(-1) ethanol in 48 h in a simple mineral salts medium, and convert various lignocellulosic materials to ethanol. Mutations contributing to ethanologenesis are discussed. The ethanologenic biocatalyst design approach was applied to other commodity chemicals, including optically pure D: (-)- and L: (+)-lactic acid, succinate and L: -alanine with similar success. This review also describes recent progress in growth medium development, the reduction of hemicellulose hydrolysate toxicity and reduction of the demand for fungal cellulases.

Low salt medium for lactate and ethanol production by recombinant Escherichia coli B.

Individual nutrient salts were experimentally varied to determine the minimum requirements for efficient L (+)-lactate production by recombinant strains of Escherichia coli B. Based on these results, AM1 medium was formulated with low levels of alkali metals (4.5 mM and total salts (4.2 g l(-1)). This medium was equally effective for ethanol production from xylose and lactate production from glucose with average productivities of 18-19 mmol l(-1) h(-1) for both (initial 48 h of fermentation).

Methylglyoxal bypass identified as source of chiral contamination in l(+) and d(-)-lactate fermentations by recombinant Escherichia coli.

Two new strains of Escherichia coli B were engineered for the production of lactate with no detectable chiral impurity. All chiral impurities were eliminated by deleting the synthase gene (msgA) that converts dihydroxyacetone-phosphate to methylglyoxal, a precursor for both L: (+)- and D: (-)-lactate. Strain TG113 contains only native genes and produced optically pure D: (-)-lactate. Strain TG108 contains the ldhL gene from Pediococcus acidilactici and produced only L: (+)-lactate. In mineral salts medium containing 1 mM betaine, both strains produced over 115 g (1.3 mol) lactate from 12% (w/v) glucose, >95% theoretical yield.

Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium.

A non-recombinant mutant of Escherichia coli B, strain SZ194, was developed that produces over 1 M D-lactate from glucose (or sucrose) in 72 h using mineral salts medium supplemented with 1 mM: betaine in simple anaerobic fermentations. Rates and yields were highest at pH 7.5. Yields approached the theoretical maximum with only trace amounts of co-products. Chiral purity of D-lactate was estimated to be 95%. Specific and volumetric productivities for SZ194 in mineral salts medium (pH 7.5) with betaine were equivalent to those in Luria broth.

Betaine tripled the volumetric productivity of D(-)-lactate by Escherichia coli strain SZ132 in mineral salts medium.

Osmotic stress restricts glycolytic flux, growth (rate and yield), D-lactate productivity, and D-lactate tolerance in Escherichia coli B strain SZ132 during batch fermentation in mineral salts medium with 10% (w/v) sugar. Addition of 1 mM: betaine, a non-metabolized protective osmolyte, doubled cell yield, increased specific productivity of D-lactate and glycolytic flux by 50%, and tripled volumetric productivity (from 8.6 to 25.7 mmol l(-1) h(-1); 0.8 to 2.3 g l(-1) h(-1)). Glycolytic flux and specific productivity in mineral salts medium with betaine exceeded that in Luria broth, substantially eliminating the need for complex nutrients during D-lactate production. In mineral salts medium supplemented with betaine, SZ132 produced approximately 1 mol D-lactate (90 g) per 100 g sugar (glucose or sucrose).