Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose.

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.

Directed evolution and characterization of Escherichia coli glucosamine synthase.

Glucosamine synthase (GlmS) converts fructose-6-phosphate to glucosamine-6-phosphate. Overexpression of GlmS in Escherichia coli increased synthesis of glucosamine-6-P, which was dephosphorylated and secreted as glucosamine into the growth medium. The E. coli glmS gene was improved through error-prone polymerase chain reaction (PCR) in order to develop microbial strains for fermentation production of glucosamine. Mutants producing higher levels of glucosamine were identified by a plate cross-feeding assay and confirmed in shake flask cultures. Over 10 mutants were characterized and all showed significantly reduced sensitivity to inhibition by glucosamine-6-phosphate. Ki of mutants ranged from 1.4 to 4.0 mM as compared to 0.56 mM for the wild type enzyme. Product resistance resulted from single mutations (L468P, G471S) and/or combinations of mutations in the sugar isomerase domain. Most overexpressed GlmS protein was found in the form of inclusion bodies. Cell lysate from mutant 2123-72 contained twice as much soluble GlmS protein and enzyme activity as the strain overexpressing the wild type gene. Using the product-resistant mutant, glucosamine production was increased 60-fold.

Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine.

Glucosamine and N-acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold, reaching 60 mg l(-1). Since GlmS is strongly inhibited by glucosamine-6-P, GlmS variants were generated via error-prone PCR and screened. Over-expression of an improved enzyme led to a glucosamine titer of 17 g l(-1). Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, N-acetylglucosamine, is stable, non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions. Therefore, the glucosamine pathway was extended to N-acetylglucosamine by over-expressing a heterologous glucosamine-6-P N-acetyltransferase. Using a simple and low-cost fermentation process developed for this strain, over 110 g l(-1) of N-acetylglucosamine was produced.

The pathway of L-ascorbic acid biosynthesis in the colourless microalga Prototheca moriformis.

When mutant strain UV77-247 of Prototheca moriformis Kruger was fed d-[1-13C]Glc, it synthesized l-ascorbic acid (AA) with approximately three-quarters of the label at the C-1 position and the remaining label at the C-6 position, showing that AA is made by a non-inversion (retention) pathway, i.e. C-1 of Glc becomes C-1 of AA. The label present at C-6 is consistent with the glycolytic conversion of Glc to 3-carbon intermediates and subsequent gluconeogenesis. Compounds suggested as intermediates in inversion-type pathways were not converted to AA. Most strains converted Man to AA at a rate greater than they did Glc. Enzyme activities leading from Fru-6-P to the formation of GDP-Man were identified in all strains, but none of these activities correlated with the mutants' abilities to accumulate AA. However, there was a strong correlation between GDP-Man-3,5-epimerase activity and AA accumulation. Wild-type P. moriformis ATCC 75669 and mutant strains of varying AA-synthesizing abilities rapidly converted l-Gal or l-galactono-1,4-lactone to AA. Based on this data, a biosynthetic pathway from Glc to AA is proposed in which the epimerase is the rate-limiting activity in AA synthesis.