Crucial role of 4-deoxy-L-erythro-5-hexoseulose uronate reductase for alginate utilization revealed by adaptive evolution in engineered Saccharomyces cerevisiae

Abstract

In brown macroalgae, alginate and D-mannitol are promising carbohydrates for biorefinery. Saccharomyces cerevisiae is widely used as a microbial cell factory, but this budding yeast is unable to utilize either alginate or D-mannitol. Alginate can be depolymerized by both endo-type and exo-type alginate lyases, yielding a monouronate, 4-deoxy-L-erythro-5-hexoseulose uronate (DEH), a key intermediate in the metabolism of alginate. Here, we constructed engineered two S. cerevisiae strains that are able to utilize both DEH and D-mannitol on two different strain backgrounds, and we also improved their aerobic growth in a DEH liquid medium through adaptive evolution. In both evolved strains, one of the causal mutations was surprisingly identical, a c.50A > G mutation in the codon-optimized NAD(P)H-dependent DEH reductase gene, one of the 4 genes introduced to confer the capacity to utilize DEH. This mutation resulted in an E17G substitution at a loop structure near the coenzyme-binding site of this reductase, and enhanced the reductase activity and aerobic growth in both evolved strains. Thus, the crucial role for this reductase reaction in the metabolism of DEH in the engineered S. cerevisiae is demonstrated, and this finding provides significant information for synthetic construction of a S. cerevisiae strain as a platform for alginate utilization.

Uncovering the reactive nature of 4-deoxy-l-erythro-5-hexoseulose uronate for the utilization of alginate, a promising marine biopolymer

“…We recently created an autotrophic bioengineered S. cerevisiae DEH++ (MK5719) strain that is able to utilize DEH and mannitol 13 . During strain construction, DEH was prepared by digestion of alginate with exo-type alginate lyase Atu3025 15,16 and, when needed, endo-type alginate lyase A1-I.…”

“…During strain construction, DEH was prepared by digestion of alginate with exo-type alginate lyase Atu3025 15,16 and, when needed, endo-type alginate lyase A1-I. The formation of DEH was verified by TLC, and DEH was visualized using the sulfate and thiobarbituric acid methods 13 . In contrast, when the exo-type alginate lyase activity of Atu3025 was assayed, DEH could be visualized using the thiobarbituric acid method but not by the sulfate method 15,16 .…”

“…SD medium was a mixture of 0.67% (w/v) yeast nitrogen base without amino acids (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and with 2.0% (w/v) glucose (pH 5.6). DEH + HN medium consisted of 0.17% (w/v) yeast nitrogen base without amino acids and ammonium sulfate [AS, (NH 4 ) 2 SO 4 ] (Becton, Dickinson and Company), 690 mg/mL –Leu DO Supplement (Clontech, Mountain View, CA, USA), 20 mM 2-morpholinoethanesulfonic acid (MES) (pH 5.6), 1.0% (w/v) DEH, and 5 mM Asn 13 . DEH-N medium was DEH + HN medium lacking nitrogen sources (–Leu DO Supplement and Asn).…”

“…The DEH solution was prepared and quantitated as described previously 13 , but without endo-type alginate lyase A1-I, except when stated. Briefly, an alginate solution [1% (w/v)] was prepared by autoclaving 1.0 g of sodium alginate (Nacalai Tesque) in 100 mL of pure water (Elix, Millipore).…”

“…To determine its structure, DRP-1 was prepared by incubating 1% (w/v) DEH and 60 mM NH 4 Cl for 18 h at 30 °C. The DEH solution was prepared as described previously with endo-type alginate lyase A1-I 13 and absorbed on 20 g of Silica Gel 60 (Nacalai Tesque). The resulting gel was dried using a desiccator and packed into a glass column.…”