In the last decades, interest in medical or cosmetic applications of hyaluronic acid (HA) has increased. Size and dispersity are key characteristics of biological function. In contrast to extraction from animal tissue or bacterial fermentation, enzymatic in vitro synthesis is the choice to produce defined HA. Here we present a one-pot enzyme cascade with six enzymes for the synthesis of HA from the cheap monosaccharides glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). The combination of two enzyme modules, providing the precursors UDP–GlcA and UDP–GlcNAc, respectively, with hyaluronan synthase from Pasteurella multocida (PmHAS), was optimized to meet the kinetic requirements of PmHAS for high HA productivity and molecular weight. The Mg2+ concentration and the pH value were found as key factors. The HA product can be tailored by different conditions: 25 mM Mg2+ and 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-NaOH pH 8 result into an HA product with high Mw HA (1.55 MDa) and low dispersity (1.05). Whereas with 15 mM Mg2+ and HEPES–NaOH pH 8.5, we reached the highest HA concentration (2.7 g/L) with a yield of 86.3%. Our comprehensive data set lays the basis for larger scale enzymatic HA synthesis.

Enzymatic synthesis is a promising approach to produce hyaluronic acid (HA) products with defined molecular weight (MW) and dispersity. We here report on the evaluation of nucleoside triphosphate (NTP) regeneration system for the synthesis of the precursors uridine-5'-diphosphate α-d-N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphosphate α-dglucuronic acid (UDP-GlcA) and integration in the one-pot synthesis of HA. By integration of polyphosphate kinase from Ruegeria pomeroyi (RpPPK2-3) into the enzyme cascade to synthesize UDP-GlcA we reached 89 % yield after 1 h with a regeneration cycle number (RCN) of 17. With the UDP-GlcNAc cascade, the ATP concentration was lowered 250-fold and reached 94 % yield after 1 h with an RCN of 234. Integration of the RpPPK2-3 regeneration system in one-pot HA synthesis afforded optimization of MgCl 2 and ATP starting concentrations. With 25 mM MgCl 2 and 0.1 mM ATP, a HA concentration of 0.81 g L À 1 with an average MW of 1.17 MDa and dispersity of about 1.08 and an RCN of 75 was obtained. We conclude that integration of NTP regeneration with RpPPK2-3 expanded the enzymatic possibilities to produce HA with lower range MW.

Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

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