Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High-Molecular-Weight Hyaluronic Acid.

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge-gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain-length specificity and a twofold increase in mass-based turnover number. Seven improved pmHAS variants out of 1392 generated by error-prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell-free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N-terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.

Surface charge engineering of a Bacillus gibsonii subtilisin protease.

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. "Deamidation" in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5-10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH 8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.

Plant-derived sucrose is a key element in the symbiotic association between Trichoderma virens and maize plants.

Fungal species belonging to the genus Trichoderma colonize the rhizosphere of many plants, resulting in beneficial effects such as increased resistance to pathogens and greater yield and productivity. However, the molecular mechanisms that govern the recognition and association between Trichoderma and their hosts are still largely unknown. In this report, we demonstrate that plant-derived sucrose (Suc) is an important resource provided to Trichoderma cells and is also associated with the control of root colonization. We describe the identification and characterization of an intracellular invertase from Trichoderma virens (TvInv) important for the mechanisms that control the symbiotic association and fungal growth in the presence of Suc. Gene expression studies revealed that the hydrolysis of plant-derived Suc in T. virens is necessary for the up-regulation of Sm1, the Trichoderma-secreted elicitor that systemically activates the defense mechanisms in leaves. We determined that as a result of colonization of maize (Zea mays) roots by T. virens, photosynthetic rate increases in leaves and the functional expression of tvinv is crucial for such effect. In agreement, the steady-state levels of mRNA for Rubisco small subunit and the oxygen-evolving enhancer 3-1 were increased in leaves of plants colonized by wild-type T. virens. We conclude that during the symbiosis, the sucrolytic activity in the fungal cells affects the sink activity of roots, directing carbon partitioning toward roots and increasing the rate of photosynthesis in leaves. A discussion of the role of Suc in controlling the fungal proliferation on roots and its pivotal role in the coordination of plant-microbe associations is provided.