Sequence-Guided Redesign of an Omega-Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

The strategies to reduce cost and improve productivity in DHA production by Aurantiochytrium sp.: from biochemical to genetic respects.

The marine oleaginous protist Aurantiochytrium sp. (Schizochytrium sp.) is a well-known docosahexaenoic acid (DHA) producer and its different DHA products are the ideal substitute for the traditional fish oil resource. However, the cost of the DHA products derived from Aurantiochytrium sp. (Schizochytrium sp.) is still high, limiting their wide applications. In order to reduce the cost or improve the productivity of DHA from the microbial resource, many researches are focusing on exploring the renewable and low-cost materials as feedbacks, and/or the stimulators for biomass and DHA production. In addition, the genetic engineering is also being used in the Aurantiochytrium sp. (Schizochytrium sp.) system for further improvement. These break the bottleneck of the DHA production by Aurantiochytrium sp. (Schizochytrium sp.) in some degree. In this review, the strategies used currently to reduce cost and improve DHA productivity, mainly from the utilizations of low-cost materials and effective stimulators to the genetic engineering perspectives, are summarized, and the availabilities from the cost perspective are also evaluated. This review provides an overview about the strategies to revolve the production cost and yield of the DHA by Aurantiochytrium sp. (Schizochytrium sp.), a theoretical basis for genetic modification of Aurantiochytrium sp. (Schizochytrium sp.), and a practical basis for the development of DHA industry. KEY POINTS : • Utilizations of various low-cost materials for DHA production • Inducing the growth and DHA biosynthesis by the effective stimulators • Reducing cost and improving DHA productivity by genetic modification • The availability from cost perspective is evaluated.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.