Hongmin Ma

Proteins with high catalytic efficiency and selectivity under mild conditions have long been appreciated by industrial and medicinal fields. These proteins, which are commonly multimeric, often possess low stability, impeding wider application. Currently, strategies to improve the stability of multimeric proteins concentrate on enhancing the interaction at internal interface of the subunits. In this report, we confirmed that the largely underestimated subunit terminal ends are as significant as the internal interface for protein stability. By connecting both the terminal ends and internal interface of subunits, the tetrameric Leifsonia alcohol dehydrogenase ( Ln ADH) protein can been cyclized into a rigid form with significantly improved thermostability and resilience. The improvement in the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment ( T ₅₀ ¹⁵ ) and melting temperature ( T _m ) of the modified protein was 18°C and 23.3°C, respectively, which is superior to the results achieved by normal protein engineering. Our study provided a novel strategy to effectively improve the stability of multimeric proteins, which is suitable not only for the short-chain dehydrogenase/reductase (SDR) family but also other classes of proteins with close terminal ends. IMPORTANCE Industrially interesting proteins are generally multimeric proteins; however, their applications are often restricted due to low stability caused by the natural tendency of subunit disassociation. Current approaches targeting this problem mainly focus on enhancing the internal interfaces of the subunits to avoid their disassociation. In this study, we identified and confirmed the external interface to be significant for improving the stability of multimeric proteins. By connecting the terminal ends and internal interface with disulfide bonds, we found that the multimeric protein Ln ADH cyclized into a robust monomeric-like form, resulting in superior thermostability compared to traditional protein engineering. This intersubunit cyclization approach is efficient and easy to perform, providing a novel method for engineering many important classes of multimeric proteins.

Natural products are critical for drug discovery and development; however their discovery is challenged by the wide inactivation or silence of microbial biosynthetic pathways. Currently strategies targeting this problem are mainly concentrated on chromosome dissembling, transcription, and translation-stage regulations as well as chemical stimulation. In this study, we developed a novel approach to awake cryptic/silenced microbial biosynthetic pathways through augmentation of the conserved protein modification step-phosphopantetheinylation of carrier proteins. Overexpression of phosphopantetheinyl transferase (Pptase) genes into 33 Actinomycetes achieved a significantly high activation ratio at which 23 (70%) strains produced new metabolites. Genetic and biochemical studies on the mode-of-action revealed that exogenous PPtases triggered the activation of carrier proteins and subsequent production of metabolites. With this approach we successfully identified five oviedomycin and halichomycin-like compounds from two strains. This study provides a novel approach to efficiently activate cryptic/silenced biosynthetic pathways which will be useful for natural products discovery.

Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.

Significance: Enzymatic modifications of anthracycline antibiotics are urgently needed in the fields of biosynthesis, biocatalysis, and even medical chemistry. However, neither hydroxyl regioisomerization nor dehydroxylation of anthracycline core was described previously. Here, we discover an unprecedented hydroxyl regioisomerization process in the biosynthesis of a rare carbon-4 deoxyanthracycline, which includes three tailoring steps performed by a four-enzyme cascade: two-component hydroxylases mediated a cryptic hydroxylation, and two NmrA-like short-chain dehydrogenase/reductases catalyzed a reduction-dearomatization followed by a reduction-dehydration process. This study expands the enzymology and chemistry of type II polyketide synthase and provides tools to generate more analogs by engineering or enzymatic semisynthesis.

Piperidine and indolizidine are two basic units of alkaloids that are frequently observed in natural and synthetic compounds. Their biosynthesis in natural products is highly conserved and mostly derived from the incorporation of lysine cyclization products. Through in vitro reconstitution, we herein identified a novel pathway involving a group of polyketide-derived indolizidines, which comprises the processes of tandem two-electron thioester reduction, transamination, and imine reduction to convert acyl carrier protein (ACP)-tethered polyketide chains into the piperidine moieties of their indolizidine scaffolds. The enzymes that catalyze the imine reduction are distinct from previous known imine reductases, which have a fold of acyl-CoA dehydrogenase but do not require flavin for reduction. Our results not only provide a new way for the biosynthesis of the basic units of alkaloids but also show a novel class of imine reductases that may benefit the fields of biocatalysis and biomanufacturing.