Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield.

Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the kcat/KM ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.

Development of a yeast cell based method for efficient screening of high yield tacrolimus production strain.

Tacrolimus (FK506) is a widely used and clinically important immunosuppressant drug that can be produced by fermentation of Streptomyces tsukubaensis. The industrial strains are typically obtained through multiple rounds of mutagenesis and screening, a labor-intensive process. We have established an efficient yeast cell based screening method for the evolutionary process of high-FK506-yielding strain. The S. tsukubaensis strains of different FK506 yields were tested for zone of growth inhibition of the wild type and calcineurin mutant (cnb1∆) yeast strains. We found that different FK506 yields correspond well to altered yeast growth inhibitions. Based on the combinational inhibition effects of FK506 with different antifungals that have been frequently reported, we also tested the zone of inhibition by addition of fluconazole, amphotericin B and caspofungin to the medium. In the end, for the best screening performance, we systemically evaluated the strategy when different yeast strains and different antifungals were used according to the clarity, size, and divergence of the inhibition circles. Using different yeast strains and antifungals, we successfully broadened the screening spectrum. An efficient high-FK506-yield S. tsukubaensis screening method has been established and optimized. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03870-y.

Expression of L-phosphinothricin synthesis enzymes in Pichia pastoris for synthesis of L-phosphinothricin.

With the ban of highly toxic herbicides, such as paraquat and glyphosate, phosphinothricin (PPT) is becoming the most popular broad-spectrum and highly effective herbicide. The current PPT products in the market are usually a racemic mixture with two configurations, the D-type and L-type, of which only the L-PPT has the herbicidal activity. The racemic product is not atom economic, more toxic and may cause soil damage. Asymmetric synthesis of L-PPT has become a research focus in recent years, while biological synthesis methods are preferred for its character of environmental friendly and requiring less reaction steps when being compared to the chemical methods. We have developed a biological synthesis route to produce optically pure L-PPT from D,L-PPT in two steps using 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid as the intermediate. In this study, we expressed the glutamate dehydrogenase and glucose dehydrogenase using Pichia pastoris as the first time. After a series of optimization, the total L-PPT yield reached 84%. The developed synthesis system showed a high potential for future industrial application. Compare to the previous plasmid-carrying-E. coli expression system, the established method may avoid antibiotic usage and provided an alternative way for industrial synthesis of optically pure L-PPT.

Biosynthesis of l-phosphinothricin with enzymes from chromosomal integrated expression in E. coli.

Phosphinothricin (PPT) is one of the most prevalently using herbicides. The commercial phosphinothricin products are generally in the form of a racemic mixture, of which only the l-phosphinothricin (L-PPT) gives herbicidal function. Synthesis of optically pure L-PPT by deracemization of D/L-PPT is a promising way to cut down the environmental burden and manufacturing cost. To convert D/L-PPT to L-PPT, we expressed the catalytic enzymes by genomic integration in E. coli. The whole production was implemented in two steps in one pot using four catalytic enzymes, namely d-amino acid oxidase, catalase, glutamate dehydrogenase, and glucose dehydrogenase. Finally, after a series of process optimization, the results showed that with our system the overall L-PPT yield reached 86%. Our study demonstrated a new strategy for L-PPT synthesis, based on enzymes from chromosomal integrated expression, which does not depend on antibiotic selection, and shows a high potential for future industrial application.

Enhancement of gibberellic acid production from Fusarium fujikuroi by mutation breeding and glycerol addition.

Gibberellic acid (GA3) is a natural plant growth hormone that has been widely used in agriculture and horticulture. To obtain higher GA3 producing strains, the method of screening the strains for resistance to simvastatin was used after treatment with nitrosoguanidine (NTG) and gamma rays. The rationale for the strategy was that mutants showing simvastatin resistance were likely to be high GA3 producers, as their activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is relatively more effective. GA3 yield of mutant S109 increased by 14.2% than that of the original strain. The GA3 production ability in mutant S109 remained relatively stable after ten generations. With the addition of 0.4 g glycerol on the 5th day during the fermentation process in Erlenmeyer flask, maximum GA3 production of 2.7 g/L was achieved by this mutant, exhibiting 28.6% increase compared with original strain. Furthermore, we also achieved 2.8 g/L GA3 and had a 33.3% increase with addition 20 g glycerol on the 5th day during the fermentation process in a 5-L bioreactor. Our results indicated efficient GA3 production could be achieved on the condition that the supply of glycerol at the suitable conditions. This study would lay a foundation for industrial production of GA3.

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit.

In recent years, there has been a noticeable increase in research interests on the Fusarium species, which includes prevalent plant pathogens and human pathogens, common microbial food contaminants and industrial microbes. Taken the advantage of gibberellin synthesis, Fusarium fujikuroi succeed in being a prevalent plant pathogen. At the meanwhile, F. fujikuroi was utilized for industrial production of gibberellins, a group of extensively applied phytohormone. F. fujikuroi has been known for its outstanding performance in gibberellin production for almost 100 years. Research activities relate to this species has lasted for a very long period. The slow development in biological investigation of F. fujikuroi is largely due to the lack of efficient research technologies and molecular tools. During the past decade, technologies to analyze the molecular basis of host-pathogen interactions and metabolic regulations have been developed rapidly, especially on the aspects of genetic manipulation. At the meanwhile, the industrial fermentation technologies kept sustained development. In this article, we reviewed the currently available research tools/methods for F. fujikuroi research, focusing on the topics about genetic engineering and gibberellin production.

Recent advances in the improvement of enzyme thermostability by structure modification.

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit-subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.

Colorimetric assay for active biomass quantification of Fusarium fujikuroi.

A colorimetric assay has been developed for quantitative analysis of active biomass of Fusarium fujikuroi, based on the reduction of the tetrazolium salt 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) when menadione was present as an electron coupling agent. The optimum assay-conditions were set as 200 µg/ml XTT, 5 µM menadione and one-hour reaction time. Under these settings, the produced formazan displayed a linear relationship with F. fujikuroi biomass. This method was subsequently applied to evaluate the cell growth behavior, which showed a positive correlation with the carbon source consumption and gibberellin biosynthesis under the industrial fermentation conditions. Our results showed that the XTT-menadione assay is a valuable tool in analyzing the industrial fermentation process of F. fujikuroi, especially when the medium contains insoluble and complex components.