Zhejiang University to Transfer Mutant Short-Chain Dehydrogenase Patent for 200,000 RMB

Recently, Zhejiang University released a public notice on the transformation of scientific and technological achievements, announcing its intention to transfer“A Short-Chain Dehydrogenase Mutant, Its Encoding Gene, Method for Obtaining the Encoding Gene, and Application of the Mutant”Transfer of Relevant Patent Rights: The patent is jointly owned by Zhejiang University and Hangzhou Xinhai Enzyme Source Biotechnology Co., Ltd. In this transfer, Zhejiang University assigns its 50% share for a consideration of RMB200,000 yuan. The inventor of this patented technology isProfessor Yu Hongwei and His Team。

The present invention belongs toBiotechnology EngineeringField, specifically relating to a mutant of a short-chain dehydrogenase, wherein the mutant is mutant L104A, mutant A150Y, mutant Y155A, mutant I158A, mutant I202A, or mutant T205A; the amino acid sequence of the short-chain dehydrogenase is as shown in SEQ ID NO.1. The short-chain dehydrogenase mutants of the present invention exhibit higher enzymatic activity relative to the wild-type short-chain dehydrogenase, and can be used to prepare prednisolone from prednisone or in combination with a hydrolase using prednisone acetate as a substrate, achieving high substrate conversion rates and high product yields without by-product formation. Moreover, the reaction can be conducted at higher substrate concentration levels, making it suitable for large-scale production.

PrednisoloneAs a clinically critical class of adrenocortical hormones, these drugs are widely used in the treatment of acute severe bacterial infections, allergic diseases, and rheumatic immune disorders. They also serve as key precursors for nide-class medications such as desonide and budesonide, sustaining robust market demand. The core objectives in their manufacturing technology are to enhance substrate conversion rates and reduce production costs, while meeting the efficiency and environmental standards required for large-scale production. For pharmaceutical companies, in particular, the dual improvement of green synthesis processes and product yield is essential to strengthening competitive advantage.

Currently, the preparation of prednisolone mainly relies onChemical Synthesis vs. Traditional Biofermentation, both technologies suffer from significant industry pain points: the chemical synthesis method, which uses steroidal nuclei or hydrocortisone as raw materials, requires multiple complex synthetic steps. This results not only in a lengthy process route and prolonged production cycle with low overall yield, but also generates substantial pollutants during the reaction, posing environmental hazards. Consequently, high production costs persist, making it difficult to align with the development trends of the green pharmaceutical industry.

Although the traditional biological fermentation method is relatively environmentally friendly, its technical shortcomings are equally prominent: first,Substrate concentration is limited,The existing process can only carry out reactions under low substrate concentration conditions, failing to meet the efficiency requirements for large-scale production; secondly,Low Conversion Efficiency, the reaction cycle generally exceeds 60 hours, and both substrate conversion rate and product yield remain at low levels, increasing the difficulty and cost of subsequent separation and purification; thirdly,High raw material costs,Some enzymatic methods use hydrocortisone as the raw material, further driving up production costs and limiting the industrial application of the technology.

Furthermore, existing preparation technologies face challenges such as difficulty in controlling product purity and the formation of by-products. These issues not only compromise the safety of clinical drug applications but also necessitate additional costs for impurity removal. Meanwhile, the low degree of process standardization results in insufficient quality stability across different production batches, making it difficult to meet the stringent quality control requirements of the pharmaceutical industry.

Consequently, existing prednisolone preparation technologies suffer from multiple deficiencies in terms of efficiency, cost, environmental sustainability, and quality stability. There is an urgent need for a green synthesis technology characterized by high activity, high selectivity, and suitability for large-scale production, to overcome industry bottlenecks and meet the pharmaceutical industry’s demand for upgraded, efficient, and environmentally friendly manufacturing processes.

The core advantages and advanced nature of this patented technology lie in its ability toEnzyme Molecular Engineering and Process OptimizationDual-dimensional efforts, throughDirected Design of Short-Chain Dehydrogenase Mutants and Innovative Construction of a Supporting Preparation System, thoroughly resolving the industry pain points of low conversion rates, substrate concentration limitations, and high costs in traditional prednisolone production, thereby establishing a new technical benchmark for the biosynthesis of steroid drugs.

Dramatic Leap in Mutant Enzyme Activity

This technology overcomes the catalytic limitations of wild-type short-chain dehydrogenases, achieving a qualitative leap in enzymatic performance through precise site-directed mutagenesis. The R&D team targetedKey functional region of short-chain dehydrogenase (SEQ ID NO. 1),Six major mutants, L104A, A150Y, Y155A, I158A, I202A, and T205A, were designed, among which the preferred mutantI158AThe conversion rate to prednisone acetate is as high as98.54%, which is 1.57 times that of the wild-type enzyme, with a product yield reaching98.07%, ee value>99%. Through directed substitution of a single amino acid residue, the mutant optimizes the binding conformation between the enzyme and its substrate, significantly enhancing catalytic efficiency while achieving highly selective reactions with no by-product formation, thereby ensuring product purity at the source.

Breakthrough in Substrate Concentration and Process Compatibility

Traditional biological methods are limited by insufficient enzyme activity, making it difficult to increase substrate concentration, whereas this technology extends the upper limit of reaction substrate concentration to20–80 g/L (preferably 60 g/L), far exceeding the capabilities of traditional processes, thereby laying a core foundation for large-scale production. The preparation system supports flexible adaptation to dual substrates: it can directly catalyze the conversion using prednisone as the raw material, or synthesize the product using low-cost prednisone acetate as the substrate with the assistance of hydrolases, significantly reducing raw material costs. Furthermore, the process is compatible with common organic solvents such as isopropanol and N,N-dimethylformamide (DMF), maintaining high catalytic efficiency even at volume fractions of 8–12%, which enhances flexibility for industrial applications.

Construction of a Low-Cost, Large-Scale Production System

Technology AdoptionScaled Preparation of Fermentation-Expressed Engineered BacteriaPathway, Mutant PassagePlasmid pET-28aConstruction of a recombinant expression vector, followed by transformation into Escherichia coli BL21 and induced cultivation, enables high-level expression. The process for preparing wet cell biomass is well-established and cost-effective. The reaction system does not require complex equipment; using potassium phosphate buffer as the reaction medium, the bioconversion is completed within 16–30 hours under shaking conditions at 150–300 rpm and a temperature of 30–48°C (preferably 37°C). This streamlined operational procedure results in lower energy consumption. Compared with chemical synthesis, this process completely eliminates multi-step complex synthesis and the use of polluting reagents. Compared with traditional microbial fermentation, it shortens the reaction cycle by more than 50%, significantly reduces the difficulty of separation and purification, and substantially lowers overall production costs.

Precise Matching of Coding Genes and Preparation Methods

This technology constructs“Mutant–Encoding Gene–Specific Primers”a complete technical system, in which each mutant corresponds to a defined nucleotide sequence (SEQ ID NOs: 9–14), and highly specific primer pairs (e.g., L104A-F/R, I158A-F/R, etc.) have been designed. The target gene can be efficiently obtained through PCR amplification and DpnI digestion, ensuring a standardized and highly reproducible construction process. The workflows for constructing recombinant expression vectors and engineered bacterial strains are well-established, with clearly defined fermentation and induction expression parameters. The use of wet cell biomass or crude enzyme preparations offers flexibility, allowing adjustments according to production scale and meeting the full spectrum of needs from laboratory R&D to industrial-scale manufacturing.

These technological advantages do not exist in isolation but rather form“Enhanced Enzyme Activity – Process Adaptation Optimization – Reduced Production Costs”the synergistic innovation effect, breaking through traditional technical limitations across the entire chain from core catalytic mechanisms to industrial applications, providing comprehensive technical support for the green, large-scale production of prednisolone.

In the field of prednisolone manufacturing, as the pharmaceutical industry’s demand for greener, more efficient, and lower-cost production intensifies, leading domestic and international steroid drug companies and biotechnology firms are actively engaged in R&D breakthroughs and capacity expansion centered on core technological routes such as enzymatic conversion and biofermentation. This has created a dual-track competitive landscape characterized by “iterative chemical synthesis alongside biological method breakthroughs.” The following outlines representative companies in the industry and their technological advancements:

Taizhou Xianju PharmaceuticalFocusing on Innovation in Enzymatic Synthesis of Steroid Drugs: Invention Patent Granted in June 2025“A KstD Enzyme Mutant, Engineered Bacterial Strain, and Their Application in the Preparation of Steroid Drugs”, high-activity KstD enzyme mutant KstD/H141T/Y326G was obtained through alanine scanning and combinatorial mutagenesis. Under conditions with a final substrate concentration ≤30 g/L, the 96-hour conversion efficiency was significantly improved compared to the wild type. Meanwhile, a self-assembling dual-enzyme complex technology was developed to achieve highly efficient fused catalysis of 11α-hydroxylase and 1,2-dehydrogenase, facilitating the green synthesis of prednisolone intermediates. Currently, this technology is in the laboratory-scale optimization phase, with plans to advance to pilot-scale amplification.

Hubei Joint PharmaceuticalThrough its wholly-owned subsidiary, Gongtong Shengwu, the company has strategically positioned itself in enzymatic bioproduction projects by investing RMB 300 million to construct production and conversion lines for biological enzymes. This initiative will expand production capacity for products such as prednisone and prednisolone. Core technologies encompass genetic modification of high-end microbial strains, enzyme-catalyzed structural modifications, and efficient phytosterol conversion. The annual production capacity of steroid intermediates at the Danjiangkou production base has reached 3,000 metric tons. Leveraging the Hubei Provincial Engineering Research Center for Steroid Drugs and Intermediates, the company is conducting directed evolution research on key dehydrogenases such as KstD, aiming to increase substrate concentration to over 40 g/L and shorten the conversion cycle to within 48 hours. The project is currently in the phase of plant construction and process commissioning, with phased production expected to commence in the second half of 2026.

In summary, this technical solution achieves full-chain innovation—from core catalytic components to scalable production systems—by obtaining high-activity short-chain dehydrogenase mutants through site-directed mutagenesis and integrating them with an optimized enzymatic synthesis process. It provides a novel solution that is efficient, cost-effective, and environmentally friendly, addressing the industry’s core challenges in prednisolone preparation, including low conversion rates, substrate concentration limitations, high costs, and insufficient environmental sustainability.