The present application relates to the technical field of genetic engineering, and provides a monooxygenase mutant, a preparation method and application thereof. The monooxygenase mutant has any one of the amino acid sequences shown in (I) and (II): (I) an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO. 1; and (II) an amino acid sequence obtained by modifying, substituting, deleting, or adding one or several amino acids to the amino acids at 23 to 508 positions of the amino acid sequence shown in SEQ ID NO. 1, the substituting referring to a substitution of 1 to 34 amino acids, wherein the mutant has the activity of monooxygenase.

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

The present disclosure provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.

The invention discloses an alcohol dehydrogenase mutant and use thereof. The alcohol dehydrogenase mutant of the present invention has high thermal stability and enables high catalytic efficiency and high conversion rate (i.e. space time yield) in the asymmetric reduction of prochiral diaryl ketones to produce chiral diaryl alcohols. Therefore, the alcohol dehydrogenase mutant of the present invention has extremely high prospect of application in the production of chiral diaryl alcohols, such as (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol, (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

The invention discloses an alcohol dehydrogenase mutant and use thereof. The alcohol dehydrogenase mutant of the present invention has high thermal stability and enables high catalytic efficiency and high conversion rate (i.e. space time yield) in the asymmetric reduction of prochiral diaryl ketones to produce chiral diaryl alcohols. Therefore, the alcohol dehydrogenase mutant of the present invention has extremely high prospect of application in the production of chiral diaryl alcohols, such as (S)-(4-chlorophenyl)-(pyridin-2-yl)-methanol, (R)-(4-chlorophenyl)-(pyridin-2-yl)-methanol.

The present disclosure includes refactored thaxtomin biosynthetic gene clusters including thaxtomin modules including one or more thaxtomin genes such that the expression of the refactored thaxtomin biosynthetic gene cluster produces at least one thaxtomin compound in the absence of thaxtomin-inducing conditions. Also included are genetically engineered Streptomyces bacterium from a non-pathogenic Streptomyces strain comprising an exogenous, refactored thaxtomin biosynthetic gene cluster of the present disclosure, such that the expression of the refactored thaxtomin biosynthetic gene cluster provides the genetically engineered Streptomyces bacterium with the ability to produce at least one thaxtomin compound in the absence of thaxtomin-inducing conditions. The present disclosure also includes methods of producing thaxtomin compounds, analogs, or intermediate with the refactored thaxtomin biosynthetic gene clusters and genetically engineered bacteria of the present disclosure.

The present invention discloses a transaminase mutant and application thereof in preparation of sitagliptin intermediates, the transaminase mutant is obtained by substitution of tyrosine with proline at position 74, substitution of glutamic acid with aspartic acid at position 228, substitution of leucine with alanine at position 254 and substitution of methionine with threonine at position 290 of the amino acid sequence shown in SEQ ID NO: 2. The present invention uses wet cells or a purified transaminase as a biocatalyst and a sitagliptin precursor ketone or a prochiral carbonyl compound as a substrate to prepare a sitagliptin intermediate or a sitagliptin ester intermediate; the total yield of the method reaches about 82%, and e.e. value of the product reaches 99%.

A method for producing ectoine by fermenting recombinant Corynebacterium glutamicum. The recombinant Corynebacterium glutamicum is obtained by overexpressing, in Corynebacterium glutamicum, an aspartokinase gene lysC of which feedback inhibition is relieved, then replacing the promoter of the dihydrodipicolinate synthase in the recombinant bacterium to attenuate the activity of the dihydropyrimidine dicarboxylic acid synthase, and then transforming the recombinant bacterium with the ectoine synthetic path related gene ectABC. The recombinant Corynebacterium glutamicum can be fermented using different cheap raw materials under a low salt condition to produce ectoine, and use cheap corn slurry instead of expensive yeast powder as a nutritional component, so as to further reduce the costs of the raw materials. In addition, the recombinant Corynebacterium glutamicum solves the biosafety problem, simplifies the post-extraction process, and has a good market application prospect.

A method for producing ectoine by fermenting recombinant Corynebacterium glutamicum. The recombinant Corynebacterium glutamicum is obtained by overexpressing, in Corynebacterium glutamicum, an aspartokinase gene lysC of which feedback inhibition is relieved, then replacing the promoter of the dihydrodipicolinate synthase in the recombinant bacterium to attenuate the activity of the dihydropyrimidine dicarboxylic acid synthase, and then transforming the recombinant bacterium with the ectoine synthetic path related gene ectABC. The recombinant Corynebacterium glutamicum can be fermented using different cheap raw materials under a low salt condition to produce ectoine, and use cheap corn slurry instead of expensive yeast powder as a nutritional component, so as to further reduce the costs of the raw materials. In addition, the recombinant Corynebacterium glutamicum solves the biosafety problem, simplifies the post-extraction process, and has a good market application prospect.

Among the various aspects of the present disclosure is the provision of a biological and biochemical production of piperazic acid derived from the newly discovered production pathway for L-piperazic acid. One aspect of the present disclosure includes a transgenic microorganism (e.g., bacteria) engineered to accumulate piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product. Another aspect of the present disclosure includes biochemical and biological methods for producing piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product. Another aspect of the present disclosure includes compositions and methods of using isotopically labeled piperazic acid and derivatives thereof, including a piperazic acid (Piz)-containing product.