Optically pure 1,2-amino alcohols are highly valuable products as intermediates for chiral pharmaceutical products. Here we designed an environmentally friendly non-natural biocatalytic cascade for efficient synthesis of 1,2-amino alcohols from cheaper epoxides. A redesignated ω-transaminase PAKω-TA was tested and showed good bioactivity at a lower pH than other reported transaminases. The cascade was efficiently constructed as a single one-pot E. coli recombinant, by coupling SpEH (epoxide hydrolase), MnADH (alcohol dehydrogenase), and PAKω-TA. Furthermore, RBS regulation strategy was used to overcome the rate limiting step by increasing expression of MnADH. For cofactor regeneration and amino donor source, an interesting point was involved as that a cofactor self-sufficient system was designed by expression of GluDH. It established a “bridge” between the cofactor and the cosubstrate, such that the cofactor self-sufficient system could release cofactor (NADP+) and cosubstrate (l-Glutamine) regenerated simultaneously. The recombinant E. coli BL21 (SGMP) with cofactor self-sufficient whole-cell cascade biocatalysis showed high ee value (>99%) and high yield, with 99.6% conversion of epoxide (S)-1a to 1,2-amino alcohol (S)-1d in 10 h. It further converted (S)-2a–5a to (S)-2d–5d with varying conversion rates ranging between 65–96.4%. This study first provides one-step synthesis of optically pure 1,2-amino alcohols from (S)-epoxides employing a synthetic redox-self-sufficient cascade.

Trans-4-hydroxy-l-proline is produced by trans-proline-4-hydroxylase with l-proline through glucose fermentation. Here, we designed a thorough “from A to Z” strategy to significantly improve trans-4-hydroxy-l-proline production. Through rare codon selected evolution, Escherichia coli M1 produced 18.2 g L−1l-proline. Metabolically engineered M6 with the deletion of putA, proP, putP, and aceA, and proB mutation focused carbon flux to l-proline and released its feedback inhibition. It produced 15.7 g L−1trans-4-hydroxy-l-proline with 10 g L−1l-proline retained. Furthermore, a tunable circuit based on quorum sensing attenuated l-proline hydroxylation flux, resulting in 43.2 g L−1trans-4-hydroxy-l-proline with 4.3 g L−1l-proline retained. Finally, rationally designed l-proline hydroxylase gave 54.8 g L−1trans-4-hydroxy-l-proline in 60 hours almost without l-proline remaining—the highest production to date. The de novo engineering carbon flux through rare codon selected evolution, dynamic precursor modulation, and metabolic engineering provides a good technological platform for efficient hydroxyl amino acid synthesis.

L-Proline takes a significant role in the pharmaceutical and chemical industries as well as graziery. Typical biosynthesis of L-proline is from L-glutamate, involving three enzyme reactions as well as a spontaneous cyclization. Alternatively, L-proline can be also synthesized in L-ornithine and/or L-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving L-proline production from L-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCD K205G/M86K/T162A from Pseudomonas putida was identified with 2.85-fold increase in catalytic efficiency for the synthesis of L-proline. Furthermore, we designed and optimized RBS for the BaargI and Ppocd coupling cascade using RedLibs, as well as sRNA inhibition of argF to moderate L-proline biosynthesis in Larginine overproducing Corynebacterium crenatum. The strain PS6 with best performance reached 15.3 g/L L-proline in the shake flask and showed a titer of 38.4 g/L in a 5 L fermenter with relatively low concentration of residual L-ornithine and/or L-arginine.

Protein-glutaminase plays a significant role in future food (e.g., plant-based meat) processing as a result of its ability to improve the solubility, foaming, emulsifying, and gel properties of plant-based proteins. However, poor stability, activity, high pressure, and high shear processing environments hinder its application. Therefore, we developed an application-oriented method isothermal compressibility perturbation engineering strategy to improve enzyme performance by simulating the high-pressure environment. The best variant with remarkable improvement in specific activity and half-time, N16M/Q21H/T113E, exhibited a 4.28-fold increase compared to the wild type in specific activity (117.18 units/mg) and a 1.23-fold increase in half-time (472 min), as one of the highest comprehensive performances ever reported. The solubility of the soy protein isolate deaminated by the N16M/Q21H/T113E mutant was 55.74% higher than that deaminated by the wild type, with a tinier particle size and coarser texture. Overall, this strategy has the potential to improve the functional performance of enzymes under complex food processing conditions.

Abstract4‐Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l‐Isoleucine hydroxylase specifically converts l‐Ile to 4‐hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4‐hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l‐isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half‐life of variant T181C was 4.03 h at 50°C, 10.27‐fold the half‐life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56‐fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole‐cell biotransformation, Bacillus subtilis 168/pMA5‐idoT181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4‐hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4‐hydroxyisoleucine. Rational design of disulfide bond improved l‐isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.

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