Identification of hyperthermophilic D-allulose 3-epimerase from Thermotoga sp. and its application as a high-performance biocatalyst for D-allulose synthesis.

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production.

Designing a novel (R)-ω-transaminase for asymmetric synthesis of sitagliptin intermediate via motif swapping and semi-rational design.

The application of (R)-ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by inadequate stereoselectivity and narrow substrate spectrum. Herein, an effective evolution strategy for (R)-ω-transaminase designing for the asymmetric synthesis of sitagliptin intermediate is presented. Since natural transaminases lack activity toward bulky prositagliptin ketone, transaminase scaffolds with catalytic machinery and activity toward the truncated prositagliptin ketone were firstly screened based on substrate walking principle. A transaminase chimera was established synchronously conferring catalytic activity and (R)-selectivity toward prositagliptin ketone through motif swapping, followed by stepwise evolution. The process resulted in a "best" engineered variant MwTAM8, which exhibited 79.2-fold higher activity than the chimeric scaffold MwTAMc. Structural analysis revealed that the heightened activity is mainly due to the enlarged and adaptive substrate pocket and tunnel. The novel (R)-transaminase exhibited unsatisfied industrial operation stability, which is expected to further modify the protein to enhance its tolerance to temperature, pH, and organic solvents to meet sustainable industrial demands. This study underscores a useful evolution strategy of engineering biocatalysts to confer new properties and functions on enzymes for synthesizing high-value drug intermediates.

Multiplex modification of Yarrowia lipolytica for enhanced erythritol biosynthesis from glycerol through modularized metabolic engineering.

Erythritol is a novelty 4-carbon sugar polyol and has great potential to be used as the precursor of some platform chemicals. The increasing cost of glucose poses researchers shifting insights to the cheaper biodiesel raw materials. Herein, we engineered a non-degradation, non-byproducts Yarrowia lipolytica for the erythritol production with high-titer from glycerol. Initially, the degradation and competition modules were blocked by URA3 counter-selection marker. Subsequently, a shortened biosynthetic pathway was explored to elevate its synthetic flux by multi-modules combination expression of functional genes. Furthermore, a screened glycerol transporter ScFPS1 was integrated into ERY6 genome to promote the glycerol uptake. The constructed strain ERY8 produced 176.66 g/L erythritol in the 5-L bioreactor with a yield and productivity of 0.631 g/g and 1.23 g/L/h, respectively, which achieved the highest fermentation production efficiency till date. This study proposed a novel multi-modules combination strategy for effectively engineering Y. lipolytica to produce erythritol using glycerol.

Metabolic engineering and pathway construction for O-acetyl-L-homoserine production in Escherichia coli.

O-Acetyl-L-homoserine (OAH) is a potentially important platform metabolic intermediate for the production of homoserine lactone, methionine, 1,4-butanediol and 1,3-propanediol which have giant market value. Currently, multiple strategies have been adopted to explore sustainable production of OAH. However, the production of OAH by consuming cheap bio-based feedstocks with Escherichia coli as the chassis is still in its infancy. Construction of high yield OAH-producing strains is of great significance in industry. In this study, we introduced an exogenous metA from Bacillus cereus (metXbc) and engineered an OAH-producing strain by combinatorial metabolic engineering. Initially, exogenous metXs/metA were screened and used to reconstruct an initial biosynthesis pathway of OAH in E. coli. Subsequently, the disruption of degradation and competitive pathways combined with optimal expression of metXbc were carried out, accumulating 5.47 g/L OAH. Meanwhile, the homoserine pool was enriched by overexpressing metL with producing 7.42 g/L OAH. Lastly, the carbon flux of central carbon metabolism was redistributed to balance the metabolic flux of homoserine and acetyl coenzyme A (acetyl-CoA) in OAH biosynthesis with accumulating 8.29 g/L OAH. The engineered strain produced 24.33 g/L OAH with a yield of 0.23 g/g glucose in fed-batch fermentation. By these strategies, the key nodes for OAH synthesis were clarified and corresponding strategies were proposed. This study would lay a foundation for OAH bioproduction. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03564-5.

Multivariate modular metabolic engineering for enhanced gibberellic acid biosynthesis in Fusarium fujikuroi.

Gibberellic acid (GA3) is one of natural phytohormones, widely used in agriculture and downstream fields. Qualified for the nature productivity, Fusarium fujikuroi was currently employed for the industrial biotransformation from agriculture residues into GA3. Herein, Multivariate modular metabolic engineering (MMME) was assigned to reconstitute the metabolic balance in F. fujikuroi for enhancing GA3 production. Three modules including precursor pool, cluster-specific channel and P450-mediated oxidation in GA3 biosynthetic pathway were defined and optimized separately. The enhancement of both precursor pool and cluster-specific channel pushed metabolic flux transfer into the GA3-specific pathway. Moreover, both introduction of Vitreoscilla hemoglobin and reinforcement of NADPH-dependent cytochrome P450 reductase facilitated oxidation cofactor transfer and subsequently boosted mycelium growth and GA3 biosynthesis. Integration of three modules in the engineered strain accumulated 2.89 g/L GA3 in shake flask via submerged fermentation, presenting a promising modular metabolic engineering model for efficient microbial transformation in agro-industrial application.