Asymmetric reduction of prochiral ketones via biocatalysis is the most effective method for the synthesis of chiral alcohols. The industrial biocatalytic reduction process requires additional co-substrates and enzymes, e.g., glucose and glucose dehydrogenase, to regenerate reductive cofactors, such as nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), for efficient substrate conversion and product yield. Thus, increasing the cost in reaction and downstream treatment. Recently, algae cells, due to a combination of microorganism-like properties (rapid growth and high density culture) and plant-like properties (highly active photosynthesis), have attracted much attention as novel host cells in industrial biocatalysis. Particularly, autotrophic photosynthesis in algae is capable of regenerating NADH and NADPH rapidly, eliminating the requirement of additional enzymes and co-substrates for cofactor regeneration in normal biocatalytic processes. Recently, a variety of algae species, such as cyanobacteria, green algae, diatoms, chrysophytes, and red algae, have been applied in asymmetric biocatalysis. Herein, various asymmetric reduction reactions performed by different type of algae cells have been reviewed, and methods for improving the reaction efficiency have been discussed. We aim to attract algae microbiologists and chemists to push forward the study of algae as biocatalytic cell factories for value-added industrial chemical production in the future.

Using algae cells to drive cofactor regeneration and asymmetric reduction for the synthesis of chiral chemicals

Microalgae are excellent biocatalyst candidates for photo-biocatalytic-asymmetric reduction of prochiral ketones to produce enantiomer alcohols. In the biocatalysis asymmetric reduction of carbonyl group process, the cofactor, NADPH, plays a key role. So the current study focused on the effect of NADPH availability on the reduction. Photosynthesis is a main pathway for NADPH regeneration in microalgae, and the ferredoxin-NADP+ oxidoreductase (FNR) is the key enzyme in this process. In this work, we constructed an engineered cyanobacterium Synechocystis sp. PCC6803::Ω-PpetE-petH to overexpress FNR gene petH to strengthen NADPH regeneration. The results show that the intracellular NADPH content was increased 80.8% compared to the wild type. Applying the Synechocystis sp. PCC6803::Ω-PpetE-petH to catalyze the model asymmetric reduction reaction (reduction of acetophenone to S-phenylethanol), the yield was improved 61.2% compared to the wild-type. This work makes the microalgal biocatalysis asymmetric reduction process become more preponderant via providing a new route to enhance the regeneration of cofactor NADPH. Putative metabolic process of nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and the photo-biocatalytic-asymmetric reduction reaction of prochiral ketones in microalgal cells.

Strengthening NADPH Regeneration for Improving Photo-biocatalytic Ketones Asymmetric Reduction Reaction by Synechocystis Through Overexpression of FNR

Enantiopure alcohols have received much attention due to their widespread use as pharmaceutical intermediates. In the asymmetric biosynthesis of enantiopure alcohols, the excellent performance of carbonyl reductase makes it be the best choice as the biocatalysts. In this work, an alkali-tolerant carbonyl reductase (BsCR, encoded by yueD) from Bacillus subtilis (strain 168) was obtained through gene mining, and successfully heterologously expressed in Escherichia coli with pET-32a. BsCR showed excellent alkali resistance and even can keep more than 70% of its peak activity after incubation in Tris–HCl buffer at pH 9.0 for 40 h. The Michaelis constants and maximal velocity of the BsCR to NADPH (A) and ethyl 4-chloroacetoacetate (B) are $$K_{m}^{A}$$
  = 5.390 × 10−2 mmol/L, $$K_{m}^{B}$$
  = 1.855 mmol/L, and $$V_{max}$$
  = 147.3 μmol·min−1·mg−1, respectively. Applying the E. coli BL21(DE3)/pET-32a-yueD to catalyze asymmetric reduction of ethyl 4-chloroacetoacetate and acetophenone, the yield of S-CHBE reached 89.9% and S-1-phenyl ethanol reached 66.7%, and e.e. of both products reached more than 99%. This work provides a novel CR for asymmetric reduction. A carbonyl reductase (BsCR) and its gene were identified through gene mining, and overexpressed in Escherichia coli BL21(DE3) for whole-cell biocatalytic asymmetric reduction

An Alkali-tolerant Carbonyl Reductase from Bacillus subtilis by Gene Mining: Identification and Application

The intracellular NADPH insufficient supply is the main bottleneck to the synthesis of chiral alcohols by asymmetric reduction with whole-cell catalysis. Herein, we provide a novel strategy to strengthen intracellular NADPH supply through introducing an NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase (gapB from Bacillus subtilis 168) into the Embden-Meyerhof pathway and a NAD kinase (yfjB from E. coli MG1655) to further enhance the NADP(H) pool. A recombinant E. coli (E. coli BL21 (DE3)/pETDuet-1-gapB-yueD&pET28a-yfjB) was constructed to co-express gapB and yfjB with a carbonyl reductase gene yueD together. The result showed that the intracellular NADPH amount increased by 134.4% with the strategy. To the model reaction (asymmetric reduction of acetophenone to S-phenyl ethanol), the yield was 3.7-fold with this strategy compared to the control. This provides a technological route for strengthening the intracellular NADPH supply in E. coli for biocatalysis and biosynthesis.

Promotion of the Asymmetric Reduction of Prochiral Ketone with Recombinant E. coli Through Strengthening Intracellular NADPH Supply by Modifying EMP and Introducing NAD Kinase

Enhancing Cofactor Regeneration of Cyanobacteria for the Light-Powered Synthesis of Chiral Alcohols

A metabolic regulation method was proposed to increase the microalgal intracellular nicotinamide adenine dinucleotide phosphate (NADPH) by lowering NADPH consumption in the Calvin cycle or accelerating NADPH regeneration in photosynthesis, thereby to promote the asymmetric reduction of prochiral ketone to chiral alcohol catalyzed by microalgae cell. The NADPH consumption was efficiently decreased by inhibiting glyceraldehyde 3-phosphate dehydrogenase with inhibitors, such as iodoacetic acid and Angeli's Salt, which increased the microalgal intracellular NADPH by 80% and 96%, respectively. Correspondingly, the yields of the asymmetric reduction reaction were enhanced by 12.9% and 63.3%, respectively. On the other hand, NADPH regeneration was accelerated by promoting electron transfer and ferredoxin-NADP+ reductase activity with addition of sodium cholate, sodium acetate and sodium thiosulfate; the intracellular NADPH was increased 73.1%, 60.2% and 70.4%, respectively. The yields of the asymmetric reduction reaction were correspondingly enhanced by 118.4%, 36.7% and 26.2%, respectively. This provides a practical technique to promote microalgae intracellular NADPH amount to enhance asymmetric reduction reaction by photo-biocatalytic processes. © 2016 Curtin University of Technology and John Wiley & Sons, Ltd.

Promotion of the microalgal photo-biocatalytic asymmetric reduction of prochiral ketone by NADPH metabolic regulation: Promotion of Biocatalysis by NADPH Metabolic Regulation

Zhi-Wei Gong

Papers

Promotion of the microalgal photo-biocatalytic asymmetric reduction of prochiral ketone by NADPH metabolic regulation: Promotion of Biocatalysis by NADPH Metabolic Regulation