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Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine‐tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high‐throughput balancing (IHTB) strategy was established to thoroughly fine‐tune the (2S)‐naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high‐throughput method, which was established based on the interactions between AlCl3 and (2S)‐naringenin. The metabolic flux of the screened high‐titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)‐naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p‐coumaric acid accumulation. Chalcone synthase was speculated to be the rate‐limiting enzyme because its expression level was closely related to the production of both (2S)‐naringenin and p‐coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) play important roles in alcohol metabolism. Therefore, a possible effective way to attenuate alcoholic liver damage is the exogenous supply of these two enzymes to the stomach as they might accelerate the oxidation of ethanol into nontoxic acetate. RESULTS: ADH and ALDH were coexpressed in Lactococcus lactis NZ3900 and used as treatments for acute alcoholic liver injury in mice. Intragastric ethanol administration was carried out at 5.6 g kg‐1 body weight per day in mice for 15 consecutive days and different doses of recombinant ADH‐ALDH L. lactis treatment were administrated together with ethanol. A high dose of L. lactis recombinant ADH‐ALDH treatment (ADH activity of 2000 U kg‐1 BW and ALDH activity of 1000 U kg‐1 BW) reduced the serum alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels by 38.1%, 54.8% and 23.2%, respectively, in ethanol‐treated mice. Moreover, it also helped maintaining serum lipid levels and liver oxidative stress parameters against ethanol. Histopathological examination of mice livers revealed that L. lactis recombinant ADH‐ALDH at a high dose (ADH activity of 2000 U kg‐1 BW and ALDH activity of 1000 U kg‐1 BW) protected liver tissue from the damage induced by ethanol. CONCLUSION: Results demonstrate that L. lactis with ADH and ALDH activity exhibit a dose‐dependent protective effect on alcohol‐induced liver damage in mice. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenase (E.C. 1.2.1.x) can catalyze detoxification of acetaldehydes. A novel acetaldehyde dehydrogenase ( istALDH) from the non- Saccharomyces yeast Issatchenkia terricola strain XJ-2 has been previously characterized. In this work, Lactococcus lactis with the NIsin Controlled Expression (NICE) System was applied to express the aldehyde dehydrogenase gene ( istALDH) in order to catalyze oxidation of acetaldehyde at low pH. A recombinant L. lactis NZ3900 was obtained and applied for the detoxification of acetaldehyde as whole-cell biocatalysts . The activity of IstALDH in L. lactis NZ3900 (pNZ8148- istALDH) reached 36.4 U mL when the recombinant cells were induced with 50 ng mL nisin at 20 °C for 2 h. The IstALDH activity of recombinant L. lactis cells showed higher stability at 37 °C and pH 4.0 compared with the crude enzyme. L. lactis NZ3900 (pNZ8148- istALDH) could convert acetaldehyde at pH 2.0 while the crude enzyme could not. Moreover, the resting cells of L. lactis NZ3900 (pNZ8148- istALDH) showed a 2.5-fold higher activity and better stability in catalyzing oxidation of acetaldehyde at pH 2.0 compared with that of Escherichia coli expressing the IstALDH. Taken together, the L. lactis cells expressing recombinant IstALDH are potential whole-cell biocatalysts that can be applied in the detoxification of aldehydes. [ABSTRACT FROM AUTHOR]

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2 S )-naringenin prenylation. The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops, along with a long side chain formed by nearly 120 amino acids. SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae . A mutant expressing the truncated enzyme at K62 site, designated as SfND8T-1-t62, produced the highest 8-PN titer. Molecular docking of SfN8DT-1-t62 with (2 S )-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue. Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates, which also indicated K185 was a potentially critical residue. Besides, the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation, while the saturated mutation of K185 showed a great decrease in 8-PN production, indicating K185 is vital for the activity of SfN8DT-1. Subsequently, overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L, which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis. Finally, 44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h, which is the highest 8-PN titer reported to date.

The (2 S )-naringenin is an important natural flavonoid with several bioactive effects on human health. It is also a key precursor in the biosynthesis of other high value compounds. The production of (2S)-naringenin is significantly influenced by the acetyl-CoA available in the cytosol. In this study, we increased the acetyl-CoA supply via the β-oxidation of fatty acids in the peroxisomes of Saccharomyces cerevisiae . Several lipases from different sources and PEX11 , FOX1 , FOX2 , and FOX3 , the key genes of the fatty acid β-oxidation pathway, were overexpressed during the production of (2 S )-naringenin in yeast. The level of acetyl-CoA was 0.205 nmol higher than that in the original strain and the production of (2 S )-naringenin increased to 286.62 mg/g dry cell weight when PEX11 was overexpressed in S. cerevisiae strain L07. Remarkable (2 S )-naringenin production (1129.44 mg/L) was achieved with fed-batch fermentation, with the highest titer reported in any microorganism. Our results demonstrated the use of fatty acid β-oxidation to increase the level of cytoplasmic acetyl-CoA and the production of its derivatives.

(2 S )-Naringenin, a (2 S )-flavanone, is widely used in the food, chemical, and pharmaceutical industries because of its diverse physiological activities. The production of (2 S )-naringenin in microorganisms provides an ideal source that reduces the cost of the flavonoid. To achieve efficient production of (2 S )-naringenin in Saccharomyces cerevisiae ( S. cerevisiae ), we constructed a biosynthetic pathway from p -coumaric acid, a cost-effective and more efficient precursor. The (2 S )-naringenin synthesis pathway genes were integrated into the yeast genome to obtain a (2 S )-naringenin production strain. After gene dosage experiments, the genes negatively regulating the shikimate pathway and inefficient chalcone synthase activity were verified as factors limiting (2 S )-naringenin biosynthesis. With fed-batch process optimization of the engineered strain, the titer of (2 S )-naringenin reached 648.63 mg/L from 2.5 g/L p -coumaric acid. Our results indicate that the constitutive production of (2 S )-naringenin from p -coumaric acid in S. cerevisiae is highly promising.