Production of 9,21-dihydroxy-20-methyl-pregna-4-en-3-one from phytosterols in Mycobacterium neoaurum by modifying multiple genes and improving the intracellular environment

Background Steroid drugs are essential for disease prevention and clinical treatment. However, due to intricated steroid structure, traditional chemical methods are rarely implemented into the whole synthetic process for generating steroid intermediates. Novel steroid drug precursors and their ideal bacterial strains for industrial production have yet to be developed. Among these, 9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HP) is a novel steroid drug precursor, suitable for the synthesis of corticosteroids. In this study, a combined strategy of blocking Δ1-dehydrogenation and the C19 pathway as well as improving the intracellular environment was investigated to construct an effective 9-OH-4-HP-producing strain. Results The Δ1-dehydrogenation-deficient strain of wild-type Mycobacterium neoaurum DSM 44074 produces 9-OH-4-HP with a molar yield of 4.8%. Hsd4A, encoding a β-hydroxyacyl-CoA dehydrogenase, and fadA5, encoding an acyl-CoA thiolase, were separately knocked out to block the C19 pathway in the Δ1-dehydrogenation-deficient strain. The two engineered strains were able to accumulate 0.59 g L−1 and 0.47 g L−1 9-OH-4-HP from 1 g L−1 phytosterols, respectively. Furthermore, hsd4A and fadA5 were knocked out simultaneously in the Δ1-dehydrogenation-deficient strain. The 9-OH-4-HP production from the Hsd4A and FadA5 deficient strain was 11.9% higher than that of the Hsd4A deficient strain and 40.4% higher than that of the strain with FadA5 deficiency strain, respectively. The purity of 9-OH-4-HP obtained from the Hsd4A and FadA5 deficient strain has reached 94.9%. Subsequently, the catalase katE from Mycobacterium neoaurum and an NADH oxidase, nox, from Bacillus subtilis were overexpressed to improve the intracellular environment, leading to a higher 9-OH-4-HP production. Ultimately, 9-OH-4-HP production reached 3.58 g L−1 from 5 g L−1 phytosterols, and the purity of 9-OH-4-HP improved to 97%. The final 9-OH-4-HP production strain showed the best molar yield of 85.5%, compared with the previous reported strain with 30% molar yield of 9-OH-4-HP. Conclusion KstD, Hsd4A, and FadA5 are key enzymes for phytosterol side-chain degradation in the C19 pathway. Double deletion of hsd4A and fadA5 contributes to the blockage of the C19 pathway. Improving the intracellular environment of Mycobacterium neoaurum during phytosterol bioconversion could accelerate the conversion process and enhance the productivity of target sterol derivatives.

Aqueous chemical bleaching of 4-nitrophenol brown carbon by hydroxyl radicals; products, mechanism and light absorptivity

The reaction of hydroxyl radicals (OH) with 4-nitrophenol (4-NP) in the aqueous solution was investigated at pH = 2 and 9. As a result, the molar yield of the phenolic products was measured to be 0.20 ± 0.05 at pH = 2 and 0.40 ± 0.1 at pH = 9. The yield of 4-nitrocatechol (4-NC) was higher at pH = 9; at the same time, a lower number of phenolic products was observed due to the hydrolysis and other irreversible reactions at pH > 7. Mineralization investigated with total organic carbon (TOC) technique showed that after 4-NP was completely consumed approx. 85 % of the organic carbon remained in the aqueous solution. Hence, up to 65 % of the organic carbon that remained in the aqueous solution accounted for the open-ring non-phenolic products. The light absorptivity of the reaction solution between 250 and 600 nm decreased as a result of OH reaction with 4-NP. At the same time, 4-NP solution showed some resistance to chemical bleaching due to the formation of the light-absorbing by-products. This phenomenon effectively prolongs the time-scale of chemical bleaching or 4-NP via reaction with OH by a factor of 3–1.5 at pH 2 and 9, respectively. The experimental data acquired indicated that both photolysis and reaction with OH can be important removal processes of the atmospheric brown-carbon from the aqueous particles containing 4-NP.

Coproduction of 5-Aminovalerate and δ-Valerolactam for the Synthesis of Nylon 5 From L-Lysine in Escherichia coli

The compounds 5-aminovalerate and δ-valerolactam are important building blocks that can be used to synthesize bioplastics. The production of 5-aminovalerate and δ-valerolactam in microorganisms provides an ideal source that reduces the cost. To achieve efficient biobased coproduction of 5-aminovalerate and δ-valerolactam in Escherichia coli, a single biotransformation step from L-lysine was constructed. First, an equilibrium mixture was formed by L-lysine α-oxidase RaiP from Scomber japonicus. In addition, by adjusting the pH and H2O2 concentration, the titers of 5-aminovalerate and δ-valerolactam reached 10.24 and 1.82 g/L from 40 g/L L-lysine HCl at pH 5.0 and 10 mM H2O2, respectively. With the optimized pH value, the δ-valerolactam titer was improved to 6.88 g/L at pH 9.0 with a molar yield of 0.35 mol/mol lysine. The ratio of 5AVA and δ-valerolactam was obviously affected by pH value. The ratio of 5AVA and δ-valerolactam could be obtained in the range of 5.63:1–0.58:1 at pH 5.0–9.0 from the equilibrium mixture. As a result, the simultaneous synthesis of 5-aminovalerate and δ-valerolactam from L-lysine in Escherichia coli is highly promising. To our knowledge, this result constitutes the highest δ-valerolactam titer reported by biological methods. In summary, a commercially implied bioprocess developed for the coproduction of 5-aminovalerate and δ-valerolactam using engineered Escherichia coli.

Novel Cold-Adapted Recombinant Laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a Green Catalyst in Biotransformation

Cold-adapted enzymes are useful tools in the organic syntheses conducted in mixed aqueous-organic or non-aqueous solvents due to their molecular flexibility that stabilizes the proteins in low water activity environments. A novel psychrophilic laccase gene from Kabatiella bupleuri, G3 IBMiP, was spliced by Overlap‑Extension PCR (OE-PCR) and expressed in Pichia pastoris. Purified recombinant KbLcc1 laccase has an optimal temperature of 30 °C and pH of 3.5, 5.5, 6.0, and 7.0 in the reaction with 2,2′‑azino‑bis(3‑ethylbenzothiazoline‑6‑sulfonic acid) (ABTS), guaiacol, sinapic acid, and syringaldazine, respectively. Moreover, laccase KbLcc1 is highly thermolabile, as it loses 40% of activity after 30 min at 40 °C and is inactivated at 50 °C after the same period of incubation. The new enzyme remained active with 1 mM of Ni2+, Cu2+, Mn2+, and Zn2+ and with 2 mM of Co2+, Ca2+, and Mg2+, but Fe2+ greatly inhibited the laccase activity. Moreover, 1% ethanol had no impact on KbLcc1, although acetone and ethyl acetate decreased the laccase activity. The presence of hexane (40%, v/v) caused a 58% increase in activity. Laccase KbLcc1 could be applied in the decolorization of synthetic dyes and in the biotransformation of ferulic acid to vanillin. After 5 days of reaction at 20 °C, pH 3.5, with 1 mM ABTS as a mediator, the vanillin concentration was 21.9 mg/L and the molar yield of transformation reached 14.39%.

Understanding the NaCl-dependent behavior of hydrogen production of a marine bacterium,Vibrio tritonius

Biohydrogen is one of the most suitable clean energy sources for sustaining a fossil fuel independent society. The use of both land and ocean bioresources as feedstocks show great potential in maximizing biohydrogen production, but sodium ion is one of the main obstacles in efficient bacterial biohydrogen production.Vibrio tritoniusstrain AM2 can perform efficient hydrogen production with a molar yield of 1.7 mol H2/mol mannitol, which corresponds to 85% theoretical molar yield of H2production, under saline conditions. With a view to maximizing the hydrogen production using marine biomass, it is important to accumulate knowledge on the effects of salts on the hydrogen production kinetics. Here, we show the kinetics in batch hydrogen production ofV. tritoniusstrain AM2 to investigate the response to various NaCl concentrations. The modified Han–Levenspiel model reveals that salt inhibition in hydrogen production usingV. tritoniusstarts precisely at the point where 10.2 g/L of NaCl is added, and is critically inhibited at 46 g/L. NaCl concentration greatly affects the substrate consumption which in turn affects both growth and hydrogen production. The NaCl-dependent behavior of fermentative hydrogen production ofV. tritoniuscompared to that ofEscherichia coliJCM 1649 reveals the marine-adapted fermentative hydrogen production system inV. tritonius.V. tritoniusAM2 is capable of producing hydrogen from seaweed carbohydrate under a wide range of NaCl concentrations (5 to 46 g/L). The optimal salt concentration producing the highest levels of hydrogen, optimal substrate consumption and highest molar hydrogen yield is at 10 g/L NaCl (1.0% (w/v)).

The Role of the ydiB Gene, Which Encodes Quinate/Shikimate Dehydrogenase, in the Production of Quinic, Dehydroshikimic and Shikimic Acids in a PTS- Strain of Escherichia coli

The culture of engineered <i>Escherichia coli</i> for shikimic acid (SA) production results in the synthesis of quinic acid (QA) and dehydroshikimic acid (DHS), reducing SA yield and impairing downstream processes. The synthesis of QA by quinate/shikimate dehydrogenase (YdiB, <i>ydiB</i>) has been previously proposed; however, the precise role for this enzyme in the production of QA in engineered strains of <i>E. coli</i> for SA production remains unclear. We report the effect of the inactivation or the overexpression of <i>ydiB</i> in <i>E. coli</i> strain PB12.SA22 on SA, QA, and DHS production in batch fermentor cultures. The results showed that the inactivation of <i>ydiB </i>resulted in a 75% decrease in the molar yield of QA and a 6.17% reduction in the yield of QA (mol/mol) relative to SA with respect to the parental strain. The overexpression of <i>ydiB</i> caused a 500% increase in the molar yield of QA and resulted in a 152% increase in QA (mol/mol) relative to SA, with a sharp decrease in SA production. Production of SA, QA, and DHS in parental and derivative <i>ydiB </i>strains suggests that the synthesis of QA results from the reduction of 3-dehydroquinate by YdiB before its conversion to DHS.

Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin

ABSTRACTThe Gram-positive bacteriumAmycolatopsissp. ATCC 39116 is used for the fermentative production of natural vanillin from ferulic acid on an industrial scale. The strain is known for its outstanding tolerance to this toxic product. In order to improve the productivity of the fermentation process, the strain's metabolism was engineered for higher final concentrations and molar yields. Degradation of vanillin could be decreased by more than 90% through deletion of thevdhgene, which codes for the central vanillin catabolism enzyme, vanillin dehydrogenase. This mutation resulted in improvement of the final concentration of vanillin by more than 2.2 g/liter, with a molar yield of 80.9%. Further improvement was achieved with constitutive expression of the vanillin anabolism genesechandfcs, coding for the enzymes feruloyl-coenzyme A (CoA) synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech). The transcription of both genes was shown to be induced by ferulic acid, which explains the unwanted adaptation phase in the fermentation process before vanillin was efficiently produced by the wild-type cells. Through the constitutive and enhanced expression of the two genes, the adaptation phase was eliminated and a final vanillin concentration of 19.3 g/liter, with a molar yield of 94.9%, was obtained. Moreover, an even higher final vanillin concentration of 22.3 g/liter was achieved, at the expense of a lower molar yield, by using an improved feeding strategy. This is the highest reported vanillin concentration reached in microbial fermentation processes without extraction of the product. Furthermore, the vanillin was produced almost without by-products, with a molar yield that nearly approached the theoretical maximum.IMPORTANCEMuch effort has been put into optimization of the biotechnological production of natural vanillin. The demand for this compound is growing due to increased consumer concerns regarding chemically produced food additives. Since this compound is toxic to most organisms, it has proven quite difficult to reach high concentrations and molar yields. This study shows that improvements in the final vanillin concentrations and molar yields can be made through a combination of modification of the fermentation parameters and molecular strain engineering, without the need for methods such as continuous extraction from the fermentation broth. Using this approach, we were able to reach a final vanillin concentration of 22.3 g/liter, which is the highest vanillin concentration reported to date that was generated withAmycolatopsissp. ATCC 39116 without additional extraction of the toxic product.

OPTIMIZATION OF BIOVANILLIN PRODUCTION OF LEMONGRASS LEAVES HYDROLYSATES THROUGH PHANEROCHAETE CHRYSOSPORIUM

The recent study has demonstrated the effects of different nitrogen sources on vanillin production by Phanerochaete chrysosporium. Primary screening supported maximum biotransformation of ferulic acid (from lemongrass leaves hydrolysate) to vanillin by using ammonium chloride and yeast extract as inorganic and organic nitrogen source, respectively. With the 2-level factorial analysis, the optimum conditions of vanillin production from ferulic acid by P. chrysosporium was achieved at 0.192g/L with a molar yield of 24.5%.

Degradation Kinetics of Arabinose and Furfural in Subcritical Water

To clarify the degradation mechanism of arabinose in a subcritical water system, the degradation process kinetics of arabinose and furfural in a subcritical water system was studied for temperatures from 210 to 250°C and under pressure of 10Mpa. The activation energy and frequency factor for the degradation of each substrate were estimated from the temperature dependence of the rate constant. The molar yield of a arabinose to furfural was ca. 0.3 at any temperature, Acidic compounds were also formed from the arabinose in proportion to the amount of consumed substrates. The formation of acidic compounds resulted in a rapid decrease in pH.