Lignocellulose-Degrading Enzymes: A Biotechnology Platform for Ferulic Acid Production from Agro-Industrial Side Streams

Biorefining by enzymatic hydrolysis (EH) of lignocellulosic waste material due to low costs and affordability has received enormous interest amongst scientists as a potential strategy suitable for the production of bioactive ingredients and chemicals. In the present study, a sustainable and eco-friendly approach to the extraction of bound ferulic acid (FA) has been demonstrated using a single-step EH by a mixture of lignocellulose-degrading enzymes. For comparative purposes of the efficiency of EH, an online SFE-SFC-MS extraction and analysis approach was applied. The experimental results demonstrated up to 369.3 mg 100 g−1 FA released from rye bran after 48 h EH with Viscozyme L. The EH of wheat and oat bran with Viscoferm for 48 h resulted in 255.1 and 33.5 mg 100 g−1 of FA, respectively. The extraction of FA from bran matrix using the SFE-CO2-EtOH delivered up to 464.3 mg 100 g−1 of FA, though the extractability varied depending on the parameters used. The 10-fold and 30-fold scale-up experiments confirmed the applicability of EH as a bioprocessing method valid for industrial-scale. The highest yield of FA in both scale-up experiments was obtained from rye bran after 48 h of EH with Viscozyme L. In purified extracts, the absence of xylose, arabinose, and glucose as final degradation products of lignocellulose was proven by a HPLC-RID system. Up to 94.0% purity of FA was achieved by SPE using the polymeric reversed-phase Strata X column and 50% EtOH as eluent.

Ferulic acid production by metabolically engineered Escherichia coli

Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics. Production of FA by eco‐friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p-coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as caffeic acid O-methytransferase. Fermentation result revealed JM109(DE3) as a more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 or p15A), and the combination p15a-T5 works best. To further improve FA production, E. coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also overexpressed. Based on the flask fermentation data which show that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E. coli K-12 strains reported to the best of our knowledge.

Characterization of Feruloyl Esterase from Bacillus pumilus SK52.001 and Its Application in Ferulic Acid Production from De-Starched Wheat Bran

Feruloyl esterase (FAE; EC 3.1.1.73) catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl group in an esterified sugar to assist in waste biomass degradation or to release ferulic acid (FA). An FAE-producing strain was isolated from humus soil samples and identified as Bacillus pumilus SK52.001. The BpFAE gene from B. pumilus SK52.001 was speculated and heterogeneously expressed in Bacillus subtilis WB800 for the first time. The enzyme exists as a monomer with 303 amino acids and a molecular mass of 33.6 kDa. Its specific activity was 377.9 ± 10.3 U/ (mg protein), using methyl ferulate as a substrate. It displays an optimal alkaline pH of 9.0, an optimal temperature of 50 °C, and half-lives of 1434, 327, 235, and 68 min at 50, 55, 60, and 65 °C, respectively. Moreover, the purified BpFAE released 4.98% FA of the alkali-acidic extractable FA from de-starched wheat bran (DSWB). When the DSWB was enzymatically degraded by the synergistic effect of the BpFAE and commercial xylanase, the FA amount reached 49.47%. It suggested that the alkaline BpFAE from B. pumilus SK52.001, which was heterologously expressed in B. subtilis WB800, possesses great potential for biomass degradation and achieving high-added value FA production from food by-products.

Ferulic Acid production by metabolically engineered Escherichia coli

Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics areas. Production of FA by eco-friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding Tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p - coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as Caffeic acid O-methytransferase. Fermentation results revealed JM109(DE3) as more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 ori or p15a ori), and the combination p15a-T5 works best. To further improve FA production, E.coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also over expressed. Based on the flask fermentation data which shows that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E.coli K-12 strains reported to the best of our knowledge.

Comparison of Enzyme Secretion and Ferulic Acid Production by Escherichia coli Expressing Different Lactobacillus Feruloyl Esterases

Construction of recombinant Escherichia coli strains carrying feruloyl esterase genes for secretory expression offers an attractive way to facilitate enzyme purification and one-step production of ferulic acid from agricultural waste. A total of 10 feruloyl esterases derived from nine Lactobacillus species were expressed in E. coli BL21 (DE3) to investigate their secretion and ferulic acid production. Extracellular activity determination showed all these Lactobacillus feruloyl esterases could be secreted out of E. coli cells. However, protein analysis indicated that they could be classified as three types. The first type presented a low secretion level, including feruloyl esterases derived from Lactobacillus acidophilus and Lactobacillus johnsonii. The second type showed a high secretion level, including feruloyl esterases derived from Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus helveticus. The third type also behaved a high secretion level but easy degradation, including feruloyl esterases derived from Lactobacillus farciminis, Lactobacillus fermentum, and Lactobacillus reuteri. Moreover, these recombinant E. coli strains could directly release ferulic acid from agricultural waste. The highest yield was 140 μg on the basis of 0.1 g de-starched wheat bran by using E. coli expressed L. amylovorus feruloyl esterase. These results provided a solid basis for the production of feruloyl esterase and ferulic acid.

Growth, ferulic acid synthesis, and histochemistry of calli of Pouteria caimito (Ruiz & Pav.) Radlk under different light qualities

Interest in harnessing biological processes for the production of bioactive compounds from natural sources has increased considerably. The manipulation of light quality in callus culture is considered a promising strategy for in vitro metabolite production. The objective of this study was to investigate the influence of light quality on the growth, histochemistry, and ferulic acid production of callus cultures of P. caimito. For in vitro callus induction, 1-cm2 leaf fragments were cultured in 50% MS medium supplemented with 2,4-dichlorophenoxyacetic acid and benzylaminopurine in the absence or presence of light (white, blue, green, yellow, or red). Methanol extraction was performed with partitioning of the extract and subsequent quantification of ferulic acid using a liquid chromatograph coupled to a mass spectrometer. The presence of light promoted greater growth than the absence of light. In the interaction between light quality and culture time, linear biomass growth until 28 days was observed under yellow, red, and blue lights and in the dark. The highest callus biomass values were observed under yellow and red lights. The histochemical tests showed the presence of phenolic compounds, alkaloids, flavonoids, and terpenes. The exposure of calli cultured under white light to different light qualities and culture times did not result in significant differences in the concentration or yield of ferulic acid.