Overview of Mycelial Fungi - Lignin Destructors

In this work, the following genuses of mycelial fungi, capable of producing ligninolytic enzymes of various actions, were considered:Penicillium, Aspergillus, Fusariumand Altermaria. Fungi of the genus Aspergilluswere capable of producing laccase, manganese peroxidase and lignin peroxidase in the medium. Penicillium mostly produced laccase. Fusariumproduced laccase, aryl alcohol oxidase, manganesedependent peroxidase, manganese-independent peroxidase and lignin peroxidase. Alternariaproduced laccase, lignin peroxidase and manganese peroxidase. The results demonstrated the possibility of using specific substrates in the study of enzyme activity, as well as the influence of some factors introduced into the medium on the synthesis of enzymes. The auxiliary influence of these fungi on the synthesis of ligninolytic enzymes in symbiosis with otherswas considered. Keywords: mycelial fungi, ligninolytic enzymes, Penicillium, Aspergillus, Fusarium, Altermaria

A One-Pot Biocatalytic and Organocatalytic Cascade Delivers High Titers of 2-Ethyl-2-Hexenal from n-Butanol

Biocatalysis provides facile access to selective chemical transformations and helps satisfy sustainable chemical production criteria. However, the reaction scope of biocatalysts is significantly narrower compared to synthetic chemical transformations. Hybrid biocatalytic-chemocatalytic cascades expand the scope of products while maintaining many of the benefits associated with biocatalysis. Here, we report that single-pot systems with whole cell K. pastoris (ATCC® 28485™) or isolated enzyme alcohol oxidase (E 1.1.3.13) as oxidative biocatalysts with a lysine organocatalyst yields the commercial target, 2-ethyl-2-hexenal (2-EH) from n-butanol in a two-step hybrid cascade. Peak yields for both biocatalysts were achieved with 100 mM n-butanol at pH 8 and 30°C. The isolated enzyme slightly outperformed whole cell K. pastoris, reaching 73% conversion (4.7 g/L titers) compared to 61% (3.9 g/L titers) in whole cells systems. Titers could be improved for both biocatalysts (5.7 – 6.7 g/L) at increased butanol loading; however, this came at the expense of decreased yields. Compared to our initial results with a Gluconobactor oxidans whole cell biocatalyst, the reported system improves upon 2-EH titers by 2.8-3.3-fold at maximal yields.

Fatty Alcohol Oxidase 3 (FAO3) and FAO4b as Mediators Connect the Alcohol- and Alkane-forming Pathways in Arabidopsis Stem Wax

Alcohol- and alkane-forming pathways in cuticular wax biosynthesis are well characterized in Arabidopsis. However, potential interactions between the two pathways remain unclear. Our study revealed that mutation of CER4, the key gene in the alcohol-forming pathway, also led to a deficiency in alkane-forming pathway in distal stems. To trace the connection between these two pathways, we characterized two homologs of fatty alcohol oxidase (FAO), FAO3 and FAO4b, which were highly expressed in distal stems and localized to the endoplasmic reticulum. The amounts of waxes from the alkane-forming pathway were significantly decreased in stems of fao4b, and much lower in fao3 fao4b, indicative of an overlapping function for both proteins in wax synthesis. Additionally, overexpression of FAO3 and FAO4b in Arabidopsis resulted in a dramatic reduction of primary alcohols and significant increase of aldehydes and related waxes. Moreover, expressing FAO3 or FAO4b led to significantly decreased amounts of C18 - C26 alcohols in yeast co-expressing CER4 and FAR1. Collectively, these findings demonstrate that FAO3 and FAO4b are functionally redundant in suppression of primary alcohols accumulation and contribution to aldehyde production, which provides a missing and long sought-after link between the two pathways in wax biosynthesis.

A plasmonic gold nanofilm-based microfluidic chip for rapid and inexpensive droplet-based photonic PCR

Polymerase chain reaction (PCR) is a powerful tool for nucleic acid amplification and quantification. However, long thermocycling time is a major limitation of the commercial PCR devices in the point-of-care (POC). Herein, we have developed a rapid droplet-based photonic PCR (dpPCR) system, including a gold (Au) nanofilm-based microfluidic chip and a plasmonic photothermal cycler. The chip is fabricated by adding mineral oil to uncured polydimethylsiloxane (PDMS) to suppress droplet evaporation in PDMS microfluidic chips during PCR thermocycling. A PDMS to gold bonding technique using a double-sided adhesive tape is applied to enhance the bonding strength between the oil-added PDMS and the gold nanofilm. Moreover, the gold nanofilm excited by two light-emitting diodes (LEDs) from the top and bottom sides of the chip provides fast heating of the PCR sample to 230 °C within 100 s. Such a design enables 30 thermal cycles from 60 to 95 °C within 13 min with the average heating and cooling rates of 7.37 ± 0.27 °C/s and 1.91 ± 0.03 °C/s, respectively. The experimental results demonstrate successful PCR amplification of the alcohol oxidase (AOX) gene using the rapid plasmonic photothermal cycler and exhibit the great performance of the microfluidic chip for droplet-based PCR.

Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomyces antarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid

The development of enzymatic processes for the environmentally friendly production of 2,5-furandicarboxylic acid (FDCA), a renewable precursor for bioplastics, from 5-hydroxymethylfurfural (HMF) has gained increasing attention over the last years. Aryl-alcohol oxidases (AAOs) catalyze the oxidation of HMF to 5-formyl-2-furancarboxylic acid (FFCA) through 2,5-diformylfuran (DFF) and have thus been applied in enzymatic reaction cascades for the production of FDCA. AAOs are flavoproteins that oxidize a broad range of benzylic and aliphatic allylic primary alcohols to the corresponding aldehydes, and in some cases further to acids, while reducing molecular oxygen to hydrogen peroxide. These promising biocatalysts can also be used for the synthesis of flavors, fragrances, and chemical building blocks, but their industrial applicability suffers from low production yield in natural and heterologous hosts. Here we report on heterologous expression of a new aryl-alcohol oxidase, MaAAO, from Moesziomyces antarcticus at high yields in the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). Fed-batch fermentation of recombinant P. pastoris yielded around 750 mg of active enzyme per liter of culture. Purified MaAAO was highly stable at pH 2–9 and exhibited high thermal stability with almost 95% residual activity after 48 h at 57.5 °C. MaAAO accepts a broad range of benzylic primary alcohols, aliphatic allylic alcohols, and furan derivatives like HMF as substrates and some oxidation products thereof like piperonal or perillaldehyde serve as building blocks for pharmaceuticals or show health-promoting effects. Besides this, MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA) to FFCA, which has not been shown for any other AAO so far. Combining MaAAO with an unspecific peroxygenase oxidizing HMFCA to FFCA in one pot resulted in complete conversion of HMF to FDCA within 144 h. MaAAO is thus a promising biocatalyst for the production of precursors for bioplastics and bioactive compounds. Key points • MaAAO from M. antarcticus was expressed in P. pastoris at 750 mg/l. • MaAAO oxidized 5-hydroxymethyl-2-furancarboxylic acid (HMFCA). • Complete conversion of HMF to 2,5-furandicarboxylic acid by combining MaAAO and UPO.

Constitutive Expression in Komagataella phaffii of Mature Rhizopus oryzae Lipase Jointly with Its Truncated Prosequence Improves Production and the Biocatalyst Operational Stability

Rhizopus oryzae lipase (ROL) containing 28 C-terminal amino acids of the prosequence fused to the N-terminal mature sequence in ROL (proROL) was successfully expressed in the methylotrophic yeast Komagataella phaffii (Pichia pastoris) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP). Although the sequence encoding the mature lipase (rROL) was also transformed, no clones were obtained after three transformation cycles, which highlights the importance of the truncated prosequence to obtain viable transformed clones. Batch cultures of the K. phaffii strain constitutively expressing proROL scarcely influenced growth rate and exhibited a final activity and volumetric productivity more than six times higher than those obtained with proROL from K. phaffii under the methanol-inducible alcohol oxidase 1 promoter (PAOX1). The previous differences were less marked in fed-batch cultures. N-terminal analysis confirmed the presence of the 28 amino acids in proROL. In addition, immobilized proROL exhibited increased tolerance of organic solvents and an operational stability 0.25 and 3 times higher than that of immobilized rROL in biodiesel and ethyl butyrate production, respectively. Therefore, the truncated prosequence enables constitutive proROL production, boosts bioprocess performance and provides a more stable biocatalyst in two reactions in which lipases are mostly used at industrial level, esterification (ethyl butyrate) and transesterification (biodiesel).

Two adjacent C-terminal mutations enable expression of aryl-alcohol oxidase from Pleurotus eryngii in Pichia pastoris

Fungal aryl-alcohol oxidases (AAOs) are attractive biocatalysts because they selectively oxidize a broad range of aromatic and aliphatic allylic primary alcohols while yielding hydrogen peroxide as the only by-product. However, their use is hampered by challenging and often unsuccessful heterologous expression. Production of PeAAO1 from Pleurotus eryngii ATCC 90787 in Pichia pastoris failed, while PeAAO2 from P. eryngii P34 with an amino acid identity of 99% was expressed at high yields. By successively introducing mutations in PeAAO1 to mimic the sequence of PeAAO2, the double mutant PeAAO1 ER with mutations K583E and Q584R was constructed, that was successfully expressed in P. pastoris. Functional expression was enhanced up to 155 U/l via further replacements D361N (variant NER) or V367A (variant AER). Fed-batch cultivation of recombinant P. pastoris yielded up to 116 mg/l of active variants. Glycosylated PeAAO1 variants demonstrated high stability and catalytic efficiencies similar to PeAAO2. Interestingly, P. pastoris expressing PeAAO1 variant ER contained roughly 13 gene copies but showed similar volumetric activity as NER and AER with one to two gene copies and four times lower mRNA levels. Additional H-bonds and salt bridges introduced by mutations K583E and Q584R might facilitate heterologous expression by enhanced protein folding.Key points• PeAAO1 not expressed in P. pastoris and PeAAO2 well-expressed in Pichia differ at 7 positions.• Expression of PeAAO1 in P. pastoris achieved through mutagenesis based on PeAAO2 sequence.• Combination of K583E and Q584R is essential for expression of PeAAO1 in P. pastoris.