Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 10³ in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 10⁵ in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species.

Lactate is a valuable compound used in food, chemical, and pharmaceutical industries. High-value, optically pure L- or D-lactate can be synthesized microbially via specific dehydrogenases. The non-conventional yeast Scheffersomyces stipitis, which is known for fermenting both hexoses and pentoses, is a promising host for biochemical production from lignocellulosic biomass but does not naturally produce lactate. In this study, we engineered S. stipitis to produce lactate by expressing two codon-optimized bacterial L-lactate dehydrogenase genes under the control of strong native promoters. The engineered strain produced 7.42 g/L (0.46 g/g yield) and 11.67 g/L (0.58 g/g yield) lactate from glucose and xylose, respectively. The highest titer, 19.27 g/L (0.52 g/g yield), was achieved from 50 g/L xylose after 74 h. Increasing the fermentation temperature from 28 °C to 32 °C improved yield by 30%, while a neutralizing agent further enhanced yield by 25% and prevented lactate degradation following carbon depletion. Although the wildtype strain produced a significant amount of ethanol on both glucose and xylose, the engineered strain produced ethanol as a side product exclusively on glucose and not on xylose. This phenomenon could be advantageous for biotechnological applications and may reflect shifts in gene expression depending on the carbon source or even on the presence of lactate.

Due to the increasing demand for herbal supplements, this study investigates the effects of the ultrasound-assisted extraction (UAE) parameters (amplitude, time and temperature) on the extraction of polyphenols from laurel leaves, as this method enables the production of a range of extracts in a shorter time. UAE affects the colour of the extracts when an amplitude of 60, 80 and 100%, a time period of 3, 6 and 9 min and acetone or ethanol (30 and 70%, v/v) are used. The solvent had the greatest influence on the colour of the extract, which was positively related to the parameters b and ΔE (Std. Coeff. = 0.9696 and 0.9354) and negatively related to the values a and L (Std. Coeff. = −0.9741 and −0.5733). The solvent also influenced the recovery of total phenols and flavonoids, as well as most phenolic compounds. The highest levels of phenols and flavonoids were determined for 70% ethanol (28.04 and 10.73 mg/g) and 70% acetone (32.10 and 12.13 mg/g) at an amplitude of 100% for 9 min. Meanwhile, 70% ethanol at an amplitude of 100% for 9 min gave the highest amount of rosmarinic acid, with values of 216.32 mg/L, showing that it dominates among the phenolic compounds.