Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates.

Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production.

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by ß-1,4 and ß-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.

Epigenetic manipulation of filamentous fungi for biotechnological applications: a systematic review.

The study of the epigenetic regulation of gene function has reached pivotal importance in life sciences in the last decades. The mechanisms and effects of processes such as DNA methylation, histone posttranslational modifications and non-coding RNAs, as well as their impact on chromatin structure and dynamics, are clearly involved in physiology homeostasis in plants, animals and microorganisms. In the fungal kingdom, studies on the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe contributed enormously to the elucidation of the eukaryote epigenetic landscape. Epigenetic regulation plays a central role in the expression of virulence attributes of human pathogens such as Candida albicans. In this article, we review the most recent studies on the effects of drugs capable of altering epigenetic states and on the impact of chromatin structure-related genes deletion in filamentous fungi. Emphasis is given on plant and insect pathogens, endophytes, secondary metabolites and cellulases/xylanases producing species.

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production.

The methylotrophic yeast Hansenula polymorpha, known as a non-conventional yeast, is used for the last 30 years for the production of recombinant proteins, including enzymes, vaccines, and biopharmaceuticals. Although a large number of reviews have been published elucidating the applications of this yeast as a cell factory, the latest was released about 10 years ago. Therefore, this review aimed at summarizing available information on the use of H. polymorpha as a host for recombinant protein production in the last decade. Examples of chemicals and virus-like particles produced using this yeast also are discussed. Firstly, the aspects that feature this yeast as a host for recombinant protein production are highlighted including the techniques available for its genetic manipulation as well as strategies for cultivation in bioreactors. Special attention is given to the novel genomic editing tools, mainly CRISPR/Cas9 that was recently established in this yeast. Finally, recent examples of using H. polymorpha as an expression platform are presented and discussed. The production of human Parathyroid Hormone (PTH) and Staphylokinase (SAK) in H. polymorpha are described as case studies for process establishment in this yeast. Altogether, this review is a guideline for this yeast utilization as an expression platform bringing a thorough analysis of the genetic aspects and fermentation protocols used up to date, thus encouraging the production of novel biomolecules in H. polymorpha.