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 .

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Hyaluronic acid (HA) is a biopolymer of repeating units of glucuronic acid and Nacetylglucosamine. Its market was valued at USD 8.9 billion in 2019. Traditionally, HA has been obtained from rooster comb-like animal tissues and fermentative cultures of attenuated pathogenic streptococci. Various attempts have been made to engineer a safe micro-organism for HA synthesis; however, the HA titres obtained from these attempts are in general still lower than those achieved by natural, pathogenic producers. In this scenario, ways to increase HA molecule length and titres in already constructed strains are gaining attention in the last years, but no recent publication has reviewed the main genetic strategies applied to improve HA production on heterologous hosts. In light of that, we hereby compile the advances made in the engineering of micro-organisms to improve HA synthesis.

Summary 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.

Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.