Non-Conventional Yeasts to Produce Aroma Compounds by Using Agri-Food Waste Materials

Nowadays, biotechnological applications are emphasized to ensure sustainable development by re-utilizing of waste materials to prevent ecological problems and to produce or recover compounds that may have positive effects on health. Yeasts are fascinating microorganisms that play a key role in several traditional and innovative processes. Although Saccharomyces is the most important genus of yeasts, and they are major producers of biotechnological products worldwide, a variety of other yeast genera and species than Saccharomyces, which called ‘non-Saccharomyces’ or ‘non-conventional’ yeasts also have important potential in order to use in biotechnological applications. Some of the non-conventional yeast strains offer a unique potential for biotechnological applications to produce valuable secondary metabolites due to their characteristics of surviving and growing in such extreme conditions, e.g. wide substrate range, rapid growth, thermotolerance, etc. In this review, we aimed to summarize potential biotechnological applications of some non-conventional yeasts (Kluyveromyces spp., Yarrowia spp., Pichia spp., Candida spp., etc.) to produce industrially important aroma compounds (phenylethyl alcohol, phenylethyl acetate, isobutyl acetate, diacetyl, etc.) by re-utilizing agri-food waste materials in order to prevent ecological problems and to produce or recover compounds that may have positive effects on health.

Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae

Mycosporine-like amino acids (MAAs), microbial secondary metabolites with ultraviolet (UV) absorption properties, are promising natural sunscreen materials. Due to the low efficiency of extracting MAAs from natural producers, production in heterologous hosts has recently received attention. Shinorine is a well characterized MAA with strong UV-A absorption property. Previous, we developed Saccharomyces cerevisiae strain producing shinorine by introducing four shinorine biosynthetic genes from cyanobacterium Nostoc punctiforme. Shinorine is produced from sedoheptulose 7-phosphate (S7P), an intermediate in the pentose phosphate pathway. Shinorine production was greatly improved by using xylose as a co-substrate, which can increase the S7P pool. However, due to a limited xylose-utilizing capacity of the engineered strain, glucose was used as a co-substrate to support cell growth. In this study, we further improved shinorine production by attenuating glucose catabolism via glycolysis, which can redirect the carbon flux from glucose to the pentose phosphate pathway favoring shinorine production. Of the strategies we examined to reduce glycolytic flux, deletion of HXK2, encoding hexokinase, was most effective in increasing shinorine production. Furthermore, by additional expression Ava3858 from Anabaena variabilis, encoding a rate-limiting enzyme 2-demethyl 4-deoxygadusol synthase, 68.4 mg/L of shinorine was produced in an optimized medium containing 14 g/L glucose and 6 g/L xylose, achieving a 2.2-fold increase compared with the previous strain.

Development of an Haa1-based biosensor for acetic acid sensing in Saccharomyces cerevisiae

Acetic acid is one of the main inhibitors of lignocellulosic hydrolysates and acetic acid tolerance is crucial for the development of robust cell factories for conversion of biomass. As a precursor of acetyl-CoA, it also plays an important role in central carbon metabolism. Thus, monitoring acetic acid levels is a crucial aspect when cultivating yeast. Transcription factor-based biosensors represent useful tools to follow metabolite concentrations. Here, we present the development of an acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1 that upon binding to acetic acid relocates to the nucleus. In the biosensor, a synthetic transcription factor consisting of Haa1 and BM3R1 from Bacillus megaterium was used to control expression of a reporter gene under a promoter containing BM3R1 binding sites. The biosensor did not drive expression under a promoter containing Haa1 binding sites and responded to acetic acid over a linear range spanning from 10 to 60 mM. To validate its applicability, the biosensor was integrated into acetic acid producing strains. A direct correlation between biosensor output and acetic acid production was detected. The developed biosensor enables high-throughput screening of strains producing acetic acid and could also be used to investigate acetic acid tolerant strain libraries.

Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

ABSTRACT Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production

ABSTRACT First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G–E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4–59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G–E2G production process.

Direct proof of the amyloid nature of yeast prions [PSI+] and [PIN+] by the method of immunoprecipitation of native fibrils

ABSTRACT Prions are proteins that can exist in several structurally and functionally distinct states, one or more of which is transmissible. Yeast proteins Sup35 and Rnq1 in prion state ([PSI+] and [PIN+], respectively) form oligomers and aggregates, which are transmitted from parents to offspring in a series of generations. Several pieces of indirect evidence indicate that these aggregates also possess amyloid properties, but their binding to amyloid-specific dyes has not been shown in vivo. Meanwhile, it is the specific binding to the Congo Red dye and birefringence in polarized light after such staining that is considered the gold standard for proving the amyloid properties of a protein. Here, we used immunoprecipitation to extract native fibrils of the Sup35 and Rnq1 proteins from yeast strains with different prion status. These fibrils are detected by electron microscopy, stained with Congo Red and exhibit yellow-green birefringence after such staining. All these data show that the Sup35 and Rnq1 proteins in prion state form amyloid fibrils in vivo. The technology of fibrils extraction in combination with standard cytological methods can be used to identify new pathological and functional amyloids in any organism and to analyze the structural features of native amyloid fibrils.

Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica

In recent years, there has been a growing interest in the use of renewable sources for bio-based production aiming at developing sustainable and feasible approaches towards a circular economy. Among these renewable sources, organic wastes (OWs) can be anaerobically digested to generate carboxylates like volatile fatty acids (VFAs), lactic acid, and longer-chain fatty acids that are regarded as novel building blocks for the synthesis of value-added compounds by yeasts. This review discusses on the processes that can be used to create valuable molecules from OW-derived VFAs; the pathways employed by the oleaginous yeast Yarrowia lipolytica to directly metabolize such molecules; and the relationship between OW composition, anaerobic digestion, and VFA profiles. The review also summarizes the current knowledge about VFA toxicity, the pathways by which VFAs are metabolized, and the metabolic engineering strategies that can be employed in Y. lipolytica to produce value-added biobased compounds from VFAs.

The N.C.Yeastract and CommunityYeastract databases to study gene and genomic transcription regulation in non-conventional yeasts

ABSTRACT Responding to the recent interest of the yeast research community in non-Saccharomyces cerevisiae species of biotechnological relevance, the N.C.Yeastract (http://yeastract-plus.org/ncyeastract/) was associated to YEASTRACT + (http://yeastract-plus.org/). The YEASTRACT + portal is a curated repository of known regulatory associations between transcription factors (TFs) and target genes in yeasts. N.C.Yeastract gathers all published regulatory associations and TF-binding sites for Komagataellaphaffii (formerly Pichia pastoris), the oleaginous yeast Yarrowia lipolytica, the lactose fermenting species Kluyveromyces lactis and Kluyveromyces marxianus, and the remarkably weak acid-tolerant food spoilage yeast Zygosaccharomyces bailii. The objective of this review paper is to advertise the update of the existing information since the release of N.C.Yeastract in 2019, and to raise awareness in the community about its potential to help the day-to-day work on these species, exploring all the information available in the global YEASTRACT + portal. Using simple and widely used examples, a guided exploitation is offered for several tools: (i) inference of orthologous genes; (ii) search for putative TF binding sites and (iii) inter-species comparison of transcription regulatory networks and prediction of TF-regulated networks based on documented regulatory associations available in YEASTRACT + for well-studied species. The usage potentialities of the new CommunityYeastract platform by the yeast community are also discussed.