Comparing laboratory and industrial yeast platforms for the direct conversion of cellobiose into ethanol under simulated industrial conditions.

An engineered yeast producing all the cellulases needed for cellulose saccharification could produce ethanol from lignocellulose at a lower cost. This study aimed to express fungal ß-glucosidases in Saccharomyces cerevisiae to convert cellobiose into ethanol. Furthermore, two engineering platforms (laboratory vs industrial strain) have been considered towards the successful deployment of the engineered yeast under simulated industrial conditions. The industrial S. cerevisiae M2n strain was engineered through the δ-integration of the ß-glucosidase Pccbgl1 of Phanerochaete chrysosporium. The most efficient recombinant, M2n[pBKD2-Pccbgl1]-C1, was compared to the laboratory S. cerevisiae Y294[Pccbgl1] strain, expressing Pccbgl1 from episomal plasmids, in terms of cellobiose fermentation in a steam exploded sugarcane bagasse pre-hydrolysate. Saccharomyces cerevisiae Y294[Pccbgl1] was severely hampered by the pre-hydrolysate. The industrial M2n[pBKD2-Pccbgl1]-C1 could tolerate high inhibitors-loading in pre-hydrolysate under aerobic conditions. However, in oxygen limited environment, the engineered industrial strain displayed ethanol yield higher than the laboratory Y294[Pccbgl1] only when supplemented with supernatant containing further recombinant ß-glucosidase. This study showed that the choice of the host strain is crucial to ensure bioethanol production from lignocellulose. A novel cellobiose-to-ethanol route has been developed and the recombinant industrial yeast could be a promising platform towards the future consolidated bioprocessing of lignocellulose into ethanol.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases.

The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e., strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase.

Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese.

Four LAB strains, isolated from Bulgarian home made white brine cheese, were selected for their effective inhibition against Listeria monocytogenes. According to their biochemical and physiological characteristics, the strains were classified as members of Enterococcus genus, and then identified as Enterococcus faecium by 16S rDNA sequencing. Their bacteriocin production and inhibitory spectrum were evaluated together with the occurrence of several bacteriocin genes (entA, entB, entP, entL50B). Their virulence potential and safety was assessed both using PCR targeted to the genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for antibiotic resistance, gelatinase, lipase, DNAse and α- and ß-haemolysis. The E. faecium strains harboured at least one enterocin gene while the occurrence of virulence, antibiotic resistance and biogenic amines genes was limited. Considering their strong antimicrobial activity against L. monocytogenes strains, the four E. faecium strains exhibited promising potential as bio-preservatives cultures for fermented food productions.

Copolymers as a turning point for large scale polyhydroxyalkanoates applications.

Traditional plastics reshaped the society thanks to their brilliant properties and cut-price manufacturing costs. However, their protracted durability and limited recycling threaten the environment. Worthy alternatives seem to be polyhydroxyalkanoates, compostable biopolymers produced by several microbes. The most common 3-hydroxybutyrate homopolymer has limited applications calling for copolymers biosynthesis to enhance material properties. As a growing number of researches assess the discovery of novel comonomers, great endeavors are dedicated as well to copolymers production scale-up, where the choice of the microbial carbon source significantly affects the overall economic feasibility. Diving into novel metabolic pathways, engineered strains, and cutting-edge bioprocess strategies, this review aims to survey up-to-date publications about copolymers production, focusing primarily on precursors origins. Specifically, in the core of the review, copolymers precursors have been divided into three categories based on their economic value: the costliest structurally related ones, the structurally unrelated ones, and finally various low-cost waste streams. The combination of cheap biomasses, efficient pretreatment strategies, and robust microorganisms paths the way towards the development of versatile and circular polymers. Conceived to researchers and industries interested in tackling polyhydroxyalkanoates production, this review explores an angle often underestimated yet of prime importance: if PHAs copolymers offer advanced properties and sustainable end-of-life, the feedstock choice for their upstream becomes a major factor in the development of plastic substitutes.

Autotrophic production of polyhydroxyalkanoates using acidogenic-derived H2 and CO2 from fruit waste.

The environmental concerns regarding fossil plastics call for alternative biopolymers such as polyhydroxyalkanoates (PHAs) whose manufacturing costs are however still too elevated. Autotrophic microbes like Cupriavidus necator, able to convert CO2 and H2 into PHAs, offer an additional strategy. Typically, the preferred source for CO2 and H2 are expensive pure gases or syngas, which has toxic compounds for most PHAs-accumulating strains. In this work, for the first time, H2 and CO2 originating from an acidogenic reactor were converted autotrophically into poly(3-hydroxybutyrate) P(3HB). During the first stage, a mixed microbial community continuously catabolized melon waste into H2 (26.7 %) and CO2 (49.2 %) that were then used in a second bioreactor by C. necator DSM 545 to accumulate 1.7 g/L P(3HB). Additionally, the VFAs (13 gCOD/L) produced during acidogenesis were processed into 2.7 g/L of P(3HB-co-3HV). This is the first proof-of-concept of using acidogenic-derived H2 and CO2 from fruit waste to produce PHAs.

Renewing Lost Genetic Variability with a Classical Yeast Genetics Approach.

Due to their long domestication time course, many industrial Saccharomyces cerevisiae strains are adopted in numerous processes mostly for historical reasons instead of scientific and technological needs. As such, there is still significant room for improvement for industrial yeast strains relying on yeast biodiversity. This paper strives to regenerate biodiversity with the innovative application of classic genetic methods to already available yeast strains. Extensive sporulation was indeed applied to three different yeast strains, specifically selected for their different origins as well as backgrounds, with the aim of clarifying how new variability was generated. A novel and easy method to obtain mono-spore colonies was specifically developed, and, to reveal the extent of the generated variability, no selection after sporulation was introduced. The obtained progenies were then tested for their growth in defined mediums with high stressor levels. A considerable and strain-specific increase in both phenotypic and metabolomic variability was assessed, and a few mono-spore colonies were found to be of great interest for their future exploitation in selected industrial processes.

Codon-optimized glucoamylase sGAI of Aspergillus awamori improves starch utilization in an industrial yeast.

The development of a yeast that converts raw starch to ethanol in one step (called consolidated bioprocessing) could yield large cost reductions in the bioethanol industry. The aim of this study was to develop an efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production. A native and codon-optimized variant of the Aspergillus awamori glucoamylase gene were expressed in the S. cerevisiae Y294 laboratory strain. Codon optimization resulted to be effective and the synthetic sequence sGAI was then δ-integrated into a S. cerevisiae strain with promising industrial fermentative traits. The mitotically stable recombinant strains showed high enzymatic capabilities both on soluble and raw starch (2425 and 1140 nkat/g dry cell weight, respectively). On raw corn starch, the engineered yeasts exhibited improved fermentative performance with an ethanol yield of 0.42 (g/g), corresponding to 75 % of the theoretical maximum yield.

Effects of heat treatment on microbial communities of granular sludge for biological hydrogen production.

Dark fermentation shares many features with anaerobic digestion with the exception that to maximize hydrogen production, methanogens and hydrogen-consuming bacteria should be inhibited. Heat treatment is widely applied as an inoculum pre-treatment due to its effectiveness in inhibiting methanogenic microflora but it may not exclusively select for hydrogen-producing bacteria. This work evaluated the effects of heat treatment on microbial viability and structure of anaerobic granular sludge. Heat treatment was carried out on granular sludge at 100 °C with four residence times (0.5, 1, 2 and 4 h). Hydrogen production of treated sludges was studied from glucose by means of batch test at different pH values. Results indicated that each heat treatment strongly influenced the granular sludge resulting in microbial communities having different hydrogen productions. The highest hydrogen yields (2.14 moles of hydrogen per mole of glucose) were obtained at pH 5.5 using the sludge treated for 4 h characterized by the lowest CFU concentration (2.3 × 10(3)CFU/g sludge). This study demonstrated that heat treatment should be carefully defined according to the structure of the sludge microbial community, allowing the selection of highly efficient hydrogen-producing microbes.

Polyhydroxyalkanoate Production from Fruit and Vegetable Waste Processing.

Traditional plastics represent a tremendous threat to the environment because of increases in polluting manufacturing as well as their very extended degradation time. Polyhydroxyalkanoates (PHAs) are polymers with similar performance to plastic but are compostable and synthesizable from renewable sources and therefore could be a replacement for fossil-based plastics. However, their production costs are still too high, thus demanding the investigation of new and cheap substrates. In this sense, agricultural wastes are attractive because they are inexpensive and largely available. Specifically, fruit and vegetables are rich in sugars that could be fermented into PHAs. In this work two strains, Cupriavidus necator DSM 545 and Hydrogenophaga pseudoflava DSM 1034, well-known PHA-producing microbes, were screened for their ability to grow and accumulate PHAs. Ten different fruit and vegetable processing waste streams, never before reported in combination with these strains, were tested. Residues from red apple and melon were found to be the most suitable feedstocks for PHA production. Under specific selected conditions, C. necator DSM 545 accumulated up to 7.4 and 4.3 g/L of 3-hydroxybutyrate (3HB) from red apple and melon, respectively. Copolymer production was also obtained from melon. These results confirm the attractiveness of food processing waste as a promising candidate for PHA production. Ultimately, these novel substrates draw attention for future studies on process optimization and upscaling with C. necator.

Engineering Cupriavidus necator DSM 545 for the one-step conversion of starchy waste into polyhydroxyalkanoates.

Starch-rich by-products could be efficiently exploited for polyhydroxyalkanoates (PHAs) production. Unfortunately, Cupriavidus necator DSM 545, one of the most efficient PHAs producers, is not able to grow on starch. In this study, a recombinant amylolytic strain of C. necator DSM 545 was developed for the one-step PHAs production from starchy residues, such as broken rice and purple sweet potato waste. The glucodextranase G1d from Arthrobacter globiformis I42 and the α-amylase amyZ from Zunongwangia profunda SM-A87 were co-expressed into C. necator DSM 545. The recombinant C. necator DSM 545 #11, selected for its promising hydrolytic activity, produced high biomass levels with noteworthy PHAs titers: 5.78 and 3.65 g/L from broken rice and purple sweet potato waste, respectively. This is the first report on the engineering of C. necator DSM 545 for efficient amylase production and paves the way to the one-step conversion of starchy waste into PHAs.

Innovative co-production of polyhydroxyalkanoates and methane from broken rice.

Broken rice, a low-cost starchy residue of the rice industry, can be an interesting substrate to reduce the polyhydroxyalkanoates (PHAs) production cost. However, since the most common PHAs-producing strains lack amylases, this waste must be firstly hydrolysed by additional commercial enzymes. In this work, the acidogenesis phase of the anaerobic digestion was exploited as efficient hydrolysis step to convert broken rice into volatile fatty acids (VFAs) to be used as PHAs carbon source by Cupriavidus necator DSM 545, one of the most promising PHAs-producing microbes. Broken rice, both non-hydrolysed and enzymatically hydrolysed, was processed in two continuous stirred tank reactors, at hydraulic retention times (HRT) of 5, 4 and, 3 days, to produce VFAs. The highest VFAs levels were obtained from non-hydrolysed broken rice which was efficiently exploited for PHAs accumulation by C. necator DSM 545. PHAs contents were higher after 96 h of incubation and, noteworthy, reached the highest value of 0.95 g/L in the case of 4 days HRT without any chemicals supplementation, except vitamins. Moreover, in view of a biorefinery approach, the residual solid fraction was used for methane production resulting in promising CH4 levels. Methane yields were very promising again for 4 days HRT. As such, this HRT resulted to be the most suitable to obtain effluents with high promise in terms of both PHAs accumulation and CH4 production. In addition, these results demonstrate that broken rice could be efficiently processed into two valuable products without any costly enzymatic pre-treatment and pave the way for future biorefining approaches where this by-product can be converted in a cluster of added-value compounds. Techno-economical estimations are in progress to assess the feasibility of the entire process, in view of supporting the low-cost conversion of organic waste into valuable products.

Biodiversity and Bioprospecting of Fungal Endophytes from the Antarctic Plant Colobanthus quitensis.

Microorganisms from extreme environments are considered as a new and valuable reservoir of bioactive molecules of biotechnological interest and are also utilized as tools for enhancing tolerance to (a)biotic stresses in crops. In this study, the fungal endophytic community associated with the leaves of the Antarctic angiosperm Colobanthus quitensis was investigated as a new source of bioactive molecules. We isolated 132 fungal strains and taxonomically annotated 26 representative isolates, which mainly belonged to the Basidiomycota division. Selected isolates of Trametes sp., Lenzites sp., Sistotrema sp., and Peniophora sp. displayed broad extracellular enzymatic profiles; fungal extracts from some of them showed dose-dependent antitumor activity and inhibited the formation of amyloid fibrils of α-synuclein and its pathological mutant E46K. Selected fungal isolates were also able to promote secondary root development and fresh weight increase in Arabidopsis and tomato and antagonize the growth of pathogenic fungi harmful to crops. This study emphasizes the ecological and biotechnological relevance of fungi from the Antarctic ecosystem and provides clues to the bioprospecting of Antarctic Basidiomycetes fungi for industrial, agricultural, and medical applications.

Selection of Superior Yeast Strains for the Fermentation of Lignocellulosic Steam-Exploded Residues.

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants.

Efficient production of polyhydroxybutyrate from slaughterhouse waste using a recombinant strain of Cupriavidus necator DSM 545.

Slaughterhouse residues are greatly available and can pose a threat to the environment if not disposed of correctly. Such by-products can be proficiently processed into polyhydroxyalkanoates by accurately selected and developed bacterial strains. Cupriavidus necator DSM 545, one of the most efficient polyhydroxyalkanoates-producing strain, cannot grow well on fatty substrates. In this work, a recombinant lipolytic C. necator microbe was developed for the efficient conversion of slaughtering by-products into polyhydroxyalkanoates. Two lipase sequences, lipC and lipH of Pseudomonas stutzeri BT3, were effectively expressed in C. necator DSM 545. The engineered strain C. necator DSM 545 JR11, selected for the outstanding extracellular lipolytic activity, produced high levels of polyhydroxyalkanoates (nearly 65% of cell dry mass) from udder, jowl and membrane caul fat. This research is crucial to the cost-effective one-step processing of slaughterhouse waste into polyhydroxyalkanoates with useful applications in several industrial and medical sectors.

Natural Saccharomyces cerevisiae Strain Reveals Peculiar Genomic Traits for Starch-to-Bioethanol Production: the Design of an Amylolytic Consolidated Bioprocessing Yeast.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

Light excess stimulates Poly-beta-hydroxybutyrate yield in a mangrove-isolated strain of Synechocystis sp.

Poly-ß-hydroxybutyrate (PHB) is a biodegradable biopolymer that may replace fossil-based plastics reducing its negative environmental impact. One highly sustainable strategy to produce these biopolymers is the exploitation of photosynthetic microorganisms that use sunlight and CO2 to produce biomass and subsequently, PHB. Exploring environmental biological diversity is a powerful tool to find resilient microorganisms potentially exploitable to produce bioproducts. In this work, a cyanobacterium (Synechocystis sp.) isolated from a contaminated area close to an important industrial complex was shown to produce PHB under different culture conditions. Carbon, nutrients supply and light intensity impact on biomass and PHB productivity were assessed, showing that the highest yield of PHB achieved was 241 mg L-1 (31%dcw) under high light intensity. Remarkably this condition not only stimulated PHB accumulation by 70% compared to other conditions tested but also high cellular duplication rate, maximizing the potential of this strain for PHB production.

Conversion of Starchy Waste Streams into Polyhydroxyalkanoates Using Cupriavidus necator DSM 545.

Due to oil shortage and environmental problems, synthetic plastics have to be replaced by different biodegradable materials. A promising alternative could be polyhydroxyalkanoates (PHAs), and the low-cost abundant agricultural starchy by-products could be usefully converted into PHAs by properly selected and/or developed microbes. Among the widely available starchy waste streams, a variety of residues have been explored as substrates, such as broken, discolored, unripe rice and white or purple sweet potato waste. Cupriavidus necator DSM 545, a well-known producer of PHAs, was adopted in a simultaneous saccharification and fermentation (SSF) process through an optimized dosage of the commercial amylases cocktail STARGEN™ 002. Broken rice was found to be the most promising carbon source with PHAs levels of up to 5.18 g/L. This research demonstrates that rice and sweet potato waste are low-cost feedstocks for PHAs production, paving the way for the processing of other starchy materials into bioplastics.

Delta-Integration of Single Gene Shapes the Whole Metabolomic Short-Term Response to Ethanol of Recombinant Saccharomyces cerevisiae Strains.

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial Saccharomyces cerevisiae 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains' tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome.

A yeast metabolome-based model for an ecotoxicological approach in the management of lignocellulosic ethanol stillage.

Lignocellulosic bioethanol production results in huge amounts of stillage, a potentially polluting by-product. Stillage, rich in heavy metals and, mainly, inhibitors, requires specific toxicity studies to be adequately managed. To this purpose, we applied an FTIR ecotoxicological bioassay to evaluate the toxicity of lignocellulosic stillage. Two weak acids and furans, most frequently found in lignocellulosic stillage, have been tested in different mixtures against three Saccharomyces cerevisiae strains. The metabolomic reaction of the test microbes and the mortality induced at various levels of inhibitor concentration showed that the strains are representative of three different types of response. Furthermore, the relationship between concentrations and FTIR synthetic stress indexes has been studied, with the aim of defining a model able to predict the concentrations of inhibitors in stillage, resulting in an optimized predictive model for all the strains. This approach represents a promising tool to support the ecotoxicological management of lignocellulosic stillage.

Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-Pccbgl1]-C1 Engineered by Multiple δ-Integration of a Fungal ß-Glucosidase Gene.

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple δ-integration of the ß-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the δ-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple δ-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.