High-producing Strain Research Articles

Utilizing a high-throughput visualization screening technology to develop a genetically encoded biosensor for monitoring 5-aminolevulinic acid production in engineered Escherichia coli

5-Aminolevulinic acid (5-ALA) is a non-protein amino acid widely used in agriculture, animal husbandry and medicine. Currently, microbial cell factories are a promising production pathway, but the lack of high-throughput fermentation strain screening tools often hinders the exploration of engineering strategies to increase cell factory yields. Here, mutant AC103-3H was screened from libraries of saturating mutants after response-specific engineering of the transcription factor AsnC of L-asparagine (Asn). Based on mutant AC103-3H, a whole-cell biosensor EAC103-3H with a specific response to 5-ALA was constructed, which has a linear dynamic detection range of 1–12 mM and a detection limit of 0.094 mM, and can be used for in situ screening of potential high-producing 5-ALA strains. With its support, overexpression of the C5 pathway genes using promoter engineering assistance resulted in a 4.78-fold enhancement of 5-ALA production in the engineered E. coli. This study provides an efficient strain screening tool for exploring approaches to improve the 5-ALA productivity of engineered strains.

ARTP mutagenesis of Aureobasidium pullulans RM1603 for high pullulan production and transcriptome analysis of mutants.

Pullulan is a microbial exopolysaccharide produced by Aureobasidium spp. with excellent physical and chemical properties, resulting in great application value. In this study, a novel strain RM1603 of Aureobasidium pullulans with high pullulan production of 51.0 ± 1.0g·L- 1 isolated from rhizosphere soil was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, followed by selection of mutants to obtain pullulan high-producing strains. Finally, two mutants Mu0816 and Mu1519 were obtained, with polysaccharide productions of 58.7 ± 0.8 and 60.0 ± 0.8g∙L- 1 after 72-h fermentation, representing 15.1 and 17.6% increases compared with the original strain, respectively. Transcriptome analysis of the two mutants and the original strain revealed that the high expression of α/β-hydrolase (ABHD), α-amylase (AMY1), and sugar porter family MFS transporters (SPF-MFS) in the mutants may be related to the synthesis and secretion of pullulan. These results demonstrated the effectiveness of ARTP mutagenesis in A. pullulans, providing a basis for the investigation of genes related to pullulan synthesis and secretion.

Rational construction of a high-quality and high-efficiency biosynthetic system and fermentation optimization for A82846B based on combinatorial strategies in Amycolatopsis orientalis

BackgroundOritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production.ResultsFirstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities.ConclusionUnder approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

Penicillium brevicompactum as a New High Catalytic Laccase Producer Strain for Sugarcane Bagasse Degradation

Penicillium brevicompactum as a New High Catalytic Laccase Producer Strain for Sugarcane Bagasse Degradation

Oxidative stress resistance prompts pyrroloquinoline quinone biosynthesis in Hyphomicrobium denitrificans H4-45

Pyrroloquinoline quinone (PQQ) is a natural antioxidant with diverse applications in food and pharmaceutical industries. A lot of effort has been devoted toward the discovery of PQQ high-producing microbial species and characterization of biosynthesis, but it is still challenging to achieve a high PQQ yield. In this study, a combined strategy of random mutagenesis and adaptive laboratory evolution (ALE) with fermentation optimization was applied to improve PQQ production in Hyphomicrobium denitrificans H4-45. A mutant strain AE-9 was obtained after nearly 400 generations of UV-LiCl mutagenesis, followed by an ALE process, which was conducted with a consecutive increase of oxidative stress generated by kanamycin, sodium sulfide, and potassium tellurite. In the flask culture condition, the PQQ production in mutant strain AE-9 had an 80.4% increase, and the cell density increased by 14.9% when compared with that of the initial strain H4-45. Moreover, batch and fed-batch fermentation processes were optimized to further improve PQQ production by pH control strategy, methanol and H2O2 feed flow, and segmented fermentation process. Finally, the highest PQQ production and productivity of the mutant strain AE-9 reached 307 mg/L and 4.26 mg/L/h in a 3.7-L bioreactor, respectively. Whole genome sequencing analysis showed that genetic mutations in the ftfL gene and thiC gene might contribute to improving PQQ production by enhancing methanol consumption and cell growth in the AE-9 strain. Our study provided a systematic strategy to obtain a PQQ high-producing mutant strain and achieve high production of PQQ in fermentation. These practical methods could be applicable to improve the production of other antioxidant compounds with uncleared regulation mechanisms.Key points• Improvement of PQQ production by UV-LiCl mutagenesis combined with adaptive laboratory evolution (ALE) and fermentation optimization.• A consecutive increase of oxidative stress could be used as the antagonistic factor for ALE to enhance PQQ production.• Mutations in the ftfL gene and thiC gene indicated that PQQ production might be increased by enhancing methanol consumption and cell growth.

Combined transcriptomic and pangenomic analyses guide metabolic amelioration to enhance tiancimycins production.

Exploration of high-yield mechanism is important for further titer improvement of valuable antibiotics, but how to achieve this goal is challenging. Tiancimycins (TNMs) are anthraquinone-fused enediynes with promising drug development potentials, but their prospective applications are limited by low titers. This work aimed to explore the intrinsic high-yield mechanism in previously obtained TNMs high-producing strain Streptomyces sp. CB03234-S for the further titer amelioration of TNMs. First, the typical ribosomal RpsL(K43N) mutation in CB03234-S was validated to be merely responsible for the streptomycin resistance but not the titer improvement of TNMs. Subsequently, the combined transcriptomic, pan-genomic and KEGG analyses revealed that the significant changes in the carbon and amino acid metabolisms could reinforce the metabolic fluxes of key CoA precursors, and thus prompted the overproduction of TNMs in CB03234-S. Moreover, fatty acid metabolism was considered to exert adverse effects on the biosynthesis of TNMs by shunting and reducing the accumulation of CoA precursors. Therefore, different combinations of relevant genes were respectively overexpressed in CB03234-S to strengthen fatty acid degradation. The resulting mutants all showed the enhanced production of TNMs. Among them, the overexpression of fadD, a key gene responsible for the first step of fatty acid degradation, achieved the highest 21.7 ± 1.1mg/L TNMs with a 63.2% titer improvement. Our studies suggested that comprehensive bioinformatic analyses are effective to explore metabolic changes and guide rational metabolic reconstitution for further titer improvement of target products. KEY POINTS: • Comprehensive bioinformatic analyses effectively reveal primary metabolic changes. • Primary metabolic changes cause precursor enrichment to enhance TNMs production. • Strengthening of fatty acid degradation further improves the titer of TNMs.

Evidence for Regulation of Cordycepin Biosynthesis by Transcription Factors Krüppel-Like Factor 4 and Retinoid X Receptor Alpha in Caterpillar Medicinal Mushroom Cordyceps militaris (Ascomycetes).

Cordyceps militaris, Chinese traditional medicinal fungus, has many bioactive properties. Cordycepin (3'-deoxyadenosine) is a major bioactive component of C. militaris. Various methods can significantly elevate cordycepin production, which suggests a diverse set of metabolic regulatory mechanisms. Thus, we aimed to identify transcription factors that regulate cordycepin biosynthesis pathways. Transcriptome analysis of wild-type C. militaris, C. militaris GYS60, a cordycepin high-producing strain, and C. militaris GYS80, a low-producing strain, were used to measure expression and function of genes related to cordycepin biosynthesis. The transcriptome expression data were confirmed by quantitative real-time polymerase chain reaction. We identified 155 relevant transcription factors in 19 families that included Fork head/winged helix factors, other C4 zinc finger-type factors, C2H2 zinc finger factors, tryptophan cluster factors, nuclear receptors with C4 zinc fingers, homeodomain factors, and Rel homology region factors. Energy generation and amino acid conversion pathways were activated in GYS60 so that abundance of cordycepin precursors was increased. Genes and transcription factors for rate-limiting enzymes in these pathways were identified. Overexpression of two key transcription factors, Kruppel-like factor 4 (Klf4) and Retinoid X receptor alpha (Rxra), promoted high cordycepin production in GYS60. In GYS60, Klf4 and Rxra were responsible for upregulation of genes in cordycepin biosynthesis, namely an oxidoreductase, 3',5'-cyclic AMP phosphodiesterase, a transferase, and adenylate cyclase. Upregulation of these genes increased 3'-AMP content, thereby elevating cordycepin synthesis.

Revealing the Mechanisms of Enhanced β-Farnesene Production in Yarrowia lipolytica through Metabolomics Analysis.

β-Farnesene is an advanced molecule with promising applications in agriculture, the cosmetics industry, pharmaceuticals, and bioenergy. To supplement the shortcomings of rational design in the development of high-producing β-farnesene strains, a Metabolic Pathway Design-Fermentation Test-Metabolomic Analysis-Target Mining experimental cycle was designed. In this study, by over-adding 20 different amino acids/nucleobases to induce fluctuations in the production of β-farnesene, the changes in intracellular metabolites in the β-farnesene titer-increased group were analyzed using non-targeted metabolomics. Differential metabolites that were detected in each experimental group were selected, and their metabolic pathways were located. Based on these differential metabolites, targeted strain gene editing and culture medium optimization were performed. The overexpression of the coenzyme A synthesis-related gene pantothenate kinase (PanK) and the addition of four mixed water-soluble vitamins in the culture medium increased the β-farnesene titer in the shake flask to 1054.8 mg/L, a 48.5% increase from the initial strain. In the subsequent fed-batch fermentation, the β-farnesene titer further reached 24.6 g/L. This work demonstrates the tremendous application value of metabolomics analysis for the development of industrial recombinant strains and the optimization of fermentation conditions.

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD-hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild-type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7-fold higher kcat /KM . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high-producing strain, 8GM1, containing eight copies of AOX1 promoter-GAS1 signal peptide-MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co-expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2-fold. In the 5-L bioreactor, conventional fed-batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3-fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high-producing strains for other industrial enzymes.

High-throughput droplet microfluidics screening and genome sequencing analysis for improved amylase-producing Aspergillus oryzae

BackgroundThe exceptional protein secretion capacity, intricate post-translational modification processes, and inherent safety features of A. oryzae make it a promising expression system. However, heterologous protein expression levels of existing A. oryzae species cannot meet the requirement for industrial-scale production. Therefore, establishing an efficient screening technology is significant for the development of the A. oryzae expression system.ResultsIn this work, a high-throughput screening method suitable for A. oryzae has been established by combining the microfluidic system and flow cytometry. Its screening efficiency can reach 350 droplets per minute. The diameter of the microdroplet was enlarged to 290 µm to adapt to the polar growth of A. oryzae hyphae. Through enrichment and screening from approximately 450,000 droplets within 2 weeks, a high-producing strain with α-amylase increased by 6.6 times was successfully obtained. Furthermore, 29 mutated genes were identified by genome resequencing of high-yield strains, with 15 genes subjected to editing and validation. Two genes may individually influence α-amylase expression in A. oryzae by affecting membrane-associated multicellular processes and regulating the transcription of related genes.ConclusionsThe developed high-throughput screening strategy provides a reference for other filamentous fungi and Streptomyces. Besides, the strains with different excellent characteristics obtained by efficient screening can also provide materials for the analysis of genetic and regulatory mechanisms in the A. oryzae expression system.

Electroporation-induced mutation and transcriptome analysis for high DHA production in Schizochytrium limacinum GCD2032

The development of the DHA industry requires continuous iterative upgrading of high-yield DHA-producing strains. In this study, a DHA high-producing strain named S. limacinum GCD2032 was screened through electroporation using zeocin as the selective agent and S. limacinum BCD as the wild strain. The production enhancement mechanism was elucidated through comparative transcriptome studies between S. limacinum GCD2032 and the wild strain. The fatty acid content, DHA content and DHA yield of S. limacinum GCD2032 were 3.51 %, 95.51 % and 71.67 % higher than those of S. limacinum BCD, respectively. Transcriptome analysis indicated that genes involved in the aerobic oxidation of glucose, the pentose phosphate pathway, the citric acid pyruvate cycle, and the PKS pathway for DHA production were all upregulated and genes involved in β-oxidation in peroxisomes were downregulated, resulting in the enhanced DHA content of S. limacinum GCD2032. This study provides an efficient iterative upgrading method and key candidate genes for DHA production in Schizochytrium. S. limacinum GCD2032 is a promising strain for further industrialization.

Chromatin architecture of two different strains of Artemisia annua reveals the alterations in interaction and gene expression.

The hierarchical architecture of chromatins affects the gene expression level of glandular secreting trichomes and the artemisinin biosynthetic pathway-related genes, consequently bringing on huge differences in the content of artemisinin and its derivatives of A. annua. The plant of traditional Chinese medicine "Qinghao" is called Artemisia annua L. in Chinese Pharmacopoeia. High content and the total amount of artemisinin is the main goal of A. annua breeding, nevertheless, the change of chromatin organization during the artemisinin synthesis process has not been discovered yet. This study intended to find the roles of chromatin structure in the production of artemisinin through bioinformatics and experimental validation. Chromosome conformation capture analysis was used to scrutinize the interactions among chromosomes and categorize various scales of chromatin during artemisinin synthesis in A. annua. To confirm the effect of the changes in chromatin structure, Hi-C and RNA-sequencing were performed on two different strains to find the correlation between chromatin structure and gene expression levels on artemisinin synthesis progress and regulation. Our results revealed that the frequency of intra-chromosomal interactions was higher in the inter-chromosomal interactions between the root and leaves on a high artemisinin production strain (HAP) compared to a low artemisinin production strain (LAP). We found that compartmental transition was connected with interactions among different chromatins. Interestingly, glandular secreting trichomes (GSTs) and the artemisinin biosynthetic pathway (ABP) related genes were enriched in the areas which have the compartmental transition, reflecting the regulation of artemisinin synthesis. Topologically associated domain boundaries were associated with various distributions of genes and expression levels. Genes associated with ABP and GST in the adjacent loop were highly expressed, suggesting that epigenetic regulation plays an important role during artemisinin synthesis and glandular secreting trichomes production process. Chromatin structure could show an important status in the mechanisms of artemisinin synthesis process in A. annua.

CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories.

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/L and 0.9 g/L of CHR and ANT in 1.3 mL miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

Functional analysis of functional membrane microdomains in the biosynthesis of menaquinone-7

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Screening of Natural High-producing NMN strains and Microbial Synthesis of NMN by Nampt

Nicotinamide mononucleotide (NMN) is an endogenous substance in humans with high safety and thermal stability, and its application in cosmetics, medical health, and functional foods has received widespread attention. However, the synthesis process of NMN has problems such as high cost, time-consuming and low yield, which limits the large-scale industrial application of NMN to a certain extent. Nicotinamide phosphate ribose transferase (Nampt) is a critical enzyme in a technical route in the biological synthesis of NMN, which can catalyze the synthesis of NMN by Nicotinamide (NAM) and Phosphoribosyl pyrophosphate (PRPP). Screening and expression of a Nampt with excellent enzymatic properties and stability is the key to the synthesis of NMN by this method. At present, the main problems in the technical route of NMN production by Nampt are that the catalytic activity of Nampt is low. And the source of Nampt is small and limited, so we isolated the microorganism with high NMN production, Enterobacter chengduensis 2021T4.7, and optimized its fermentation conditions. The yield of NMN is up to 67.66 μM. In addition, we synthesized Nampt and constructed related recombinant high-yield engineering bacteria. We semi-rationally designed the Nampt structure derived from mice and obtained mutant mNampt-V365L with NMN yields as high as 135.99 μM increased by 62% compared with wild type. Here, we screened high-yield NMN natural strains, and obtained high-yield strains through the semi-rational design optimization of Nampt enzyme, which provided new chassis microorganisms and new ideas for the conversion rate of NMN.

Modulating Sensitivity of an Erythromycin Biosensor for Precise High-Throughput Screening of Strains with Different Characteristics.

Genetically encoded biosensors are powerful tools for product-driven high-throughput screening in synthetic biology and metabolic engineering. However, most biosensors can only properly function in a limited concentration cutoff, and the incompatible performance characteristics of biosensors will lead to false positives or failure in screening. The transcription factor (TF)-based biosensors are usually organized in modular architecture and function in a regulator-depended manner, whose performance properties can be fine-tuned by modifying the expression level of the TF. In this study, we modulated the performance characteristics, including sensitivity and operating range, of an MphR-based erythromycin biosensor by fine-adjusting regulator expression levels via ribosome-binding site (RBS) engineering and obtained a panel of biosensors with varied sensitivities by iterative fluorescence-assisted cell sorting (FACS) in Escherichia coli to accommodate different screening purposes. To exemplify their application potential, two engineered biosensors with 10-fold different sensitivities were employed in the precise high-throughput screening by microfluidic-based fluorescence-activated droplet sorting (FADS) of Saccharopolyspora erythraea mutant libraries with different starting erythromycin productions, and mutants representing as high as 6.8 folds and over 100% of production improvements were obtained starting from the wild-type strain and the high-producing industrial strain, respectively. This work demonstrated a simple strategy to engineer biosensor performance properties, which was significant to stepwise strain engineering and production improvement.

Systematic modulating carbon metabolism to improve α-farnesene production in Pichia pastoris

α-farnesene, a simple acyclic sesquiterpenes, has significant economic value in medicine, condiments, cosmetics and bioenergy. In this study, we increased α-farnesene production through modifying three module pathways: the main MVA biosynthesis module, the farnesyl pyrophosphate (FPP) branch module and the cytosolic acetyl-CoA supply module. To do these, the appropriate promoters were used to control the expression level of the key enzymes to strengthen the main MVA biosynthesis module and the cytosolic acetyl-CoA supply module as well as to weaken FPP branch module in P. pastoris X33 with expression of α-farnesene synthase (αFS) from Malus domestica. As a result, a α-farnesene high-producing strain P. pastoris XF22 was obtained, which produced 17.92 ± 1.35 g/L of α-farnesene with a productivity of 0.107 g/(L·h) and a yield of 0.078 g/(g glucose) after 168 h in fed-batch fermentation. Although the titer of α-farnesene is not the highest, the productivity and the yield in this study are the highest among the reported literatures, demonstrating that strain XF22 is a competitive platform strain for α-farnesene production.

TFBMiner: A User-Friendly Command Line Tool for the Rapid Mining of Transcription Factor-Based Biosensors

Transcription factors responsive to small molecules are essential elements in synthetic biology designs. They are often used as genetically encoded biosensors with applications ranging from the detection of environmental contaminants and biomarkers to microbial strain engineering. Despite our efforts to expand the space of compounds that can be detected using biosensors, the identification and characterization of transcription factors and their corresponding inducer molecules remain labor- and time-intensive tasks. Here, we introduce TFBMiner, a new data mining and analysis pipeline that enables the automated and rapid identification of putative metabolite-responsive transcription factor-based biosensors (TFBs). This user-friendly command line tool harnesses a heuristic rule-based model of gene organization to identify both gene clusters involved in the catabolism of user-defined molecules and their associated transcriptional regulators. Ultimately, biosensors are scored based on how well they fit the model, providing wet-lab scientists with a ranked list of candidates that can be experimentally tested. We validated the pipeline using a set of molecules for which TFBs have been reported previously, including sensors responding to sugars, amino acids, and aromatic compounds, among others. We further demonstrated the utility of TFBMiner by identifying a biosensor for S-mandelic acid, an aromatic compound for which a responsive transcription factor had not been found previously. Using a combinatorial library of mandelate-producing microbial strains, the newly identified biosensor was able to distinguish between low- and high-producing strain candidates. This work will aid in the unraveling of metabolite-responsive microbial gene regulatory networks and expand the synthetic biology toolbox to allow for the construction of more sophisticated self-regulating biosynthetic pathways.

Third Generation Lactic Acid Production by Lactobacillus pentosus from the Macroalgae Kappaphycus alvarezii Hydrolysates

The evaluation of macroalgae as a new raw material for diverse bioprocesses is of great interest due to their fast growth rate and low environmental impact. Lactic acid has a high value in the bio-based industry and is mainly produced via fermentation. The anaerobic lactic acid fermentation of Kappaphycus alvarezii hydrolysates using the high-producing strain Lactobacillus pentosus was evaluated for detoxified and non-treated hydrolysates prepared from concentrated algal biomass and dilute acid solution mixtures. A novel hydrolysate detoxification procedure, combining activated charcoal and over-liming, for 5-hydroxymethylfurfural (HMF) removal was used. L. pentosus was found to successfully ferment detoxified and untreated hydrolysates produced in up to 30% and 20% w/v solutions, respectively. Significant production rates (1.88 g/L.h) and short lag phases were achieved in bioreactor fermentation operating at 37 °C and pH 6 with 150 rpm impeller velocity. A 0.94 g/g yield from fermentable sugars (galactose and glucose) was achieved, indicating that K. alvarezii could be used as a raw material for lactic acid production, within the context of Third Generation (3G) biorefinery.

Pilot Scale Evaluation of Wild Saccharomyces cerevisiae Strains in Aglianico

In winemaking, the influence of Saccharomyces cerevisiae strains on the aromatic components of wine is well recognized on a laboratory scale, but few studies deal with the comparison of numerous strains on a pilot scale fermentation. In this scenario, the present work aimed to validate the fermentative behavior of seven wild S. cerevisiae strains on pilot-scale fermentations to evaluate their impact on the aromatic profiles of the resulting wines. The strains, isolated from grapes of different Italian regional varieties, were tested in pilot-scale fermentation trials performed in the cellar in 1 hL of Aglianico grape must. Then, wines were analyzed for their microbiological cell loads, main chemical parameters of enological interest (ethanol, total sugars, fructose, glucose, total and volatile acidity, malic and lactic acids) and volatile aroma profiles by GC/MS/SPME. Seventy-six volatile compounds belonging to six different classes (esters, alcohols, terpenes, aldehydes, acids, and ketones) were identified. The seven strains showed different trends and significant differences, and for each class of compounds, high-producing and low-producing strains were found. Since the present work was performed at a pilot-scale level, mimicking as much as possible real working conditions, the results obtained can be considered as a validation of the screened S. cerevisiae strains and a strategy to discriminate in real closed conditions strains able to impart desired wine sensory features.