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Ethanol stress in Saccharomyces cerevisiae is a well-studied phenomenon, but pinpointing specific genes or polymorphisms governing ethanol tolerance remains a subject of ongoing debate. Naturally found in sugar-rich environments, this yeast has evolved to withstand high ethanol concentrations, primarily produced during fermentation in the presence of suitable oxygen or sugar levels. Originally a defense mechanism against competing microorganisms, yeast-produced ethanol is now a cornerstone of brewing and bioethanol industries, where customized yeasts require high ethanol resistance for economic viability. However, yeast strains exhibit varying degrees of ethanol tolerance, ranging from 8 to 20%, making the genetic architecture of this trait complex and challenging to decipher. In this study, we introduce a novel QTL mapping pipeline to investigate the genetic markers underlying ethanol tolerance in an industrial bioethanol S. cerevisiae strain. By calculating missense mutation frequency in an allele located in a prominent QTL region within a population of 1011 S. cerevisiae strains, we uncovered rare occurrences in gene IRA2. Following molecular validation, we confirmed the significant contribution of this gene to ethanol tolerance, particularly in concentrations exceeding 12% of ethanol. IRA2 pivotal role in stress tolerance due to its participation in the Ras-cAMP pathway was further supported by its involvement in other tolerance responses, including thermotolerance, low pH tolerance, and resistance to acetic acid. Understanding the genetic basis of ethanol stress in S. cerevisiae holds promise for developing robust yeast strains tailored for industrial applications., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Protein folding both promotes and constrains adaptive evolution. We uncover this surprising duality in the role of the protein-folding chaperone heat shock protein 90 (Hsp90) in maintaining the integrity of yeast metabolism amid proteotoxic stressors within industrial domestication niches. Ethanol disrupts critical Hsp90-dependent metabolic pathways and exerts strong selective pressure for redundant duplications of key genes within these pathways, yielding the classical genomic signatures of beer and bread domestication. This work demonstrates a mechanism of adaptive canalization in an ecology of major economic importance and highlights Hsp90-dependent variation as an important source of phantom heritability in complex traits.

Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Molinet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Alginates are natural polysaccharides widely participating in food, pharmaceutical, and environmental applications due to their excellent gelling capacity. Their excellent biocompatibility and biodegradability further extend their application to biomedical fields. The low consistency in molecular weight and composition of algae-based alginates may limit their performance in advanced biomedical applications. It makes microbial alginate production more attractive due to its potential for customizing alginate molecules with stable characteristics. Production costs remain the primary factor limiting the commercialization of microbial alginates. However, carbon-rich wastes from sugar, dairy, and biodiesel industries may serve as potential substitutes for pure sugars for microbial alginate production to reduce substrate costs. Fermentation parameter control and genetic engineering strategies may further improve the production efficiency and customize the molecular composition of microbial alginates. To meet the specific needs of biomedical applications, alginates may need functionalization, such as functional group modifications and crosslinking treatments, to achieve enhanced mechanical properties and biochemical activities. The development of alginate-based composites incorporated with other polysaccharides, gelatin, and bioactive factors can integrate the advantages of each component to meet multiple requirements in wound healing, drug delivery, and tissue engineering applications. This review provided a comprehensive insight into the sustainable production of high-value microbial alginates. It also discussed recent advances in alginate modification strategies and alginate-based composites for representative biomedical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

DsrC is a key protein in dissimilatory sulfur metabolism, where it works as co-substrate of the dissimilatory sulfite reductase DsrAB. DsrC has two conserved cysteines in a C-terminal arm that are converted to a trisulfide upon reduction of sulfite. In sulfate-reducing bacteria, DsrC is essential and previous works suggested additional functions beyond sulfite reduction. Here, we studied whether DsrC also plays a role during fermentative growth of Desulfovibrio vulgaris Hildenborough, by studying two strains where the functionality of DsrC is impaired by a lower level of expression (IPFG07) and additionally by the absence of one conserved Cys (IPFG09). Growth studies coupled with metabolite and proteomic analyses reveal that fermentation leads to lower levels of DsrC, but impairment of its function results in reduced growth by fermentation and a shift towards more fermentative metabolism during sulfate respiration. In both respiratory and fermentative conditions, there is increased abundance of the FlxABCD-HdrABC complex and Adh alcohol dehydrogenase in IPFG09 versus the wild type, which is reflected in higher production of ethanol. Pull-down experiments confirmed a direct interaction between DsrC and the FlxABCD-HdrABC complex, through the HdrB subunit. Dissimilatory sulfur metabolism, where sulfur compounds are used for energy generation, is a key process in the ecology of anoxic environments, and is more widespread among bacteria than previously believed. Two central proteins for this type of metabolism are DsrAB dissimilatory sulfite reductase and its co-substrate DsrC. Using physiological, proteomic and biochemical studies of Desulfovibrio vulgaris Hildenborough and mutants affected in DsrC functionality, we show that DsrC is also relevant for fermentative growth of this model organism and that it interacts directly with the soluble FlxABCD-HdrABC complex that links the NAD(H) pool with dissimilatory sulfite reduction., (© 2023 Applied Microbiology International and John Wiley & Sons Ltd.)

Background: The lipopeptide herbicolin A (HA) secreted by the biocontrol agent Pantoea agglomerans ZJU23 is a promising antifungal drug to combat fungal pathogens by targeting lipid rafts, both in agricultural and clinical settings. Improvement of HA production would be of great significance in promoting its commercialization. This study aims to enhance the HA production in ZJU23 by combining fermentation optimization and strain engineering., Results: Based on the results in the single-factor experiments, corn steep liquor, temperature and initial pH were identified as the significant affecting factors by the Plackett-Burman design. The fermentation medium and conditions were further optimized using the Box-Behnken response surface method, and the HA production of the wild type strain ZJU23 was improved from ~ 87 mg/mL in King's B medium to ~ 211 mg/mL in HA induction (HAI) medium. A transposon library was constructed in ZJU23 to screen for mutants with higher HA production, and two transcriptional repressors for HA biosynthesis, LrhA and PurR, were identified. Disruption of the LrhA gene led to increased mRNA expression of HA biosynthetic genes, and subsequently improved about twofold HA production. Finally, the HA production reached ~ 471 mg/mL in the ΔLrhA mutant under optimized fermentation conditions, which is about 5.4 times higher than before (~ 87 mg/mL). The bacterial suspension of the ΔLrhA mutant fermented in HAI medium significantly enhanced its biocontrol efficacy against gray mold disease and Fusarium crown rot of wheat, showing equivalent control efficacies as the chemical fungicides used in this study. Furthermore, HA was effective against fungicide resistant Botrytis cinerea. Increased HA production substantially improved the control efficacy against gray mold disease caused by a pyrimethanil resistant strain., Conclusions: This study reveals that the transcriptional repressor LrhA negatively regulates HA biosynthesis and the defined HAI medium is suitable for HA production. These findings provide an extended basis for large-scale production of HA and promote biofungicide development based on ZJU23 and HA in the future., (© 2023. The Author(s).)

Although numerous studies exist relating ruminal volatile fatty acid (VFA) concentrations to diet composition and animal performance, minimal information is available describing how VFA dynamics respond to diets within the context of the whole rumen environment. The objective of this study was to characterize how protein and fiber sources affect dry matter intake, rumen pH, fluid dynamics, fermentation parameters, and epithelial gene expression. Four diet treatments (soybean meal or heat-treated soybean meal and beet pulp or timothy hay) were delivered to 10 wethers. The soybean meals served as crude protein (CP) sources while the beet pulp and timothy hay represented neutral detergent fiber (NDF) sources. Feed intake, rumen pH, fluid pool size, and fluid passage rate were unaffected by treatment. Butyrate synthesis and absorption were greater on the beet pulp treatment whereas synthesis and absorption of other VFA remained unchanged. Both CP and NDF treatment effects were associated with numerous VFA interconversions. Expression levels of rumen epithelial genes were not altered by diet treatment. These results indicate that rumen VFA dynamics are altered by changes in dietary sources of nutrients but that intake, rumen environmental parameters, and the rumen epithelium may be less responsive to such changes., (© 2022. The Author(s).)

Kiwifruit plants have a fleshy, shallow root system which is sensitive to waterlogging stress, which results in a decrease in crop yield or even plants death. Although the waterlogging stress responses in kiwifruit have attracted much attention, the underlying molecular mechanism remains unclear. In this study, waterlogging led to drastic inhibition of root growth of 'Donghong' kiwifruit (Actinidia chinensis) plants grown in vitro, which was accompanied by significant elevation of endogenous acetaldehyde and ethanol contents. RNA-seq of roots of plants waterlogged for 0, 1 and 2 days revealed that a total of 149 genes were up- or down-regulated, including seven biosynthetic genes related to the glycolysis/gluconeogenesis pathway and 10 transcription factors. Analyses with real-time PCR, dual-luciferase assays and EMSA demonstrated that AcERF74 and AcERF75, two members of the ERF-VII subfamily, directly upregulated AcADH1 (alcohol dehydrogenase). Moreover, the overexpression of AcERF74/75 in transgenic calli resulted in dramatic increase of endogenous ethanol contents through the triggering of AcADH1 and AcADH2 expression. Although the AcPDC2 (pyruvate decarboxylase) expression was also enhanced in transgenic lines, the endogenous acetaldehyde contents showed no significant changes. These results illustrated that AcERF74/75 are two transcriptional activators on alcoholic fermentation related genes and are responsive to waterlogging stress in kiwifruit., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Wine fermentation is an ancient biotechnological process mediated by different microorganisms such as yeast and bacteria. Understanding of the metabolic and physiological phenomena taking place during this process can be now attained at a genome scale with the help of metabolic models. In this chapter, we present a detailed protocol for modeling wine fermentation using genome-scale metabolic models. In particular, we illustrate how metabolic fluxes can be computed, optimized and interpreted, for both yeast and bacteria under winemaking conditions. We also show how nutritional requirements can be determined and simulated using these models in relevant test cases. This chapter introduces fundamental concepts and practical steps for applying flux balance analysis in wine fermentation, and as such, it is intended for a broad microbiology audience as well as for practitioners in the metabolic modeling field., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Bacillus licheniformis 2709 is a potential cell factory for the production of alkaline protease AprE, which has important value in industrial application but still lacks sufficient production capacity. To address this problem, we investigated the effects of the secretory viscous materials on the synthesis of AprE, which might seriously affect the industrial fermentation. Furthermore, an iterative chromosomal integration strategy at various chromosomal loci was implemented to achieve stable high-level expression of AprE in B. licheniformis 2709. The host was genetically modified by disrupting the native pgs cluster controlling the biosynthesis of viscous poly-glutamic acid identified in the study by GC/MS, generating a mutant with significantly higher biomass and better bioreactor performance. We further enhanced the expression of alkaline protease by integrating two additional aprE expression cassettes into the genome, generating the integration mutant BL ∆UEP-3 with three aprE expression cassettes, whose AprE enzyme activity in shake flasks reached 25,736 ± 997 U/mL, which was 136% higher than that of the original strain, while the aprE transcription level increased 4.05 times. Thus, an AprE high-yielding strain with excellent fermentation traits was engineered, which was more suitable for bulk-production. Finally, the AprE titer was further increased in a 5-L fermenter, reaching 57,763 ± 1039 U/mL. In summary, genetic modification is an enabling technology for enhancing enzyme production by eliminating the unfavorable characteristics of the host and optimizing the expression of aprE through iterative chromosomal integration. We believe that the protocol developed in this study provides a valuable reference for chromosomal overexpression of proteins or bioactive molecules in other Bacillus species., (Copyright © 2021. Published by Elsevier B.V.)

Fermentation-derived alcohols have gained much attention as an alternate fuel due to its minimal effects on atmosphere. Besides its application as biofuel it is also used as raw material for coating resins, deicing fluid, additives in polishes, etc. Among the liquid alcohol type of fuels, isobutanol has more advantage than ethanol. Isobutanol production is reported in native yeast strains, but the production titer is very low which is about 200 mg/L. In order to improve the production, several genetic and metabolic engineering approaches have been carried out. Genetically engineered organism has been reported to produce maximum of 50 g/L of isobutanol which is far more than the native strain without any modification. In bacteria mostly last two steps in Ehrlich pathway, catalyzed by enzymes ketoisovalerate decarboxylase and alcohol dehydrogenase, are heterologously expressed to improve the production. Native Saccharomyces cerevisiae can produce isobutanol in negligible amount since it possesses the pathway for its production through valine degradation pathway. Further modifications in the existing pathways made the improvement in isobutanol production in many microbial strains. Fermentation using cost-effective lignocellulosic biomass and an efficient downstream process can yield isobutanol in environment friendly and sustainable manner. The present review describes the various genetic and metabolic engineering practices adopted to improve the isobutanol production in microbial strains and its downstream processing.

Bioconversion of acetate, a byproduct generated in industrial processes, into microbial lipids using oleaginous yeasts offers a promising alternative for the economic utilization of acetate-containing waste streams. However, high acetate concentrations will inhibit microbial growth and metabolism. In this study, the acetate utilization capability of Yarrowia lipolytica PO1f was successively improved by overexpressing the key enzyme of acetyl-CoA synthetase (ACS), which resulted in an accumulation of 9.2% microbial lipids from acetate in shake flask fermentation. By further overexpressing the second key enzymes of acetyl-CoA carboxylase (ACC1) and fatty acid synthase (FAS) in Y. lipolytica , the lipid content was increased to 25.7% from acetate. Finally, the maximum OD 600 of 29.2 and a lipid content of 41.7% were obtained with the engineered strain by the adoption of cosubstrate (glycerol and acetate) fed-batch fermentation, which corresponded to an increase of 68 and 95%, respectively. These results presented a promising strategy for economic and efficient microbial lipid production from the waste acetate.

Chromosomal integration of exogenous genes is preferred for industrially related fermentation, as plasmid-mediated fermentation leads to extra metabolic burden and genetic instability. Moreover, with the development and advancement of genome engineering and gene editing technologies, inserting genes into chromosomes has become more convenient; integration expression is extensively utilized in microorganisms for industrial bioproduction and expected to become the trend of recombinant protein expression. However, in actual research and application, it is important to enhance the expression of heterologous genes at the host genome level. Herein, we summarized the basic principles and characteristics of genomic integration; furthermore, we highlighted strategies to improve the expression of chromosomal integration of genes and pathways in host strains from three aspects, including chassis cell optimization, regulation of expression elements in gene expression cassettes, optimization of gene dose level and integration sites on chromosomes. Moreover, we reviewed and summarized the relevant studies on the application of integrated expression in the exploration of gene function and the various types of industrial microorganism production. Consequently, this review would serve as a reference for the better application of integrated expression.

Aluminum (Al)-tolerant tobacco cell line ALT301 derived from SL (wild-type) hardly exhibits Al-triggered reactive oxygen species (ROS) compared with SL. Molecular mechanism leading to this phenotype was investigated comparatively with SL. Under normal growth condition, metabolome data suggested the activation of glycolysis and lactate fermentation but the repression of the tricarboxylic acid (TCA) cycle in ALT301, namely aerobic fermentation, which seemed to be transcriptionally controlled partly by higher expression of genes encoding lactate dehydrogenase and pyruvate dehydrogenase kinase. Microarray and gene ontology analyses revealed the upregulation of the gene encoding related to APETALA2.3 (RAP2.3)-like protein, one of the group VII ethylene response factors (ERFVIIs), in ALT301. ERFVII transcription factors are known to be key regulators for hypoxia response that promotes substrate-level ATP production by glycolysis and fermentation. ERFVIIs are degraded under normoxia by the N-end rule pathway of proteolysis depending on both oxygen and nitric oxide (NO), and NO is produced mainly by nitrate reductase (NR) in plants. In ALT301, levels of the NR gene expression (NIA2), NR activity and NO production were all lower compared with SL. Consistently, the known effects of NO on respiratory pathways were also repressed in ALT301. Under Al-treatment condition, NO level increased in both lines but was lower in ALT301. These results suggest that the upregulation of the RAP2.3-like gene and the downregulation of the NIA2 gene and resultant NO depletion in ALT301 coordinately enhance aerobic fermentation, which seems to be related to a higher capacity to prevent ROS production in mitochondria under Al stress., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: VIB and LRD have submitted a patent application (June. 15, 2020; EP 20179988.9.) on commercial use of the results.

Hybrids between species can harbor a combination of beneficial traits from each parent and may exhibit hybrid vigor, more readily adapting to new harsher environments. Interspecies hybrids are also sterile and therefore an evolutionary dead end unless fertility is restored, usually via auto-polyploidisation events. In the Saccharomyces genus, hybrids are readily found in nature and in industrial settings, where they have adapted to severe fermentative conditions. Due to their hybrid sterility, the development of new commercial yeast strains has so far been primarily conducted via selection methods rather than via further breeding. In this study, we overcame infertility by creating tetraploid intermediates of Saccharomyces interspecies hybrids to allow continuous multigenerational breeding. We incorporated nuclear and mitochondrial genetic diversity within each parental species, allowing for quantitative genetic analysis of traits exhibited by the hybrids and for nuclear-mitochondrial interactions to be assessed. Using pooled F12 generation segregants of different hybrids with extreme phenotype distributions, we identified quantitative trait loci (QTLs) for tolerance to high and low temperatures, high sugar concentration, high ethanol concentration, and acetic acid levels. We identified QTLs that are species specific, that are shared between species, as well as hybrid specific, in which the variants do not exhibit phenotypic differences in the original parental species. Moreover, we could distinguish between mitochondria-type-dependent and -independent traits. This study tackles the complexity of the genetic interactions and traits in hybrid species, bringing hybrids into the realm of full genetic analysis of diploid species, and paves the road for the biotechnological exploitation of yeast biodiversity., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Objectives: Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus., Results: Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower., Conclusions: Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

Tobramycin is a broad-spectrum aminoglycoside antibiotic agent. The compound is obtained from the base-catalyzed hydrolysis of carbamoyltobramycin (CTB), which is naturally produced by the actinomycete Streptoalloteichus tenebrarius . However, the strain uses the same precursors to synthesize several structurally related aminoglycosides. Consequently, the production yields of tobramycin are low, and the compound's purification is very challenging, costly, and time-consuming. In this study, the production of the main undesired product, apramycin, in the industrial isolate Streptoalloteichus tenebrarius 2444 was decreased by applying the fermentation media M10 and M11, which contained high concentrations of starch and dextrin. Furthermore, the strain was genetically engineered by the inactivation of the aprK gene (∆ aprK ), resulting in the abolishment of apramycin biosynthesis. In the next step of strain development, an additional copy of the tobramycin biosynthetic gene cluster (BGC) was introduced into the ∆ aprK mutant. Fermentation by the engineered strain ( ∆aprK _1-17L) in M11 medium resulted in a 3- to 4-fold higher production than fermentation by the precursor strain (∆ aprK ). The phenotypic stability of the mutant without selection pressure was validated. The use of the engineered S. tenebrarius 2444 facilitates a step-saving, efficient, and, thus, more sustainable production of the valuable compound tobramycin on an industrial scale.

Bioproduction of renewable chemicals is considered as an urgent solution for fossil energy crisis. However, despite tremendous efforts, it is still challenging to generate microbial strains that can produce target biochemical to high levels. Here, we report an example of biosynthesis of high-value and easy-recoverable derivatives built upon natural microbial pathways, leading to improvement in bioproduction efficiency. By leveraging pathways in solventogenic clostridia for co-producing acyl-CoAs, acids and alcohols as precursors, through rational screening for host strains and enzymes, systematic metabolic engineering-including elimination of putative prophages, we develop strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl butyrate. Techno-economic analysis results suggest the economic competitiveness of our developed bioprocess. Our principles of selecting the most appropriate host for specific bioproduction and engineering microbial chassis to produce high-value and easy-separable end products may be applicable to other bioprocesses., (© 2021. The Author(s).)

The relationship between different levels of integration is a key feature for understanding the genotype-phenotype map. Here, we describe a novel method of integrated data analysis that incorporates protein abundance data into constraint-based modeling to elucidate the biological mechanisms underlying phenotypic variation. Specifically, we studied yeast genetic diversity at three levels of phenotypic complexity in a population of yeast obtained by pairwise crosses of eleven strains belonging to two species, Saccharomyces cerevisiae and S. uvarum. The data included protein abundances, integrated traits (life-history/fermentation) and computational estimates of metabolic fluxes. Results highlighted that the negative correlation between production traits such as population carrying capacity (K) and traits associated with growth and fermentation rates (Jmax) is explained by a differential usage of energy production pathways: a high K was associated with high TCA fluxes, while a high Jmax was associated with high glycolytic fluxes. Enrichment analysis of protein sets confirmed our results. This powerful approach allowed us to identify the molecular and metabolic bases of integrated trait variation, and therefore has a broad applicability domain., Competing Interests: The authors have declared that no competing interests exist.

Microbial natural products are an invaluable resource for the biotechnological industry. Genome mining studies have highlighted the huge biosynthetic potential of fungi, which is underexploited by standard fermentation conditions. Epigenetic effectors and/or cultivation-based approaches have successfully been applied to activate cryptic biosynthetic pathways in order to produce the chemical diversity suggested in available fungal genomes. The addition of Suberoylanilide Hydroxamic Acid to fermentation processes was evaluated to assess its effect on the metabolomic diversity of a taxonomically diverse fungal population. Here, metabolomic methodologies were implemented to identify changes in secondary metabolite profiles to determine the best fermentation conditions. The results confirmed previously described effects of the epigenetic modifier on the metabolism of a population of 232 wide diverse South Africa fungal strains cultured in different fermentation media where the induction of differential metabolites was observed. Furthermore, one solid-state fermentation (BRFT medium), two classic successful liquid fermentation media (LSFM and YES) and two new liquid media formulations (MCKX and SMK-II) were compared to identify the most productive conditions for the different populations of taxonomic subgroups.

Bioethanol produced by fermentative microorganisms is regarded as an alternative to fossil fuel. Bioethanol to be used as a viable energy source must be produced cost-effectively by removing expense-intensive steps such as the enzymatic hydrolysis of substrate. Consolidated bioprocessing (CBP) is believed to be a practical solution combining saccharification and fermentation in a single step catalyzed by a microorganism. Bacillus subtills with innate ability to grow on a diversity of carbohydrates seems promising for affordable CBP bioethanol production using renewable plant biomass and wastes. In this study, the genes encoding alcohol dehydrogenase from Z. mobilis (adh Z ) and S. cerevisiae (adh S ) were each used with Z. mobilis pyruvate decarboxylase gene (pdc Z ) to create ethanologenic operons in a lactate-deficient (Δldh) B. subtilis resulting in NZ and NZS strains, respectively. The S. cerevisiae adh S caused significantly more ethanol production by NZS and therefore was used to make two other operons including one with double copies of both pdc Z and adh S and the other with a single pdc Z but double adh S genes expressed in N(ZS)2 and NZS2 strains, respectively. In addition, two fusion genes were constructed with pdc Z and adh S in alternate orientations and used for ethanol production by the harboring strains namely NZ:S and NS:Z, respectively. While the increase of gene dosage was not associated with elevated carbon flow for ethanol production, the fusion gene adh S :pdc Z resulted in a more than two times increase of productivity by strain NS:Z as compared with NZS during 48 h fermentation. The CBP ethanol production by NZS and NS:Z using potatoes resulted in 16.3 g/L and 21.5 g/L ethanol during 96 h fermentation, respectively. For the first time in this study, B. subtilis was successfully used for CBP ethanol production with S. cerevisiae alcohol dehydrogenase. The results of the study provide insights on the potentials of B. subtilis for affordable bioethanol production from inexpensive plant biomass and wastes. However, the potentials need to be improved by metabolic and process engineering for higher yields of ethanol production and plant biomass utilization.

Hadal trenches are the deepest but underexplored ecosystems on the Earth. Inhabiting the trench bottom is a group of micro-organisms termed obligate piezophiles that grow exclusively under high hydrostatic pressures (HHP). To reveal the genetic and physiological characteristics of their peculiar lifestyles and microbial adaptation to extreme high pressures, we sequenced the complete genome of the obligately piezophilic bacterium Moritella yayanosii DB21MT-5 isolated from the deepest oceanic sediment at the Challenger Deep, Mariana Trench. Through comparative analysis against pressure sensitive and deep-sea piezophilic Moritella strains, we identified over a hundred genes that present exclusively in hadal strain DB21MT-5. The hadal strain encodes fewer signal transduction proteins and secreted polysaccharases, but has more abundant metal ion transporters and the potential to utilize plant-derived saccharides. Instead of producing osmolyte betaine from choline as other Moritella strains, strain DB21MT-5 ferments on choline within a dedicated bacterial microcompartment organelle. Furthermore, the defence systems possessed by DB21MT-5 are distinct from other Moritella strains but resemble those in obligate piezophiles obtained from the same geographical setting. Collectively, the intensive comparative genomic analysis of an obligately piezophilic strain Moritella yayanosii DB21MT-5 demonstrates a depth-dependent distribution of energy metabolic pathways, compartmentalization of important metabolism and use of distinct defence systems, which likely contribute to microbial adaptation to the bottom of hadal trench.

With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli .

The mechanism of pyruvate-underproduction of aneuploid sake yeast was investigated in this study. In our previous report, we revealed that an increase in chromosome XI decreases pyruvate productivity of sake yeast. In this report, we found that increased copy number of CCP1, which is located on chromosome XI and encodes cytochrome-c peroxidase, decreased the pyruvate productivity of sake yeasts. Introducing an extra copy of CCP1 activated respiratory metabolism governed by Hap4 and increased reactive oxygen species. Therefore, it was concluded that increased copy number of CCP1 on chromosome XI activated respiratory metabolism and decreased pyruvate levels in an aneuploid sake yeast. This is the first report that describes a mechanism underlying the improvement of brewery yeast by chromosomal aneuploidy., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Sake yeast is mostly diploid, so the introduction of recessive mutations to improve brewing characteristics requires considerable effort. To construct sake yeast with multiple excellent brewing characteristics, we used an evidence-based approach that exploits genome editing technology. Our breeding targeted the AWA1 , CAR1 , MDE1 , and FAS2 genes. We introduced eight mutations into standard sake yeast to construct a non-foam-forming strain that makes sake without producing carcinogens or an unpleasant odor, while producing a sweet ginjo aroma. Small-scale fermentation tests showed that the desired sake could be brewed with our genome-edited strains. The existence of a few unexpected genetic perturbations introduced during breeding proved that genome editing technology is extremely effective for the serial breeding of sake yeast.

Non-lactose-fermenting Escherichia coli (NLFEC) has a few descriptive studies restricted to human infections. In the present study, isolates of NLFEC obtained from urine samples of dogs with hyperadrenocorticism were characterized regarding their virulence ability, biofilm formation capacity and antimicrobial susceptibility profile. Escherichia coli lactose-fermenting strains from urinary infection in dogs with the same conditions were analysed to provide comparisons. The non-lactose-fermenting E. coli strains were classified as belonging to clade I E. coli, whereas the lactose-fermenting strains were classified in phylogroup B2. All strains presented virulence markers to adhesion, iron acquisition, toxins, colicin and cytotoxin production, and biofilm regulation. Components of the extracellular matrix in addition to the in vitro biofilm formation ability were observed in the strains. Multidrug resistance (MDR) profiles were observed by in vitro susceptibility tests to all NLFEC strains. In summary, non-lactose-fermenting uropathogenic E. coli from dogs behaves similar to lactose-fermenting E. coli, exhibiting MDR profile, and pathogenic potential of promote animal infections., (© 2021 The Society for Applied Microbiology.)

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans , that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

In response to environmental stress, microorganisms adapt to drastic changes while exerting cellular functions by controlling gene expression, metabolic pathways, enzyme activities, and protein-protein interactions. Microbial cells that undergo a fermentation process are subjected to stresses, such as high temperature, freezing, drying, changes in pH and osmotic pressure, and organic solvents. Combinations of these stresses that continue over long terms often inhibit cells' growth and lead to their death, markedly limiting the useful functions of microorganisms (eg their fermentation ability). Thus, high stress tolerance of cells is required to improve productivity and add value to fermented/brewed foods and biofuels. This review focuses on stress tolerance mechanisms, including l-proline/l-arginine metabolism, ubiquitin system, and transcription factors, and the functional development of the yeast Saccharomyces cerevisiae, which has been used not only in basic science as a model of higher eukaryotes but also in fermentation processes for making alcoholic beverages, food products, and bioethanol., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Thermotolerant microorganisms are useful for high-temperature fermentation. Several thermally adapted strains were previously obtained from Acetobacter pasteurianus in a nutrient-rich culture medium, while these adapted strains could not grow well at high temperature in the nutrient-poor practical culture medium, "rice moromi." In this study, A. pasteurianus K-1034 originally capable of performing acetic acid fermentation in rice moromi was thermally adapted by experimental evolution using a "pseudo" rice moromi culture. The adapted strains thus obtained were confirmed to grow well in such the nutrient-poor media in flask or jar-fermentor culture up to 40 or 39 °C; the mutation sites of the strains were also determined. The high-temperature fermentation ability was also shown to be comparable with a low-nutrient adapted strain previously obtained. Using the practical fermentation system, "Acetofermenter," acetic acid production was compared in the moromi culture; the results showed that the adapted strains efficiently perform practical vinegar production under high-temperature conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.

In spite of increased complexity in eukaryotes compared to prokaryotes, several basic metabolic and regulatory processes are conserved. Here we explored analogies in the eubacteria Escherichia coli and the unicellular fission yeast Schizosaccharomyces pombe transcriptomes under two carbon sources: 2% glucose; or a mix of 2% glycerol and 0.2% sodium acetate using the same growth media and growth phase. Overall, twelve RNA-seq libraries were constructed. A total of 593 and 860 genes were detected as differentially expressed for E. coli and S. pombe, respectively, with a log2 of the Fold Change ≥ 1 and False Discovery Rate ≤ 0.05. In aerobic glycolysis, most of the expressed genes were associated with cell proliferation in both organisms, including amino acid metabolism and glycolysis. In contrast in glycerol/acetate condition, genes related to flagellar assembly and membrane proteins were differentially expressed such as the general transcription factors fliA, flhD, flhC, and flagellum assembly genes were detected in E. coli, whereas in S. pombe genes for hexose transporters, integral membrane proteins, galactose metabolism, and ncRNAs related to cellular stress were overexpressed. In general, our study shows that a conserved "foraging behavior" response is observed in these eukaryotic and eubacterial organisms in gluconeogenic carbon sources., Competing Interests: The authors have declared that no competing interests exist.

Amid known microbial bioethanol producers, the yeast Scheffersomyces (Pichia) stipitis is particularly promising in terms of alcoholic fermentation of both glucose and xylose, the main constituents of lignocellulosic biomass hydrolysates. However, the ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a feasible industrial technology; therefore, the construction of more efficient S. stipitis ethanol producers is of great significance. The aim of this study was to isolate the insertional mutants of S. stipitis with altered ethanol production from glucose and xylose and to identify the disrupted gene(s). Mutants obtained by random insertional mutagenesis were screened for their growth abilities on solid media with different sugars and for resistance to 3-bromopyruvate. Of more than 1,300 screened mutants, 17 were identified to have significantly changed ethanol yields during the fermentation. In one of the best fermenting strains (strain 4.6), insertion was found to occur within the ORF of a homolog to the Saccharomyces cerevisiae gene HEM25 (YDL119C), encoding a mitochondrial glycine transporter required for heme synthesis. The role of HEM25 in heme accumulation, respiration, and alcoholic fermentation in the yeast S. stipitis was studied using strain 4.6, the complementation strain Comp-a derivative from the 4.6 strain with expression of the WT HEM25 allele and the deletion strain hem25Δ. As hem25Δ produced lower amounts of ethanol than strain 4.6, we assume that the phenotype of strain 4.6 may be caused not only by HEM25 disruption but additionally by some point mutation., (© 2019 International Federation for Cell Biology.)

Fermentative production of L-cysteine has been established using Escherichia coli. In that procedure, thiosulfate is a beneficial sulfur source, whereas repressing sulfate utilization. We first found that thiosulfate decreased transcript levels of genes related to sulfur assimilation, particularly whose expression is controlled by the transcription factor CysB. Therefore, a novel approach, i.e. increment of expression of genes involved in sulfur-assimilation, was attempted for further improvement of L-cysteine overproduction. Disruption of the rppH gene significantly augmented transcript levels of the cysD, cysJ, cysM and yeeE genes (≥1.5-times) in medium containing sulfate as a sole sulfur source, probably because the rppH gene encodes mRNA pyrophosphohydrolase that triggers degradation of certain mRNAs. In addition, the ΔrppH strain appeared to preferentially uptake thiosulfate rather than sulfate, though thiosulfate dramatically reduced expression of the known sulfate/thiosulfate transporter complexes in both ΔrppH and wild-type cells. We also found that both YeeE and YeeD are required for the strain without the transporters to grow in the presence of thiosulfate as a sole sulfur source. Therefore, yeeE and yeeD are assigned as genes responsible for thiosulfate uptake (tsuA and tsuB, respectively). In final, we applied the ΔrppH strain to the fermentative production of L-cysteine. Disruption of the rppH gene enhanced L-cysteine biosynthesis, as a result, a strain producing approximately twice as much L-cysteine as the control strain was obtained.

Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT (microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000 Bacillus subtilis cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Yeast plays a key role in the production of fermented foods and beverages, such as bread, wine, and other alcoholic beverages. They are able to produce and release from the fermentation environment large numbers of volatile organic compounds (VOCs). This is the reason for the great interest in the possibility of adapting these microorganisms to fermentation at reduced temperatures. By doing this, it would be possible to obtain better sensory profiles of the final products. It can reduce the addition of artificial flavors and enhancements to food products and influence other important factors of fermented food production. Here, we reviewed the genetic and physiological mechanisms by which yeasts adapt to low temperatures. Next, we discussed the importance of VOCs for the food industry, their biosynthesis, and the most common volatiles in fermented foods and described the beneficial impact of decreased temperature as a factor that contributes to improving the composition of the sensory profiles of fermented foods.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum . This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H . uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Lactiplantibacillus plantarum can adapt to a variety of niches and is widely distributed in many sources. We used comparative genomics to explore the differences in the genome and in the physiological characteristics of L. plantarum isolated from pickles, fermented sauce, and human feces. The relationships between genotypes and phenotypes were analyzed to address the effects of isolation source on the genetic variation of L. plantarum . The comparative genomic results indicate that the numbers of unique genes in the different strains were niche-dependent. L. plantarum isolated from fecal sources generally had more strain-specific genes than L. plantarum isolated from pickles. The phylogenetic tree and average nucleotide identity (ANI) results indicate that L. plantarum in pickles and fermented sauce clustered independently, whereas the fecal L. plantarum was distributed more uniformly in the phylogenetic tree. The pan-genome curve indicated that the L. plantarum exhibited high genomic diversity. Based on the analysis of the carbohydrate active enzyme and carbohydrate-use abilities, we found that L. plantarum strains isolated from different sources exhibited different expression of the Glycoside Hydrolases (GH) and Glycosyl Transferases (GT) families and that the expression patterns of carbohydrate active enzymes were consistent with the evolution relationships of the strains. L. plantarum strains exhibited niche-specific characteristicsand the results provided better understating on genetics of this species.

Saccharomyces cerevisiae is the primary yeast species responsible for most fermentations in winemaking. However, other yeasts, including Saccharomyces uvarum, have occasionally been found conducting commercial fermentations around the world. S. uvarum is typically associated with white wine fermentations in cool-climate wine regions, and has been identified as the dominant yeast in fermentations from France, Hungary, northern Italy, and, recently, Canada. However, little is known about how the origin and genetic diversity of the Canadian S. uvarum population relates to strains from other parts of the world. In this study, a highly diverse S. uvarum population was found dominating uninoculated commercial fermentations of Chardonnay grapes sourced from two different vineyards. Most of the strains identified were found to be genetically distinct from S. uvarum strains isolated globally. Of the 106 strains of S. uvarum identified in this study, four played a dominant role in the fermentations, with some strains predominating in the fermentations from one vineyard over the other. Furthermore, two of these dominant strains were previously identified as dominant strains in uninoculated Chardonnay fermentations at the same winery two years earlier, suggesting the presence of a winery-resident population of indigenous S. uvarum. This research provides valuable insight into the diversity and persistence of non-commercial S. uvarum strains in North America, and a stepping stone for future work into the enological potential of an alternative Saccharomyces yeast species., Competing Interests: The authors have declared that no competing interests exist.

Objective: Lipases are often used in immobilized form, but commercial immobilized lipases are costly. An alternative is to produce lipases in solid-state fermentation, dry the solids and then use the "dry fermented solids" (DFS) directly. We produced DFS by growing Burkholderia contaminans on a mixture of sugarcane bagasse and sunflower seed meal and used the DFS to esterify oleic acid with ethanol in subcritical and supercritical CO 2 at 40 °C., Results: Compared to a control without CO 2 at atmospheric pressure, subcritical CO 2 at 30 bar improved esterification activity 1.2-fold. Higher pressures, including supercritical pressures up to 150 bar, reduced activity to less than 80% of the control. At 30 bar, the esterification activity was improved a further 1.8-fold with the addition of 9% water (i.e. 9 g water per 100 g oleic acid) to the reaction medium., Conclusion: A subcritical CO 2 atmosphere, with the addition of a small amount of water, improved the esterification activity of DFS containing lipases of Burkholderia contaminans.

Objectives: A redox potential-driven fermentation, maintaining dissolved oxygen at a prescribed level while simultaneously monitoring the changes of fermentation redox potential, was developed to guide the cultivation progress of recombinant protein expression., Results: A recombinant E. coli harboring prolinase-expressing plasmid (pKK-PepR2) was cultivated using the developed process. Two distinct ORP valleys were noticeable based on recorded profile. The first ORP valley is equivalent to the timing for the addition of inducing agent, and the second ORP valley serves to guide the timing for cell harvesting. The final prolinase activity is 0.172 μmol/mg/min as compared to that of 0.154 μmol/mg/min where the optical density was employed to guide the timing of inducer addition and an empirically determined length of the cultivation., Conclusion: The developed process can be further modified to become an automatic operation.

Objective: O-acetylhomoserine (OAH) is an important platform chemical to produce high-valuable chemicals. To improve the production of O-acetylhomoserine from glycerol, the glycerol-oxidative pathway was investigated and the optimization of fermentation with crude glycerol was carried out., Results: The glycerol-uptake system and glycerol-oxidative pathway were modified and O-acetyltransferase from Corynebacterium glutamicum was introduced into the engineered strain to produce O-acetylhomoserine. It was found that overexpression of glycerol 3-phosphate dehydrogenase improved the OAH production to 6.79 and 4.21 g/L from pure and crude glycerol, respectively. And the higher OAH production depending on higher level of transcription of glpD. Two-step statistical approach was employed to optimize the fermentation conditions. The significant effects of glycerol, ammonium chloride and yeast extract were screened applying Plackett-Burman design and were optimized further by employing the Response Surface Methodology. Under optimized conditions, the OAH production was up to 9.42 and 7.01 g/L when pure and crude glycerol were used in shake flask cultivations, respectively., Conclusions: The enzymatic step catalyzing the oxidation of glycerol through GlpD was the key step for OAH production, which served the foundation for realization of a consistent OAH production from crude glycerol in the future.

The main aim of this study was to investigate the effects of the initial pH values of the buffer on photo-fermentation biohydrogen production. Hydrogen production and the kinetics of it under different initial pH values were analyzed. Effects of initial pH values on reducing sugar consumption, hydrogen production rate, and byproduct production were evaluated at initial pH values of 5-7. The results showed that initial pH values of phosphate buffer had a significant effect on biohydrogen production via photo-fermentation. With the initial pH value of phosphate buffer at 6.0, the cumulative hydrogen production reached its maximum, 569.6 mL. The maximum hydrogen production rate was 23.96 mL/h at the initial pH value of 6.5. With the initial pH values at 5.0 and 7.5, the maximum hydrogen production rates were becoming lower, only 5.59 mL/h and 5.42 mL/h, respectively. And with the increase in pH values, the peak period of hydrogen production was gradually delayed, indicating that the alkaline environment had a negative effect on the ability of photosynthetic bacteria. This study revealed the influence of phosphate buffer initial pH values on the biohydrogen production via photo-fermentation and aimed to provide a scientific reference for further improving the theory and technology for biohydrogen production from biomass.

L-tyrosine is a widely used aromatic amino acid with an increasing market demand. Improving the parameters of L-tyrosine production results in a more cost-effective process that is of great interest for industrial applications. E. coli GHLTYR-168 was used to ferment L-tyrosine, with a productivity of 1.73 g/(L·h) and a yield of 17.6%. To further increase its production efficiency, repeated batch fermentation was applied to L-tyrosine, in which both replacement time points and ratios were studied during different fermentations. The broth substitution time point had no significant effect on L-tyrosine subjected to repeated batch fermentation, and 70% broth replacement ratio was the best choice. Repeated batch fermentation was performed in 5 batches within 100 h, among which the efficiency of the third batch fermentation was the highest. In the third batch fermentation, the productivity and yield were 2.53 g/(L·h) and 30.1%, respectively. Compared with that during fed-batch fermentation, the productivity and yield of L-tyrosine increased by 43.8% and 74.0%, respectively, during repeated batch fermentation. This is the highest level of L-tyrosine fermentation reported so far. Thus, repeated batch fermentation of L-tyrosine can improve its production efficiency.

As essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. For example, in this study, we engineered campesterol production in yeast using plant enzymes; although we were able to enhance the titer of campesterol to ∼40 mg/L by upregulating the mevalonate pathway, no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, many heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through repeated culture. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to reconstruct the multienzyme pathways for the synthesis of a set of phytosterols: campesterol (∼7 mg/L), β-sitosterol (∼2 mg/L), 22-hydroxycampesterol (∼1 mg/L), and 22-hydroxycampest-4-en-3-one (∼4 mg/L). This work identified and addressed some of the technical bottlenecks in phytosterol-derived pathway reconstitution in the baker's yeast and opens up opportunities for efficient bioproduction and metabolic pathway elucidation of this group of phytochemicals.

Mining biosynthetic genes for the exploration of hybrid metabolic pathways is a promising approach in heterologous production of natural and unnatural products. Here, we developed an integrative biosynthetic gene cluster (BGC) mining strategy to engineer the biosynthesis of l-homophenylalanine (l-Hph), an important intermediate for the synthesis of angiotensin-converting enzyme inhibitors. We assembled the putative l-Hph BGCs and integrated phylogenetic analysis with target metabolite abundance mapping to prioritize candidate BGCs. To obtain an effective l-Hph pathway, various combinations of candidate genes from different species were screened in an iterative design-build-test stepwise manner. After the pathway was strength balanced and the metabolic flux was enhanced, engineered Escherichia coli produced 1.41 g/L of l-Hph from glucose in feeding shake-flask fermentation. Our cluster mining strategy enabled optimization of the target metabolic pathway, and it would be promising for production of other valuable products in the postgenomic era.

Lactic acid bacteria (LAB) play an important role in kimchi fermentation by metabolizing raw materials into diverse metabolites. Bacterial adaptation is therefore a crucial element of fermentation. In this study, we investigated the transcriptional changes of Lactobacillus plantarum under acidic conditions to evaluate the elements of bacterial adaptation critical for fermentation. Differentially expressed genes (DEGs) have shown that transport function is primarily affected by acidic conditions. Five of the 13 significantly down-regulated genes and 7 of the 25 significantly up-regulated genes were found to have transport-related functions. We quantified the intracellular leucine content of bacteria grown at different pH ranges, determining that optimal bacterial leucine transport could be controlled by acidity during fermentation. Inhibition of L. plantarum growth was investigated and compared with other LAB at a pH range of 6.2-5.0. Interestingly, valinomycin inhibited L. plantarum growth from pH 6.2 to 5.0. This showed that L. plantarum had a wider range of transport functions than other LAB. These results suggested that L. plantarum had robust transport functions, and that this was the crucial factor for bacterial adaptation during fermentation.

Utilising one-carbon substrates such as carbon dioxide, methane, and methanol is vital to address the current climate crisis. Methylotrophic metabolism enables growth and energy generation from methanol, providing an alternative to sugar fermentation. Saccharomyces cerevisiae is an important industrial microorganism for which growth on one-carbon substrates would be relevant. However, its ability to metabolize methanol has been poorly characterised. Here, using adaptive laboratory evolution and 13 C-tracer analysis, we discover that S. cerevisiae has a native capacity for methylotrophy. A systems biology approach reveals that global rearrangements in central carbon metabolism fluxes, gene expression changes, and a truncation of the uncharacterized transcriptional regulator Ygr067cp supports improved methylotrophy in laboratory evolved S. cerevisiae. This research paves the way for further biotechnological development and fundamental understanding of methylotrophy in the preeminent eukaryotic model organism and industrial workhorse, S. cerevisiae.

A grand challenge of biological chemical production is the competition between synthetic circuits and host genes for limited cellular resources. Quorum sensing (QS)-based dynamic pathway regulations provide a pathway-independent way to rebalance metabolic flux over the course of the fermentation. Most cases, however, these pathway-independent strategies only have capacity for a single QS circuit functional in one cell. Furthermore, current dynamic regulations mainly provide localized control of metabolic flux. Here, with the aid of engineering synthetic orthogonal quorum-related circuits and global mRNA decay, we report a pathway-independent dynamic resource allocation strategy, which allows us to independently controlling two different phenotypic states to globally redistribute cellular resources toward synthetic circuits. The strategy which could pathway-independently and globally self-regulate two desired cell phenotypes including growth and production phenotypes could totally eliminate the need for human supervision of the entire fermentation.