High-level Production of Isoleucine and Fusel alcohol by expression of the Feedback Inhibition-insensitive Threonine deaminase in Saccharomyces cerevisiae

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2-3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Overexpression of SRO_3163, a homolog of Streptomyces antibiotic regulatory protein, induces the production of novel cyclohexene-containing enamide in Streptomyces rochei

Streptomyces antibiotic regulatory proteins (SARPs) are well characterized as transcriptional activators for secondary metabolites in Streptomyces species. Streptomyces rochei 7434AN4 harbors 15 SARP genes, among which 3 were located on a giant linear plasmid pSLA2-L and others were on the chromosome. Some SARP genes were cloned into an integrative thiostrepton-inducible vector pIJ8600, and their recombinants were cultivated. The recombinant of SARP gene, SRO_3163, accumulated a UV-active compound YM3163-A, which was not detected in the parent strain and other SARP recombinants. Its molecular formula was established to be C8H11NO. Extensive NMR analysis revealed that YM3163-A is a novel enamide, 2-(cyclohex-2-en-1-ylidene)acetamide, and its structure was confirmed by chemical synthesis including Horner-Wadsworth-Emmons reaction and ammonolysis.

Three orphan histidine kinases inhibit Clostridioides difficile sporulation

The ability of the anaerobic gastrointestinal pathogen, Clostridioides difficile, to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a conserved phosphotransfer protein, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional conserved orphan phosphotransfer proteins, CD2492, CD1579, and CD1949 which are hypothesized to regulate Spo0A phosphorylation. Disruption of the conserved phosphotransfer protein, CD2492, also increased sporulation frequency, similarly to the CD1492 mutant, and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of the conserved CD1579 phosphotransfer protein also variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492 and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant negative hypersporulation phenotype. Altogether, at least three phosphotransfer proteins, CD1492, CD2492 and CD1579 (herein, PtpA, PtpB and PtpC) repress C. difficile sporulation initiation by regulating activity of Spo0A.

Roles of OmpA in Type III Secretion System-Mediated Virulence of Enterohemorrhagic Escherichia coli

Outer membrane proteins are commonly produced by gram-negative bacteria, and they have diverse functions. A subgroup of proteins, which includes OmpA, OmpW and OmpX, is often involved in bacterial pathogenesis. Here we show that OmpA, rather than OmpW or OmpX, contributes to the virulence of enterohemorrhagic Escherichia coli (EHEC) through its type III secretion system (T3SS). Deletion of ompA decreased secretion of the T3SS proteins EspA and EspB; however, the expression level of the LEE genes that encode a set of T3SS proteins did not decrease. The ompA mutant had less abilities to form A/E lesions in host epithelial cells and lyse human red blood cells than the parent strain. Moreover, the virulence of an ompA mutant of Citrobacter rodentium (traditionally used to estimate T3SS-associated virulence in mice) was attenuated. Mice infected with the ompA mutant survived longer than those infected with the parent strain. Furthermore, mice infected with ompA developed symptoms of diarrhea more slowly than mice infected with the parent strain. Altogether, these results suggest that OmpA sustains the activity of the T3SS and is required for optimal virulence in EHEC. This work expands the roles of outer membrane proteins in bacterial pathogenesis.

1275. Dynamics of Enterococcus faecalis Cardiolipin Synthase Gene Expression Reveal Compensatory Roles in Daptomycin Resistance

Background Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been associated with activation of the E. faecalis (Efs) LiaFSR response and redistribution of APL microdomains (predicted to contain cardiolipin) away from the septum. Efs encodes two putative cardiolipin synthase genes, cls1 and cls2. While changes in Cls1 are associated with DAP-R, the exact roles of each enzyme in resistance are unknown. This work aims to establish the contributions for both enzymes in the development of DAP-R. Methods cls1 and cls2 were deleted individually and in tandem from Efs OG117∆liaX (a DAP-R strain with an activated LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange staining. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117. Membrane lipid content was analyzed using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS). Results cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant phenotypic changes from the parent strain. Interestingly, qRT-PCR showed that cls2 expression was upregulated upon deletion of cls1 (and vice-versa), suggesting a compensatory role for one enzyme upon deletion of the other (Fig 1). When comparing membrane lipid content between Efs OG117∆liaX∆cls1 and Efs OG117∆liaX∆cls2, there were no significant differences in both the overall amount or species of cardiolipin generated, further supporting a potential redundancy between the cardiolipin synthases (Fig 2). Ultimately, double deletion of both cls genes lowered the DAP MIC relative to the parent strain and restored septal localization of APL microdomains. Conclusion Overall, Cls1 has a predominant role in the development of DAP-R in E. faecalis. However, here, we describe a novel compensatory role for Cls2 under conditions in which there is no functional Cls1 to maintain the DAP-R phenotype. Disclosures Truc T. Tran, PharmD, Merck (Grant/Research Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

Analysis of six tonB gene homologs in Bacteroides fragilis revealed that tonB3 is essential for survival in experimental intestinal colonization and intra-abdominal infection

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp) and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB’s role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro . Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1 , ΔtonB2 , ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1 , ΔtonB2 , ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo . The genetic organization of tonB1 , tonB2 , tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space - in contrast to an oxidative environment in aerobes - it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

Metabolic Engineering of Enterobacter aerogenes for Improved 2,3-Butanediol Production by Manipulating NADH Levels and Overexpressing the Small RNA RyhB

Biotechnological production of 2,3-butanediol (2,3-BD), a versatile platform bio-chemical and a potential biofuel, is limited due to by-product toxicity. In this study, we aimed to redirect the metabolic flux toward 2,3-BD in Enterobacter aerogenes (E. aerogenes) by increasing the intracellular NADH pool. Increasing the NADH/NAD+ ratio by knocking out the NADH dehydrogenase genes (nuoC/nuoD) enhanced 2,3-BD production by up to 67% compared with wild-type E. aerogenes. When lactate dehydrogenase (ldh) was knocked out, the yield of 2,3-BD was increased by 71.2% compared to the wild type. Metabolic flux analysis revealed that upregulated expression of the sRNA RyhB led to a noteworthy shift in metabolism. The 2,3-BD titer of the best mutant Ea-2 was almost seven times higher than that of the parent strain in a 5-L fermenter. In this study, an effective metabolic engineering strategy for improved 2,3-BD production was implemented by increasing the NADH/NAD+ ratio and blocking competing pathways.

Personal care formulations demonstrate virucidal efficacy against multiple SARS-CoV-2 variants of concern: implications for hand hygiene

The second and third waves of COVID-19 pandemic have largely been driven by the surge of successive SARS-CoV-2 variants of concern (VOC). These VOC have rapidly spread through multiple geographies being enabled by high transmission rates and/or high viral load compared to the original parent strain. Consequently, the altered phenotypes of these VOC have posed greater challenges to diagnostic and clinical management of COVID-19. Despite considerable progress being made on vaccine roll out, practicing proper hand hygiene has been advocated as a consistent precautionary intervention as more virulent VOC continue to emerge and spread across geographies.Two variants of concern, namely beta and delta, have recently been shown to escape antibody-mediated neutralization by virtue of acquired mutations in the receptor-binding domain of the viral spike protein which binds to the human ACE2 receptor for cellular entry. In this report we have empirically determined the efficacy of a range of personal care formulations in inactivating the beta and delta variants of SARS-CoV-2. High titres of these variants were exposed to marketed personal care formulations from Unilever under standard in-vitro suspension test-based conditions relevant to end-user habits. All the formulations demonstrated greater than 99.9% reduction in viral infective titres. The rate of inactivation by these products were comparable to that of the original strain of SARS-CoV-2 virus tested under the same conditions. Therefore, it can be concluded that well-designed personal care formulations when tested under consumer-centric conditions, and with proven efficacy against the parent strain of SARS-CoV-2 will continue to be effective against extant and emerging variants of SARS-CoV-2. This is through their broad-spectrum mode of action (disruption of lipid bilayer of the host-derived viral envelope, denaturation of envelop and nucleocapsid proteins, and disruption of genome) which is independent of the escape mutations that facilitate immune evasion or enhanced transmissibility.

Klebsiella Pneumoniae Metabolic Regulation and its Application in 1,3-Propanediol Production

Klebsiella pneumoniae is a well-known model organism for glycerol metabolism to produce 1,3-propanediol (1,3-PD), a valuable chemical intermediate for materials, such as polyesters. However, the relatively low conversion rate and productivity, as well as the accumulation of by-products such as lactic acid, ethanol and acetic acid, inhibit the production of 1,3-PD. Hereby, the 1,3-PD metabolism in K. pneumoniae was regulated through pathway engineering by using CRISPR-Cas9 technology for the first time to knock out the ldhA gene of lactate dehydrogenase,the adhE gene of alcohol dehydrogenase and the ack gene of acetate kinase respectively as needed and constructed recombinant bacteria ldhA(−), ldhA(−)-ack(−), ldhA(−)-adhE(−) and ldhA(−)-adhE(−)-ack(−), all of which showed a decrease in by-product production, leading to a higher NADH availability, and 1,3-BD production was significantly increased. In the shake flask fermentation, the 1,3-PD yield and conversion rate of the recombinant strain ldhA(−), ldhA(−)-ack(−), ldhA(−)-adhE(−), ldhA(−)-adhE(−)-ack(−) were higher than those of the parent strain. In the fed-batch fermentation, the 1,3-PD yield and conversion rate of the recombinant strain ldhA(−) were higher than those of the parent strain. The biomass of the recombinant strain ldhA(−)-adhE(−)-ack(−) was reduced due to the accumulation of acetic acid, but its 1,3-PD conversion rate was still higher than that of the parent strain. The higher productivity and fewer by-products concluded that the four Klebsiella pneumoniae recombinant strains could be promising industrial strain for economical production of 1,3-PD.

Improving the Production of Riboflavin by Introducing a Mutant Ribulose 5-Phosphate 3-Epimerase Gene in Bacillus subtilis

Ribulose 5-phosphate (Ru5P) and guanosine 5′-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5′-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29 ± 63.44 mg/L), showing the potential for further engineering of this strain.