Industrial Microbiology & Biotechnology

Synthetic microbial consortia for biosynthesis and biodegradation: promises and challenges Abstract Functional differentiation and metabolite exchange enable microbial consortia to perform complex metabolic tasks and efficiently cycle the nutrients. Inspired by the cooperative relationships in environmental microbial consortia, synthetic microbial consortia have great promise for studying the microbial interactions in nature and more importantly for various engineering applications. However, challenges coexist with promises, and the potential of consortium-based technologies is far from being fully harnessed. Thorough understanding of the underlying molecular mechanisms of microbial interactions is greatly needed for the rational design and optimization of defined consortia. These knowledge gaps could be potentially filled with the assistance of the ongoing revolution in systems biology and synthetic biology tools. As current fundamental and technical obstacles down the road being removed, we would expect new avenues with synthetic microbial consortia playing important roles in biological and environmental engineering processes such as bioproduction of desired chemicals and fuels, as well as biodegradation of persistent contaminants.

Promoting microbial utilization of phenolic substrates from bio-oil Abstract The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70 wt% Tween® 20 and 30 wt% Span® 80. Pseudomonas putida KT2440 grew to an optical density (OD600) of 1.0 ± 0.2 when supplied with 1.6 wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002 g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization.

Stable N -acetyltransferase Mpr1 improves ethanol productivity in the sake yeast Saccharomyces cerevisiae Abstract N-Acetyltransferase Mpr1 was originally discovered as an enzyme that detoxifies l-azetidine-2-carboxylate through its N-acetylation in the yeast Saccharomyces cerevisiae Σ1278b. Mpr1 protects yeast cells from oxidative stresses possibly by activating a novel l-arginine biosynthesis. We recently constructed a stable variant of Mpr1 (N203K) by a rational design based on the structure of the wild-type Mpr1 (WT). Here, we examined the effects of N203K on ethanol fermentation of the sake yeast S. cerevisiae strain lacking the MPR1 gene. When N203K was expressed in the diploid Japanese sake strain, its fermentation performance was improved compared to WT. In a laboratory-scale brewing, a sake strain expressing N203K produced more ethanol than WT. N203K also affected the contents of flavor compounds and organic acids. These results suggest that the stable Mpr1 variant contributes to the construction of new industrial yeast strains with improved fermentation ability and diversity of taste and flavor.

Influence of R and S enantiomers of 1-octen-3-ol on gene expression of Penicillium chrysogenum Abstract Inhibition of spore germination offers an attractive and effective target for controlling fungal species involved in food spoilage. Mushroom alcohol (1-octen-3-ol) functions as a natural self-inhibitor of spore germination for many fungi and, therefore, provides a useful tool for probing the molecular events controlling the early stages of fungal growth. In Penicillium spp., the R and S enantiomers of 1-octen-3-ol delayed spore germination and sporulation in four species of Penicillium involved in soils of fruit and grains, but to different degrees. Because of its well-annotated genome, we used Penicillium chrysogenum to perform a comprehensive comparative transcriptomic analysis of cultures treated with the two enantiomers. Altogether, about 80% of the high-quality reads could be mapped to 11,396 genes in the reference genome. The top three active pathways were metabolic (978 transcripts), biosynthesis of secondary metabolites (420 transcripts), and microbial metabolism in diverse environments (318 transcripts). When compared to the control, treatment with (R)-(-)-1-octen-3-ol affected the transcription levels of 91 genes, while (S)-(+)-1-octen-3-ol affected only 41 genes. Most of the affected transcripts were annotated and predicted to be involved in transport, establishment of localization, and transmembrane transport. Alternative splicing and SNPs' analyses indicated that, compared to the control, the R enantiomer had greater effects on the gene expression pattern of Penicillium chrysogenum than the S enantiomer. A qRT-PCR analysis of 28 randomly selected differentially expressed genes confirmed the transcriptome data. The transcriptomic data have been deposited in NCBI SRA under the accession number SRX1065226.

Metabolic engineering of glucose uptake systems in Corynebacterium glutamicum for improving the efficiency of l -lysine production Abstract Traditional amino acid producers typically exhibit the low glucose uptake rate and growth deficiency, resulting in a long fermentation time because of the accumulation of side mutations in breeding of strains. In this study, we demonstrate that the efficiency of l-lysine production in traditional l-lysine producer Corynebacterium glutamicum ZL-9 can be improved by rationally engineering glucose uptake systems. To do this, different bypasses for glucose uptake were investigated to reveal the best glucose uptake system for l-lysine production in traditional l-lysine producer. This study showed that overexpression of the key genes in PTSGlc or non-PTSGlc increased the glucose consumption, growth rate, and l-lysine production. However, increasing the function of PTSGlc in glucose uptake led to the increase of by-products, especially for plasmid-mediated expression system. Increasing the participation of non-PTSGlc in glucose utilization showed the best glucose uptake system for l-lysine production. The final strain ZL-92 with increasing the expression level of iolT1, iolT2 and ppgK could produce 201.6 ± 13.8 g/L of l-lysine with a productivity of 5.04 g/L/h and carbon yield of 0.65 g/(g glucose) in fed-batch culture. This is the first report of a rational modification of glucose uptake systems that improve the efficiency of l-lysine production through increasing the participation of non-PTSGlc in glucose utilization in traditional l-lysine producer. Similar strategies can be also used for producing other amino acids or their derivatives.

Over-expression of Isu1p and Jac1p increases the ethanol tolerance and yield by superoxide and iron homeostasis mechanism in an engineered Saccharomyces cerevisiae yeast Abstract The ethanol stress response in ethanologenic yeast during fermentation involves the swishing of several adaptation mechanisms. In Saccharomyces cerevisiae, the Jac1p and Isu1p proteins constitute the scaffold system for the Fe–S cluster assembly. This study was performed using the over-expression of the Jac1p and Isu1p in the industrially utilized S. cerevisiae UMArn3 strain, with the objective of improving the Fe–S assembly/recycling, and thus counteracting the toxic effects of ethanol stress during fermentation. The UMArn3 yeast was transformed with both the JAC1-His and ISU1-His genes-plasmid contained. The Jac1p and Isu1p His-tagged proteins over-expression in the engineered yeasts was confirmed by immunodetection, rendering increases in ethanol tolerance level from a DL50 = ~ 4.5% ethanol (v/v) to DL50 = ~ 8.2% ethanol (v/v), and survival up 90% at 15% ethanol (v/v) comparing to ~ 50% survival in the control strain. Fermentation by the engineered yeasts showed that the ethanol production was increased, producing 15–20% more ethanol than the control yeast. The decrease of ROS and free-iron accumulation was observed in the engineered yeasts under ethanol stress condition. The results indicate that Jac1p and Isu1p over-expression in the S. cerevisiaeUMArn3.3 yeast increased its ethanol tolerance level and ethanol production by a mechanism that involves ROS and iron homeostasis related to the biogenesis/recycling of Fe–S clusters dependent proteins.

Effect of applied voltage and temperature on methane production and microbial community in microbial electrochemical anaerobic digestion systems treating swine manure Abstract Microbial electrochemical technology (MET) that can harvest electricity/valuable materials and enhance the efficiency of conventional biological processes through the redox reaction of organic/inorganic compounds has attracted considerable attention. MET-based anaerobic digestion (AD) systems treating swine manure were operated at different applied voltages (0.1, 0.3, 0.5, 0.7, and 0.9 V) and temperatures (25, 35, and 45 °C). Among the MET-based AD systems with different applied voltages at 35 °C, M4 at 0.7 V showed the highest methane production (2.96 m3-CH4/m3) and methane yield (0.64 m3-CH4/kg-VS). The methane production and yield increased with increasing temperature at an applied voltage of 0.7 V. Nevertheless, the MET-based AD systems (LM at 25 °C and 0.7V) showed competitive AD performance (2.33 m3-CH4/m3 and 0.53 m3-CH4/VS) compared with the conventional AD system (35 °C). The microbial community was affected by the applied voltage and temperature, and hydrogenotrophic methanogens such as M. flavescens, M. hungatei, and M. thermautotrophicus were mainly responsible for methane production in MET-based AD systems. Therefore, the methane production can be enhanced by an applied voltage or by direct interspecies electron transfer because M. flavescens and M. thermautotrophicus were especially predominant in cathode of MET-based AD systems. The MET-based AD systems can help enhance biogas production from swine manure with no significant change in methane content. Furthermore, MET-based AD systems will be a promising AD system through low material development and the optimal operation.

Characterization and engineering of the Lrp/AsnC family regulator SACE_5717 for erythromycin overproduction in Saccharopolyspora erythraea Abstract In this work, we found that the Lrp/AsnC family protein SACE_5717 negatively regulated erythromycin biosynthesis in S. erythraea. Disruption of SACE_5717 led to a 27% improvement in the yield of erythromycin in S. erythraea A226. SACE_5717 directly repressed its own gene expression, as well as that of the adjacent gene SACE_5716 by binding to the target sequence 5′-GAACGTTCGCCGTCACGCC-3′. The predicted LysE superfamily protein SACE_5716 directly influenced the export of lysine, histidine, threonine and glycine in S. erythraea. Arginine, tyrosine and tryptophan were characterized as the effectors of SACE_5717 by weakening the binding affinity of SACE_5717. In the industrial S. erythraea WB strain, deletion of SACE_5717 (WBΔSACE_5717) increased erythromycin yield by 20%, and by 36% when SACE_5716 was overexpressed in WBΔSACE_5717 (WBΔSACE_5717/5716). In large-scale 5-L fermentation experiment, erythromycin yield in the engineered strain WBΔSACE_5717/5716 reached 4686 mg/L, a 41% enhancement over 3323 mg/L of the parent WB strain.

Inactivation of the uptake hydrogenase in the purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS enables a biological water–gas shift platform for H 2 production Abstract Biological H2 production has potential to address energy security and environmental concerns if produced from renewable or waste sources. The purple non-sulfur photosynthetic bacterium Rubrivivax gelatinosus CBS produces H2 while oxidizing CO, a component of synthesis gas (Syngas). CO-linked H2 production is facilitated by an energy-converting hydrogenase (Ech), while a subsequent H2 oxidation reaction is catalyzed by a membrane-bound hydrogenase (MBH). Both hydrogenases contain [NiFe] active sites requiring 6 maturation factors (HypA-F) for assembly, but it is unclear which of the two annotated sets of hyp genes are required for each in R. gelatinosus CBS. Herein, we report correlated expression of hyp1 genes with Ech genes and hyp2 expression with MBH genes. Moreover, we find that while Ech H2 evolving activity is only delayed when hyp1 is deleted, hyp2 deletion completely disrupts MBH H2 uptake, providing a platform for a biologically driven water–gas shift reaction to produce H2 from CO.

CRISPR/Cas9-mediated engineering of Escherichia coli for n -butanol production from xylose in defined medium Abstract Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1–xylose medium.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com