Organic Wastes as Feedstocks for Non-Conventional Yeast-Based Bioprocesses

Non-conventional yeasts are efficient cell factories for the synthesis of value-added compounds such as recombinant proteins, intracellular metabolites, and/or metabolic by-products. Most bioprocess, however, are still designed to use pure, ideal sugars, especially glucose. In the quest for the development of more sustainable processes amid concerns over the future availability of resources for the ever-growing global population, the utilization of organic wastes or industrial by-products as feedstocks to support cell growth is a crucial approach. Indeed, vast amounts of industrial and commercial waste simultaneously represent an environmental burden and an important reservoir for recyclable or reusable material. These alternative feedstocks can provide microbial cell factories with the required metabolic building blocks and energy to synthesize value-added compounds, further representing a potential means of reduction of process costs as well. This review highlights recent strategies in this regard, encompassing knowledge on catabolic pathways and metabolic engineering solutions developed to endow cells with the required metabolic capabilities, and the connection of these to the synthesis of value-added compounds. This review focuses primarily, but not exclusively, on Yarrowia lipolytica as a yeast cell factory, owing to its broad range of naturally metabolizable carbon sources, together with its popularity as a non-conventional yeast. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

Published ArticleEndocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa

Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene-expression differences between Mut+ and MutS strains of Pichia pastoris.

peer reviewedPichia pastoris is a very popular yeast for recombinant protein production, mainly due to the strong, methanol-inducible P(AOX1) promoter. Methanol induction however poses several drawbacks. One approach to improve processes is to use MutS strains with reduced methanol catabolic ability. Various reports claim that MutS allows higher recombinant protein production levels than Mut+ but scarcely elaborate on reasons for differences. In this study, enhanced green fluorescent protein was used as a P(AOX1) -driven reporter for the investigation of expression differences between Mut+ and MutS strains. Mut+ exhibited higher responses to methanol, with faster growth (0.07 vs. 0.01 hr(-1) ) and higher consumption of methanol (2.25 vs. 1.81 mmol/g(DCW) .hr) and oxygen (2.2 vs. 0.66 mmol/g(DCW) .hr) than MutS. Mut+ yielded more biomass than MutS (2.3 vs. 1.3 g(DCW) /L), and carbon dioxide analysis of bioreactor off-gas suggested that considerable amounts of methanol were consumed by Mut+ via the dissimilatory pathway. In contrast, it was demonstrated that the MutS population switched to an induced state more rapidly than Mut+. In addition, MutS exhibited 3.4-fold higher fluorescence levels per cell (77,509 vs. 23,783 SFU) indicative of higher recombinant protein production. The findings were verified by similar results obtained during the expression of a lipase. Based on the differences in response to methanol versus recombinant protein production, it was proposed that higher energy availability occurs in MutS for recombinant protein synthesis, contrary to Mut+ that uses the energy to maintain high levels of methanol catabolic pathways and biomass production

Role of Dissimilative Pathway of Komagataella phaffii (Pichia pastoris): Formaldehyde Toxicity and Energy Metabolism

Komagataella phaffii (aka Pichia pastoris) is a yeast able to grow in methanol as the sole carbon and energy source. This substrate is converted into formaldehyde, a toxic intermediary that can either be assimilated to biomass or dissimilated to CO2 through the enzymes formaldehyde dehydrogenase (FLD) and formate dehydrogenase, also producing energy in the form of NADH. The dissimilative pathway has been described as an energy producing and a detoxifying route, but conclusive evidence has not been provided for this. In order to elucidate this theory, we generated mutants lacking the FLD activity (Δfld1) and used flux analysis to evaluate the metabolic impact of this disrupted pathway. Unexpectedly, we found that the specific growth rate of the Δfld1 strain was only slightly lower (92%) than the control. In contrast, the sensitivity to formaldehyde pulses (up to 8mM) was significantly higher in the Δfld1 mutant strain and was associated with a higher maintenance energy. In addition, the intracellular flux estimation revealed a high metabolic flexibility of K. phaffii in response to the disrupted pathway. Our results suggest that the role of the dissimilative pathway is mainly to protect the cells from the harmful effect of formaldehyde, as they were able to compensate for the energy provided from this pathway when disrupted

Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

Endocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa. Significance:  • First report of endocrine disruptor nonylphenol-using bacteria from South Africa • Six bacterial species capable of using nonylphenol as a carbon source were isolated • Results will pave the way for further exploration of endocrine disruptors degrading microbes from South Afric

Isolation and characterisation of endocrine disruptor nonylphenol-using bacteria from South Africa

Endocrine disrupting chemicals (EDCs) are synthetic chemicals that alter the function of endocrine systems in animals including humans. EDCs are considered priority pollutants and worldwide research is ongoing to develop bioremediation strategies to remove EDCs from the environment. An understanding of indigenous microorganisms is important to design efficient bioremediation strategies. However, much of the information available on EDCs has been generated from developed regions. Recent studies have revealed the presence of different EDCs in South African natural resources, but, to date, studies analysing the capabilities of microorganisms to utilise/degrade EDCs have not been reported from South Africa. Here, we report for the first time on the isolation and enrichment of six bacterial strains from six different soil samples collected from the Mpumalanga Province, which are capable of utilising EDC nonylphenol as a carbon source. Furthermore, we performed a preliminary characterisation of isolates concerning their phylogenetic identification and capabilities to degrade nonylphenol. Phylogenetic analysis using 16S rRNA gene sequencing revealed that four isolates belonged to Pseudomonas and the remaining two belonged to Enterobacteria and Stenotrophomonas. All six bacterial species showed degradation of nonylphenol in broth cultures, as HPLC analysis revealed 41–46% degradation of nonylphenol 12 h after addition. The results of this study represent the beginning of identification of microorganisms capable of degrading nonylphenol, and pave the way for further exploration of EDC-degrading microorganisms from South Africa. Significance:  First report of endocrine disruptor nonylphenol-using bacteria from South Africa Six bacterial species capable of using nonylphenol as a carbon source were isolated Results will pave the way for further exploration of endocrine disruptors degrading microbes from South Afric

The Potential Use of Bacteriophage Therapy as a Treatment Option in a Post-Antibiotic Era

The impending postantibiotic era creates an urgent requirement for alternative treatments of infectious diseases in humans and animals. Bacteriophages are viruses that infect and kill bacteria. The application of bacteriophages as a treatment option was investigated before the development of antibiotics. However, the initial success of antibiotic therapy soon shifted the focus from bacteriophage research. The revitalization of phage therapy has received increased global attention since the appearance of multidrug-resistant bacteria. Bacteriophages replicate via either the lytic or lysogenic cycle. While both life cycles have potential applications in bacteriophage therapy, the lytic cycle seems most suited to antibacterial therapy. The most striking advantage of bacteriophage therapy is the high degree of host specificity exhibited by these viruses, which enables the formulation of tailored treatments that kill only pathogenic bacteria. However, the high specificity of such treatments requires highly accurate diagnostic procedures in order to succeed. Other restrictions of bacteriophage therapy, such as limitations with the registration of phage therapy options, may possibly be overcome by the expression and engineering of phage lytic enzymes, which break the bacterial cell wall. The problem of bacterial immunity to phage infection also cannot be ignored, although it is more solvable than resistance to antibiotics. Considering the available information, phage therapy holds promise as an alternative treatment option, although the road ahead is not without obstacles. © 2016 Elsevier Inc. All rights reserved