Fermentation technology has unprecedented potential to upgrade state-of-art biotechnology and refine the processes used in existing ones, taking into account of complex technical, economic and environmental factors. Given the economic importance and ongoing challenges of biotech sector, multidisciplinary engineering technologies is poised to become an increasingly important tool along with the emergence of modern technology and innovation. This article reviews recent technology advancement in the field of fermentation using Saccharomyces cerevisiae. Interesting research progress has been made by leveraging multiple engineering fields such as electrical engineering, information engineering, electrochemical engineering and new material development, leading to recent development of novel real-time probes (electronic nose technology, analysis of yeast morphology and metabolites, timely control of glucose feed), improved understanding of electro-fermentation (enhanced electronic transfer provision), as well as application of cost-effective and sustainable materials (bioreactor vessel manufactured from textile, and yeast immobilisation support matrix made from abundant natural biomass). To the best of our knowledge, the subject is reviewed for the first time in recent years. Furthermore, this review also constitutes a futuristic S. cerevisiae fermentation process based on the recent advancement discussed.

Pigments are compounds with highly diverse structures and wide uses, which production is increasing worldwide. An eco-friendly method of bioproduction is to use the ability of some microorganisms to ferment on renewable carbon sources. Wheat bran (WB) is a cheap and abundant lignocellulosic co-product of low recalcitrance to biological conversion. Microbial candidates with theoretical ability to degrade WB were first preselected using specific databases. The microorganisms were Ashbya gossypii (producing riboflavin), Chitinophaga pinensis (producing flexirubin), Chromobacterium vaccinii (violacein) and Gordonia alkanivorans (carotenoids). Growth was shown for each on minimal salt medium supplemented with WB at 5 g.L-1. Activities of the main enzymes consuming WB were measured, showing leucine amino-peptidase (up to 8.45 IU. mL-1) and β-glucosidase activities (none to 6.44 IU. mL-1). This was coupled to a FTIR (Fourier Transform Infra-Red) study of the WB residues that showed main degradation of the WB protein fraction for C. pinensis, C. vaccinii and G. alkanivorans. Production of the pigments on WB was assessed for all the strains except Ashbya, with values of production reaching up to 1.47 mg.L-1. The polyphasic approach used in this study led to a proof of concept of pigment production from WB as a cheap carbon source.

Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31-64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims.

Biogas production through co-digestion of second and third generation substrates is an environmentally sustainable approach. Green willow biomass, chicken manure waste and microalgae biomass substrates were combined in the anaerobic digestion experiments. Biochemical methane potential test showed that biogas yields of co-digestions were significantly higher compared to the yield when energy willow was the sole substrate. To scale up the experiment continuous stirred-tank reactors (CSRTs) are employed, digestion parameters are monitored. Furthermore, genome-centric metagenomics approach was employed to gain functional insight into the complex anaerobic decomposing process. This revealed the importance of Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes phyla as major bacterial participants, while Methanomicrobia and Methanobacteria represented the archaeal constituents of the communities. The bacterial phyla were shown to perform the carbohydrate hydrolysis. Among the representatives of long-chain carbohydrate hydrolysing microbes Bin_61: Clostridia is newly identified metagenome assembled genome (MAG) and Bin_13: DTU010 sp900018335 is common and abundant in all CSTRs. Methanogenesis was linked to the slow-growing members of the community, where hydrogenotrophic methanogen species Methanoculleus (Bin_10) and Methanobacterium (Bin_4) predominate. A sensitive balance between H2 producers and consumers was shown to be critical for stable biomethane production and efficient waste biodegradation.

The shape of wool yarns was changed by laccase-assisted grafting of tyrosine. Prior to tyrosine grafting a cysteine pre-treatment was optimized aiming to increase the amount of thiol reaction groups available. The best operational conditions for laccase-assisted tyrosine grafting were: i) pre-treatment with cysteine (2.2 mM) in a solution of 20 % ethanol, 15 % propylene glycol and 0.5 % benzyl alcohol, pH = 10, 40 °C; ii) tyrosine grafting with 3.0 mM tyrosine, 18 U/mL laccase, pH = 5, 40 °C. The shape modification was evaluated by number of curly twists determination on the grafted yarn samples. The thermal and mechanical properties of the grafted wool yarns was evaluated by TGA, DSC and breaking strength determination. The amount of free thiols and weight gain were assessed aiming to infer the role of the cysteine pre-treatment on the final tyrosine grafting and shape modification. The laccase-assisted grafting of tyrosine onto wool yarns have influenced the thermal and mechanical properties of the yarns however without compromising their structural integrity for the final application purposes. The developed methodology to impart new shape to wool yarns is presented herein as an environmentally friendly alternative to chemical methods. The new findings revealed great potentialities for application in similar fibers like hair.

Genetic manipulation of plant genes in prokaryotes has been widely used in molecular biology, but the function of a DNA sequence is far from being fully known. Here, we discovered that a plant protein-coding gene containing the CRAL_TRIO domain serves as a promoter in bacteria. We firstly characterized CitPITP1 from Citrus, which contains the CRAL_TRIO domain, and identified a 64-bp sequence (key64) that is critical for prokaryotic promoter activity. In vitro experiments indicated that the bacterial RNA polymerase subunit RpoD specifically binds to key64. We then expanded our research to fungi, plant and animal species to identify key64-like sequences. Five such prokaryotic promoters were isolated from Amborella, Rice, Arabidopsis and Citrus. Two conserved motifs were identified, and mutation analysis indicated that the nucleotides at positions 7, 29 and 30 are crucial for key64-like transcription activity. We detected full-length recombinant CitPITP1 from E. coli, and visualized a CitPITP1-GFP fusion protein in plant cells, supporting the idea that CitPITP1 encodes a protein. However, although exon 4 of CitPITP1 contained key64, it did not demonstrate promoter activity in plants. Our study describes a new basal promoter, provides evidence for neofunction of gene elements across different kingdoms, and provides new knowledge for the modular design of promoters.

D-pantothenic acid (D-PA) is an essential vitamin that has been widely used in medicine, food, and animal feed. Microbial production of D-PA from natural renewable resources is attractive and challenging. In this study, both strain improvements and fermentation process strategies were applied to achieve high-level D-PA production in Escherichia coli. First, a D-PA-producing strain was developed through deletion of the aceF and mdh genes combined with the overexpression of the gene ppnk. The obtained engineered E. coli DPA02/pT-ppnk accumulated 6.89 ± 0.11 g/L of D-PA in shake flask fermentation, which was 79.9 % higher than the control strain. Moreover, the cultivation process contributed greatly to D-PA production with respect to titer and productivity by betaine supplementation and dissolved oxygen (DO)-feedback feeding framework. Under optimal conditions, 68.3 g/L of D-PA, the specific productivity of 0.794 g/L h and the yield of 0.36 g/g glucose in 5 L fermenter were achieved. Overall, this research successfully exploited advanced strategies to lay the foundation for bio-based D-PA production in industrial applications.

New Yarrowia lipolytica strains for the co-expression of steroidogenic mammalian proteins were obtained in this study. For this purpose, a two-step approach for constructing recombinant strains that permits the simple introduction of several expression cassettes encoding heterologous proteins into the yeast genome was successfully applied. This study tested two series of integrative multi-copy expression vectors containing cDNAs for the mature forms of P450scc system components (cytochrome P450scc (CYP11A1), adrenodoxin reductase, adrenodoxin, or fused adrenodoxin-P450scc) or for P45017α (CYP17A1) under the control of the isocitrate lyase promoter pICL1, which were constructed using the basic plasmids p64PT or p67PT (rDNA or the long terminal repeat (LTR) zeta of Ylt1 as integration targeting sequences and ura3d4 as a multi-copy selection marker). This study demonstrated the integration of up to three expression vectors containing different heterologous cDNA via their simultaneous transformation into haploid recipient strains. Additionally, further combinations of the different expression cassettes in one strain were obtained by subsequent diploidisation using selected haploid multi-copy transformants. Thus, recombinant strains containing three to five different expression cassettes were obtained, as demonstrated by Southern blotting. Expression of P450scc system proteins was identified by western blotting. The presented method for recombinant strain construction is a useful tool for the heterologous expression of multi-component enzyme systems in Y. lipolytica.

Atmospheric and room temperature plasma (ARTP) is a new and efficient mutation breeding technique. In this study, we discuss a strategy combining ARTP mutagenesis and high-throughput screening to engineer Corynebacterium glutamicum towards high yield production of heterologous proteins. First, three target strains, MC2, MA8, and MA6, were screened from the mutant library with enhanced green fluorescent protein (EGFP) as the reporter protein, and their growth stability and the influence of heterologous protein production were verified. Second, genes encoding three high-value medicinal proteins (glycoprotein D, gD; endoxylanase, XynA; and variable domain of heavy chain of heavy-chain antibody, VHH) were expressed in the mutagenized strain, which confirmed its applicability for an increased biosynthesis of other heterologous proteins. During the large-scale fermentation of C. glutamicum for VHH production, the fermentation characteristics of the best mutant MA6 were verified. Compared to the original strain, the yield of VHH obtained with strain MA6 was increased by nearly 91 % to approximately 862 mg/L. Finally, through systematic genome analysis mutations in five genes were obtained. These genes code for putative proteases or are potentially related to the bacterial restriction repair systems. These findings will help to obtain optimized chassis cells and provide a direction for in-depth research on genetic targets that can increase protein production.

This paper describes a simplified affinity precipitation process for the purification of mAbs from complex mixtures using elastin-like polypeptide fused to a single Z domain of protein A (ELP-Z). This approach eliminates several steps in the original process by directly extracting the mAb from the affinity precipitate, without the need for resolubilization. The efficacy of this elution without resolubilization (EWR) approach for obtaining pure mAb is demonstrated and the effects of mixing are examined. This simplification of the affinity precipitation process may facilitate the implementation of ELP-Z based mAb bioprocessing, particularly in a continuous scenario.

With the advent of global industrialisation and adaptation of smart life there is rise in anthropogenic pollution especially in water. Remediation of the pollutants (such as metals, and dyes) present in industrial effluents is possible via microbes and algae present in the environment. Microbes are used in a microbial fuel cell (MFC) for remediation of various organic and inorganic pollutants. However, for industrial scale application coupling the MFCs with photocatalytic and photoelectric fuel cell has a potential in improving the output of power. It can also be used for remediation of pollutants more expeditiously, conserving fossil fuels, cleaning environment, hence making the coupled hybrid fuel cell to run economically. Furthermore, such MFC inbuilt with algae in living or powder form give additional value addition products like biofuel, polysaccharides, biopolymers, and polyhydroxy alkanoates etc. This review provides bird's eye view on the removal of environmental pollutants by different biological sources like bacteria and algae. The article is focussed on diatoms as potential algae since they are rich source of crude oil and high value added products in a hybrid photocatalytic MFC. It also covers bottle necks, challenges and future in this field of research.

Gluconobacter oxydans is an important industrial bacterial strain widely used to produce a lot of useful products. However, very few gene editing tools are available for G. oxydans. This study aimed to develop an efficient genome editing method for G. oxydans using SacB as a counter-selectable marker. A plasmid that could express the kanamycin resistance gene in both E. coli and G. oxydans was constructed using the screened shuttle promoter P116. After optimizing the genome editing conditions, the derivative plasmids could be effectively utilized for diverse genome editing, such as gene deletion, insertion, replacement, and in situ modification in G. oxydans WSH-003. In addition, the SacB-based system also achieved multiple gene editing in G. oxydans. Moreover, the genome of the industrial strain G. oxydans WSH-003 was modified and the growth rate and substrate conversion rate of the strain successfully increased using this system. The system could also have potential to be applied in different G. oxydans strains. The process established in this study also provides a reference for constructing genetic tools for many other genetically recalcitrant bacteria.

Bixin is an apocarotenoid derived from Bixa orellana L. well known as a food colorant along with its numerous industrial and therapeutic applications. With the current surge in usage of natural products, bixin has contributed immensely to the world carotenoid market and showcases a spike in its requirement globally. To bridge the gap between bixin availability and utility, owed to its bioactivity and demand as a colouring agent in industries the sustainable production of bixin is critical. Therefore, to meet up this challenge effective use of multidisciplinary strategies is a promising choice to enhance bixin quantity and quality. Here we report, an optimal blend of approaches directed towards manipulation of bixin biosynthesis pathway with an insight into the impact of regulatory mechanisms and environmental dynamics, engineering carotenoid degradation in plants other than annatto, usage of tissue culture techniques supported with diverse elicitations, molecular breeding, application of in silico predictive tools, screening of microbial bio-factories as alternatives, preservation of bixin bioavailability, and promotion of eco-friendly extraction techniques to play a collaborative role in promoting sustainable bixin production.

Microalgae are a promising feedstock for carbon-neutral biofuel production due to their superior cellular composition. Alternatively, oxidative torrefaction has been recognized as a potential thermochemical technique for microalgal solid biofuel upgrading. Herein, by using microalga N. oceanica as a feedstock, several characterizations are adopted for evaluating the potential of oxidative torrefaction towards microalgal solid biofuel production. The oxidatively torrefied microalgae can be upgraded as lignite. After in-depth analysis, significant change in the surface microstructure of oxidatively torrefied microalgae is largely changed (via wrinkle and fragmentation) The hydrophobicity, thermal decomposition, thermal stability, and aromatization of oxidatively torrefied microalgae can be largely enhanced as the oxidative torrefaction severity increase. With the increasing torrefaction temperature, the hydrophobicity of oxidative torrefied microalgae gradually improved. The decomposition of C-2/3/5, and -OCH3, the CO bonds of CH3CO-, and the aromatization occurs via oxidative torrefaction according to the NMR analysis. For XPS analysis, torrefaction operation significantly decreases the carbide carbon and enhances the graphitization. As a result, the thermal stability of oxidatively torrefied microalgae is improved. Conclusively, the information obtained in this study can provide insights into the evaluation of oxidative torrefaction performance and fuel properties of microalgal solid biofuel, which may help accelerate the advancement of oxidative torrefaction industrialization.

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω-3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh-sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2-3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene β-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and β-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms.

Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy, which has been produced on several platforms. In this study, a novel single-chain anti-VEGF monoclonal antibody (scVEGFmAb) was produced in the goat mammary gland by adenoviral transduction. scVEGFmAb was purified by affinity chromatography. N-glycans were analyzed by exoglycosidase digestion and hydrophilic interaction ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The biological activity of scVEGFmAb was assessed by scratch and mouse aortic ring assays. scVEGFmAb was produced at 0.61 g/L in the goat milk, and its purification rendered 95 % purity. N-glycans attached to scVEGFmAb backbone were mainly neutral biantennary core fucosylated with Galβ1,4GlcNAc motif, and charged structures were capped with Neu5Ac and Neu5Gc. The chimeric molecule significantly prevented cell migration and suppressed microvessel sprouting. These results demonstrated for the first time the feasibility of producing an anti-VEGF therapeutic antibody in the milk of non-transgenic goats with the potential to counteract tumor angiogenesis.

The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40-95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70-95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.