Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture.

Hyaluronic acid (HA) is a biopolymer of repeating units of glucuronic acid and N-acetylglucosamine. Its market was valued at USD 8.9 billion in 2019. Traditionally, HA has been obtained from rooster comb-like animal tissues and fermentative cultures of attenuated pathogenic streptococci. Various attempts have been made to engineer a safe micro-organism for HA synthesis; however, the HA titres obtained from these attempts are in general still lower than those achieved by natural, pathogenic producers. In this scenario, ways to increase HA molecule length and titres in already constructed strains are gaining attention in the last years, but no recent publication has reviewed the main genetic strategies applied to improve HA production on heterologous hosts. In light of that, we hereby compile the advances made in the engineering of micro-organisms to improve HA synthesis.

Exploring the Brazilian diversity of Aspergillus sp. strains for lovastatin and itaconic acid production.

Filamentous fungi are well known for producing secondary metabolites applied in various industrial segments. Among these, lovastatin and itaconic acid, produced by Aspergillus terreus, have applications in the pharmaceutical and chemical industries. Lovastatin is primarily used for the control of hypercholesterolemia, while itaconic acid is a building block for the production of synthetic fibers, coating adhesives, among others. In this study, for the first time, 35 strains of Aspergillus sp. from four Brazilian culture collections were evaluated for lovastatin and itaconic acid production and compared to a reference strain, ATCC 20542. From an initial screening, the strains ATCC 20542, URM 224, URM1876, URM 5061, URM 5254, URM 5256, URM 5650, and URM 5961 were selected for genomic comparison. Among tested strains, the locus corresponding to the lovastatin genomic cluster was assembled, showing that all genes essential for lovastatin biosynthesis were present in producing URM 5961 and URM 5650 strains, with 100% and 98.5% similarity to ATCC 20542, respectively. However, in the no producing URM 1876, URM 224, URM 5254, URM 5061, and URM 5256 strains, this cluster was either fragmented or missing. Among the 35 strains evaluated for itaconic acid production in this study, only three strains had titers above 0.5 g/L, 16 strains had production below 0.5 g/L, and the remaining 18 strains had no production, with the highest production of itaconic acid observed in the URM 5254 strain with 2.2 g/L. The essential genes for itaconic acid production, mttA, cadA msfA were also mapped, where all three genes linked to itaconic acid production were found in a single contig in the assembly of each strain. In contrast to lovastatin loci, there is no correlation between the level of itaconic acid production and genetic polymorphisms in the genes associated with its biosynthesis.

Comparative assessment of fermentative capacity of different xylose-consuming yeasts.

BACKGROUND: Understanding the effects of oxygen levels on yeast xylose metabolism would benefit ethanol production. In this work, xylose fermentative capacity of Scheffersomyces stipitis, Spathaspora passalidarum, Spathaspora arborariae and Candida tenuis was systematically compared under aerobic, oxygen-limited and anaerobic conditions. RESULTS: Fermentative performances of the four yeasts were greatly influenced by oxygen availability. S. stipitis and S. passalidarum showed the highest ethanol yields (above 0.44 g g-1) under oxygen limitation. However, S. passalidarum produced 1.5 times more ethanol than S. stipitis under anaerobiosis. While C. tenuis showed the lowest xylose consumption rate and incapacity to produce ethanol, S. arborariae showed an intermediate fermentative performance among the yeasts. NAD(P)H xylose reductase (XR) activity in crude cell extracts correlated with xylose consumption rates and ethanol production. CONCLUSIONS: Overall, the present work demonstrates that the availability of oxygen influences the production of ethanol by yeasts and indicates that the NADH-dependent XR activity is a limiting step on the xylose metabolism. S. stipitis and S. passalidarum have the greatest potential for ethanol production from xylose. Both yeasts showed similar ethanol yields near theoretical under oxygen-limited condition. Besides that, S. passalidarum showed the best xylose consumption and ethanol production under anaerobiosis.

Genetic basis for hyper production of hyaluronic acid in natural and engineered microorganisms.

Hyaluronic acid, or HA, is a rigid and linear biopolymer belonging to the class of the glycosaminoglycans, and composed of repeating units of the monosaccharides glucuronic acid and N-acetylglucosamine. HA has multiple important functions in the human body, due to its properties such as bio-compatibility, lubricity and hydrophilicity, it is widely applied in the biomedical, food, health and cosmetic fields. The growing interest in this molecule has motivated the discovery of new ways of obtaining it. Traditionally, HA has been extracted from rooster comb-like animal tissues. However, due to legislation laws HA is now being produced by bacterial fermentation using Streptococcus zooepidemicus, a natural producer of HA, despite it being a pathogenic microorganism. With the expansion of new genetic engineering technologies, the use of organisms that are non-natural producers of HA has also made it possible to obtain such a polymer. Most of the published reviews have focused on HA formulation and its effects on different body tissues, whereas very few of them describe the microbial basis of HA production. Therefore, for the first time this review has compiled the molecular and genetic bases for natural HA production in microorganisms together with the main strategies employed for heterologous production of HA.

Novel homologous lactate transporter improves L-lactic acid production from glycerol in recombinant strains of Pichia pastoris.

BACKGROUND: Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous L-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for L-lactic acid production. RESULTS: Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. CONCLUSIONS: We showed that P. pastoris has a great potential as a fermentative organism for producing L-lactic acid using glycerol as the carbon source at limited oxygenation conditions (below 0.05 % DO in the bioreactor). The best strain had both the LDHb and the homologous lactate transporter encoding genes expressed, and reached a titer 1.5 times higher than the strain with the S. cerevisiae transporter. Finally, it was also shown that increased lactic acid production was concomitant to reduction of acetic acid formation by half.

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae.

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.

Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates.

Whole-cell bioconversion of technical lignins using Pseudomonas putida strains overexpressing amine transaminases (ATAs) has the potential to become an eco-efficient route to produce phenolic amines. Here, a novel cell growth-based screening method to evaluate the in vivo activity of recombinant ATAs towards vanillylamine in P. putida KT2440 was developed. It allowed the identification of the native enzyme Pp-SpuC-II and ATA from Chromobacterium violaceum (Cv-ATA) as highly active towards vanillylamine in vivo. Overexpression of Pp-SpuC-II and Cv-ATA in the strain GN442ΔPP_2426, previously engineered for reduced vanillin assimilation, resulted in 94- and 92-fold increased specific transaminase activity, respectively. Whole-cell bioconversion of vanillin yielded 0.70 ± 0.20 mM and 0.92 ± 0.30 mM vanillylamine, for Pp-SpuC-II and Cv-ATA, respectively. Still, amine production was limited by a substantial re-assimilation of the product and formation of the by-products vanillic acid and vanillyl alcohol. Concomitant overexpression of Cv-ATA and alanine dehydrogenase from Bacillus subtilis increased the production of vanillylamine with ammonium as the only nitrogen source and a reduction in the amount of amine product re-assimilation. Identification and deletion of additional native genes encoding oxidoreductases acting on vanillin are crucial engineering targets for further improvement.

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production.

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by ß-1,4 and ß-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.

Flotation as a tool for indirect DNA extraction from soil.

Nowadays, soil diversity is accessed at molecular level by the total DNA extraction of a given habitat. However, high DNA yields and purity are difficult to achieve due to the co-extraction of enzyme-inhibitory substances that inhibit downstream applications, such as PCR, restriction enzyme digestion, and DNA ligation. Therefore, there is a need for further development of sample preparation methods that efficiently can result in pure DNA with satisfactory yield. In this study, the buoyant densities of soil microorganisms were utilized to design a sample preparation protocol where microbial cells could be separated from the soil matrix and enzyme-inhibitory substances by flotation. A discontinuous density gradient was designed using a colloidal solution of non-toxic silanised silica particles (BactXtractor). The method proved to be an efficient alternative to direct extraction protocols where cell lysis is performed in the presence of soil particles. The environmental DNA extracted after flotation had high molecular weight and comparable yield as when using available commercial kits (3.5 microg DNA/g soil), and neither PCR nor restriction enzyme digestion of DNA were inhibited. Furthermore, specific primers enabled recovery of both prokaryotic and eukaryotic sequences.

Evaluation of Product Distribution in Chemostat and Batch Fermentation in Lactic Acid-Producing Komagataella phaffii Strains Utilizing Glycerol as Substrate.

Lactic acid is the monomeric unit of polylactide (PLA), a bioplastic widely used in the packaging, automotive, food, and pharmaceutical industries. Previously, the yeast Komagataella phaffii was genetically modified for the production of lactate from glycerol. For this, the bovine L-lactate dehydrogenase- (LDH)-encoding gene was inserted and the gene encoding the pyruvate decarboxylase (PDC) was disrupted, resulting in the GLp strain. This showed a yield of 67% L-lactic acid and 20% arabitol as a by-product in batches with oxygen limitation. Following up on these results, the present work endeavored to perform a detailed study of the metabolism of this yeast, as well as perturbing arabitol synthesis in an attempt to increase lactic acid titers. The GLp strain was cultivated in a glycerol-limited chemostat at different dilution rates, confirming that the production of both lactic acid and arabitol is dependent on the specific growth rate (and consequently on the concentration of the limiting carbon source) as well as on the oxygen level. Moreover, disruption of the gene encoding arabitol dehydrogenase (ArDH) was carried out, resulting in an increase of 20% in lactic acid and a 50% reduction in arabitol. This study clarifies the underlying metabolic reasons for arabitol formation in K. phaffii and points to ways for improving production of lactic acid using K. phaffii as a biocatalyst.

Bioprospecting Microbial Diversity for Lignin Valorization: Dry and Wet Screening Methods.

Lignin is an abundant cell wall component, and it has been used mainly for generating steam and electricity. Nevertheless, lignin valorization, i.e. the conversion of lignin into high value-added fuels, chemicals, or materials, is crucial for the full implementation of cost-effective lignocellulosic biorefineries. From this perspective, rapid screening methods are crucial for time- and resource-efficient development of novel microbial strains and enzymes with applications in the lignin biorefinery. The present review gives an overview of recent developments and applications of a vast arsenal of activity and sequence-based methodologies for uncovering novel microbial strains with ligninolytic potential, novel enzymes for lignin depolymerization and for unraveling the main metabolic routes during growth on lignin. Finally, perspectives on the use of each of the presented methods and their respective advantages and disadvantages are discussed.

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production.

The methylotrophic yeast Hansenula polymorpha, known as a non-conventional yeast, is used for the last 30 years for the production of recombinant proteins, including enzymes, vaccines, and biopharmaceuticals. Although a large number of reviews have been published elucidating the applications of this yeast as a cell factory, the latest was released about 10 years ago. Therefore, this review aimed at summarizing available information on the use of H. polymorpha as a host for recombinant protein production in the last decade. Examples of chemicals and virus-like particles produced using this yeast also are discussed. Firstly, the aspects that feature this yeast as a host for recombinant protein production are highlighted including the techniques available for its genetic manipulation as well as strategies for cultivation in bioreactors. Special attention is given to the novel genomic editing tools, mainly CRISPR/Cas9 that was recently established in this yeast. Finally, recent examples of using H. polymorpha as an expression platform are presented and discussed. The production of human Parathyroid Hormone (PTH) and Staphylokinase (SAK) in H. polymorpha are described as case studies for process establishment in this yeast. Altogether, this review is a guideline for this yeast utilization as an expression platform bringing a thorough analysis of the genetic aspects and fermentation protocols used up to date, thus encouraging the production of novel biomolecules in H. polymorpha.

Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.

Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.

Comparison of Yeasts as Hosts for Recombinant Protein Production.

Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns.

Rhamnolipids production from sucrose by engineered Saccharomyces cerevisiae.

Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway's overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway's intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.

Antibiotic combinations for controlling colistin-resistant Enterobacter cloacae.

Enterobacter cloacae is a Gram-negative bacterium associated with high morbidity and mortality in intensive care patients due to its resistance to multiple antibiotics. Currently, therapy against multi-resistant bacteria consists of using colistin, in spite of its toxic effects at higher concentrations. In this context, colistin-resistant E. cloacae strains were challenged with lower levels of colistin combined with other antibiotics to reduce colistin-associated side effects. Colistin-resistant E. cloacae (ATCC 49141) strains were generated by serial propagation in subinhibitory colistin concentrations. After this, three colistin-resistant and three nonresistant replicates were isolated. The identity of all the strains was confirmed by MALDI-TOF MS, VITEK 2 and MicroScan analysis. Furthermore, cross-resistance to other antibiotics was checked by disk diffusion and automated systems. The synergistic effects of the combined use of colistin and chloramphenicol were observed via the broth microdilution checkerboard method. First, data here reported showed that all strains presented intrinsic resistance to penicillin, cephalosporin (except fourth generation), monobactam, and some associations of penicillin and ß-lactamase inhibitors. Moreover, a chloramphenicol and colistin combination was capable of inhibiting the induced colistin-resistant strains as well as two colistin-resistant clinical strains. Furthermore, no cytotoxic effect was observed by using such concentrations. In summary, the data reported here showed for the first time the possible therapeutic use of colistin-chloramphenicol for infections caused by colistin-resistant E. cloacae.

Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with Automatically Simulated Executable Documentation.

We have developed the Yeast Pathway Kit (YPK) for rational and random metabolic pathway assembly in Saccharomyces cerevisiae using reusable and redistributable genetic elements. Genetic elements are cloned in a suicide vector in a rapid process that omits PCR product purification. Single-gene expression cassettes are assembled in vivo using genetic elements that are both promoters and terminators (TP). Cassettes sharing genetic elements are assembled by recombination into multigene pathways. A wide selection of prefabricated TP elements makes assembly both rapid and inexpensive. An innovative software tool automatically produces detailed self-contained executable documentation in the form of pydna code in the narrative Jupyter notebook format to facilitate planning and sharing YPK projects. A d-xylose catabolic pathway was created using YPK with four or eight genes that resulted in one of the highest growth rates reported on d-xylose (0.18 h(-1)) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improve the yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool.

Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans.

BACKGROUND: The conversion of lignocellulosic biomass to biofuels (second-generation biofuel production) is an environmentally friendlier alternative to petroleum-based energy sources. Enzymatic deconstruction of lignocellulose, catalyzed by filamentous fungi such as Aspergillus nidulans, releases a mixture of mono- and polysaccharides, including hexose (glucose) and pentose (xylose) sugars, cellodextrins (cellobiose), and xylooligosaccharides (xylobiose). These sugars can subsequently be fermented by yeast cells to ethanol. One of the major drawbacks in this process lies in the inability of yeast, such as Saccharomyces cerevisiae, to successfully internalize sugars other than glucose. The aim of this study was, therefore, to screen the genome of A. nidulans, which encodes a multitude of sugar transporters, for transporters able to internalize non-glucose sugars and characterize them when introduced into S. cerevisiae. RESULTS: This work identified two proteins in A. nidulans, CltA and CltB, with roles in cellobiose transport and cellulose signaling, respectively. CltA, when introduced into S. cerevisiae, conferred growth on low and high concentrations of cellobiose. Deletion of cltB resulted in reduced growth and extracellular cellulase activity in A. nidulans in the presence of cellobiose. CltB, when introduced into S. cerevisiae, was not able to confer growth on cellobiose, suggesting that this protein is a sensor rather than a transporter. However, we have shown that the introduction of additional functional copies of CltB increases the growth in the presence of low concentrations of cellobiose, strongly indicating CltB is able to transport cellobiose. Furthermore, a previously identified glucose transporter, HxtB, was also found to be a major xylose transporter in A. nidulans. In S. cerevisiae, HxtB conferred growth on xylose which was accompanied by ethanol production. CONCLUSIONS: This work identified a cellobiose transporter, a xylose transporter, and a putative cellulose transceptor in A. nidulans. This is the first time that a sensor role for a protein in A. nidulans has been proposed. Both transporters are also able to transport glucose, highlighting the preference of A. nidulans for this carbon source. This work provides a basis for future studies which aim at characterizing and/or genetically engineering Aspergillus spp. transporters, which, in addition to glucose, can also internalize other carbon sources, to improve transport and fermentation of non-glucose sugars in S. cerevisiae.

Cell cycle, DNA replication, repair, and recombination in the dimorphic human pathogenic fungus Paracoccidioides brasiliensis.

DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.

Production of a modified peptide clavanin in Pichia pastoris: cloning, expression, purification and in vitro activities.

Antimicrobial peptides are one of the most promising peptide-based drugs due to their enormous potential as novel biopharmaceuticals in both human and animal industries. In order to develop strategies to over produce such molecules, heterologous production of a modified version of clavanin A, here named clavanin MO (clavMO), was successfully achieved in the methylothopic yeast Pichia pastoris. ClavMO was fused to thioredoxin as a carrier protein and the construction was tested using two promoters, PAOX1 and PGAP, based on either induced or constitutive expression systems, respectively. After growth in 5 L Bioreactor, clavMO-thio was recovered and purified through size exclusion chromatography. Our findings show that both constitutive and inducible expression systems produce active clavMO fused to thioredoxin against both Gram-negative Klebsiella pneumoniae and Gram-positive Staphylococcus aureus microorganisms.