Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production.

Lactic acid is the monomer unit of the bioplastic poly-lactic acid (PLA). One candidate organism for lactic acid production is Pichia pastoris, a yeast widely used for heterologous protein production. Nevertheless, this yeast has a poor fermentative capability that can be modulated by controlling oxygen levels. In a previous study, lactate dehydrogenase (LDH) activity was introduced into P. pastoris, enabling this yeast to produce lactic acid. The present study aimed to increase the flow of pyruvate towards the production of lactic acid in P. pastoris. To this end, a strain designated GLp was constructed by inserting the bovine lactic acid dehydrogenase gene (LDHb) concomitantly with the interruption of the gene encoding pyruvate decarboxylase (PDC). Aerobic fermentation, followed by micro-aerophilic culture two-phase fermentations, showed that the GLp strain achieved a lactic acid yield of 0.65 g/g. The distribution of fermentation products demonstrated that the acetate titer was reduced by 20% in the GLp strain with a concomitant increase in arabitol production: arabitol increased from 0.025 g/g to 0.174 g/g when compared to the GS115 strain. Taken together, the results show a significant potential for P. pastoris in producing lactic acid. Moreover, for the first time, physiological data regarding co-product formation have indicated the redox balance limitations of this yeast.

Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag.

An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti-infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa-MAP 2, a polyalanine AMP. Pa-MAP 2 was fused to an ELP-intein tag where the ELP (Elastin-like polypeptide) was used to promote aggregation and fast and cost-effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa-MAP 2 fused to the N-termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa-MAP 2 showed a MIC of 25µM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa-MAP-2 can be produced in both rich and defined media at yields 50-fold higher than reported for other AMPs produced by the ELP-intein system, and in comparable yields to expression systems with protease or chemical cleavage.

Lovastatin production: From molecular basis to industrial process optimization.

Lovastatin, composed of secondary metabolites produced by filamentous fungi, is the most frequently used drug for hypercholesterolemia treatment due to the fact that lovastatin is a competitive inhibitor of HMG-CoA reductase. Moreover, recent studies have shown several important applications for lovastatin including antimicrobial agents and treatments for cancers and bone diseases. Studies regarding the lovastatin biosynthetic pathway have also demonstrated that lovastatin is synthesized from two-chain reactions using acetate and malonyl-CoA as a substrate. It is also known that there are two key enzymes involved in the biosynthetic pathway called polyketide synthases (PKS). Those are characterized as multifunctional enzymes and are encoded by specific genes organized in clusters on the fungal genome. Since it is a secondary metabolite, cultivation process optimization for lovastatin biosynthesis has included nitrogen limitation and non-fermentable carbon sources such as lactose and glycerol. Additionally, the influences of temperature, pH, agitation/aeration, and particle and inoculum size on lovastatin production have been also described. Although many reviews have been published covering different aspects of lovastatin production, this review brings, for the first time, complete information about the genetic basis for lovastatin production, detection and quantification, strain screening and cultivation process optimization. Moreover, this review covers all the information available from patent databases covering each protected aspect during lovastatin bio-production.

Current scenario of peptide-based drugs: the key roles of cationic antitumor and antiviral peptides.

Cationic antimicrobial peptides (AMPs) and host defense peptides (HDPs) show vast potential as peptide-based drugs. Great effort has been made in order to exploit their mechanisms of action, aiming to identify their targets as well as to enhance their activity and bioavailability. In this review, we will focus on both naturally occurring and designed antiviral and antitumor cationic peptides, including those here called promiscuous, in which multiple targets are associated with a single peptide structure. Emphasis will be given to their biochemical features, selectivity against extra targets, and molecular mechanisms. Peptides which possess antitumor activity against different cancer cell lines will be discussed, as well as peptides which inhibit virus replication, focusing on their applications for human health, animal health and agriculture, and their potential as new therapeutic drugs. Moreover, the current scenario for production and the use of nanotechnology as delivery tool for both classes of cationic peptides, as well as the perspectives on improving them is considered.

Construction of an artificial secYEG operon allowing high level secretion of α-amylase.

The key components of the major secretion pathway in bacteria, the Sec pathway, are the proteins SecA, an ATPase that generates the energy required for protein translocation, and the heterotrimeric protein complex SecYEG, which functions as the preprotein-conducting channel through the cytoplasmic membrane, named translocon. Overexpression of exoproteins can cause jamming of the membrane, e.g., due to a shortage of translocons. Therefore, we decided to increase the number of translocons by first creating an artificial secYEG operon and then fusing it to an inducible promoter. By Western- and Northern-blot analysis, we could first show that the amount of the SecY protein and the secYEG transcript can be increased after addition of the inducer. Next, we proved by immunoblot experiments that the amount of α-amylase secreted in the presence of increased amounts of SecYEG proteins is enhanced. Therefore, increasing the number of translocons is accompanied by a concomitant increase in the amount exoenzymes. This finding will be of importance for high-level secretion of recombinant proteins.

Exploring the pharmacological potential of promiscuous host-defense peptides: from natural screenings to biotechnological applications.

In the last few years, the number of bacteria with enhanced resistance to conventional antibiotics has dramatically increased. Most of such bacteria belong to regular microbial flora, becoming a real challenge, especially for immune-depressed patients. Since the treatment is sometimes extremely expensive, and in some circumstances completely inefficient for the most severe cases, researchers are still determined to discover novel compounds. Among them, host-defense peptides (HDPs) have been found as the first natural barrier against microorganisms in nearly all living groups. This molecular class has been gaining attention every day for multiple reasons. For decades, it was believed that these defense peptides had been involved only with the permeation of the lipid bilayer in pathogen membranes, their main target. Currently, it is known that these peptides can bind to numerous targets, as well as lipids including proteins and carbohydrates, from the surface to deep within the cell. Moreover, by using in vivo models, it was shown that HDPs could act both in pathogens and cognate hosts, improving immunological functions as well as acting through multiple pathways to control infections. This review focuses on structural and functional properties of HDP peptides and the additional strategies used to select them. Furthermore, strategies to avoid problems in large-scale manufacture by using molecular and biochemical techniques will also be explored. In summary, this review intends to construct a bridge between academic research and pharmaceutical industry, providing novel insights into the utilization of HDPs against resistant bacterial strains that cause infections in humans.