Salvador Peiru

Acid-Inducible Transcription of the Operon Encoding the Citrate Lyase Complex of Lactococcus lactis Biovar diacetylactis CRL264

Although Lactococcus is one of the most extensively studied lactic acid bacteria and is the paradigm for biochemical studies of citrate metabolism, little information is available on the regulation of the citrate lyase complex. In order to fill this gap, we characterized the genes encoding the subunits of the citrate lyase of Lactococcus lactis CRL264, which are located on an 11.4-kb chromosomal DNA region. Nucleotide sequence analysis revealed a cluster of eight genes in a new type of genetic organization. The citM-citCDEFXG operon (cit operon) is transcribed as a single polycistronic mRNA of 8.6 kb. This operon carries a gene encoding a malic enzyme (CitM, a putative oxaloacetate decarboxylase), the structural genes coding for the citrate lyase subunits (citD, citE, and citF), and the accessory genes required for the synthesis of an active citrate lyase complex (citC, citX, and citG). We have found that the cit operon is induced by natural acidification of the medium during cell growth or by a shift to media buffered at acidic pHs. Between the citM and citC genes is a divergent open reading frame whose expression was also increased at acidic pH, which was designated citI. This inducible response to acid stress takes place at the transcriptional level and correlates with increased activity of citrate lyase. It is suggested that coordinated induction of the citrate transporter, CitP, and citrate lyase by acid stress provides a mechanism to make the cells (more) resistant to the inhibitory effects of the fermentation product (lactate) that accumulates under these conditions.

Expression of codon optimized genes in microbial systems: current industrial applications and perspectives

The efficient production of functional proteins in heterologous hosts is one of the major bases of modern biotechnology. Unfortunately, many genes are difficult to express outside their original context. Due to their apparent “silent” nature, synonymous codon substitutions have long been thought to be trivial. In recent years, this dogma has been refuted by evidence that codon replacement can have a significant impact on gene expression levels and protein folding. In the past decade, considerable advances in the speed and cost of gene synthesis have facilitated the complete redesign of entire gene sequences, dramatically improving the likelihood of high protein expression. This technology significantly impacts the economic feasibility of microbial-based biotechnological processes by, for example, increasing the volumetric productivities of recombinant proteins or facilitating the redesign of novel biosynthetic routes for the production of metabolites. This review discusses the current applications of this technology, particularly those regarding the production of small molecules and industrially relevant recombinant enzymes. Suggestions for future research and potential uses are provided as well.

Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family

The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the K m value for oxaloacetate was 0.52 ± 0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram‐positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.

Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum

BackgroundSynthetic biology approaches can make a significant contribution to the advance of metabolic engineering by reducing the development time of recombinant organisms. However, most of synthetic biology tools have been developed for Escherichia coli. Here we provide a platform for rapid engineering of C. glutamicum, a microorganism of great industrial interest. This bacteria, used for decades for the fermentative production of amino acids, has recently been developed as a host for the production of several economically important compounds including metabolites and recombinant proteins because of its higher capacity of secretion compared to traditional bacterial hosts like E. coli. Thus, the development of modern molecular platforms may significantly contribute to establish C. glutamicum as a robust and versatile microbial factory.ResultsA plasmid based platform named pTGR was created where all the genetic components are flanked by unique restriction sites to both facilitate the evaluation of regulatory sequences and the assembly of constructs for the expression of multiple genes. The approach was validated by using reporter genes to test promoters, ribosome binding sites, and for the assembly of dual gene operons and gene clusters containing two transcriptional units. Combinatorial assembly of promoter (tac, cspB and sod) and RBS (lacZ, cspB and sod) elements with different strengths conferred clear differential gene expression of two reporter genes, eGFP and mCherry, thus allowing transcriptional “fine-tuning”of multiple genes. In addition, the platform allowed the rapid assembly of operons and genes clusters for co-expression of heterologous genes, a feature that may assist metabolic pathway engineering.ConclusionsWe anticipate that the pTGR platform will contribute to explore the potential of novel parts to regulate gene expression, and to facilitate the assembly of genetic circuits for metabolic engineering of C. glutamicum. The standardization provided by this approach may provide a means to improve the productivity of biosynthetic pathways in microbial factories for the production of novel compounds.

High-level production of Bacillus cereus phospholipase C in Corynebacterium glutamicum.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme.

An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

BackgroundBiodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.ResultsA steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.ConclusionsThe results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

Design and synthesis of pathway genes for polyketide biosynthesis.

In this chapter we describe novel methods for the design and assembly of synthetic pathways for the synthesis of polyketides and tailoring sugars. First, a generic design for type I polyketide synthase genes is presented that allows their facile assembly for the expression of chimeric enzymes in an engineered Escherichia coli host. The sequences of the synthetic genes are based on naturally occurring polyketide synthase genes but they are redesigned by custom-made software to optimize codon usage to maximize expression in E. coli and to provide a standard set of restriction sites to allow combinatorial assembly into unnatural enzymes. The methodology has been validated by building a large number of bimodular mini-PKSs that make easily assayed triketide products. Learning from the successful bimodules, a conceptual advance was made by assembling genes encoding functional trimodular enzymes, capable of making tetraketide products. Second, methods for the rapid assembly and exchange of sugar pathway genes into functional operons are described. The approach was validated by the assembly of the 15 genes for the synthesis of mycarose and desosamine in two operons, which yielded erythromycin C when coexpressed with the corresponding PKS genes. These methods are important enabling steps toward the goals of making designer drugs by polyketide synthase and sugar pathway engineering and, in the shorter term, producing by fermentation advanced intermediates for the synthesis of compounds that otherwise require large numbers of chemical steps.

Pilot-scale process development for low-cost production of a thermostable biodiesel refining enzyme in Escherichia coli

Biodiesels produced from vegetable oils have a major quality problem due to the presence of steryl glucosides (SGs), which form precipitates that clog filters and cause engine failures. Recently, we described an enzymatic process for removing SGs from biodiesel. However, industrial adoption of this technology was hindered by the cost of the steryl glucosidase (SGase) enzyme used. Here we report the development and validation at the pilot scale of a cost-efficient process for manufacturing the SGase. First, we tested various low-cost carbon sources for the Escherichia coli producing strain, ultimately developing a fed-batch fermentation process that utilizes crude glycerol as a feedstock. Next, we designed an efficient process for isolating the SGase. That process uses a novel thermolysis approach in the presence of a non-ionic detergent, centrifugation to separate the solids, and ultrafiltration to concentrate and formulate the final product. Our cost analysis indicates that on a large scale, the dose of enzyme required to eliminate SGs from each ton of biodiesel will have a manufacturing cost below

Recombinant approaches to large polyketide molecules as potential drugs.

1. The new process for manufacturing the SGase, which will lead to biodiesels of a higher quality, should contribute to facilitate the global adoption of this renewable fuel. Our technology could also be used to manufacture other thermostable proteins in E. coli.

The production, properties, and applications of thermostable steryl glucosidases

Combinatorial biosynthesis of polyketide synthases through genetic engineering should provide complex compounds as drug development candidates. Different strategies are currently being evaluated to create libraries of new compounds. The number of different compounds in such libraries and the quantities that can be made using combinatorial strategies will determine the impact of this technology on the pharmaceutical industry.