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Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficiant to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86 % within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

Background Arbutin is a plant-derived glycoside with potential antioxidant, antibacterial and anti-inflammatory activities. Currently, it is mainly produced by plant extraction or enzymatic processes, which suffers from expensive processing cost and low product yield. Metabolic engineering of microbes is an increasingly powerful method for the high-level production of valuable biologicals. Since Pseudomonas chlororaphis has been widely engineered as a phenazine-producing platform organism due to its well-characterized genetics and physiology, and faster growth rate using glycerol as a renewable carbon source, it can also be engineered as the cell factory using strong shikimate pathway on the basis of synthetic biology. Results In this work, a plasmid-free biosynthetic pathway was constructed in P . chlororaphis P3 for elevated biosynthesis of arbutin from sustainable carbon sources. The arbutin biosynthetic pathway was expressed under the native promoter P phz using chromosomal integration. Instead of being plasmid and inducer dependent, the metabolic engineering approach used to fine-tune the biosynthetic pathway significantly enhanced the arbutin production with a 22.4-fold increase. On the basis of medium factor optimization and mixed fed-batch fermentation of glucose and 4-hydroxybenzoic acid, the engineered P. chlororaphis P3-Ar5 strain led to the highest arbutin production of 6.79 g/L with the productivity of 0.094 g/L/h, with a 54-fold improvement over the initial strain. Conclusions The results suggested that the construction of plasmid-free synthetic pathway displays a high potential for improved biosynthesis of arbutin and other shikimate pathway derived biologicals in P. chlororaphis .

Salinicoccus salsiraiae IM408 (=CGMCC13032) is a novel halophilic bacterium that we isolated from the saline soil of Da Gang Oilfield. It tolerates 60 g/l sodium chloride and up to 123 g/l (1.5 M) sodium acetate and has shown a potential application in bioremediation of wastewater with high salt and high chemical oxygen demand (COD). Two plasmids, pS408-1 and pS408-2, were identified in S. salsiraiae IM408, and the sequences and copy numbers of the plasmids were determined. Based on these plasmids, two shuttle vectors containing a replicon for Escherichia coli , ampicillin, and chloramphenicol resistance genes, as well as the replicon from pS408-1 or pS408-2, were constructed and named as pTCS101 and pTCS201, respectively. A suitable host strain, named S. salsiraiae PE01, was also developed from the wild-type by plasmid elimination. Using the plasmid pTCS101 as an expression vector, l -lactate dehydrogenase from Staphylococcus aureus was expressed successfully in S. salsiraiae PE01. This is the first gene expression system for the Salinicoccus genus. It has provided the potential for expression of desired proteins or for establishment of desired pathways in Salinicoccus strains, which would make these halophiles more advantageous in future biotechnological applications.