BackgroundD-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT.ResultsIntracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold.ConclusionsThis is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems.

BackgroundGenetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression.ResultsInitially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5–7.5% of transformants with 2–4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2–3-fold and 10–12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1–2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture.ConclusionsHere, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.Electronic supplementary materialThe online version of this article (10.1186/s12896-018-0497-z) contains supplementary material, which is available to authorized users.

Transcriptome analysis requires a large quantity of high-quality DNase-treated RNA for poly(A)+ mRNA isolation and sequencing. This could be problematic in many oleaginous microalgal species that harbor strong cell walls and accumulate high lipid content. Using Scenedesmus obliquus, a microalga with high oil content and potential as a source of algal biofuel, we assessed the efficiency of four RNA isolation methods: direct extraction using TriPure, mechanical breakage using either freeze-thawed with bead beating or grinding in liquid nitrogen followed by TriPure, and grinding in liquid nitrogen before using Qiagen RNeasy Plant Mini Kit. Liquid nitrogen grinding with TriPure method gave the best RNA yields at 15.15 µg mg -1 cell dry weight and ~148.9 µg total RNA from 100 ml culture of S. obliquus. Despite lower yields, RNA isolation of oil accumulating cells (~22% w/w lipid content) provided ~68.1 µg total RNA with the yield of 1.70 µg mg -1 cell dry weight. Transcriptome sequencing and de novo assembly with the average contig length of 824 bp reflected high quality of RNA obtained using this method. The RNA isolation protocol was tested on six other oleaginous microalgae including Chlamydomonas reinhardtii, S. acuminatus, Chorella vulgaris, Chlorococcum humicola, Tetradesmus cumbricus and Coelastrum sp. and yielded 0.86 -5.42 µg mg -1 cell dry weight. For large scale RNA isolation from microalgae, grinding with liquid nitrogen before TriPure provided the best yield and quality. This finding helps simplify RNA isolation for upcoming transcriptome analyses in microalgae.