Cleavage of delta and epsilon tubulin PCR products is observed upon PCR purification.

In this study, we report an unusual phenomenon of the self-cleavage of purified PCR products of codon-optimized Chlamydomonas reinhardtii delta tubulin ( uni3 ) and epsilon tubulin ( bld2 ) genes through an unknown mechanism. Our studies revealed that intact PCR products for both these genes could be obtained upon PCR amplification from plasmid templates carrying these genes. However, interestingly, purification of these PCR products led to their cleavage through an unidentified mechanism. This cleavage persisted despite using different PCR purification kits. Deleting a synthetic intron within the delta tubulin gene also did not have any effect on this cleavage.

Light intensity and spectral quality modulation for improved growth kinetics and biochemical composition of Chlamydomonas reinhardtii.

Effective strategies to optimize algal growth and lipid productivity are critical for the sustainable production of biomass for various applications. Light management has emerged as a promising approach, but the intricate relationship between light intensity, spectral quality, and algal responses remains poorly understood. This study investigated the effects of different light qualities (blue, red-orange, and white-yellow) and intensities (45-305 µmol/m2·s) on Chlamydomonas reinhardtii. Red-orange light exhibited the highest promotion of biomass growth and lipid productivity, with specific growth rates of 1.968 (d-1) and biomass productivity of 0.284 (g/L/d) at 155 µmol/m2·s and 205 µmol/m2·s, respectively. Within the intensity range of 205 µmol/m2·s to 305 µmol/m2·s, lipid mass fractions ranged from 10.5% w/w to 11.0% w/w, accompanied by lipid concentrations ranging from 68.6 mg/L to 74.9 mg/L. Red-orange light positively influenced carbohydrate accumulation, while blue light promoted protein synthesis. These findings highlight the importance of optimizing light quality and intensity to enhance algal biomass productivity and manipulate biochemical composition. Understanding the complex relationship between light parameters and algal physiology will contribute to sustainable algal cultivation practices and the use of microalgae as a valuable bioresource.

LED alternating between blue and red-orange light improved the biomass and lipid productivity of Chlamydomonas reinhardtii.

Light management is important for improving algae cultivation, specifically by enhancing the productivity of biomass and valued bioproducts. In this study, we present evidence that alternating blue and red-orange light can improve the algal growth kinetics and lipid production in a photobioreactor. Blue (430-445, 460-470 nm) and red-orange light (580-660 nm) from a LED were set at the light saturation point (B: 65 µmol/m2s; RO: 155 µmol/m2s) and alternated for the cultivation of the green alga Chlamydomonas reinhardtii. Growth kinetics, lipid, carbohydrate, and protein content were measured as a function of alternating illumination time. Results reveal that the first illumination light and illumination time had a significant impact on the growth kinetics and nutrient composition. When the red-orange light illumination was used at the beginning of cultivation (RO/B alternation), the biomass concentration and productivity increased 8% and 18% on average, respectively; lipid mass fraction and concentration increased 21-27% and 24-26% when 0.25-0.50 h per day of blue light illumination was used; no significant change of carbohydrate and protein content were observed. Relative to blue light alone, the improvement of growth kinetics, lipid mass fraction and concentration, and the carbohydrate concentration was significant. Under B/RO alternation (when the blue light was used first), on average, the protein content was significantly higher than RO/B alternation.

Interactive effects of light quality and culturing temperature on algal cell size, biomass doubling time, protein content, and carbohydrate content.

Light management strategy can be used to improve algal biomass and nutrient production. However, the response of algal metabolism to different light qualities, especially their interaction with other environmental factors, is not well understood. This study focuses on the interactive effects of light quality and culturing temperature on algal protein content and carbohydrate content of C. reinhardtii. Three LED light sources (blue light, red-orange light, and white-yellow light) were applied to grow algae in batch cultures with a light intensity of 105 µmol/m2s under the temperatures of 24 °C to 32 °C. The protein and carbohydrate content were measured in both the late exponential growth phase and the late stationary growth phase. The results revealed that there was an interactive effect of light quality and culturing temperature on the protein and carbohydrate content. The combined conditions of blue light and a temperature of 24 °C or 28 °C, which induced a larger algal cell size with a prolonged cell cycle and a low division rate, resulted in the highest protein content; the protein mass fraction and concentration were 32% and 52% higher than that under white-yellow light at 32 °C. The combined conditions of red-orange light and a temperature of 24 °C, which promoted both the cell division and size growth, enhanced the carbohydrate content; the carbohydrate mass fraction and concentration were 161% and 155% higher than that under white-yellow light at 24 °C. When there was temperature stress (32 °C) or nutrient stress, the effect of light quality reduced, and the difference of protein and carbohydrate content among the three light qualities decreased. KEY POINTS: • Studied light quality-temperature interactive effect on protein, carbohydrate synthesis. • Protein content was high under low cell division rate. • Carbohydrate content was high under high cell division and cell size growth rate.

Characterization of the flavin monooxygenase involved in biosynthesis of the antimalarial FR-900098.

The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto 3-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal structure and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a similar overall structure with flavin-containing monooxygenases (FMOs). Notably, we also show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-binding domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.

Production and characterization of algae extract from Chlamydomonas reinhardtii

Background: Algae offer many advantages as biofuel sources including: high growth rates, high lipid content, the ability to grow on non-agricultural land, and the genetic versatility to improve strains rapidly and produce co-products. Research is ongoing to make algae biofuels a more financially attractive energy option; however, it is becoming evident that the economic viability of algae-based fuels may hinge upon high-value co-products. This work evaluated the feasibility of using a co-product, algae extract, as a nutrient source in cell culture media. Results: Algae extract prepared from autolysed Chlamydomonas reinhardtii was found to contain 3.0% protein, 9.2% total carbohydrate, and 3.9% free α-amino acid which is similar to the nutrient content of commercially available yeast extract. The effects of algae extract on the growth and metabolism of laboratory strains of Escherichia coli and Saccharomyces cerevisiae were tested by substituting algae extract for yeast extract in LB and YPAD growth media recipes. Complex laboratory media supplemented with algae extract instead of yeast extract showed markedly improved effects on the growth and metabolism of common laboratory microorganisms in all cases except ethanol production rates in yeast. Conclusions: This study showed that algae extract derived from C. reinhardtii is similar, if not superior, to commercially available yeast extract in nutrient content and effects on the growth and metabolism of E. coli and S. cerevisiae. Bacto™ yeast extract is valued at USD $0.15-0.35 per gram, if algae extract was sold at similar prices, it would serve as a high-value co-product in algae-based fuel processes.

Method for assembling and expressing multiple genes in the nucleus of microalgae.

The green alga, Chlamydomonas reinhardtii, is a model organism used in the study of photosynthesis and biotechnological research. Despite its importance, a complete set of genetic tools has yet to be developed. Here, we report the development of a new method for constructing a multi-gene pathway in Saccharomyces cerevisiae and integrating the assembled pathway into the nuclear genome of C. reinhardtii. To demonstrate the use of this method, we assembled and functionally expressed up to three reporter proteins (Ble, AphVIII, and GFP) simultaneously in the nucleus of C. reinhardtii. This new molecular tool should aid efforts to engineer microalgae for biofuel and biopharmaceutical production.

Production of xylitol by recombinant microalgae.

Microalgae have received significant attention recently as a potential low-cost host for the production of next-generation biofuels and natural products. Here we show that the chloroplast genome of the eukaryotic green microalga Chlamydomonas reinhardtii can be genetically engineered to produce xylitol through the introduction of a gene encoding a xylose reductase (XR) from the fungi Neurospora crassa. Increased levels of heterologous protein accumulation and xylitol production were achieved by synthesizing the XR gene in the chloroplast codon bias and by driving expression of the codon-optimized XR gene using a 16S/atpA promoter/5'-UTR fusion. These results demonstrate the feasibility of engineering microalgae to produce xylitol, and show the importance of codon optimizing the XR gene and using the 16S/atpA promoter/5'-UTR fusion to express XR in the chloroplast of C. reinhardtii.

Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii.

Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated region selection when constructing a target pathway. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products.

Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase.

Phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri catalyzes the nicotinamide adenine dinucleotide-dependent oxidation of phosphite to phosphate. The enzyme belongs to the family of D-hydroxy acid dehydrogenases (DHDHs). A search of the protein databases uncovered many additional putative phosphite dehydrogenases. The genes encoding four diverse candidates were cloned and expressed, and the enzymes were purified and characterized. All oxidized phosphite to phosphate and had similar kinetic parameters despite a low level of pairwise sequence identity (39-72%). A recent crystal structure identified Arg301 as a residue in the active site that has not been investigated previously. Arg301 is fully conserved in the enzymes shown here to be PTDHs, but the residue is not conserved in other DHDHs. Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ~100-fold decrease in k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher k(cat) than the parent PTDH, and a more modest increase in K(m) for phosphite (nearly 40-fold). Given these results, Arg301 may be involved in the binding and orientation of the phosphite substrate and/or play a catalytic role via electrostatic interactions. Three other residues in the active site region that are conserved in the PTDH orthologs but not DHDHs were identified (Trp134, Tyr139, and Ser295). The importance of these residues was also investigated by site-directed mutagenesis. All of the mutants had k(cat) values similar to that of the wild-type enzyme, indicating these residues are not important for catalysis.

Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration.

The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD(+)-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD(+) (1.7 Å resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 Å resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.

Directed evolution: selection of the host organism.

Directed evolution has become a well-established tool for improving proteins and biological systems. A critical aspect of directed evolution is the selection of a suitable host organism for achieving functional expression of the target gene. To date, most directed evolution studies have used either Escherichia coli or Saccharomyces cerevisiae as a host; however, other bacterial and yeast species, as well as mammalian and insect cell lines, have also been successfully used. Recent advances in synthetic biology and genomics have opened the possibility of expanding the use of directed evolution to new host organisms such as microalgae. This review focuses on the different host organisms used in directed evolution and highlights some of the recent directed evolution strategies used in these organisms.

Deciphering the late biosynthetic steps of antimalarial compound FR-900098.

FR-900098 is a potent chemotherapeutic agent for the treatment of malaria. Here we report the heterologous production of this compound in Escherichia coli by reconstructing the entire biosynthetic pathway using a three-plasmid system. Based on this system, whole-cell feeding assays in combination with in vitro enzymatic activity assays reveal an unusual functional role of nucleotide conjugation and lead to the complete elucidation of the previously unassigned late biosynthetic steps. These studies also suggest a biosynthetic route to a second phosphonate antibiotic, FR-33289. A thorough understanding of the FR-900098 biosynthetic pathway now opens possibilities for metabolic engineering in E. coli to increase production of the antimalarial antibiotic and combinatorial biosynthesis to generate novel derivatives of FR-900098.

Cloning and heterologous expression of the spectinabilin biosynthetic gene cluster from Streptomyces spectabilis.

Spectinabilin is a rare nitrophenyl-substituted polyketide metabolite. Here we report the cloning and heterologous expression of the spectinabilin gene cluster from Streptomyces spectabilis. Unexpectedly, this gene cluster is evolutionarily closer to the aureothin gene cluster than to the spectinabilin gene cluster from Streptomyces orinoci. Moreover, the two nearly identical spectinabilin gene clusters use a distinctly different regulation mechanism.

Cloning, expression, and biochemical characterization of Streptomyces rubellomurinus genes required for biosynthesis of antimalarial compound FR900098.

The antibiotics fosmidomycin and FR900098 are members of a unique class of phosphonic acid natural products that inhibit the nonmevalonate pathway for isoprenoid biosynthesis. Both are potent antibacterial and antimalarial compounds, but despite their efficacy, little is known regarding their biosynthesis. Here we report the identification of the Streptomyces rubellomurinus genes required for the biosynthesis of FR900098. Expression of these genes in Streptomyces lividans results in production of FR900098, demonstrating their role in synthesis of the antibiotic. Analysis of the putative gene products suggests that FR900098 is synthesized by metabolic reactions analogous to portions of the tricarboxylic acid cycle. These data greatly expand our knowledge of phosphonate biosynthesis and enable efforts to overproduce this highly useful therapeutic agent.

Further improvement of phosphite dehydrogenase thermostability by saturation mutagenesis.

Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.

Efficient regeneration of NADPH using an engineered phosphite dehydrogenase.

The in situ regeneration of reduced nicotinamide cofactors (NAD(P)H) is necessary for practical synthesis of many important chemicals. Here, we report the engineering of a highly stable and active mutant phosphite dehydrogenase (12x-A176R PTDH) from Pseudomonas stutzeri and evaluation of its potential as an effective NADPH regeneration system in an enzyme membrane reactor. Two practically important enzymatic reactions including xylose reductase-catalyzed xylitol synthesis and alcohol dehydrogenase-catalyzed (R)-phenylethanol synthesis were used as model systems, and the mutant PTDH was directly compared to the commercially available NADP(+)-specific Pseudomonas sp. 101 formate dehydrogenase (mut Pse-FDH) that is widely used for NADPH regeneration. In both model reactions, the two regeneration enzymes showed similar rates of enzyme activity loss; however, the mutant PTDH showed higher substrate conversion and higher total turnover numbers for NADP(+) than mut Pse-FDH. The space-time yields of the product with the mutant PTDH were also up to fourfold higher than those with mut Pse-FDH. In particular, a space-time yield of 230 g L(-1) d(-1) xylitol was obtained with the mutant PTDH using a charged nanofiltration membrane, representing the highest productivity compared to other existing biological processes for xylitol synthesis based on yeast D-xylose converting strains or similar in vitro enzyme membrane reactor systems.

Directed evolution of enzymes and biosynthetic pathways.

Directed evolution is an important tool for overcoming the limitations of natural enzymes as biocatalysts. Recent advances have focused on applying directed evolution to a variety of enzymes, such as epoxide hydrolase, glyphosate N-acetyltransferase, xylanase and phosphotriesterase, in order to improve their activity, selectivity, stability and solubility. The focus has also shifted to manipulating biosynthetic pathways for the production of many naturally synthesized compounds, as well as the production of novel 'unnatural' compounds. A combined directed evolution and computational design approach is becoming increasingly important in exploring enzyme sequence-space and creating improved or novel enzymes. Fueled by recent breakthroughs in genomics and metagenomics, these developments should help expand the use of biocatalysts in industry.

Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration.

NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T50 is 20 degrees C higher and half-life of thermal inactivation at 45 degrees C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50 degrees C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to l-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.