Secretion of Acetylxylan Esterase From Chlamydomonas reinhardtii Enables Utilization of Lignocellulosic Biomass as a Carbon Source.

Microalgae offer a promising biological platform for sustainable biomanufacturing of a wide range of chemicals, pharmaceuticals, and fuels. The model microalga Chlamydomonas reinhardtii is thus far the most versatile algal chassis for bioengineering and can grow using atmospheric CO2 and organic carbons (e.g., acetate and pure cellulose). Ability to utilize renewable feedstock like lignocellulosic biomass as a carbon source could significantly accelerate microalgae-based productions, but this is yet to be demonstrated. We observed that C. reinhardtii was not able to heterotrophically grow using wheat straw, a common type of lignocellulosic biomass, likely due to the recalcitrant nature of the biomass. When the biomass was pretreated with alkaline, C. reinhardtii was able to grow using acetate that was released from the biomass. To establish an eco-friendly and self-sustained growth system, we engineered C. reinhardtii to secrete a fungal acetylxylan esterase (AXE) for hydrolysis of acetylesters in the lignocellulosic biomass. Two transgenic strains (CrAXE03 and CrAXE23) secreting an active AXE into culture media were isolated. Incubation of CrAXE03 with wheat straw resulted in an eight-fold increase in the algal cell counts with a concomitant decrease of biomass acetylester contents by 96%. The transgenic lines showed minor growth defects compared to the parental strain, indicating that secretion of the AXE protein imposes limited metabolic burden. The results presented here would open new opportunities for applying low-cost renewable feedstock, available in large amounts as agricultural and manufacturing by-products, for microalgal cultivation. Furthermore, acetylesters and acetate released from them, are well-known inhibitors in lignocellulosic biofuel productions; thus, direct application of the bioengineered microalga could be exploited for improving renewable biofuel productions.

High-yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii.

Microalga-based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post-translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C-terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)n , wherein n = 10 or 20]. The yields of the (SP)n -fused Venus were higher than Venus without the glycomodule by up to 12-fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus-(SP)n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)n glycomodule to promote a more efficient biomanufacturing of microalgae-based recombinant proteins.

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria.

BACKGROUND: Cyanobacteria are photosynthetic bacteria that thrive in diverse ecosystems and play major roles in the global carbon cycle. The abilities of cyanobacteria to fix atmospheric CO2 and to allocate the fixed carbons to chemicals and biofuels have attracted growing attentions as sustainable microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls, lysis of cyanobacterial cells is inefficient and often laborious. In some cases radioisotope-labeled substrates can be fed directly to intact cells; however, label-free assays are often favored due to safety and practical reasons. RESULTS: Here we show an easy and highly efficient method for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism in cyanobacteria. Incubation of the cyanobacterial cells in the commercially available B-PER reagent for 10 min permeabilized the cells, as confirmed by the SYTOX Green staining. There was no significant change in the cell shape and no major loss of intracellular proteins was observed during the treatment. When used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process and subsequent activity assays were successfully adapted to the 96-well plate system. CONCLUSIONS: An easy, efficient and scalable permeabilization protocol was established for cyanobacteria. The permeabilized cells can be directly applied for measurement of G6PDH and Rubisco activities without using radioisotopes and the protocol may be readily adapted to studies of other cyanobacterial species and other intracellular enzymes. The permeabilization and enzyme assays can be performed in 96-well plates in a high-throughput manner.