Simultaneous CAS9 editing of cpSRP43, LHCA6, and LHCA7 in Picochlorum celeri lowers chlorophyll levels and improves biomass productivity.

High cellular pigment levels in dense microalgal cultures contribute to excess light absorption. To improve photosynthetic yields in the marine microalga Picochlorum celeri, CAS9 gene editing was used to target the molecular chaperone cpSRP43. Depigmented strains (>50% lower chlorophyll) were generated, with proteomics showing attenuated levels of most light harvesting complex (LHC) proteins. Gene editing generated two types of cpSRP43 transformants with distinct lower pigment phenotypes: (i) a transformant (Δsrp43) with both cpSRP43 diploid alleles modified to encode non-functional polypeptides and (ii) a transformant (STR30309) with a 3 nt in-frame insertion in one allele at the CAS9 cut site (non-functional second allele), leading to expression of a modified cpSRP43. STR30309 has more chlorophyll than Δsrp43 but substantially less than wild type. To further decrease light absorption by photosystem I in STR30309, CAS9 editing was used to stack in disruptions of both LHCA6 and LHCA7 to generate STR30843, which has higher (5-24%) productivities relative to wild type in solar-simulating bioreactors. Maximal productivities required frequent partial harvests throughout the day. For STR30843, exemplary diel bioreactor yields of ~50 g m-2 day-1 were attained. Our results demonstrate diel productivity gains in P. celeri by lowering pigment levels.

Cas9 deletion of lutein biosynthesis in the marine alga Picochlorum celeri reduces photosynthetic pigments while sustaining high biomass productivity.

Domestication of algae for food and renewable biofuels remains limited by the low photosynthetic efficiencies of processes that have evolved to be competitive for optimal light capture, incentivizing the development of large antennas in light-limiting conditions, thus decreasing efficient light utilization in cultivated ponds or photobioreactors. Reducing the pigment content to improve biomass productivity has been a strategy discussed for several decades and the ability to reduce pigment significantly is now fully at hand thanks to the widespread use of genome editing tools. Picochlorum celeri is one of the fastest growing marine algae identified and holds particular promise for outdoor cultivation, especially in saline water and warm climates. We show that while chlorophyll b is essential to sustain high biomass productivities under dense cultivation, removing Picochlorum celeri's main carotenoid, lutein, leads to a decreased total chlorophyll content, higher a/b ratio, reduced functional LHCII cross section and higher maximum quantum efficiencies at lower light intensities, resulting in an incremental increase in biomass productivity and increased PAR-to-biomass conversion efficiency. These findings further strengthen the existing strategies to improve photosynthetic efficiency and biomass production in algae.

Picochlorum celeri as a model system for robust outdoor algal growth in seawater.

With fast growth rates, broad halotolerance and the ability to thrive at high temperatures, algae in the genus Picochlorum are emerging as promising biomass producers. Recently, we isolated a remarkably productive strain, Picochlorum celeri, that attains > 40 g m-2 day-1 productivities using simulated outdoor light. To test outdoor productivities, Picochlorum celeri was cultivated in 820 L raceway ponds at the Arizona Center for Algae Technology and Innovation. Picochlorum celeri demonstrated the highest outdoor biomass productivities reported to date at this testbed averaging ~ 31 g m-2 day-1 over four months with a monthly (August) high of ~ 36 g m-2 day-1. Several single day productivities were > 40 g m-2 day-1. Importantly for sustainability, Picochlorum celeri achieved these productivities in saline water ranging from seawater to 50 parts per thousand sea salts, without any biocides or pond crashes, for over 143 days. Lastly, we report robust genetic engineering tools for future strain improvements.

Phased Diploid Genome Sequence for the Fast-Growing Microalga Picochlorum celeri.

Picochlorum celeri is a fast-growing marine microalga with high biomass productivity. Here, we report the use of PacBio sequencing to assemble the phased diploid genome of P. celeri.