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

Abstract

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.

Advances in light system engineering across the phototrophic spectrum

“…These mutants showed reduced pigmentation due to incomplete, but still functional, light harvesting complexes (LHCs). This result contrasted both the authors’ expectations, and results found in a variety of other studies ( Baek et al., 2016 ; Caddell et al., 2023 ; Krishnan et al., 2023 ). The authors proposed two possible explanations, first that the mutants could have higher susceptibility to photodamage, second that the mutation strategies used resulted in off target gene editing with negative side effects.…”

“…Recently Krishan et al. found that the pigment content of Picochlorum celeri can be heavily reduced by CRISPR/Cas9 gene editing ( Krishnan et al., 2023 ). This work builds off previous studies done with Picochlorum celeri ( Krishnan et al., 2020 ; Cano et al., 2021 ), and focuses on the cpSRP43, LHCA6, and LHCA7 proteins.…”

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

“…Cultures were grown in a custom-built solar-simulating automated photobioreactor system (the ALGiSIM system, ALGi, Inc.) as described previously ( Cano et al, 2021 ) at a constant temperature of 33°C in 500-mL glass bottles bubbled with 2% CO 2 /air balance at a flow rate of 400 mL min −1 helping maintain a constant pH at ∼7.2. The solar day used had a maximum irradiance of 2,400 µmoles PAR m −2 s −1 at mid-day and represents a simulated summer day in Mesa, Arizona, excluding cloud cover, fog, rain, or other dynamic environmental fluctuations ( Krishnan et al, 2023 ). Cultures followed a daily dilution regime with a prescribed volume of culture (60% unless otherwise noted) dilution occurring 1 h before sunset.…”

“…The idea is not new ( Kok, 1953 ; Radmer Richard and Kok Bessel 1977b ) and has been used somewhat successfully to improve the solar-to-biomass conversion efficiency in mutants with truncated light-harvesting chlorophyll antenna (or tla ) ( Nakajima and Ueda, 1997 ; 2000 ; 1999 ; Polle et al, 2002 ; Polle, Kanakagiri, and Melis, 2003 ; Kirst et al, 2012 ; Jeong et al, 2017 ). In particular, mutants targeting genes of the chloroplast signal recognition particle (CpSRP) pathway affecting the assembly of LHC proteins display a smaller chlorophyll (Chl) antenna size, a higher Chl a/ b ratio, as well as a higher light intensity for the saturation of photosynthesis ( Kirst et al, 2012 ; Kirst and Melis, 2014 ; Jeong et al, 2017 ; Kirst et al, 2017 ; Krishnan et al, 2023 ). These concepts are actively being explored in microalgae and hold promising potential for verification in outdoor systems in the future.…”