MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering

Abstract

Photosynthetic microalgae are promising green cell factories for the sustainable production of high-value chemicals and biopharmaceuticals. The chloroplast organelle is being developed as a chassis for synthetic biology as it contains its own genome (the plastome) and some interesting advantages, such as high recombinant protein titers and a diverse and dynamic metabolism. However, chloroplast engineering is currently hampered by the lack of standardized cloning tools and Design-Build-Test-Learn workflows to ease genomic and metabolic engineering. The MoClo (Modular Cloning) toolkit based on Golden Gate assembly was recently developed in the model eukaryotic green microalgae Chlamydomonas reinhardtii to facilitate nuclear transformation and engineering. Here, we present MoCloro as an extension of the MoClo that allows chloroplast genome engineering. Briefly, a Golden Gate-compatible chloroplast transformation vector (pWF.K.4) was constructed, which contains homologous arms for integration at the petA site in the plastome. A collection of standardized parts (promoters, terminators, reporter and selection marker genes) was created following the MoClo syntax to enable easy combinatorial assembly of multi-cassettes in the destination pWF.K.4 vector. The functionality of the biobricks was assayed by constructing and assessing the expression of several multigenic constructs. Finally, a generic vector pK4 was constructed for easy Golden Gate cloning of 5' and 3' homologous arms, allowing targeting at alternative plastome integration sites. This work represents a significant advancement in technology aimed at facilitating more efficient and rapid chloroplast transformation and engineering of green microalgae.

FIGURE 1

Construction of MoCloro Level 0 biobricks and pWF.K.4 destination vector for Level 2 assembly and Chlamydomonas plastome transformation. (A) Five promoter‐5′UTRs, four 3′UTR‐terminators, and one reporter gene (uidA‐GUS) Level 0 parts were generated for multigene assembly using standard Golden Gate Assembly syntax. (B) Level 0 parts are assembled in transcriptional units or modules upon Level 1 MoClo reactions. A module for the expression of the selection marker was also constructed (atpA‐SpecR‐rbcL). Modules are then cloned using Level 2 MoClo reactions in the constructed destination vector pWF.K.4. This vector was constructed using standard syntax and contains pWF 5′ and 3′ arms to integrate into the pWF locus of the Chlamydomonas plastome by homologous recombination upon microbombardment transformation.

FIGURE 2

Construction and analysis of GUS reporter combinations to test the functionality of all biobricks. (A) Transcriptional units constructed to drive GUS reporter expression, including combinations of five different promoters‐5′UTR and four different 3′UTR‐terminators. (B) GUS‐specific activity was assayed from protein extracts obtained from six independent lines showing homoplasmy and grown in deep‐well plates. C‐ is a wild type 137c, whereas C+ is a stable line containing rcbL‐GUS‐psbA cassette that was generated and maintained in the laboratory for more than 7 passages. Bars represent average ± standard error (SE), dots represent individual data points, except for C+, which represent technical triplicates. A Tukey–Kramer test was performed, and significant differences are shown as letters above each bar.

FIGURE 3

Assessment of position and orientation effect in multigene constructs. (A) Scheme showing the six multigenic constructs in pWF.K.4 vector including SpecR and RhlA expression cassettes in forward orientation, and the GUS expression cassette in position one, two or three in forward (1F, 2F, 3F) or reverse (1R, 2R, 3R) orientation. (B) GUS‐specific activity was assayed from protein extracts obtained from six independent lines grown in deep‐well plates. Bars represent average ± SE, dots represent individual data points. C‐ and C+ lines are as in Figure 2 and represent a single technical replicate. Tukey–Kramer test was performed, and significant differences are shown as the letters above each bar. (C) Western‐blot using α‐HA antibody to detect RhlA‐HA protein (34.4 kDa) of one biological replicate for each construct and the 137c as a negative control. Coomassie Blue staining of the membrane (lower panel) is shown as loading control. Mk: prestained protein marker.

FIGURE 4

Construction and testing the functionality of pK4, a Level 2 destination vector facilitating cloning of 5′ and 3′ homology arms for customized plastome integration sites. (A) Schematic representation of the pK4 vector (left). It contains the LacZ‐alpha cassette flanked by standardized BbsI sites for MoCloro Level 2 assemblies and by four BsaI sites strategically located upstream and downstream of the BsbI‐LacZ‐BsbI cassette. The BsaI sites allow golden gate cloning of 5′ and 3′ arms to construct customized p”XX”K4 vector (middle and right), allowing targeting at desired plastome sites. (B) Schematic representation of constructed vectors targeting plastome pLM20 site. These vectors were constructed in two steps. First, pK4 was used as a backbone to clone the 5′ (20–5′) and two 3′ (short, 20‐3′S; and long, 20‐3′L) homology arms from pLM20, to generate pLM20.K.4‐3′S and pLM20.K.4‐3′L vectors. Then golden gate Level 2 reactions were performed to clone 16SrRNA‐GUS‐atpB and specR cassettes to construct pLM20.K.4‐3′L + GUS‐specR and pLM20.K.4‐3′S + GUS‐specR vectors. (C) GUS‐specific activity was assayed from protein extracts obtained from six independent lines grown in deep‐well plates that had been previously subjected to five rounds of selection on spectinomycin as lines in Figure 2, so they are probably homoplasmic or close to homoplasmic. Bars represent average ± SE, dots represent individual data points. C‐ and C+ lines are as in Figure 2 and represent a single technical replicate. Tukey–Kramer test was performed, and significant differences are shown as the letters above each bar.

FIGURE 5

Pipeline and timing for construct generation, Chlamydomonas transformation and selection and line characterization. The pipeline is illustrated by the construction of pLM20.K.4‐3′L + GUS‐specR. Level 0 biobricks and customized 5′ and 3′ homology arms can either be ordered as synthetic DNA or by PCR amplification. Level 1 modules and pK4‐derived destination vector with customized 5′ and 3′ homology arms are assembled in Golden Gate reactions using BsaI. Level 2 devices are constructed from modules and the destination vector pXX.K.4 using BbsI. Constructs are then confirmed by Oxford nanopore technology (ONT) sequencing and are used to transform Chlamydomonas cells by microbombardment. Transformants are selected for several re‐streaking rounds until homoplasmy is reached. Transplastomic lines can be screened or characterized by physiological, molecular, biochemical or analytical methods. Level 2 constructs can be obtained as short as in one week starting from Level 0 biobricks, whereas the whole process up to line characterization can be completed in less than two months.