RECOMBINANT ALGAE WITH HIGH LIPID PRODUCTIVITY

Abstract

The invention involves providing recombinant algal mutants that have a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme and / or a genetic modification in a nucleic acid encoding an RNA-binding domain. In some embodiments, any of these algal mutants may also have a genetic mutation in a nucleic acid sequence encoding an SGI1 polypeptide. The attenuation of one, two, or all three genes results in a mutant organism with increased lipid productivity. It was also discovered that one, two, three, or more genetic mutations can accumulate or "stack" in a particular mutant cell or organism to result in further increases in the production of lipid products. The lipid products of these mutants are useful as biofuels or for other specialty chemicals.

Description

RECOMBINANT ALGAE WITH HIGH LIPID PRODUCTIVITY Cross-reference to related applications This application claims the benefit of priority pursuant to 35 USC §119(e) of US serial number 62 / 949.378, filed on December 17, 2019, the full contents of which are incorporated by reference in full in this memorandum. Field of invention The invention involves providing recombinant algal mutant organisms for lipid production. Incorporation of sequence list The attached sequence listing material is incorporated herein by reference. The attached sequence listing text file, named SGI2240_1 WO_Sequence_Listing, was created on December 14, 2020, and is 129 KB in size. It can be accessed using Microsoft Word on a computer running the Windows operating system. Background of the invention The production of biodiesel fuels presents significant opportunities for developing environmentally sound energy sources that can be obtained at a reasonable cost. Efforts have focused on using algae or other microorganisms to produce hydrocarbons that can be used as biodiesel due to their high lipid content. Additional specialized chemicals can also be obtained from these organisms for use in consumer products. Since algae use sunlight to combine water and carbon dioxide to produce biomass, achieving higher productivity offers the possibility of a carbon-neutral fuel source. Therefore, developing algal strains with very high lipid productivity for biofuel production from algae presents the potential for a significant reduction in carbon dioxide released into the atmosphere and a consequent mitigation of global warming. Strategies to increase the production of algae for biofuels and other products have included modifying the nutrition provided to the organisms, such as cultivating them in media deficient in nitrogen, phosphorus, or silicon. Other strategies have included modifying environmental or cultivation protocols, or various efforts aimed at genetically engineering the organisms. However, wild-type algal strains have not been sufficiently productive to allow for the economically viable development of this resource. Higher levels of cell productivity are needed to efficiently utilize this energy source, and achieving sufficient productivity remains a significant barrier. Summary of the invention The invention involves methods and recombinant algal mutants that have a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme and / or a genetic modification in a nucleic acid encoding an RNA-binding domain. The attenuation of either or both of these genes results in a mutant organism with increased lipid productivity. It was also discovered that one, two, three, or more genetic mutations can be accumulated or “stacked” in a particular mutant cell or organism to result in further increases in the production of lipid products. The lipid products from these mutants can be used as biofuels or for other specialty chemicals.In some embodiments, recombinant algal mutants have a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme or a genetic modification in a nucleic acid sequence encoding an RNA-binding domain. In other embodiments, the algal mutants may have both genetic modifications. And in some embodiments, any of these algal mutants may additionally (and optionally) have a genetic mutation in a nucleic acid sequence encoding an SGI1 polypeptide. Each of these algal mutants exhibits increased lipid productivity compared to a control alga. In a first aspect, the invention provides a recombinant algal cell having a genetic modification in a nucleic acid sequence encoding an enzyme of the trehalose biosynthesis pathway; and / or a genetic modification in a nucleic acid sequence encoding an RNA-binding domain; wherein the recombinant alga exhibits increased lipid productivity compared to a corresponding control algal cell. In one embodiment, the genetic modification results in attenuation of the expression of the nucleic acid sequence bearing the genetic modification. In another embodiment, the recombinant alga may have a genetic modification in the nucleic acid sequence encoding the enzyme of the trehalose biosynthesis pathway and a genetic modification in the nucleic acid sequence encoding the RNA-binding domain.In any of the embodiments, the recombinant alga can be a chlorophyte alga and, optionally, from the Class Trebouxiophyceae. nefr / nn / zznz / E / YiAi In various embodiments, the enzyme of the trehalose biosynthesis pathway can be a trehalose-6-phosphate synthase, a trehalose-6-phosphate phosphatase, or a trehalose-6-phosphate synthase / phosphatase. In any of these embodiments, the recombinant alga may also have an attenuation of a nucleic acid sequence encoding an SGI1 polypeptide. In one embodiment, the recombinant alga has a genetic modification in a nucleic acid sequence encoding an enzyme of the trehalose biosynthesis pathway (e.g., trehalose-6-phosphate synthase / phosphatase), a genetic modification in a nucleic acid sequence encoding an RNA-binding domain, and an attenuation of a nucleic acid sequence encoding an SGI1 polypeptide. The recombinant alga exhibits increased lipid productivity compared to a corresponding control algal cell. In one embodiment, the genetic modification of the nucleic acid sequence encoding the RNA-binding domain is a functional deletion. In another embodiment, the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase may have a substitution mutation relative to the wild-type sequence. In either embodiment, the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase may have at least 90% sequence identity with the wild-type sequence. ID No. 2. In either embodiment, the nucleic acid sequence encoding the RNA-binding domain may have at least 90% sequence identity with the wild-type sequence. ID No. 1. In some embodiments, the substitution mutation in the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase is an E723V mutation compared to the wild-type sequence, and the recombinant algal cell is an alga of the genus Parachlorella. Genetic modification of the nucleic acid sequence encoding the trehalose biosynthesis pathway and the nucleic acid sequence encoding the RNA-binding domain can result in attenuation of the expression of each of these nucleic acid sequences. In various embodiments, the recombinant alga has at least 50%, 60%, 70%, 80%, 90%, or at least 2-fold increased lipid productivity compared to a control alga.In some embodiments, the recombinant algae may have (alone or in addition to increased lipid productivity) at least 50%, 60%, 70%, 80%, 90%, or at least twice the biomass productivity of a control algae. In some embodiments, the recombinant algae has at least 5 grams per square meter per day of lipid production. The recombinant algae may also have increased biomass productivity per unit time compared to a control algae. The recombinant algae may have the highest indicated biomass productivity and / or the highest indicated total organic carbon production under nitrogen-deficient conditions. nefr / nn / zznz / E / YiAi In various embodiments, the recombinant alga can be a chlorophyte alga from any of the selected genera Chlorella, Parachlorella, Picochlorum, Tetraselmis, and Oocystis. In another aspect, the invention provides a method for producing a lipid-containing composition. The methods involve cultivating an algal organism having a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme and / or a genetic modification in a nucleic acid sequence encoding an RNA-binding domain, and thereby producing a lipid-containing composition. In any embodiment, the algal organism may also have attenuated expression of a nucleic acid sequence encoding an SGI1 polypeptide. Any organism described herein may be cultivated or used in the methods. In another aspect, the invention involves methods for producing a recombinant lipid-producing algal organism. The methods involve introducing a genetic modification into a nucleic acid sequence encoding a trehalose biosynthetic enzyme in an algal organism and / or introducing a genetic modification into a nucleic acid sequence encoding an RNA-binding domain in an algal organism, wherein the genetic modification(s) is / are relative to a corresponding control algal organism, thereby producing a recombinant lipid-producing algal organism. The recombinant algal organism exhibits increased lipid productivity compared to a corresponding control algal organism lacking the modification(s). Any organism described herein can be produced using these methods. In one embodiment, the methods involve introducing a genetic modification into the nucleic acid sequence encoding the enzyme of the trehalose biosynthetic pathway and also introducing a genetic modification into the nucleic acid sequence encoding the RNA-binding domain. The methods may also involve culturing the recombinant algal organism to produce a lipid-containing composition. In one embodiment, the genetic modification(s) are introduced by mutagenesis. In all embodiments, the algal organism is a chlorophyte alga. In various embodiments, the enzyme of the trehalose biosynthesis pathway may be trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase, or trehalose-6-phosphate synthase / phosphatase.In any of the embodiments, the methods may also involve the introduction of a genetic modification in a nucleic acid sequence that encodes an SGI1 polypeptide. In some embodiments, the genetic modification(s) of any one or more of the nucleic acid sequences is a functional deletion. In one embodiment of nefr / nn / zznz / E / YiAi, the trehalose-6-phosphate biosynthetic enzyme is a synthase / phosphatase, and its genetic modification is a substitution mutation against the wild-type sequence. In one embodiment, the nucleic acid sequence encoding the trehalose-6-phosphate synthase / phosphatase has at least 90% sequence identity with the SEQ. ID No. 2. In any embodiment, the nucleic acid sequence of the algal organism encoding the RNA-binding domain is a functional deletion. The nucleic acid sequence encoding the RNA-binding domain in the algal organism may have at least 90% sequence identity with the SEQ. ID No. 1. In any embodiment, the alga may be of the Class Trebouxiophyceae. In one embodiment, the substitution mutation in the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase is an E723V mutation, and the recombinant algal cell is an alga of the genus Parachlorella. In any embodiment, the genetic modification(s) may be an attenuation in the expression of the nucleic acid sequence(s). In various embodiments, the algal organism may have at least 50% greater lipid productivity than the corresponding control alga, or at least 75% greater lipid productivity than the corresponding control alga. The algal organism may also have at least 5 grams per square meter per day of lipid production.The algal organism may have a higher biomass productivity per unit of time and / or a higher biomass productivity under nitrogen deficiency conditions and / or a higher total organic carbon production under nitrogen deficiency conditions. In any of the embodiments, the recombinant alga may be a chlorophyll-containing alga from a genus selected from the group consisting of: Chlorella, Parachlorella, Picochlorum, Tetraselmis, and Oocystis. In one embodiment, the recombinant alga is an alga of the class Chlorellales. In one embodiment, the method further involves a step of treating the algal organism with L)V radiation before the cultivation step. The method may also involve collecting a lipid composition from the algal organism. Brief description of the figures Figure 1A provides a graphical illustration of a FAME / TOC productivity diagram for strains isolated from BODIPY-FACS enrichment after 2 days of nitrogen-depleted conditions, showing the amount of fixed carbon allocated to lipids and the lipid productivity under nitrogen-depleted conditions. Figure 1B shows the airborne lipid productivity (as TOC) as the average for the first two days under nitrogen-depleted conditions. nefr / nn / zznz / E / YiAi Figures 2A to 2C show that in Figure 2A, the Tre6P recapitulation (SGI1 + Tre6P) showed higher FAME productivity per area compared to the SGI1-KO strain (SGI1 “blocked” only) in the early stages of nitrogen deficiency. By Day 6, levels fell to the same level as the SGI1-KO strain. In Figure 2B, RBD repair in the SGI1+Tre6P+RBD strain resulted in higher FAME productivity during the first few days of nitrogen deficiency, falling to the same levels as the SGI1-KO strain by Day 6. Figure 2C shows the results for the semi-continuous batch urea assay under nitrogen depletion conditions for 2 days for the triple mutation strain (STR0600, i.e., SGI1+RBD+Tre6P mutations) compared to wild-type Parachlorella sp. FAME's production for the STR600 was 53% higher. Figures 3A to 3B show late improvements in lipid productivity driven by RBD removal. In Figure 3A, the RBD recapitulation strain (SGI1 + RBD) shows FAME productivity equivalent to the SGI1 mutant in the early stages of nitrogen deprivation, but increases to almost the levels of the triple mutant (STR00600) by Day 6. In the SGI1 + RBD strain, the RBD SNP was introduced into an SGI1 strain only. In Figure 3B, Tre6P repair in the STR0600 triple mutant showed lower FAME productivity relative to the STR0600 triple mutant at the beginning of nitrogen deprivation, but approaches the same level as the triple mutant by Day 5. Figures 4A to 4B show assay data for stacked RBD and Tre6P mutations in an SGI1 strain only. The data represent the mean and standard deviation for duplicate biological cultures, in which FAME and TOC productivities are determined during the first two days under nitrogen-depleted conditions. Figure 4A shows increased FAME productivity for the SGI1-KO / RBD / Tre6P stacked mutation strains, similar to the triple mutant STR0600. Figure 4B shows TOC data for the same. Figures 5A to 5B show mutation-repairing strains compared to SGI1 alone as the background strain. Figure 5A shows the triple mutant STR600 (SGI1 + RBD + Tre6P) compared to the SGI1 repair strains 680 and 681 (which have the repaired SGI1 mutation) and the repair strain 682 (which has the repaired RBD mutation). FAME accumulation after 2 days is reduced for the repair strains compared to the triple mutant (STR600), but is still higher than for the SGI1-only mutants. Figure 5B shows similar data regarding TOC accumulation after 2 days. FIGURE 6 provides a graphical illustration of a biosynthetic pathway for the conversion of glucose-6-phosphate and UDP-glucose to trehalose-6-phosphate and then to trehalose. nefr / nn / zznz / E / YiAi Detailed description of the invention The invention provides recombinant algal mutants having a genetic modification in a nucleic acid sequence encoding an enzyme of the trehalose biosynthetic pathway and / or a genetic modification in a nucleic acid encoding an RNA-binding domain. A genetic modification of either or both of these genes, as described herein, results in a recombinant or mutant cell or organism with increased productivity, for example, increased lipid productivity. The recombinant cells or organisms may also have increased biomass productivity. The recombinant algal mutants may also optionally have reduced chlorophyll content and / or reduced PSII antenna size. Any of the algal mutants described herein may also optionally have an attenuation of a gene encoding an SGI1 polypeptide.Therefore, in some embodiments, the algal mutants have 1) a genetic modification in a nucleic acid sequence encoding an enzyme of the trehalose biosynthetic pathway and / or 2) a genetic modification in a nucleic acid encoding an RNA-binding domain; and additionally and optionally 3) a genetic modification in a gene encoding an SGI1 polypeptide. Any of the recombinant cells or organisms disclosed herein may be photosynthetic mutant organisms. It was unexpectedly discovered that the genetic mutations disclosed herein can accumulate or “stack” in a cell or organism to result in further significant increases in the production of lipid products manufactured by the cells or organisms—additional increases that may be additive, or rather, synergistic or exponential.Stacking can be achieved by recapitulating more than one of the mutations in a wild-type or other type of cell or organism. The resulting recombinant algal cells or organisms may have one, two, three, or more of the two or more than three genetic mutations described herein and, therefore, may possess the desirable characteristics disclosed herein. The recombinant cells or organisms of the invention may have higher FAME productivity and / or biomass than the corresponding control cells or organisms that lack the corresponding attenuation(s) of the nucleic acid sequence encoding an RBD domain and / or a nucleic acid sequence encoding an enzyme of the trehalose biosynthetic pathway and, optionally, one or both, a nucleic acid sequence encoding an SGI1 polypeptide, or any combination or subcombination of these attenuations, and that are cultured under the same or substantially the same conditions. Biomass productivity can be measured as the rate of biomass accumulation, for example, the total organic carbon content of the respective cells or organisms, which in one embodiment can be in batch cultures.Batch culture is a culture method in which nutrients are neither renewed nor replenished during the period in which the cells or organisms are cultured. Any of the mutant cells or organisms disclosed herein may be photosynthetic. Any of the recombinant cells or organisms described herein may exhibit increased lipid productivity and / or biomass under photoautotrophic conditions. The corresponding (control) cells or organisms are useful for evaluating the effect of one or more genetic modifications.The corresponding cells or organisms (control) do not have one or more of the genetic modifications being evaluated and are subject to the same or substantially the same culture conditions as the test cells or organisms, such that the evaluation is based solely on a difference in the performance of the cells or organisms with the genetic modification(s) being evaluated. The corresponding cells or organisms (control) may be of the same species as the test organism. They may also be identical or similar in all respects except for the genetic modification(s) being evaluated. In some embodiments, the corresponding cell or organism (control) is a wild-type cell or organism. In one embodiment, the recombinant cells or organisms are algal cells. In one embodiment, the recombinant alga has a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme. In another embodiment, the recombinant alga has a genetic modification in a nucleic acid sequence encoding an RNA-binding domain. In yet another embodiment, the recombinant alga may have a genetic modification in a nucleic acid encoding an enzyme of the trehalose biosynthetic pathway and a genetic modification in a nucleic acid sequence encoding an RNA-binding domain.Additionally and optionally, any of the recombinant algae may also have a genetic modification in a nucleic acid sequence that encodes an SGI1 polypeptide with the genetic modification that encodes an RNA-binding domain and / or a genetic modification in a nucleic acid that encodes an enzyme of the trehalose biosynthetic pathway. The lipid products of these mutants can be further processed into biofuels or used in the production of other specialty chemicals. The nucleic acid sequences encoding the trehalose biosynthetic pathway enzyme, the RNA-binding domain, or the SGI1 polypeptide can be any of the nucleic acid sequences described herein, disclosed herein in all possible combinations and subcombinations. In some embodiments, any of the recombinant cells or organisms of the invention may have a reduced amount of chlorophyll and may have an increased chlorophyll a:chlorophyll b ratio compared to a corresponding control cell or organism. The recombinant cells or organisms may have a reduced photosynthetic antenna size, for example, a reduced photosystem II (PSII) and / or a reduced photosystem I (PSI). In various embodiments, the cross-sectional unit size of the PSII and / or PSI antenna of the recombinant cells or organisms disclosed herein may be reduced by at least 10%, at least 20%, at least 30%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 60% compared to the PSII and / or PSI antenna size of a corresponding control cell or organism.Recombinant cells or organisms may have a higher growth rate and / or greater biomass productivity than a corresponding control cell or organism without the genetic modification; for example, higher biomass productivity per hour, per day, or over a period of 2, 3, 4, 5, or 6 days. “Biomass refers to cell mass, whether of living or dead cells. Biomass productivity, or biomass accumulation, or growth rate, can be measured by any means accepted in the art, for example, as ash-free dry weight (AFDW), dry weight, wet weight, or total organic carbon (TOO) productivity. In any embodiment, biomass productivity, or biomass accumulation, or growth rate, can be measured as total organic carbon (TOO) productivity.” The recombinant cells or organisms of the invention can produce a greater quantity of a bioproduct per period of time (e.g., per minute, per hour, per day, or per period of 2, 3, 4, 5, or 6 days), for example, a lipid product, FAME profile, carbohydrate, protein product, polyketide, terpenoid, pigment, antioxidant, vitamin, one or more nucleotides, one or more nucleic acids, one or more amino acids, one or more carbohydrates, alcohol, hormone, cytokine, peptide, or polymer than a corresponding (control) organism that does not have the genetic modification(s) and that is analyzed and cultured under substantially the same conditions for the same period of time. The quantity of product can be expressed as g / period of time, mg / period of time, µg / period of time, or any other quantity defined per defined period of time described herein.These bioproducts can be isolated from a lysate of any of the recombinant cells or organisms of the invention. In some embodiments, the recombinant cells or organisms of the invention produce at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% more of a bioproduct than a corresponding control alga grown under substantially the same conditions, which may be batch, semi-continuous, or continuous culture conditions and may be nutrient-rich culture conditions or nitrogen-depleted conditions and may be photoautotrophic conditions. Without intending to limit ourselves to any particular theory, it is believed that the genetic modification(s) described herein result in an attenuation of the expression of a nucleic acid sequence encoding the enzyme of the trehalose biosynthetic pathway and / or a nucleic acid sequence encoding the RNA-binding domain and, optionally with one or both, a nucleic acid sequence encoding the SGI1 polypeptide. These attenuations result in a significant increase in the amount of lipids produced by the cell, as demonstrated by the total FAME produced by the cell. They may also result in a significant increase in biomass productivity, as demonstrated by the organic carbon produced by the cell (as measured, for example, by total organic carbon). As used in this document, “exogenous” with respect to a nucleic acid or gene indicates that the nucleic acid or gene has been introduced (e.g., “transformed”) into an organism, microorganism, or cell through human intervention. Typically, such an exogenous nucleic acid is introduced into a cell or organism via a recombinant nucleic acid construct. An exogenous nucleic acid may be a sequence from one species introduced into another species—that is, a heterologous nucleic acid. A “heterologous” nucleic acid may also be an exogenous synthetic sequence not found in the species into which it is introduced. An exogenous nucleic acid may also be a sequence homologous to an organism (i.e., the nucleic acid sequence occurs naturally in that species or encodes a polypeptide that occurs naturally in the host species) that has been isolated and subsequently reintroduced into cells of that organism.An exogenous nucleic acid containing a homologous sequence can often be distinguished from the native sequence by the presence of non-native sequences attached to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking the homologous gene sequence in a recombinant nucleic acid construct. Alternatively, or additionally, a stably transformed exogenous nucleic acid can be detected and / or distinguished from a native gene by its juxtaposition to sequences in the genome into which it has been integrated. Furthermore, a nucleic acid is considered exogenous if it has been introduced into a progenitor of the cell, organism, or strain in question. A “recombinant” or “engineered” nucleic acid molecule is a nucleic acid molecule that has been altered through human manipulation. By way of non-limiting examples, a recombinant nucleic acid molecule includes any nucleic acid molecule that: 1) has been partially or totally synthesized or modified in vitro, for example, by the use of chemical or enzymatic techniques (e.g., by the use of chemical synthesis of nucleic acids, or by the use of enzymes for replication, polymerization, digestion (exonucleolytic or endonucleolytic), ligation, reverse transcription, transcription, base modification (including, for example,1) incorporates the following characteristics of nucleic acid molecules: 1) methylation), integration, or recombination (including homologous and site-specific recombination) of nucleic acid molecules; 2) includes linked nucleotide sequences that are not linked in nature; 3) has been engineered using molecular biology techniques such that it lacks one or more nucleotides compared to the sequence of the naturally occurring nucleic acid molecule; and / or 4) has been manipulated using molecular biology techniques such that it has one or more sequence changes or rearrangements compared to the sequence of the naturally occurring nucleic acid. By way of non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or which has been integrated into a vector, such as a cloning vector or an expression vector. When applied to organisms, the terms “transgenic,” “transformed,” “recombinant,” “engineered,” or “genetically engineered” refer to organisms that have been manipulated by introducing an exogenous or recombinant nucleic acid sequence into the organism, or by genetically manipulating native sequences (which are then recombinant). In some embodiments, the exogenous or recombinant nucleic acid may express a heterologous protein product. Non-limiting examples of such manipulations include gene inactivation, targeted mutations and gene replacement, promoter replacement, deletions or insertions, interruptions in a gene or regulatory sequence, and the introduction of transgenes into the organism.For example, a transgenic microorganism may include an introduced exogenous regulatory sequence that enables transcription in the organism, operatively linked to an endogenous gene of the transgenic microorganism. Recombinant or genetically modified organisms may also be organisms in which constructs for the “downgraded,” deleted, attenuated, or altered gene have been introduced to accomplish the stated manipulation. Such constructs include, but are not limited to, RNAi, microRNA, shRNA, antisense, and ribozyme constructs. Organisms whose genomes have been altered by the activity of meganucleases or zinc-finger nucleases are also included.A heterologous or recombinant nucleic acid molecule may be integrated into the genome of a genetically modified / recombinant organism or, in other cases, not be integrated into the genome of a recombinant / genetically modified organism, or it may be present in a vector or other nucleic acid construct. As used herein, “recombinant microorganism” or “recombinant host cell” includes the progeny or derivatives of the recombinant microorganisms disclosed herein. Because certain modifications may occur in successive generations due to mutations or environmental influences, such progeny or derivatives may, in fact, not be identical to the parent cell, but are still included within the scope of the term as used herein. The term “Pfam” refers to a large collection of protein domains and protein families maintained by the Pfam Consortium and available on several sponsored websites worldwide, including: pfam.sanger.ac.uk / (Welcome Trust, Sanger Institute); pfam.sbc.su.se (Stockholm Centre for Bioinformatics); pfam.janelia.org / (Janelia Farm, Howard Hughes Medical Institute); pfam.jouy.inra.fr / (National Institute for Agricultural Research); and pfam.ccbb.re.kr. Pfam domains and families are identified through multiple sequence alignments and hidden Markov models (HMMs). Pfam-A domain or family assignments are high-quality assignments generated by a seed alignment selected using representative members of a protein family and hidden Markov profile models based on the seed alignment.(Unless otherwise specified, matches of a queried protein to a Pfam domain or family are Pfam-A matches.) All identified sequences belonging to the family are then used to automatically generate a complete alignment for the family (Sonnhammer (1998) Nucleic Acids Research 26, 320 to 322, Bateman (2000) Nucleic Acids Research 26, 263 to 266; Bateman (2004) Nucleic Acids Research 32, Database Number, DI38-D141; Finn (2006) Nucleic Acids Research 34, Database Number, D247-251; Finn (2010) Nucleic Acids Research 38, Database Number, D211-D222). By accessing the Pfam database, for example, by using any of the reference websites above, the protein sequences can be compared to the HMMs by using the HMMER homology search software (e.g., HMMER2, HMMER3 or a higher version, hmmer.janelia.org / ).Significant matches that identify a queried protein as part of a pfam family (or with a particular pfam domain) are those in which the bit score is greater than or equal to the collection threshold for the pfam domain. Expected values ​​(e-values) can also be used as a criterion for including a queried protein in a pfam or for determining whether a queried protein has a particular pfam domain, where low e-values ​​(much less than 1.0, for example less than 0.1, or less than or equal to 0.01) represent low probabilities that a match is due to chance. The recombinant cells or organisms described herein can be generated by human intervention, for example, through classical mutagenesis or genetic engineering, or by any feasible mutagenesis method, including, but not limited to, UV radiation, CRISPR / Cas9, cre / lox, gamma radiation, or chemical mutagenesis. Screening methods can be used to identify mutants with desirable characteristics (e.g., reduced chlorophyll and increased productivity). Methods for generating nefr / nn / zznz / E / YiAi mutants of photosynthetic organisms using classical mutagenesis, genetic engineering, and phenotype or genotype screening are well known in the field. Algal cell or organism The recombinant algal cell or organism of the invention may be a mutant microalga, a mutant photosynthetic organism, or a mutant green alga. The recombinant alga may be any eukaryotic microalga such as, but not limited to, a chlorophyte, ochrophyte, or charophyte alga. In some embodiments, the mutant microalga may be a chlorophyte alga of the taxonomic class Chlorophyceacea, or of the class Chlorodendrophyceae, or of the class Prasinophyceacea, or of the class Trebouxiophyceae, or of the class Eustigmatophyceae. In some embodiments, the mutant microalga may be a member of the Class Chlorophyceace, such as a species of any one or more of the genera Asteromonas, Ankistrodesmus, Cartería, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorodendrales, Chloroellales, Chrysosphaera, Dunaliella, Haematococcus, Monoraphidium, Neochloris, Oedogonium, Pelagomonas, Pleurococcus, Pyrobotrys, Scenedesmus, or Volvox.In other embodiments, the mutant microalga may be a member of the class Chlorodendrophyceae, such as a species of any one or more of the genera Prasinocladus, Scherffelia, or Tetraselmis. In further alternative embodiments, the mutant alga may be a member of the class Prasinophyceae, optionally a species of any one or more of the genera Ostreococcus or Micromonas. More alternatively, the mutant microalga can be a member of the Class Trebouxiophyceae, and optionally of the Order Chlorellales, and optionally a genus selected from any or more of Botryococcus, Chlorella, Auxenochlorella, Heveochlorella, Marinichlorella, Oocystis, Parachlorella, Pseudochlorella, Tetrachlorella, Eremosphaera, Franceia, Micractinium, Nannochloris, Picochlorum, Prototheca, Stichococcus or Viridiella, or any of all possible combinations or subcombinations of the genera.In another embodiment, the recombinant alga is a chlorophyte alga of the Class Trebouxiophyceae, the Order Chlorellales, the Family Oocystaceae, Chlorellaceae, or Eustigmatophyceae, and optionally a genus selected from one or more of Oocystis, Parachlorella, Picochlorum, Nannochloropsis, and Tetraselmis. The recombinant alga may also be of the genus Oocystis, or of the genus Parachlorella, or of the genus Picochlorum, or of the genus Tetraselmis, or of any of all possible combinations and subcombinations of the genera. In various embodiments, the recombinant alga of the invention may have a genetic modification in a nucleic acid encoding a trehalose biosynthetic enzyme, an RNA-binding protein, or both. Any of the recombinant algae of the invention may also, optionally, have a genetic modification in a nucleic acid encoding an SGI1 polypeptide. In one embodiment, the recombinant alga nefr / nn / zznz / E / YiAi of the invention has a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme, a genetic modification in a nucleic acid sequence encoding an RNA-binding protein, and a genetic modification in a nucleic acid encoding an SGI1 polypeptide. In one embodiment, each of these genetic modifications is to a native or endogenous sequence of the cell or organism. A “genetic modification” can denote one or more of the following: a deletion, a mutation, a disruption, an insertion, an inactivation, an attenuation, a rearrangement, one or more point mutations, a frameshift mutation, an inversion, a “lock,” or an “incursion,” resulting in a physical change to the modified gene and reducing or eliminating the expression of one or more gene products. The genetic modification (e.g., to a gene or nucleic acid sequence encoding an enzyme of the trehalose biosynthetic pathway, an RBD domain, or the SGI1 polypeptide) can occur at any sequence that affects gene expression or the nature or amount of its product, such as the coding or non-coding sequence, regulatory sequence, promoter, terminator, exon, intron, or 3' or 5' UTR. The genetic modification can be to the native genome of the host cell.In some embodiments, for example, a recombinant cell or organism having attenuated expression of a gene as disclosed herein may have one or more mutations, which may be one or more nucleobase changes and / or one or more nucleobase deletions and / or one or more nucleobase insertions, in the region of a gene 5' from the transcription start site, such as, in non-limiting examples, within approximately 2 kb, within approximately 1.5 kb, within approximately 1 kb, or within approximately 0.5 kb from the known or putative transcription start site, or within approximately 3 kb, within approximately 2.5 kb, within approximately 2 kb, within approximately 1.5 kb, within approximately 1 kb, or within approximately 0.5 kb from the translation start site. In one embodiment, the genetic modification(s) may be an attenuation (but the genetic modification(s) may also be a deletion or an alteration). An “attenuation” refers to a nucleic acid sequence or gene whose function, activity, or expression is reduced compared to the amount of function, activity, or expression in a corresponding (control) organism that does not have the genetic modification being examined, in which the cell is cultured under the same or substantially the same conditions; that is, the decreased function, activity, or expression is due to the genetic modification.In various embodiments, an attenuated nucleic acid sequence or gene produces less than 70%, 50%, 30%, 20%, 10%, 5%, or 1% of its function, activity, or expression compared to a corresponding cell lacking the genetic modification under the same or substantially the same culture conditions. The terms deletion cassette and disruption cassette are used interchangeably. Substantially the same conditions may be the same conditions or slightly different conditions in which the change does not materially affect the function, activity, or expression of the modified nucleic acid sequence. In various forms, genetic modification can be a deletion or an alteration. An unmodified nucleic acid sequence naturally present in the organism indicates a natural, endogenous, or wild-type sequence. In a deletion, at least part of the nucleic acid sequence is removed, but a deletion can also be achieved by interrupting a gene (e.g., a "blocking" mutation) or by, for example, inserting (insertion mutation) another sequence (e.g., a selection marker), or a combination of deletion and insertion. However, a deletion can also be carried out by other genetic modifications known to those skilled in the technique that result in the gene not being functionally expressed.A gene “disruption” is a functional deletion by insertion or deletion of a nucleotide sequence in or from the coding, non-coding, or regulatory region of a gene, resulting in partial or complete loss of gene function, activity, or expression. Functional expression refers to the expression of a nucleic acid product or functional activity. When the expressed nucleic acid product is a polypeptide, a functional polypeptide has at least some of the normal activity of the encoded polypeptide. For a nucleic acid, functional activity is at least some of the nucleic acid's normal activity.A functional deletion or disruption removes at least so much of the expression or activity of a nucleic acid sequence that the resulting product or activity has no significant effect on the cell or organism compared to the natural or normal level of expression. In other words, the cell exhibits the same behavior as a "blocked" deletion or disruption (e.g., with respect to lipid productivity or biomass productivity). When the nucleic acid sequence encodes a polypeptide, the encoded polypeptide will not be expressed in a quantity that would make a significant difference in the cell or organism compared to its expression in the unmodified cell or organism. When the nucleic acid sequence has an activity other than encoding a polypeptide, the activity is insufficient to show a significant effect compared to its activity in the unmodified cell or organism.In some embodiments, functional deletion can eliminate all expression or activity of the nucleic acid sequence. In some embodiments, functional deletion is a blocking deletion. Therefore, deletions, functional deletions, and interruptions can also be attenuations. The recombinant cells or organisms of the invention may have a reduced functional absorption cross-section of PSII or a reduced PSII antenna size. For example, the PSII antenna cross-sectional unit size may be reduced by at least 10%, at least 20%, at least 30%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least approximately 70%, or at least approximately 80% compared to the PSII antenna size of the corresponding (control) cell or organism. The recombinant cells or organisms of the invention may further have a reduced functional absorption cross-section of PSI or a reduced PSI antenna size.For example, the cross-sectional unit size of the PSI antenna can be reduced by at least 10%, at least 20%, at least 30%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 60% compared to the PSI antenna size of a control photosynthetic organism. In various embodiments, a mutant photosynthetic organism as provided herein may have an increased Fv / Fm ratio compared to a corresponding control photosynthetic organism. For example, the mutant photosynthetic organism may have an Fv / Fm increase of at least 5%, at least 10%, at least 12%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a corresponding (control) photosynthetic organism. In various embodiments, the Fv / Fm ratio may be increased between approximately 5% and approximately 50%, or between approximately 5% and 30%, or between 5% and 20% compared to a control photosynthetic organism. Furthermore, a mutant photosynthetic organism as provided herein may have an increased electron transport rate on the acceptor side of photosystem II compared to a control or wild-type cell. The rate may be at least approximately 20%, 30%, 40%, 50%, 60%, 80%, or 100% higher compared to a corresponding control or wild-type organism. Additionally, the mutant photosynthetic cells or organisms of the invention may have a carbon fixation rate (Pmax(O)) in a recombinant cell or organism, as provided herein, that may be elevated compared to a control organism. For example, Pmax(14C) may be increased by at least approximately 20%, 30%, 40%, 50%, 60%, 80%, or 100% compared to a corresponding control or wild-type organism. In some embodiments, the recombinant cells or organisms of the invention have a reduced PSI and / or PSII antenna size and, optionally, may also have a greater amount of ribulose bisphosphate carboxylase activase (Rubisco activase or “RA”) than a corresponding (control) or wild-type organism, for example, at least 1.2, 1.4, 1.6, 1.8, 2, 2.2, or 2.5 times the amount of AR as the control organism. In some embodiments, the mutants exhibit reduced expression of the 6, 8, 10, 12, or 14 nefr / nn / zznz / E / YiAi LHCP genes and increased expression of an RA gene, such as an RA-a or RA-P gene. Therefore, the recombinant cells or organisms of the invention may be mulant photosynthetic organisms having reduced chlorophyll and reduced PSII antenna size in which the mulants have a higher amount of rubisco activase than the control photosynthetic organisms. The LHC supergene family encodes the light-harvesting chlorophyll α / β-binding proteins (LHCs) that constitute the antenna system of the photosynthetic apparatus. A recombinant algal mockingbird of the invention may also have reduced expression of LHC genes. Therefore, in some embodiments, the recombinant cells or organisms of the invention have at least 6, at least 8, at least 10, or at least 12 LHC genes that are attenuated or downregulated relative to their expression level in a corresponding (control) cell or organism. In various embodiments, the reduction in LHC gene expression may be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% at the level of LHC transcripts. Enzymes of the trehalose biosynthetic pathway Trehalose biosynthesis is an important process, and several biosynthetic pathways have been developed to supply trehalose. The most common biosynthetic pathways involve enzymes of the trehalose biosynthetic pathway, including: 1) trehalose-6-phosphate synthase (T6PS), which converts glucose-6-phosphate and UDP-glucose to trehalose-6-phosphate (T6P); 2) trehalose-6-phosphate phosphatase (T6PP), which dephosphorylates T6P to trehalose; 3) trehalase, which converts trehalose to glucose; 4) trehalose phosphate hydrolase, which converts T6P to glucose and UDP-glucose-6-phosphate; and 5) trehalose-6-phosphate synthase / phosphatase (T6PS / P), which has the synthase and phosphatase activity of the previous two enzymes in the same molecule. The recombinant algae of the invention may have a genetic modification in a gene or nucleic acid sequence encoding one or more of the enzymes of the trehalose biosynthetic pathway, or any combination thereof, as disclosed herein in all possible combinations and subcombinations, as fully set forth herein. In one embodiment, the genetic modification is for one or more genes or nucleic acid sequences encoding a trehalose-6-phosphate synthase (T6PS). In another embodiment, the genetic modification is for one or more nucleic acid sequences or genes encoding a trehalose-6-phosphate phosphatase (T6PP). In yet another embodiment, the genetic modification is for one or more nucleic acid sequences or genes encoding a trehalose-6-phosphate synthase / phosphatase (T6PS / P).In another embodiment, the genetic modification is to one or more nucleic acid sequences or genes encoding a trehalose phosphate hydrolase. In another embodiment, the genetic modification nefr / nn / zznz / E / YiAi is to one or more nucleic acid sequences or genes encoding a trehalase. In some embodiments, the genetic modification may be to a promoter, terminator, binding site, or other regulatory sequence for a gene encoding the aforementioned biosynthetic pathway enzyme. The regulatory sequence may control the transcription or translation of the encoded enzyme. In another embodiment, the genetic modification is to any combination or subcombination of the above nucleic acid sequence(s) or gene(s), e.g., for T6PP and T6PS. However, any combination or subcombination of the aforementioned nucleic acid sequences (or genes) may be genetically modified to achieve the desired effect.In one specific embodiment, the genetic modification is an attenuation (e.g., to T6PS / P, or to T6PS and T6PP). In another embodiment, the attenuation is a deletion. In one embodiment, the recombinant cells or organisms of the invention have a genetic modification in a gene or nucleic acid sequence encoding a trehalose-6-phosphate synthase / phosphatase (T6PS / P) in a trehalose biosynthetic pathway. For example, a modified organism of the invention (a chlorophyte alga, Parachlorella sp.) was found to have a nucleic acid sequence encoding a T6PS / P from SEC. ID No. 2. This enzyme shows approximately 30% sequence identity with T6PP from Candida albicans. SEC. ID No. 2 has a genetic modification (compared to the unmodified “wild-type” organism) at position 273, in which a glutamic acid (E273) in the wild-type was replaced by valine in the modified organism, which has increased biomass and / or lipid productivity. E273 is conserved in all species, but in some species the corresponding amino acid residue is an Asp.This residue can also be changed to Val in conserved sequences of other species or can be changed to another amino acid of similar chemical class in the corresponding position to achieve the high-lipid phenotype exhibited by the mutant cells or organisms of the invention. For example, instead of Val, another nonpolar amino acid could be substituted, such as any of Gly, Ala, Leu, He, Ser, Asn, Gln, Asp, or Met. Therefore, in some embodiments, the mutant cells or organisms of the invention have an E273V mutation, or a D273V mutation, or an E273X or D273X mutation, where X is any of Gly, Ala, Leu, He, Ser, Asn, Gln, Asp, or Met. In various embodiments, the encoded enzyme of the trehalose biosynthetic pathway has at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% amino acid sequence identity with SEQ ID No. 2 (trehalose-6-phosphate synthase / phosphatase). In some embodiments, the encoded trehalose biosynthetic pathway enzyme has at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity with an amino acid sequence of at least 50, or at least 60, or at least 70, or at least 100, or at least 300, or at least 400, or at least 500, or at least 600, or at least 700, or at least 750, or at least 800 contiguous amino acids within any of the SEC. ID No.: 2 or 4 or 5.In other embodiments, the trehalose-6-phosphate synthase / phosphatase may be encoded by a nucleic acid sequence that has at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 97%, or at least 98% sequence identity with SEC. ID No.: 49. In one embodiment, the genetic modification inserts a stop codon into a coding or regulatory sequence of one or more nucleic acid sequences encoding an enzyme of the trehalose biosynthesis pathway to effect gene deletion or interruption. In one embodiment, the genetic modification is a Glu723 to Val (E723V) mutation in the polypeptide encoding SEC ID No. 2 (trehalose 6-phosphate synthase / phosphatase) or in a nucleic acid encoding a polypeptide that has at least 60%, 70%, 80%, 90%, 95%, or 98% sequence identity with SEC ID No. 2.Those with ordinary experience will understand that a stop mutation or other mutation can be inserted at many other locations or loci within the nucleotide sequence, including at a promoter or other regulatory sequence of the gene, and result in attenuation of gene expression or the activity of the encoded polypeptide. Such an attenuation or other mutation can also lead to a loss of function in the enzyme of the trehalose biosynthetic pathway and result in the effect of increased lipid productivity. RNA-binding domain RNA-binding proteins (RBPs) are involved in RNA metabolism. The function of RBPs is varied and can include transient binding to RNA sequences to aid in splicing, regulation of alternative splicing, a component of hnRNP (heterogeneous nuclear ribonucleoprotein) proteins, processing, transport, or localization. Most RBPs have multiple RNA-binding domains that include different types of RNA-binding motifs that recognize RNA sequences or targets. The RNA recognition motif known as RRM is the most abundant RNA-binding domain. In the invention, the RNA-binding domain can be an RRM from any or more of the organisms described herein. In one embodiment, the RNA-binding domain can be a protein from the RRM superfamily, for example, RRM_1.In other embodiments, the RNA-binding domain may be a protein from the PFAM 0076 family. SEC. ID No.: 1 is the polypeptide sequence of an RNA-binding domain with two RNA recognition motif (RRM) domains in the N-terminal half of the coding sequence. The orthologs nefr / nn / zznz / E / YiAi are found in many green algae (chlorophytes) and plants. The recombinant algal cell of the invention may have a genetic modification in a nucleotide sequence encoding an RNA-binding domain having at least 50% sequence identity, or at least 60% sequence identity, or at least 70% sequence identity, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity with SEQ. ID No.: 1, or with the RRM domain of SEQ. ID No.3, or a sequence of at least 100, or at least 150, or at least 200, or at least 250, or at least 300 contiguous amino acids within SEQ. ID No.: 1 or SEQ. ID No.: 3. In various embodiments, the RNA-binding domain is encoded by a nucleotide sequence that has at least 60% sequence identity, or at least 70% sequence identity, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% amino acid sequence identity with SEQ. ID No.: 50. In some embodiments, the nucleotide orthologs may encode an RRM domain that has at least 70% sequence identity, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% (and, optionally, in any of the embodiments less than 100%) amino acid sequence identity with SEQ ID No.: 1 or SEQ ID No.: 3. In some embodiments, the genetic modification of the nucleic acid sequence is an attenuation or a deletion, but in other embodiments it may be an insertion, a point mutation, an alteration, or any of the genetic modifications described herein. In one embodiment, the genetic modification inserts a stop codon into a nucleic acid sequence or gene encoding an RNA-binding domain described herein, or into a nucleic acid sequence or gene encoding a trehalose biosynthesis enzyme described herein, or into a nucleic acid sequence or gene encoding an SGI1 polypeptide. In one embodiment, the genetic modification is a stop mutation Lys36 (L36Stop or L36*) inserted into a nucleic acid sequence encoding SEQ ID No. 1 (or, for example, a Lys* inserted into a nucleic acid sequence encoding SEQ ID No. 1).: 3), or in a nucleic acid sequence or gene having at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98% (and, optionally, less than 100% in any embodiment) sequence identity with SEQ ID No.: 1 or 3 or, similarly, with a sequence encoding at least 100 or 150 or 200 or 250 or 300 contiguous amino acids of SEQ ID No.: 1 or 3. The nefr / nn / zznz / E / YiAi stop codon or other modification can also be carried out at many other loci or locations within a nucleic acid sequence or gene or regulatory sequence that encodes an RNA-binding domain or enzyme of the trehalose biosynthetic pathway, e.g., at a promoter, terminator, or other regulatory sequence.Such modifications can attenuate gene expression or the activity of the encoded polypeptide. Analogous modifications to the sequence(s) can achieve a similar effect. Such attenuation or other mutations can also lead to loss of function in the RNA-binding domain, the trehalose biosynthetic pathway enzyme, or the SGI1 polypeptide, resulting in increased lipid productivity. SGI1 Polypeptide As described herein, SGI1, or “Significant Growth Enhancement 1” polypeptide, is a polypeptide comprising a Response Regulator receptor or “RR” domain (pfam PF00072) and a Myb-like binding domain, referred to herein simply as a “myb” domain (pfam PF00249), wherein the RR domain is located at the N-terminus of the myb domain or the myb domain is located at the C-terminus of the RR domain. The amino acid sequence of an SGI1 polypeptide spanning the RR domain and the myb domain may include a stretch of amino acids occurring between the RR and myb domains that may be sparsely conserved or non-conserved among SGI1 polypeptides. The amino acid sequence occurring between the RR domain and the myb domain may be referred to herein as a link between the two domains.The linker can be of any length and, in several examples, can range from one to approximately 300 amino acids, from 10 to approximately 200 amino acids, or from 20 to approximately 150 amino acids in length. The linker region may optionally include a nuclear localization sequence (NLS). An RR domain within an SGI1 protein can be characterized as pfam PF00072, or as a “signal receptor domain” or simply a “receptor domain,” and / or can be classified as cd00156 in the Conserved Domains Database (CDD), as COG0784 in the Clusters of Orthologous Groups of Proteins database, or as an Interpro “CheY-like superfamily” domain, IPR011006. The RR domain is found in two-component bacterial regulatory systems (such as the two-component bacterial chemotaxis system that includes a polypeptide known as CheY), in which it receives a signal from an associated sensor. The RR domain of such systems is often located at the N-terminus of a DNA-binding domain and may include a phosphoacceptor site. The alignment of RR domains from SGI1 attenuation mutant strains of algae can be shown. The RR domain subsequences of Parachlorella sp.WT-1185, Coccomyxa subellipsoidea, Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Chromochloris zofingiensis, Volvox carteri, nefr / nn / zznz / E / YiAi. Tetraselmis sp. 105, Oocystis sp. WT-4183 and Micromonas sp. RCC299 shows substantial homology. A myb domain within an SGI1 protein can be characterized, for example, as pfamPF00249: “Myb-like DNA-binding domain,” and / or can be identified as the conserved domain TIGR01557 “myb-like DNA-binding domain, SHAQKYF class,” or as an Interpro Homeobox-like domain superfamily domain (IPR009057) and / or an Interpro Myb domain (IPROI 7930). Substantial alignment and homology of Myb domains from SGI1-KO algal strains was also shown. Subsequences of the Myb domains of Parachlorella sp. WT-1185, Coccomyxa subellipsoidea, Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Chromochloris zofingiensis, Volvox carteri, Tetraselmis sp. 105, and Oocystis sp. are shown. WT-4183 and Micromonas sp. RCC299. In addition to having an N-terminal RR domain to a myb domain, an SGI1 protein, as provided herein, may score 300 or higher, 320 or higher, 340 or higher, 350 or higher, 360 or higher, or 370 or higher with an e-value of less than approximately le-10, le-50, le-70, or le-100, when scanned with a Hidden Markov Model (HMM) designed to rate proteins based on how well the protein's amino acid sequence matches the conserved amino acids of a region of SGI1 homologs in algae. The SGI1 polypeptide region used to develop the HMM is the amino acid sequence that includes (proceeding from the N-terminal to the C-terminal direction) the RR domain, the linker, and the myb domain.In a HMM, highly conserved amino acid positions are weighted more heavily than poorly conserved amino acid positions within a compared region of polypeptides to arrive at the score. Polypeptides that have scores of at least approximately 300, or 350 or more, such as 370 or more, when explored with an HMM based on SGI1 protein sequences of algal polypeptides that include a single continuous sequence comprising the RR domain, connector, and myb domain developed using [the HMM model], include, without limitation, polypeptides from the algal and plant species Parachlorella sp. 1185 (SEQ ID NO: 8), Coccomyxa subellipsoiclea (SEQ ID NO: 9), Ostreococcus lucimarinus (SEQ ID NO: 10), Chlamydomonas reinharcltii (SEQ ID NO: 11), Chromochloris zofingiensis (SEQ ID NO: 12), Volvox carteri (SEQ ID NO: 13), Tetraselmis sp. 105 (SEQ ID NO: 14 to 16, Oocystis sp. (SEQ ID NO: 17), Micromonas sp.RCC299 (SEC. ID No.: 18) and Micromonas pusilla (SEC. ID No.: 19), Sphagnumfallax (SEC. ID No.: 20), and Physcomitrella patens (SEC. ID No.: 21). Additional SGI1 orthologs of additional algal species are identifiable by those with ordinary experience in the technique. The SGI1 polypeptide encoded by a nucleic acid comprising the recombinant plant or algal cells of the invention may have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, nefr / nn / zznz / E / YiAi at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (and, optionally, in any embodiment less than 100%) amino acid sequence identity with any SGI1 polypeptide sequence of SEC. ID No.6 to 21, or with fragments of any of them comprising a consecutive sequence of at least 100, or at least 125, or at least 150, or 200 or more amino acid residues of the complete protein, wherein the polypeptide has an RR domain and a myb domain, and the RR domain may be N-terminal to the myb domain, wherein the SGI1 polypeptide is a naturally occurring polypeptide or a variant thereof. In various embodiments, the SGI1 polypeptide is derived from a plant or algal species, i.e., it is a naturally occurring polypeptide from a plant or algal species. A gene or nucleotide sequence encoding an SGI1 polypeptide as provided herein, e.g., a gene that is disrupted or whose expression is attenuated in a mutant as provided herein, may be a naturally occurring gene from a plant or algal species encoding a polypeptide as disclosed herein. In various embodiments, the encoded SGI1 polypeptide may have an amino acid subsequence (myb domain) that has at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98% (and, optionally, in any embodiment less than 100%) sequence identity with a Myb domain sequence from any of SEQ. ID No. 22 to 30, or with a consecutive sequence of at least 25, or at least 30, or at least 50, or at least 75 amino acid residues from the complete sequence. In various embodiments, any of these myb domains may be present in an SGI1 polypeptide with any of the RR domains described herein (e.g., SEQ. ID No. 31 to 48). An SGI1 gene that encodes a polypeptide having the sequence of a naturally occurring algal SGI polypeptide may be a gene having a naturally occurring gene sequence, or it may have a sequence that varies from the sequence of a naturally occurring gene.In various embodiments, an SGI1 gene that is attenuated, mutated, or disrupted in a mutant photosynthetic organism as disclosed herein may be a gene identified through homology search, for example, by using one or more sequences disclosed herein as queries, and / or by means of HMM scanning, wherein the HMM is constructed from amino acid sequences, for example, following multiple alignment of at least six SGI1 polypeptides, wherein the amino acid sequences include an RR domain and a myb domain, wherein the RR domain is N-terminal to the myb domain, and wherein there is a linker sequence between the RR and myb domains that does not belong to either domain. nefr / nn / zznz / E / YiAi In some embodiments, an SGI1 polypeptide may be the sequence of an SGI1 polypeptide from algae or plants, or is a variant of a natural SGI1 polypeptide from algae or plants, and may contain a receptor response regulatory domain as a subsequence, e.g., a subsequence of any of SEC. ID No.: 6 to 21, which may be a consecutive sequence of at least 25, or at least 30, or at least 50, or at least 75 amino acid residues from the complete sequence.The response regulator's receiving domain may contain an amino acid subsequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% (and, optionally, in any embodiment less than 100%) sequence identity with any of the response regulator receptor domains of SEC ID No. 31 to 48, or with a consecutive sequence of at least 25, or at least 30, or at least 50, or at least 75 amino acid residues from the complete sequence. Those experienced in the technique know how to calculate the percentage of “sequence identity” between two sequences. In one embodiment, the percentage of sequence identity can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389–3402 and Karlin (1990), Proc. Nati. Acad. Sel. USA 87, 2264–2268). In one embodiment, the search parameters for histogram, descriptions, alignments, expectations (i.e., the statistical significance threshold for reporting matches with database sequences), cutoff, matrix, and filter (low complexity) can be set to the default configuration. The default scoring matrix used by blastp, blastx, tblastn, and tblastx may be the BLOSUM62 matrix (Henikoff (1992), Proc. Nati. Acad. Sel. USA 89, 10915 to 10919).For blastn, the scoring matrix can be set using the ratios of M (i.e., the reward score for a matching residue pair) to N (i.e., the penalty score for non-matching residues), where the default values ​​for M and N can be +5 and -4, respectively. Four blast parameters can be set as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word matches at each wink position throughout the query); yw=gap16 (sets the width of the window within which gap alignments are generated). Equivalent Blastp parameter settings for amino acid sequence matching can be: Q=9; R=2; wink=1; and gapw=32.A Bestfit comparison between sequences, available in version 10.0 of the GCG package, can use the DNA parameters GAP=50 (gap creation penalty) and nefr / nn / zznz / E / YiAi. LEN=3 (gap extension penalty), and the equivalent configuration in protein comparisons can be GAP=8 and LEN=2. Recombinant algae The recombinant mulant algae of the invention demonstrate a significant increase in lipid production in the organism, which can be measured (for example) by using fatty acid methyl ester (FAME) assays. The increase in lipid production can be measured as an increase in the total FAME produced by the organisms.The recombinant cells of the invention having a genetic modification in a nucleic acid sequence or gene encoding an enzyme of the trehalose biosynthesis pathway disclosed herein and / or a genetic modification in a nucleic acid sequence encoding an RNA-binding domain disclosed herein and, optionally, for either of them, a genetic modification to a nucleic acid sequence encoding an SGI1 polypeptide disclosed herein, may exhibit at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% greater lipid productivity compared to a corresponding control alga.In other embodiments, the increase in lipid productivity can be 15 to 35%, 15 to 40%, 25 to 45%, 15 to 50%, 25 to 70%, 25 to 90%, 25 to 100%, 25 to 150%, or 25 to 200%. In one embodiment, lipid productivity is measured using the total fatty acid methyl ester (FAME) assay, familiar to those experienced in the technique. In another embodiment, the recombinant cells of the invention having the genetic modification in one or more enzymes of the trehalose biosynthesis pathway and / or a genetic modification in one or more RNA-binding domains and, optionally, for any of them, a genetic modification of one or more nucleic acid sequences encoding the SGI1 polypeptide, exhibit at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% greater biomass productivity compared to a control alga. In other embodiments, the increase in biomass productivity can be from 15 to 35%, or from 15 to 40%, or from 25 to 45%, or from 15 to 50%, or from 25 to 70%, or from 50 to 100%, or from 50 to 200%.In one embodiment, biomass productivity can be measured as total organic carbon (TOC) by using assays known to those experienced in the technique. The recombinant cells or organisms of the invention may have the highest revealed lipid productivity and / or the highest revealed biomass productivity. nefr / nn / zznz / E / YiAi Increased lipid productivity Any of the recombinant algal cells disclosed herein may exhibit increased lipid productivity. For example, recombinant algal cells that have a genetic modification in one or more nucleic acid sequences encoding a trehalose biosynthetic enzyme (e.g., trehalose-6-phosphate synthase / phosphatase) and / or in one or more nucleic acid sequences encoding an RNA-binding domain and, optionally with either or both, a genetic modification in one or more nucleic acid sequences encoding the SGI1 polypeptide, exhibit increased lipid productivity. In any embodiment, lipid productivity can be measured using the fatty acid methyl ester (FAME) profile, which is known to those skilled in the art. In various embodiments, any of the recombinant algal cells or organisms of the invention can produce at least 20% more, or at least 25% more, or at least 30% more, or at least 35% more, or at least 50% more, or at least 60% more, or at least 70% more, or at least 80% more, or at least 90% more, or at least 100% more, or at least 125% more, or at least 150% more, or at least 200% more lipid product than a corresponding (control) cell or organism. In one embodiment, lipid productivity can be measured by using the FAME profile of the respective cells or organisms. An increase in lipid production or lipid productivity can also be measured in grams per square meter per day of the surface area of ​​a culture vessel (e.g., a flask, a photobioreactor, a culture pond). In various embodiments, the recombinant algae of the invention produces at least 3, 4, 5, 6, or 7 grams per square meter per day of lipids, which can be measured using the FAME profile. In any embodiment, the high lipid productivity and / or high biomass phenotype can be obtained under nitrogen-depleted conditions, which can be achieved with semi-continuous dilutions (e.g., dilution by 30%, 40%, or 50% once a day, followed by replacement with fresh medium). In one embodiment, the lipid product is a fatty acid and / or a fatty acid derivative.In one embodiment, fatty acids and / or fatty acid derivatives comprise one or more species of molecules having a carbon chain between C8-C18 or C8-C20 or C8-C22 or C8-C24. In any of the embodiments, the genetic modification of a gene or nucleic acid sequence encoding an RBD domain described herein and / or a gene or nucleic acid sequence encoding an enzyme of the trehalose biosynthetic pathway described herein, and optionally with either or both, a genetic modification of a nucleic acid sequence encoding a polypeptide SGI1 nefr / nn / zznz / E / YiAi described herein, may result in attenuation of the expression of the respective genes. The genetic modification of one or more of these genes or nucleic acid sequences may be a blocking, a targeted mutation and gene replacement, a gene replacement, a promoter replacement, a deletion, an insertion, a substitution, a functional deletion, a disruption in a gene or its regulatory sequence, as well as the introduction of transgenes into the organism. Biomass productivity The recombinant algal cells of the invention may also have higher biomass productivity than a corresponding organism that does not have a genetic modification in the gene or nucleic acid sequence encoding one or more enzymes of the trehalose biosynthetic pathway described herein and / or in one or more genes or nucleic acid sequences encoding an RBD domain described herein and, optionally with one or both, a genetic modification of a gene or nucleic acid sequence encoding one or more SGI1 polypeptides described herein. Biomass can be measured using total organic carbon (TOC) analysis, which is familiar to those skilled in the art.Recombinant cells can have at least 20%, 25%, 30%, 35%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or at least 200% greater biomass productivity than a corresponding (control) cell or organism, which can be measured by total organic carbon analysis. Biomass productivity can be measured as mg / ml of culture per time period (e.g., 1, 2, 3, 4, or 5 days). In any embodiment, the recombinant algae can exhibit the higher biomass productivity and / or higher lipid productivity specified herein under nitrogen-depleted conditions. Therefore, in one embodiment, the recombinant algae of the invention can produce a higher total organic carbon yield than a corresponding cell or organism (control), a higher yield than can be produced under nitrogen-depleted or low-nitrogen conditions. In one embodiment, biomass productivity can be assessed by measuring the increase in total organic carbon in the cells. Lipid production methods The invention also provides methods for producing a lipid-containing composition. The methods involve subjecting a culture of algal organisms described herein to at least one UV radiation treatment (or gamma radiation, or both) to produce a recombinant algal organism described herein, culturing the recombinant algal organisms in a suitable medium (such as any described herein), and thereby producing a lipid-containing composition. Optionally, the lipids can be isolated from the recombinant algal organisms. The recombinant alga can be cultured in any suitable medium, such as any described herein. The UV treatment may involve, for example, subjecting the culture to UV light (or gamma radiation, or both) for a suitable period of time or under a suitable UV regime.The recombinant algae can be cultivated for at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 10 days, or at least 20 days, or from 2 to 10 days, or from 2 to 20 days, or from 2 to 25 days. Any of the recombinant cells or organisms of the invention can be grown in batch, semi-batch, or continuous culture. In some embodiments, the culture medium may be nutrient-rich or nitrogen-depleted (-N). In some embodiments, the culture is carried out under photoautotrophic conditions, and inorganic carbon (e.g., carbon dioxide or carbonate) may be the sole or substantially sole carbon source in the culture medium. Nitrogen-depleted conditions can be achieved by using a culture medium that does not have a significant source of nitrogen available for cell growth. In various embodiments, nitrogen-depleted conditions may involve culture in a buffer containing less than 0.5 mM nitrogen in any form available externally to the cell or organism.In some embodiments, the cells can be grown in 0.5 mM or less of KNO3 or urea as a nitrogen source. The invention also provides methods for producing a biofuel involving the cultivation of a recombinant algal organism described herein. The methods may also include a step of harvesting a biofuel from a recombinant algal organism of the invention. The recombinant organism can be cultivated in any growth medium, such as any described herein. In one embodiment, the recombinant organism is cultivated in a nitrogen-free medium. In various embodiments, the cultivation may occur for a period of at least 3 days, or at least 5 days, or at least 7 days, or at least 15 days, or at least 20 days. FAME and TOC analysis methods The lipid productivity of cells or organisms can be measured by any method accepted in the technique, for example, as an increase or decrease in the fatty acid methyl esters (FAME) contained in the cell, i.e., analysis of the nefr / nn / zznz / E / YiAi profile FAME of the cell or organism. In some embodiments, any of the recombinant algal cells or organisms of the invention may have higher biomass productivity compared to the corresponding control cells or organisms. In some embodiments, any of the recombinant algal cells or organisms of the invention may have higher lipid productivity and higher biomass productivity compared to a corresponding control cell or organism. Biomass productivity can be measured by any method accepted in the art, for example, by measuring the total organic carbon (TOC) content of a cell. The Examples provide embodiments of both methods. The terms “FAME lipids” or “FAME” refer to lipids that have acyl groups that can be derivatized into fatty acid methyl esters, such as monoacylglycerols, diacylglycerols, triacylglycerols, wax esters, and membrane lipids such as phospholipids, galactolipids, etc. In some embodiments, lipid productivity is evaluated as FAME productivity in milligrams per liter (mg / L), and for algae, it can be reported as grams per square meter per day (g / m² / day). In semi-continuous assays, mg / L values ​​are converted to g / m² / day, taking into account the incident irradiation area (the opening of the SOPA flask rack, 1.5 in x 3.3 / 8 in, or 0.003145 m²) and the culture volume (550 ml). To obtain productivity values ​​in g / m2 / day, the mg / L values ​​are multiplied by the daily dilution rate (30%) and a conversion factor of 0.175.When lipids or their subcategories (e.g., TAGs or FAMEs) are referred to as percentages, the percentage is a percentage by weight unless otherwise stated. The term “fatty acid product” includes free fatty acids, mono-, di-, or triglycerides, fatty aldehydes, fatty alcohols, fatty acid esters (including, but not limited to, wax esters); and hydrocarbons, including, but not limited to, alkanes and alkenes. EXAMPLES EXAMPLE 1- PRODUCTION OF SGI1 MUTANTS The production of algal strains containing a genetic modification in a nucleic acid sequence encoding an SGI1 polypeptide is known in the art and is detailed in document US 2018 / 0186842, published on July 5, 2018, and which is incorporated herein by reference in its entirety, including all tables, figures and claims. Briefly, Parachlorella sp. (a species of chlorophyll or green algae) obtained from marine environments was mutagenized with UV radiation in a STRATALINKER® 2400 UV crosslinker (Agilent Technologies, Santa Clara, CA) and selected based on low chlorophyll fluorescence after low light acclimation. nefr / nn / zznz / E / YiAi Cells were cultured to the mid-log phase and then diluted to 1 x 10⁶ cells / mL with nutrient-rich growth medium. Cell suspensions were transferred to a Petri dish and placed inside a STRATALINKER® 2400 UV crosslinker (Agilent Technologies, Santa Clara, CA) without the lid. UV irradiation was performed at 10,000, 25,000, and 50,000 pJ / cm². After irradiation, cell suspensions were pipetted into a foil-wrapped shaking flask to prevent light exposure for 24 hours during recovery. After mutagenesis and recovery, cells were selected from pale-colored colonies and allowed to grow for one to five days in low light (100 pmol photons nr2sec-1), after which they were sorted by flow cytometry to select cells with low chlorophyll fluorescence levels. A further primary screening of isolated reduced-antenna lines was performed by flow cytometry through visual selection of pale green or yellow colonies after seeding the sorted cells. To filter out presumed reduced-antenna lines from other reduced-pigment mules and false positives, the selected colonies were subjected to a medium-yield secondary culture screen to acclimate the isolates to low-light conditions before photophysiological measurements. Chlorophyll fluorescence was monitored during low-light acclimation to select colonies that retained the reduced-chlorophyll fluorescence characteristic of the high-light acclimated state. The selected clones showed only small increases in chlorophyll (relative to wild-type cells) when transferred from high to low light. Semi-continuous culture assays were conducted under constant intense light (approximately 1,700 pmol photons nr2s-1) using 165 ml cultures in 75 cm2 tissue culture flasks to identify strains with increased productivity (increased rate of biomass production, measured as TOC accumulation) compared to the wild-type parent. Two 75 cm2 flasks were inoculated with seed culture of a given mutant strain using CO2-enriched air (1% CO2) bubbled through the cultures. Samples for TOC analysis were taken from the culture extracted for dilution. Isolates with higher productivity were identified. Genome sequencing and genotyping of the resulting strains revealed mutations involving various SNPs. The effect of increased biomass productivity was found to be related to a SNP in the SGI1 polypeptide sequence at amino acid 250, which was changed from Leu to Pro (i.e., a Leu250Pro SNP). The gene encoding the SGI1 polypeptide in Parachlorella sp. has the nucleotide sequence in SEC. ID No. 6, and the coding sequence in SEC. ID No. 7, which encodes the amino acid sequence in SEC. ID No. 8. The SNP was found to result in a nefr / nn / zznz / E / YiAi Leu250Pro mutation in the SGI1 polypeptide. This mutation was recapitulated in a wild-type Cas9-editing strain of Parachlorella sp. to produce “SGI1 mutants”, and these cells were then used in subsequent procedures. EXAMPLE 2- MUTAGENESIS The SGI1 mutants from Example 1 were irradiated with UV light using a STRATALINKER® 2400 UV crosslinker (Agilent Technologies®, Santa Clara, CA). Irradiation was performed in four doses with duplicates per dose. Cells were diluted to a concentration of 5 x 10⁶ (5e⁶) cells / ml and irradiated on agar plates with approximately 5 x 10⁷ (5e⁷) cells total per Petri dish. Irradiation doses included 16 seconds at 27,000 µJ / cm², 12 seconds at 20,000 µJ / cm², 8 seconds at 13,000 µJ / cm², and 6 seconds at 10,000 µJ / cm². EXAMPLE 3 - BODY GROWTH AND STAINING Next, the mutagenized cells were cultured to an appropriate concentration measured by an OD730 of 3.0 in flasks containing PM074 medium. The cells were then transferred to media containing PM123 media (aquarium salts, PROLINE A®, and PROLINE B® (Pentair Aquatic Eco-Systems®, Inc.)) and a final OD730 of 0.1 (PROLINE A® and PROLINE B® together include 8.8 mM NaNOs, 0.36 mM NaH2PO4.H2O, 10x F / 2 Trace Metals, and 10x F / 2 Vitamins (Guillard (1975) “Culture of phytoplankton for feed marine invertebrates”, eds. Smith, WL and Chanley, MH, Plenum Press, New York, pp. 26–60)). After growing to a suitable concentration (OD730 of 2.8), the cells were centrifuged and resuspended in flasks containing nitrogen-free PM67 medium (aquarium salts, K2HPO4, vitamin mix, and a mixture of chelated trace metals).The cells were placed in a glass tank flask and a final OD730 cell concentration of 1.4 was achieved and the cells were placed under a constant stream of 1% CO2 in air. After 48 hours of batch growth in nitrogen-free media (i.e., the culture medium lacked a nitrogen source), an aliquot of cells was extracted and stained with the lipid-specific dye BODIPY (boron-dipyrromethane). Mutant cells with the highest level of BODIPY staining were enriched by fluorescence-activated cell sorting (FACS). The enriched cell populations were cultured and re-inoculated and allowed to grow as described above, and were again subjected to BODIPY and FACS staining at 48 hours under nitrogen-free (-N) batch growth. This iterative process was repeated for a total of five sorting rounds, with the final sorting resulting in individual cell isolates. nefr / nn / zznz / E / YiAi EXAMPLE 4 - ANALYSIS OF FAME AND TOC Once rendered axenic by bacterial contamination through treatment of cultures with streptomycin at 0.6 mg / ml, isolates with the SGI1 mutation plus the RNA-binding domain (RBD) and trehalose-6-phosphate synthase / phosphatase (Tre6P) attenuations were cultured under nitrogen-depleted growth conditions and compared to the original single-mutant SGI1-KO strain for nitrogen-depleted batch biomass and lipid productivity. FAME and TOC measurements show the amount of fixed carbon that is allocated to lipids and the nitrogen-depleted lipid productivity. Figure 1a shows that the isolates exhibited an increased FAME / TOC ratio (an indicator of the amount of fixed carbon allocated to lipids) compared to the SGI1-KO-only mutants, and Figure 1b shows increased TOC productivity.Therefore, mutants that had the SGI1+RBD+Tre6P attenuations were isolated and had improved lipid productivity compared to mutants that only had SGI1-KO. The total organic carbon (TOC) of the algal culture samples was determined by diluting 2 mL of cell culture to a total volume of 20 mL with DI water. Three injections per measurement were administered into a high-sensitivity TOC analyzer for the determination of Total Carbon (TC) and Total Inorganic Carbon (TIC). The combustion oven was set to 720 °C, and TOC was determined by subtracting TIC from TC. The 4-point calibration range was 2 ppm to 200 ppm, corresponding to 20 to 2000 ppm for undiluted cultures with a correlation coefficient (r²) greater than 0.999. To determine lipid content, a FAME analysis was performed on 2 mL samples that were dried using an evaporator. The following were added to the dried pellets: 500 mL of 500 mM KOH in methanol, 200 mL of tetrahydrofuran containing 0.05% butylated hydroxytoluene, 40 mL of a 2 mg / mL C11:0 free fatty acid / C13:0 / C23:0 triglyceride internal standard mixture of fatty acid methyl ester, and 500 mL of glass beads (425–600 µm in diameter). The vials were capped with open-top PTFE septum-lined caps and placed in a tissue homogenizer at 1.65 krpm for 7.5 minutes. The samples were then heated to 80 °C for five minutes and allowed to cool. For derivatization, 500 µL of 10% boron trifluoride in methanol were added to the samples before heating them to 80 °C for 30 minutes. The tubes were allowed to cool before adding 2 mL of heptane and 500 µL of 5 M NaCl.The samples were then shaken for five minutes at 2 krpm and finally centrifuged for three minutes at 1 krpm. The heptane layer was sampled using an autosampler. Quantification was performed using 80 pg of C23:0 internal FAME standard. nefr / nn / zznz / E / YiAi EXAMPLE 5 - GENOTYPING OF MUTANTS Genomic DNA was isolated from these mutants and from the single-mutant parental strain SGI1 (STR0012) and the corresponding wild-type control strain (STR0010). The isolated gDNA was sequenced using next-generation sequencing on an Illumina® instrument (Illumina, Inc., San Diego, CA). The sequence reads were processed, mapped to the wild-type reference genome, and analyzed using a small variant caller algorithm (derived from the FreeBayes polymorphism detection software, which is a Bayesian genetic variant detector designed to find small polymorphisms, specifically SNPs, indels (insertions and deletions), MNPs (multinucleotide polymorphisms), and complex events (composite insertion and substitution events) smaller than the length of a short-read alignment sequence).Analysis of small nucleotide polymorphisms (SNPs) and small insertions / deletions (InDels) revealed that the sequenced strains were genetically sister to each other and had a core set of 28 polymorphisms shared between the strains. The SNPs shown in Table 1 revealed candidate genes in which the underlying causative mutations for the high-lipid phenotype might occur. Fifteen SNPs were intergenic or present in the introns of a gene; evaluation of transcriptomic and RNA sequence data from the new mutants indicated that these SNPs had no impact on the expression of neighboring genes or on intron splicing. However, thirteen of the 28 SNPs were prioritized for genetic recapitulation based on the deduced change in the coding sequence of the encoded gene. One of the 13 sister mutants with high lipid content, designated Strain 600 (STR0600), was selected for all subsequent experiments. The 13 mutants were identified according to Table I. nefr / nn / zznz / E / YiAi Table 1 Downstream genetic variant Protein 2 containing the haloacid dehalogenase-like hydrolase domain SNP GA Downstream genetic variant abalo predicted conserved protein MNP GG AA Downstream genetic variant abaio Glucuronoxylan 4α-methyltransferase 1 SNP mi T Intron variant Protein containing SNF2 domain / protein containing helicase domain / F-box family protein isoform 3 SNP T e Intron variant Transmembrane amino acid transporter SNP mi T Intron variant Kinase containing AarF domain SNP G UN Upstream genetic variant Conserved predicted protein SNP T UN Upstream genetic variant Conserved predicted protein CTCATCAC deletion CTC AC Upstream genetic variant Protein 29 containing ccch domain with zinc fingers SNP G UN Upstream genetic variant D-amino acid aminotransferase SNP T UN Intron variant Exostosin family protein isoform 1 SNP mi UN Upstream genetic variant Conserved predicted protein SNP TG Intron variant Exostosin family protein isoform 1 ATT insertion ATT T EXAMPLE 6 - GENETIC RECAPITULATION To determine the mutation(s) responsible for the high-lipid phenotype, the inactivations and / or exact recapitulation of the SNPs observed in STR0600 were recreated in a markerless Cas9 / Cre expression strain containing an SGI1 knockout (SGI1-KO). Additionally, a variant of STR00600 was generated incorporating markerless Cas9 / Cre expression cassettes to examine the effect of reverting the SNPs to the wild type. This allowed for the evaluation of individual SNPs to determine which were necessary to produce the high-lipid phenotype and whether the phenotype might have a complex genetic basis where a single SNP is insufficient to recapitulate it. All generated strains were analyzed to determine the high lipid content and / or high biomass productivity phenotype under nitrogen-depleted (-N) batch conditions in a simplified assay carried out in T25 flasks containing nefr / nn / zznz / E / YiAi buffer PM153 for an OD730 of 0.1. PM153 is a nutrient-rich medium based on PM074 but includes urea instead of nitrate as the nitrogen source. It is prepared by adding 1.3 ml of Proline® F / 2 Algae Feed Part A (Pentair Aquatic Eco-Systems) and 1.3 ml of 'Solution C' to a final volume of 1 liter of aquarium salt solution (17.5 g / L), and then adding 4 ml of filter-sterilized 1.1 M urea. Solution C contains 38.75 g / L of NaH2PO4.H2O, 758 mg / L of thiamine HCl, 3.88 mg / L of vitamin B12, and 3.84 mg / L of biotin. PM074 is a nutrient-rich medium. While any suitable algae growth medium may be used in the invention, PM074 is prepared by adding 1.3 ml of Proline® F / 2 Algae Feed Part A (Pentair Aquatic Eco-Systems, Inc., Apopka, FL) and 1.3 ml of Proline® F / 2 Algae Feed Part B to a final volume of 1 liter of an Instant Ocean Salts (35 g / L) solution (Pentair Aquatic Eco Systems Inc., Apopka, FL). Proline A® and Proline B® together include 8.8 mM NaNOs, 0.361 mM NaH2PO4.H2O, 10x F / 2 of trace metals and 10x F / 2 of vitamins (Guillará (1975) Culture of phytoplankton for feeding marine invertebrates in “Culture of Marine Invertebrate Animals”, (eds: Smith WL and Chanley MH) Plenum Press, New York, USA pp. 26 to 60). For biomass and lipid productivities, strains were pre-acclimated in a 1% CO2 incubator with a 14:10 diel and scaled up to 1000 mL in PM153 medium. Cultures were normalized to approximately 350 mg / L of TOO, which is 60% of the empirically determined steady-state standing biomass density for a 40% daily dilution rate. The total culture volume was 420 mL in a 500 mL square polycarbonate flask. Flasks were maintained at 30 °C using a water bath, stirred with a magnetic stir bar, and bubbled with 1% CO2 at 300 mL / min. Light was supplied by LED panels through a 0.0875 m² aperture programmed with a 14:10 diel cycle. For nitrogen-rich biomass productivity measurements, samples were taken for OD730, flow cytometry, FAME, and TOC analysis at dusk, and the cultures were diluted to 40% with PM153 semi-continuously for 8 to 9 days.Following the semi-continuous nitrogen replacement method, the flask was removed at dusk, the sediment was collected, and the supernatant was discarded. The strains were resuspended in PM152 medium (nitrogen-free) and normalized to approximately 250 mg / L of TOC, as empirically determined to yield maximum lipid productivity. The cultures were then transferred back into new 500 mL bottles with a volume of 420 mL, as before. The cultures were grown in batch mode to test lipid productivity during nitrogen depletion induction, with sampling again at dusk. Two mutations were identified that resulted in the high-lipid phenotype. The identified mutations were a Glu723Val (E723V) mutation in the encoded Tre6P enzyme (SEC. ID No.: 2) and a Lys36Stop (Lys36*) mutation in the encoded RBD (SEC. ID No.: 1). It was observed that 1) the introduction of the Tre6P SNP into an SGI1-KO mutant for nefr / nn / zznz / E / YiAi produced a mutant with attenuated SGI1-KO+Tre6P, resulting in a strain with increased lipid productivity, as shown in Figure 2a; 2) Repairing the wild-type RBD attenuation in Strain 600 (SGI1+RBD+Tre6P) to produce a strain having the SGI1+Tre6P attenuation resulted in a strain with reduced lipid productivity (Figure 2b); 3) Introducing the RBD attenuation into the SGI1-only mutant strain resulted in increased lipid productivity under nitrogen-depleted batch growth, as shown in Figure 3a;4) Repair of Tre6P in the SGI1+Tre6P+RBD strain (STR0600) resulted in reduced lipid productivity in batch N, as shown in Figure 3b, but it was still higher than the wild-type strain (STR0010) and reached almost as high as STR0600 with the three modifications; 5) Repair of the SGI1 mutation in STR00600 resulted in reduced lipid productivity in batch -N (nitrogen depletion) and reduced TOC productivity, as shown in Figures 5a and 5b. Figure 2c also shows significantly higher FAME productivity for the mutant strain SGI1+Tre6P+RBD (STR0600) under batch conditions. According to the information provided, the strain designated STR0600 has all three genetic modifications: 1) the SG11 mutation, 2) the RBD mutation, and 3) the Tre6P mutations. Figures 4a and 4b show that the SGI1-only mutant (STR012) exhibited approximately 30% higher FAME and TOC production than the wild type. Figures 4a and 4b also show three independent lines generated with the SGI1 mutation as well as the RBD and Tre6P attenuations; all three lines showed lipid productivity and TOC equivalent to STR0600. Figures 5a and 5b show two strains (680, 681) that have only the RBD and Tre6P mutations, and one that has only the SGI1 and Tre6P mutations. These strains showed a higher accumulation of FAME on day 2 (55%, 59%, and 49% for FAME, respectively) than the wild type (STR010). The strains also showed a TOC accumulation of 19%, 15% higher than the wild type for 2-day TOC accumulation.RBD repair resulted in a drop in lipid productivity to a level below STR0600 and similar to that of strains 680 and 681 (RBD+Tre6p). Therefore, it was shown that the RBD and Tre6P mutations can be “stacked” in an SGI1 mutation strain to recapitulate the high-lipid phenotype of STR0600, and the mutations that give rise to the phenotype were resolved. EXAMPLE 7 - IDENTIFICATION OF MUTATIONS The genomes of the organisms were sequenced, and the amino acid sequences of the encoded polypeptides RBD and Tre6P were determined to be from Parachlorella sp. (SEC. ID No. 1 and SEC. ID No. 2, respectively). Functional annotation and ortholog analysis in other organisms revealed that the gene encoding the RNA-binding domain protein (SEC. ID No. 1) has two RNA recognition motif (RRM) domains in the N-terminal half of the coding sequence. BLAST search revealed that the orthologs are widely distributed in green algae, with approximately 50% sequence identity and approximately 75% positive amino acid identity within the amino acid chemical classes (aliphatic, hydroxyl- or sulfur-containing, cyclic, aromatic, basic, and acidic and their amides), with the RRM domain observed in maize. Musashi-like proteins of this class are well-characterized RBD proteins (J Cell Sel.April 1, 2002; 115 (Pt 7): 1355 to 1359), and the function of this class of proteins is thought to be the directed regulation of mRNA translation, and the RNA-binding domains encoded in the present case may therefore have similar characteristics or the same function. Sequence ID No. 2 is the sequence of a trehalose-6-phosphate synthase / phosphatase from Parachlorella sp. that could catalyze reactions analogous to the Tsp1 and Tsp2 reactions of Candida albicans, presented as an example in Figure 6. Glu723 was found to be well conserved in homologs such as Glu or, in some cases, aspartic acid. However, in the recombinant alga of the invention, it was found to have mutated to valine and was found to be an E723V mutation. Although the invention has been described with reference to the preceding examples, it is understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A recombinant algal cell comprising: a) a genetic modification in a nucleic acid sequence encoding an enzyme of the trehalose biosynthesis pathway; b) a genetic modification in a nucleic acid sequence encoding an RNA-binding domain; wherein the recombinant alga exhibits increased lipid productivity compared to a corresponding control algal cell lacking the genetic modification.

2. The recombinant algal cell according to claim 1, wherein the genetic modification results in an attenuation of the expression of the nucleic acid sequence having the genetic modification.

3. The recombinant algae according to any of claims 1 to 2 comprising a genetic modification in the nucleic acid sequence encoding the enzyme of the trehalose biosynthesis pathway and a genetic modification in the nucleic acid sequence encoding the RNA-binding domain.

4. The recombinant algae according to any of claims 1 to 3, wherein the recombinant algae is a chlorophyll algae.

5. The recombinant algae according to any of claims 1 to 4, wherein the enzyme of the trehalose biosynthesis pathway is trehalose-6-phosphate synthase or a trehalose-6-phosphate phosphatase.

6. The recombinant algae according to any of claims 1 to 5, wherein the enzyme of the trehalose biosynthesis pathway is trehalose-6-phosphate synthase / phosphatase.

7. The recombinant algae according to any of claims 1 to 5, wherein the enzyme of the trehalose biosynthesis pathway is selected from one or more enzymes from the group consisting of: trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase and trehalose-6-phosphate nefr / nn / zznz / E / YiAi synthase / phosphatase.

8. The recombinant algae according to any of claims 1 to 7, further comprising an attenuation of a nucleic acid sequence encoding an SGI1 polypeptide.

9. The recombinant algae according to any of claims 1 to 8, wherein the genetic modification of the nucleic acid sequence encoding the RNA-binding domain is a functional deletion.

10. The recombinant alga according to any of claims 1 to 9, further comprising that the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase comprises a substitution mutation against a trehalose-6-phosphate synthase / phosphatase sequence in the corresponding control algal cell.

11. The recombinant algae according to claim 10, further comprising that the nucleic acid sequence encoding the trehalose-6-phosphate synthase / phosphatase has at least 80% sequence identity with SEC. ID No.:

2.

12. The recombinant algae according to any of claims 1 to 11, further comprising that the genetic modification of the nucleic acid sequence encoding the RNA-binding domain is a functional deletion or interruption.

13. The recombinant alga according to any of claims 1 to 12, wherein the nucleic acid sequence encoding the RNA-binding domain has at least 80% sequence identity with SEC. ID No.:

1.

14. The recombinant algae according to any of claims 1 to 13, wherein the algae is of the Class Trebouxiophyceae.

15. The recombinant alga according to claim 10, wherein the substitution mutation in the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase comprises an E723V mutation versus the wild-type sequence, and the recombinant algal cell is an alga of the genus Parachlorella.

16. The recombinant algae according to claim 3, wherein the genetic modification of the nucleic acid sequence encoding the trehalose biosynthetic enzyme and the nefr / nn / zznz / E / YiAi nucleic acid sequence encoding the RNA-binding domain results in an attenuation in the expression of each of the nucleic acid sequences.

17. The recombinant algae according to any of claims 1 to 16, wherein the recombinant algae has at least 50% more lipid productivity than a control algae.

18. The recombinant algae according to claim 17, wherein the recombinant algae has at least 75% more lipid productivity compared to a control algae.

19. The recombinant algae according to any of claims 1 to 18, wherein the recombinant algae has at least 5 grams per square meter per day of lipid production.

20. The recombinant algae according to any of claims 1 to 19, wherein the recombinant algae has a higher biomass productivity per unit time.

21. The recombinant algae according to any of claims 1 to 20, wherein the recombinant algae has increased biomass productivity under nitrogen deficiency conditions.

22. The recombinant algae according to any of claims 1 to 21, wherein the recombinant algae has a higher total organic carbon production under nitrogen deficiency conditions.

23. The recombinant algae according to any of claims 1 to 22, wherein the recombinant algae is a chlorophyll algae of a genus selected from the group consisting of: Chlorella, Parachlorella, Picochlorum, Tetraselmisy, and Oocystis.

24. The recombinant algae according to any of claim 23, wherein the recombinant algae is an algae of the genus Parachlorella.

25. The recombinant algae according to any of claims 1 to 22, wherein the recombinant algae is a chlorophyte algae. nefr / nn / zznz / E / YiAi 26. A method for producing a lipid-containing composition comprising: cultivating an algal organism comprising a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme; or a genetic modification in a nucleic acid sequence encoding an RNA-binding domain; and thereby producing a lipid-containing composition.

27. The method according to claim 28, wherein the algal organism comprises a genetic modification in a nucleic acid sequence encoding a trehalose biosynthetic enzyme, and a genetic modification in a nucleic acid sequence encoding an RNA-binding domain.

28. The method according to any of claims 26 to 27, wherein the algal organism further comprises an attenuation in the expression of a nucleic acid sequence encoding an SGI1 polypeptide.

29. The method according to any of claims 26 to 28, wherein the enzyme of the trehalose biosynthesis pathway is selected from one or more enzymes from the group consisting of: trehalose-6-phosphate synthase, trehalose-6-phosphate phosphatase, and trehalose-6-phosphate synthase / phosphatase.

30. The method according to claim 29, wherein the trehalose biosynthetic enzyme is trehalose-6-phosphate synthase / phosphatase.

31. The method according to any of claims 26 to 30, further comprising a step of treating the algal organism with UV radiation prior to the cultivation step.

32. The method according to any of claims 26 to 31, further comprising collecting a lipid composition from the algal organism.

33. The recombinant algae according to any of claims 26 to 32, wherein the genetic modification of the sequence encoding an RNA-binding domain is a functional deletion.

34. The recombinant alga according to any of claims 26 to 33, wherein the genetic modifications result in an attenuation of the expression of the nucleic acid sequence encoding the enzyme of the trehalose biosynthesis pathway. nefr / nn / zznz / E / YiAi 35. The recombinant alga according to any of claims 26 to 34, wherein the genetic modification in the nucleic acid sequence encoding trehalose-6-phosphate synthase / phosphatase comprises an E723V mutation and the recombinant algal cell is an alga of the genus Parachlorella.

36. The recombinant algae according to any of claims 26 to 35, wherein the recombinant algae has at least 50% more lipid productivity than a control algae.